Socio-demographical Profile of 7285 SARS-Cov-2 Positive Early Cases; Comparison with National Four Epidemic Waves.

Background: Objectives were to investigate aspects of the COVID-19 epidemics via testing the individuals who were referred to Aramesh Medical Laboratory in Tehran and to integrate the molecular results with epidemiological data since the beginning of the epidemic. Methods: In this cross-sectional Study 77528 outpatients were referred to Aramesh Medical laboratory by physicians for the diagnosis of SARS-CoV-2 infection between March 2019 and May 2021. Viral acid nucleic extracted from nasal and throat specimens and subsequently amplified using Reverse Transcriptase Real-Time PCR. Laboratory data including Ct values compared with epidemic peaks of COVID-19 countrywide. Statistical Analysis was done by SPSS 21 Software. Results: 14312 (18.46%) tested positive.36.5% of the positive cases were in the 30 to 39 years old age group. The positive result rate was significantly different based on months, ranging from 6% to 28%, compatible with four recognized epidemic peaks encompassing the end of March through the first week of April (first epidemic peak), from June to July 2020 (second epidemic peak), October until mid of November 2020 (third epidemic wave) followed by the end of April to May 2021 (until the end period of study, in the middle of 4th peak). In 37.8% of cases, the Ct value was between 21 and 28. Two separate trends were seen for Ct ≤ 25 and Ct ≤ 20 for the first and fourth epidemic peaks, respectively. There was an association between the number of total monthly positive results and total deaths in the country, especially with the second to third peaks (in the course of summer 2020) and fourth epidemic peak. Conclusion: It might be useful to consider laboratory admission rates as an indicator for changes in the epidemic level in the country to continue the SARS-CoV-2 surveillance in accordance with public decision-makers.

Inhibitory effects of postbiotic consisting sonication-killed Bifidobacterium bifidum on experimental triple negative breast neoplasm in mice: a preliminary study.

Background and Objectives: Breast cancer is the second leading cause of death and one of the most common malignancies among women in the world. The aim of this study was to investigate the preventive effects of postbiotic consisting of sonicated Bifidobacterium bifidum cells on triple negative breast cancer. Materials and Methods: Thirty-six female BALB/c mice aged 5-7 weeks were randomly divided into 3 groups (n=12): Ctrl-, healthy mice; Ctrl+, mice with breast cancer with no treatment; and Postbiotic, mice with orally gavage postbiotic before and after 4T1 cell line transplantation. Cancer progress and the effects of postbiotic were assayed by histological, immunohistochemical and gene expression quantification. Results: The histological results showed that administration postbiotic consisting of B. bifidum significantly decreased carcinogenesis in terms of tumor incidence, multiplicity and volume. The tumor progress was suppressed by oral intake of B. bifidum as showed by p53 and Ki-67 expression. Furthermore, Oral intake of postbiotic resulted in extended survival of mice and inhibited sever weigh loss. Conclusion: Pretreatment with sonication killed B. bifidum, as a postbiotic, inhibited breast cancer progress and malignancy.

The Association of EBV and HHV-6 Viral Load with Different NK and CD8+ T Cell Subsets in The Acute Phase of Relapsing-Remitting Multiple Sclerosis.

