Characterization of local SARS-CoV-2 isolates and pathogenicity in IFNAR-/- mice.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) recently a global pandemic with unprecedented public health, economic and social impact. The development of effective mitigation strategies, therapeutics and vaccines relies on detailed genomic and biological characterization of the regional viruses. This study was carried out to isolate SARS-CoV-2 viruses circulating in Anatolia, and to investigate virus propagation in frequently-used cells and experimental animals. We obtained two SARS-CoV-2 viruses from nasopharngeal swabs of confirmed cases in Vero E6 cells, visualized the virions using atomic force and scanning electron microscopy and determined size distribution of the particles. Viral cytopathic effects on Vero E6 cells were initially observed at 72 h post-inoculation and reached 90% of the cells on the 5th day. The isolates displayed with similar infectivity titers, time course and infectious progeny yields. Genome sequencing revealed the viruses to be well-conserved, with less than 1% diversity compared to the prototype virus. The analysis of the viral genomes, along with the available 62 complete genomes from Anatolia, showed limited diversity (up to 0.2% on deduced amino acids) and no evidence of recombination. The most prominent sequence variation was observed on the spike protein, resulting in the substitution D614G, with a prevalence of 56.2%. The isolates produced non-fatal infection in the transgenic type I interferon knockout (IFNAR-/-) mice, with varying neutralizing antibody titers. Hyperemia, regional consolidation and subpleural air accumulation was observed on necropsy, with similar histopathological and immunohistochemistry findings in the lungs, heart, stomach, intestines, liver, spleen and kidneys. Peak viral loads were detected in the lungs, with virus RNA present in the kidneys, jejunum, liver, spleen and heart. In conclusion, we characterized two local isolates, investigated in vitro growth dynamics in Vero E6 cells and identified IFNAR-/- mice as a potential animal model for SARS-CoV-2 experiments.

[Multilocus sequence analysis, biofilm production, antibiotic susceptibility and synergy tests of Burkholderia species in patients with and without cystic fibrosis]. / Kistik fibrozisli ve kistik fibrozisi olmayan hasta grubunda Burkholderia türlerinin multilokus sekans analizi, biyofilm olusturma, antibiyotik duyarlilik ve sinerji testleri.

Burkholderia spp. emerged as important pathogens in the airways of immunocompromised humans, especially those with cystic fibrosis (CF). Failure of identification with conventional techniques, high intrinsic resistance to most antibiotics and biofilm formation can cause difficulties in the treatment of these infections. The aim of this study was to identify Burkholderia spp. strains isolated from CF and non-CF patients with with routine microbiological methods, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and multilocus sequence analysis (MLSA), to determine of the antibiotic susceptibility and synergies, and to evaluate biofilm formation of these isolates. A total of 38 Burkholderia spp. (25 CF, 13 non-CF) from 26 patients were identified by biochemical, phenotypical and matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and sequence types were revealed by multilocus sequence analysis (MLSA). Sequence types of isolates were identified using the PubMLST database. Characteristics of biofilm formation of clinical isolates were evaluated by microplate method. Antibiotic susceptibilities of ceftazidime, meropenem, trimethoprim-sulfamethoxazole (TMP-SXT) and levofloxacin were determined by broth microdilution method according to CLSI (2017) guidelines. Synergy tests were performed by checkerboard method. Clinical isolates were identified as Burkholderia cenocepacia (n= 16), Burkholderia contaminans (n= 11), Burkholderia gladioli (n= 4), Burkholderia dolosa (n= 4), Burkholderia multivorans (n= 2) and Burkholderia seminalis (n= 1). Sequence types of these isolates were determined as ST19, ST72, ST102, ST180, ST482, ST602, ST629, ST740, ST839 and ST1392. The correct identification at the species-level with MALDI-TOF MS was 94-100% for all isolates except B.contaminans. Biofilm formation among the identified species in the study was determined as 53% (n= 20). There was no statistical difference when the biofilm production was evaluated separately among Burkholderia species and biofilm production rates between CF (56%, 14/25) and non-CF (46%, 6/13) Burkholderia isolates (p> 0.05). Overall rates of resistance to ceftazidime, meropenem, TMP-SXT, and levofloxacin of the isolates were 35%, 66%, 50% and 40%, respectively. The antibiotic resistance against Burkholderia spp., isolates obtained from CF patients were more susceptible to ceftazidime, but no significant difference was found for other antibiotics. Synergy was determined between meropenem and TMP-SXT in two isolates. Antagonism was detected in 15 isolates, 12 of them were between meropenem and ceftazidime, three of them were between ceftazidime and TMP-SXT. Numerous resistance mechanisms may lead to higher resistance in this bacteria, whereas the antagonism between meropenem and ceftazidime in this study might be attributed to the expression of beta-lactamases. In this study, the distinctness of sequence types between Burkholderia spp. isolated from CF and non-CF patient, provided a better understanding about the importance of biofilm formation for the infections with these bacteria and emphasized that the management of therapy should be driven by the antibiotic test results.

PPAR-alpha agonist treatment increases trefoil factor family-3 expression and attenuates apoptosis in the liver tissue of bile duct-ligated rats.

BACKGROUND/AIMS: Peroxisome proliferators-activated receptor alpha activation modulates cholesterol metabolism and suppresses bile acid synthesis. The trefoil factor family comprises mucin-associated proteins that increase the viscosity of mucins and help protect epithelial linings from insults. We evaluated the effect of short-term administration of fenofibrate, a peroxisome proliferators activated receptor alpha agonist, on trefoil factor family-3 expression, degree of apoptosis, generation of free radicals, and levels of proinflammatory cytokines in the liver tissue of bile duct-ligated rats. MATERIALS AND METHODS: Forty male Wistar rats were randomly divided into four groups: 1 = sham operated, 2 = bile duct ligation, 3 = bile duct-ligated + vehicle (gum Arabic), and 4 = bile duct-ligated + fenofibrate (100 mg/kg/day). All rats were sacrificed on the 7 th day after obtaining blood samples and liver tissue. Liver function tests, tumor necrosis factor-alpha and interleukin 1 beta in serum, and trefoil factor family-3 mRNA expression, degree of apoptosis (TUNEL) and tissue malondialdehyde (malondialdehyde, end-product of lipid peroxidation by reactive oxygen species) in liver tissue were evaluated. RESULTS: Fenofibrate administration significantly reduced serum total bilirubin, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, gamma-glutamyl transferase, and tumor necrosis factor-alpha and interleukin-1ß levels. Apoptosis and malondialdehyde were significantly reduced in the fenofibrate group. Trefoil factor family-3 expression increased with fenofibrate treatment in bile duct-ligated rats. CONCLUSIONS: The peroxisome proliferators-activated receptor alpha agonist fenofibrate significantly increased trefoil factor family-3 expression and decreased apoptosis and lipid peroxidation in the liver and attenuated serum levels of proinflammatory cytokines in bile duct-ligated rats. Further studies are needed to determine the protective role of fenofibrate in human cholestatic disorders.

Differential identification of atypical pneumonia pathogens in aorta and internal mammary artery related to ankle brachial index and walking distance.

BACKGROUND: We studied the existence of agents in aorta biopsies, such as Chlamydia pneumoniae, cytomegalovirus, and Mycoplasma pneumoniae, that are thought to have a role in atherosclerosis etiopathogenesis role, and their association with peripheral artery disease. MATERIALS AND METHODS: We examined aorta wall and internal mammarian artery (IMA) biopsies taken from two different places in 63 patients in whom coronary artery bypass was performed. In these biopsies, we evaluated the deoxyribonuclease (DNA) of these microorganisms using polymerase chain reaction. From the same patients, we recorded the ankle brachial index, road walking distance information, lipid profile, C-reactive proteins, blood parameters such as fibrinogen, and the patient's operation data. RESULTS: In the nine aorta biopsies taken from 63 patients, we isolated C pneumoniae DNA. In IMA biopsies taken from the same patients, we detected no microorganism DNA (P < 0.001). In the same aorta biopsies, we found no cytomegalovirus or M pneumoniae DNA. We examined 12 patients using an index value of 0.9 in the ankle brachial index evaluation; eight had C pneumoniae in the aorta biopsies (P < 0.001). CONCLUSIONS: We found a significant relationship between C pneumoniae DNA and the existence of peripheral artery disease. In the development of atherosclerosis with C pneumoniae, there may be a determinant pathogen in both the aorta and the peripheral arteries. The nonexistence of C pneumoniae DNA in the IMA biopsies may indicate infectious agents because of the predominant endothelial functions in this artery, and thus its resistance to atherosclerosis.