OBJECTIVE: Epstein-Barr virus (EBV) and Human Herpes virus 6 (HHV-6) are believed to involve in multiple sclerosis (MS) pathogenesis. Natural killer (NK) and CD8+ T cells have essential roles in handling viral infections and their phenotypic and functional properties may be influenced following exposure to viral infections. Here, we investigated the association of NK and CD8+ T cells subpopulations frequency with EBV and HHV-6 viral load in MS patients. MATERIALS AND METHODS: In this case-control study, EBV and HHV-6 viral load were evaluated in plasma of newly diagnosed relapsing-remitting MS (RRMS) patients at relapse phase (n=23), who were not on disease-modifying therapy (DMT), and sex- and age-matched healthy controls (n=19) using real-time polymerase chain reaction (PCR). The frequency of NK and CD8+ T cells subsets were assessed by CD27, CD28, CD45RO, CD56, and CD57 markers using flow cytometry. RESULTS: Despite the increased level of EBV viral load in RRMS patients compared to the control group, there was no statistically significant difference in EBV and HHV-6 copy numbers between the studied groups. In addition, a significant decrease was observed in the percentages of CD56bright CD57- and CD56dim CD57+ CD8low CD45RO- NK cells in RRMS patients in comparison to healthy controls. Analysis of CD8+ T cell subsets showed a substantially high proportion of CD27+ CD28+ CD45RO+ CD57- CD8hi T cells in patients at relapse phase compared to controls. The frequency of NK and T cells subtypes was not associated with EBV and HHV6 plasma viral loads. CONCLUSION: These findings further highlight the variation of NK and CD8+ T cells subsets frequency in clinically active RRMS patients. Since the composition of cells was not associated with EBV and HHV-6 viral load, perhaps other viral infections may be involved in altered NK and CD8+ T cells subpopulation. Larger cohort studies are needed to confirm these results.

Survey of HCMV in allogenic and autologous stem cell transplantation by real-time PCR in Kermanshah, west of Iran.

INTRODUCTION: Human Cytomegalovirus (HCMV) is the most important viral pathogen in people undergoing bone marrow transplantation (BMT). HCMV detection in the early stages makes is possible to save the patients' lives through immediate and timely treatment. The aim of this study was to investigate the status of HCMV using the real-time PCR method in BMT patients in Kermanshah, west of Iran. METHODS: HCMV monitoring was done in 120 patients who underwent BMT, 38 allogeneic cases and 82 autologous cases, using the ELISA serology test before transplantation. The participants were followed up 100 days after transplantation for HCMV detection in blood samples using real-time PCR. Preemptive therapy started with Ganciclovir and Foscarnet when the viral load was > 200 HCMV DNA copies/ml. RESULTS: Despite preemptive therapy, infection recurred in less than 1 month. HCMV recurred more frequently in patients undergoing allogenic transplation versus those receiving autologous transplantation. Recurrence was seen in 5 patients receiving allogenic transplantation. HCMV recurrence occurred in five patients with allogeneic transplantation. Twelve patients undergoing allogeneic or autologous transplantation (83%) and a virus load of > 1000 copies/ml showed HCMV-related symptoms. Three patients died, two due to HCMV-related pneumonia and the other one due to a fungal infection. CONCLUSION: Real-time PCR may be a useful method for quantification and monitoring of HCMV recurrence and may be helpful in choosing more efficient HCMV preemptive treatment in BMT recipients.

Toxigenic and non-toxigenic patterns I, II and III and biofilm-forming ability in Bacteroides fragilis strains isolated from patients diagnosed with colorectal cancer.

BACKGROUND: Enterotoxigenic Bacteroides fragilis (ETBF) associated with the initiation and progression of colorectal cancer (CRC) has been alarmingly reported all over the world. In this study, simultaneous investigation of toxigenic and non-toxigenic patterns I, II and III and biofilm formation ability of Bacteroides fragilis isolated from patients with colorectal cancer was performed. METHODS: Thirty-one patients diagnosed with CRC and thirty-one control subjects were recruited in this study. Specimens were cultured on BBE and BBA culture media. Classical phenotypic identification tests and PCR was performed to verify Bacteroides fragilis presence. Also, biofilm-forming ability and expression of bft gene were assessed under biofilm and planktonic forms. RESULTS: A total of 68 B.fragilis was isolated from all colorectal tissue, of which 13 isolates (19.1%) (11 isolates from CRC and 2 from normal tissue) were positive for bft gene. The abundance patterns of I, II and III were as follow in descending order; pattern I > pattern III > pattern II in CRC subjects and pattern II > pattern III > pattern I in normal tissues. Also, pattern I showed higher biofilm formation ability compared to other patterns. Toxin expression was significantly reduced in biofilm form comparing with planktonic form. CONCLUSIONS: Based on our findings, there was a difference between the abundance of patterns I, II, and III and biofilm formation in isolates obtained from CRC and normal tissues. Biofilm formation ability and toxin encoding gene (bft) are two main virulence factors in B. fragilis pathogenicity which require more investigation to treat B. fragilis infections effectively.