Bacterial adhesion and colonization differences between zirconium oxide and titanium alloys: an in vivo human study.

PURPOSE: The purpose of this study was to compare zirconium oxide and titanium alloys with respect to their tendency to adhesion and colonization of two periodontal pathogens on both hard surfaces and on soft tissues in vivo. MATERIALS AND METHODS: The present study was designed as a prospective stratified randomized controlled clinical trial. Patients were scheduled to receive two implants with different types of abutments in the posterior mandible. Three months after implant placement, titanium and zirconium abutments were connected. Five weeks after abutment connections, the abutments were removed, probing depth measurements were recorded, and gingival biopsy samples were obtained. Abutments and biopsy specimens were analyzed by reverse-transcriptase polymerase chain reaction to compare the DNA copy numbers of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and total bacteria. The surface free energy of the abutments was calculated by sesile water drop before replacement. RESULTS: No statistically significant differences were found between probing depths or DNA copy numbers of A actinomycetemcomitans, P gingivalis, and total bacteria both for both titanium alloys and zirconium oxide surfaces and the biops specimens obtained from their buccal gingival. With respect to the surface free energy of zirconium and titanium abutments, zirconium abutments showed lower surface free energy than titanium abutments. CONCLUSION: The results of this study showed that zirconium oxide surfaces have comparable properties to titanium alloy surfaces in their tendency to adhesion and colonization of two periodontal pathogens on both hard surfaces and in soft tissues.

Topical N-acetylcysteine reduces interleukin-1-alpha in tear fluid after laser subepithelial keratectomy.

PURPOSE: To evaluate the effect of topical N-acetylcysteine (NAC) on interleukin 1-alpha (IL-1alpha) levels in tear fluid after myopic laser subepithelial keratectomy (LASEK) and its possible role in modulating corneal wound healing. METHODS: Twenty-six eyes of 13 patients who underwent myopic LASEK were divided into 2 groups. Group 1 (n=10 eyes) was used as a control group. All patients received topical lomefloxacin and dexamethasone postoperatively. Additionally, patients in Group 2 received topical NAC for 1 month postoperatively. Tear fluid samples were collected with microcapillary tubes preoperatively, on the first and on the fifth postoperative day, and the release of IL-1alpha in tear fluid was calculated. Haze grading and confocal microscopic examination were performed at 1 and 3 months postoperatively. RESULTS: The mean IL-1-alpha release values were 0.285-/+0.159 pg/min in Group 1 and 0.235-/+0.142 pg/min in Group 2 preoperatively. In Group 1, the values were 0.243-/+0.155 pg/min on day 1 and 0.164-/+0.125 pg/min on day 5. In Group 2, the mean IL-1alpha release values were 0.220-/+0.200 pg/min on day 1 and 0.080-/+0.079 pg/min on day 5. The difference between the groups was significant only for day 5 (p<0.05). Mean corneal haze score and grey scale value in confocal microscopy were significantly higher (p<0.05) in Group 1 at 1 month. However, at 3 months there was no difference between groups (p>0.05). CONCLUSIONS: NAC seems to have an additive effect to steroids in suppressing IL-1alpha levels in tear fluid and may be clinically advantageous in modulating corneal wound healing during the early postoperative period after LASEK.

Could the simplified (14)C urea breath test be a new standard in noninvasive diagnosis of Helicobacter pylori infection?

OBJECTIVE: The carbon-14 ((14)C) urea breath test (UBT) is a reliable and noninvasive technique for the diagnosis of Helicobacter pylori (HP) infection. The diagnostic performance of a new practical and low dose (14)C UBT system (Heliprobe, Stockholm, Sweden) was compared with those of other diagnostic tests, namely, rapid urease test (RUT), histopathology, and DNA detection using polymerase chain reaction (PCR). METHODS: Eighty-nine patients (mean age = 45 +/- 13, 30 men) with dyspeptic complaints who underwent an endoscopic procedure were studied. Biopsy specimens acquired during the procedure were subjected to RUT, histopathological examination using hematoxylin and eosin (HP-HE) and PCR. All patients underwent UBT using the Heliprobe system on a different day. The gold standard for HP positivity was defined as any two of the three tests being positive, excluding UBT, and the sensitivity and specificity of any single test alone were determined using this gold standard. Whenever only one test was positive, it was considered to be a false-positive one. RESULTS: With the gold standard used in this study, 59 (66%) patients were diagnosed HP positive. The Heliprobe method detected HP infection with 96.6% sensitivity and 100% specificity and had the best diagnostic performance when compared with all the other methods. The sensitivity and specificity of the other methods for the detection of HP positivity were 89.8% and 100% for RUT, 93.2% and 63.3% for PCR, and 93.2% and 76.6% for HP-HE, respectively. Areas under the receiver-operating characteristic were 0.977 for UBT, 0.947 for RUT, 0.84 for HP-HE, and 0.775 for PCR. CONCLUSIONS: Using a combination of invasive diagnostic tests as the gold standard, Heliprobe UBT was found to be highly sensitive and specific for the diagnosis of HP infection in patients with dyspeptic complaints.