Detection of enterotoxigenic Bacteroides fragilis in patients with ulcerative colitis.

PURPOSE: Ulcerative colitis (UC) as a type of inflammatory bowel disease (IBD), presumed to occur as a consequence of increased immune responses to intestinal microbiota in genetically susceptible individuals. Enterotoxigenic Bacteroides fragilis (ETBF) strains are important intestinal bacteria that can be involved in IBD. The aim of this study was to design a quantitative assay for detection of B. fragilis and ETBF and also to find their association with UC. METHODS: Ninety-five biopsies were collected from patients with UC (n = 35) and with no IBD (nIBD, n = 60). All the specimens were cultured in Bacteroides bile esculin agar medium. Specific primers and probes were designed for quantitative real-time PCR (QRT-PCR) based on 16S rRNA and bft genes sequences of ETBF. RESULTS: The bft genes were detected in 51.4% of UC samples and 1.6% of nIBD samples, respectively. In UC patients, 37.1% of samples with diarrhea and 11.4% of samples without diarrhea, harbored the bft gene. Mean value of the number of ETBF with bft gene in UC and nIBD samples were 4.46 ×Y 102 and 1.96, respectively. Likewise these result for 16S rRNA gene in UC and nIBD samples were 2.0 × 103 and 8.4 × 103, respectively. CONCLUSIONS: There was no significant association between presence and numbers of 16S rRNA gene of B. fragilis and UC. ETBF was detected more in UC specimens and biopsies of UC patients with diarrhea than in the control group. These data demonstrated that ETBF is associated with development of UC and as a causative agent for the development of diarrhea in these patients.

Identification and differentiation of Campylobacter species by high-resolution melting curve analysis.

BACKGROUND: Campylobacter jejuni and Campylobacter coli are the important food-born zoonotic pathogen, also are leading causes of human food borne illnesses worldwide. cadF gene is expressed in all C. jejuni and C. coli strains and mediates cell binding to the cell matrix protein, Fibronectin. High-resolution melting (HRM) analysis is emerging as an efficient and robust method for discriminating DNA sequence variants. The goal of this study was to apply HRM analysis to identification of C. jejuni and C. coli. MATERIALS AND METHODS: A total of 100 samples were obtained from chicken in Kermanshah, Iran. HRM analysis based on cadF gene and Eva Green was developed to the identification of Campylobacter to the species level. RESULTS: Fifty-five of 100 samples were positive as campylobacter (7 C. jejuni, 29 C. coli, 16 mixes and 3 unknown). Minor variations were observed in melting point temperatures of C. coli or C. jejuni isolates and this variation can be used to the differentiation between C. coli or C. jejuni isolates. CONCLUSIONS: The results of this study indicated that HRM curve analysis can be a suitable technique and rapid technique for distinguishing between C. jejuni and C. coli isolates.

Prevalence and Clonal Dissemination of Metallo-Beta-Lactamase-Producing Pseudomonas aeruginosa in Kermanshah.

BACKGROUND: Pseudomonas aeruginosa is an opportunistic pathogen associated with nosocomial infections. The emergence and dissemination of metallo-beta-lactamases (MBLs) has contributed to the high rate of resistance among P. aeruginosa isolates. OBJECTIVES: The purpose of this study was to describe the prevalence and the clonal dissemination of MBL- producing P. aeruginosa isolates collected from major hospitals in Kermanshah. MATERIALS AND METHODS: Antibiotic susceptibility testing was performed using the minimal inhibitory concentrations. The MBLs were investigated using the Double-Disk Synergy Test (DDST) and Polymerase Chain Reaction. Molecular typing was performed by Pulsed-Field Gel Electrophoresis (PFGE). RESULTS: Of the 60 P. aeruginosa isolates included in this study, 30 (50%) were resistant to Gentamicin, 38 (63.3%) to Piperacillin, 42 (70%) to Ceftazidime, and 45 (75%) to Cefepime. Twenty-nine (48.3%) isolates were MBL producers in the DDST test. Five (8.3%) isolates were positive for the VIM gene. PFGE analysis among the MBL producers revealed 12 distinct clonal patterns. CONCLUSIONS: The inter- and intra-hospital dissemination of resistant clones is a matter of concern and is an indicator of the level of the improvement and surveillance of standard hygiene, particularly disinfection and hand washing before and after contact with patients. Given the emergence of MBL-producing strains, surveillance has become an important procedure to control the transmission of resistant strains.

Comparison of smear microscopy, culture, and real-time PCR for quantitative detection of Mycobacterium tuberculosis in clinical respiratory specimens.

BACKGROUND: As a rapid diagnostic technique, the real-time polymerase chain reaction (PCR) can detect Mycobacterium tuberculosis with a high sensitivity and specificity. However, further studies are needed to confirm it as a standard method. In this study, we evaluated the cyp141 gene for the detection and quantification of M. tuberculosis in respiratory specimens and compared the results with direct microscopy and culture. METHODS: Sputum samples (n = 247) were collected from patients of the different provinces of Iran. DNA was extracted from clinical specimens and H37Rv strain. After measuring the standard strain DNA concentration by NanoDrop and using the Avogadro number, the DNA was diluted 6 times in order to obtain 1 × 10(6) to 10 template copies. A Taqman probe was designed for detection of the target in a real-time PCR using the specific primers. RESULTS: Of 247 samples, 135 (55%) were culture-negative. Of 112 (45%) culture-positive samples, 88 were positive by both smear and culture and 24 were smear-negative but culture-positive. The real-time PCR enumerated 1.5E + 02 to 4.3E+ 03, 8.5E + 03 to 5.5E + 04, 7.2E + 04 to 1.1E + 06, and 1.2E + 06 to 8.1E + 07 M. tuberculosis cells in the specimens with smear-negative, 1-plus, 2-plus, and 3-plus codes, respectively. The overall sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the real-time PCR were 90.2% (101/112), 97.8% (132/135), 97.1%, and 92.3%, respectively. CONCLUSIONS: The overall sensitivity and specificity, the results in comparison with those of the Xpert MTB/RIF kit, and the good correlation with molecular and phenotypic methods, show that cyp141 could be a good target for the quantification of M. tuberculosis in sputum and possibly other clinical specimens.

Hospital acquired pneumonia: comparison of culture and real-time PCR assays for detection of Legionella pneumophila from respiratory specimens at Tehran hospitals.

Legionella pneumophila is an important etiological agent in both hospital and community acquired pneumonia. The sensitivity of culture for isolation of L. pneumophila from clinical specimens is low and time consuming. Similar problem also exists when the method of direct immunofluorescence is used. To detect this organism quantitatively from respiratory specimens, a Taq Man based real-time PCR targeting the mip sequence was developed. Both real-time PCR and culture methods were applied on 262 respiratory specimens from 262 ICU patients with pneumonia admitted to 5 different hospitals in Tehran. The results of real-time PCR were compared with those obtained by culture. Real-time PCR and culture found 12 and 4 specimens, respectively, as positive for L. pneumophila. Its technical specificity (100%) was checked against a panel of microorganisms consisting of both Gram-positive and Gram-negative bacteria. Our real-time PCR assay showed high sensitivity (100%) and specificity (96.9%) and could detect 200 organisms per ml from respiratory specimens. Using real-time PCR as a screening method, the frequency of nosocomial pneumonia with L. pneumophila at Tehran hospitals was estimated as 4.58%.

Single tube real time PCR for detection of Streptococcus pneumoniae, Mycoplasma pneumoniae, Chlamydophila pneumoniae and Legionella pneumophila from clinical samples of CAP.

We designed a multiplex real time PCR for rapid, sensitive and specific detection of Streptococcus pneumoniae, Legionella pneumophila, Chlamydophila pneumoniae and Mycoplasma pneumoniae. The study cases consisted of 129 patients with community acquired pneumonia (CAP). Bacteriological techniques were implemented for detection of the cultivable organisms. DNA were extracted from sputa, throat swabs, bronchoalveolar lavages and tracheal aspirates and used as templates in real time PCR. The primers and probes were designed for cbpA (S. pneumoniae), p1adhesin (M. pneumoniae), mip (L. pneumophila) and ompA (C. pneumoniae). After optimization of real time PCR for every organism, the experiments were continued in multiplex in a single tube. Of 129 CAP specimens, the positive cultures included 14 (10.85%) for S. pneumoniae, 9 (6.98%) for L. pneumophila and 3 (2.33%) for M. pneumoniae. Four specimens (3.10%) were positive for C. pneumoniae by real time PCR. The sensitivity of our real time PCR was 100% for all selected bacteria. The specificity of the test was 98.26%, 98.34%, 100% and 100% for S. pneumoniae, L. pneumophila, M. pneumoniae and C. pneumoniae, respectively. This is the first report on the use of multiplex real time PCR for detection of CAP patients in the Middle East. The method covers more than 90% of the bacterial pathogens causing CAP.

Application of fnbA gene as new target for the species-specific and quantitative detection of Staphylococcus aureus directly from lower respiratory tract specimens by real time PCR.

Staphylococcus aureus is a significant cause of hospital-acquired pneumonia (HAP), particularly in mechanically ventilated patients. We used the fibronectin-binding protein A gene (fnbA) for the species-specific and quantitative detection of S. aureus directly from lower respiratory tract (LRT) specimens by a Taq Man real time PCR. For this reason, a total of 269 lower respiratory tract (LRT) specimens collected from patients with hospital-acquired pneumonia were assayed. Amplification of fnbA in serial dilutions ranged from 10(9) CFU/ ml to 10(2) CFU/ml. Standard curve of triplicate every dilution had slope 3.34±0.1 and R2>0.99 with SD 0.1. Based on these data, the sensitivity and specificity of the newly developed real time PCR targeting the fnbA gene were both 100%. The Cohen's Kappa test showed the Kappa value of 1.0. The fnbA gene is a potential marker for the species-specific detection of S. aureus and can be used to detect this bacterium in any clinical specimens by real time PCR. Moreover, this method reduces the time needed for quantitative detection of Staphylococcus aureus from LRT specimens to nearly 2 hours compared to 1 to 4 days for culture and provided sensitivity equal to or greater than culture.

Development of a modified DNA extraction method for pulsed-field gel electrophoresis analysis of Staphylococcus aureus and enterococci without using lysostaphin.

A modified pulsed-field gel electrophoresis (PFGE) protocol was developed and applied to clinical isolates of Staphylococcus aureus and enterococci to reduce the cost of using lysostaphin. This protocol reduces the expenses of PFGE typing of S. aureus and enterococci as it removes the use of lysostaphin during the spheroplast formation from these bacteria.

Direct detection of Pseudomonas aeruginosa from patients with healthcare associated pneumonia by real time PCR.

Using oprL sequences, a TaqMan real time PCR was developed and used for quantitative detection of Pseudomonas aeruginosa from 99 broncoalveolar lavage and 11 sputum specimens collected from patients with health care associated pneumonia. All specimens were cultured on appropriate media to isolate bacteria. Twenty five specimens were positive by both methods. Polymicrobial infections were found in 13 specimens. Amplification of oprL in serial dilutions ranged from 10(9)CFU/ml to 10(2)CFU/ml. Standard curve of duplicated every dilution had slope 3.25±0.1 and R(2)>0.99 with SD 0.1. Our real time PCR assay showed high sensitivity (100%) and specificity (98.85%). This technique could detect and enumerate 100 bacteria directly from clinical specimens and showed that the threshold is 10(3)CFU/ml in cases with clinical symptoms. Our method can be used for quantitative detection of P. aeruginosa from BAL and sputum specimens in 1h and 10min.