Identification of Helicobacter species by 16S rDNA PCR and sequence analysis in human liver samples from patients with various etiologies of benign liver diseases.

BACKGROUND/AIMS: Several reports indicated an increased prevalence of the Helicobacter species in hepatocellular cancer tissue and in liver samples infected with hepatitis viruses. The frequency of Helicobacter spp. in benign liver diseases was, however, not thoroughly investigated. METHODS: Seventy-five consecutive patients with suspected liver disease were enrolled. The indications were hepatitis B virus (n=30), C virus (n=8), B and C dual infection (n=1), nonalcoholic steatohepatitis (n=27), autoimmune hepatitis (n=3), primary biliary cirrhosis (n=1) and idiopathic elevation of liver enzymes (n=5). PCR detection of 16S recombinant RNA gene of Helicobacter spp. was performed on liver samples. PCR products of positive samples were further identified by DNA sequencing. The patients also had upper gastrointestinal endoscopy and gastric biopsy for the detection of H. pylori using histopathology and PCR. RESULTS: Helicobacter spp. DNA was detected in two out of 75 liver biopsy samples (2.6%), which were typed as H. pylori by DNA sequencing. One of these patients had chronic hepatitis C infection (man, 51 years old) and the other had nonalcoholic steatohepatitis (woman, 44 years old). Fifty-two out of 75 of the patients (69.3%) had H. pylori infection in their stomachs. CONCLUSION: We have found that H. pylori infection is much less prevalent in benign liver diseases. The presence of H. pylori in nonalcoholic steatohepatitis (NASH) patients is a novel finding and this finding should be confirmed in a larger series.

Infliximab "TNF-alpha antagonist" decreases intraabdominal adhesions.

OBJECTIVE: To evaluate the effect of infliximab on adhesion formation and it's associated morbidity and complications. METHODS: This study was performed in the Faculty of Medicine, Gazi University, Turkey between July 2005 and October 2005. Thirty-five rats were randomly divided into 4 groups. Laparotomy was performed in the Sham group (n=5), whereas cecal abrasion was carried out in all other groups. After cecal abrasion 0.9% sodium chloride was administered in the saline group (n=10), infliximab was administered to the study group (n=10) and nothing was administered to the last group (n=10). Adhesion formation was evaluated with macroscopic and microscopic adhesion scoring systems. Peritoneal fluid samples and mesenteric lymph node biopsies were taken to rule out bacterial peritonitis. Blood and peritoneal irrigation fluid samples were taken to measure the Tumor necrosis factor-alpha (TNF-alpha) levels. RESULTS: Macroscopic adhesion scores showed fewer adhesions in the infliximab group. The infliximab group had significantly fewer adhesions than the abrasion control and saline groups. According to the histological findings, there were no statistically significant differences between the groups. CONCLUSION: Early blocking of the activity of TNF-alpha after cecal abrasion resulted in lower rates of adhesion formation, macroscopically. The TNF-alpha, a proinflammatory cytokine appears to be an important mediator for postoperative adhesion formation.

[Comparison of agar screening plates and double disk synergy method in detection of extended-spectrum beta-lactamase production]. / Genislemis spektrumlu beta-laktamaz uretiminin saptanmasinda agar tarama plaklari ve çift disk sinerji yönteminin karsilastirilmasi.

In this study, extended-spectrum beta-lactamase (ESBL) production of 83 enteric isolates has been investigated by using a new agar screening method described by Storenburg et al. and double disk synergy (DDS) method. Agar screening method has also been evaluated in terms of presumptive bacterial identification. ESBL production was shown in 15 (18.1%) and 17 (20.5%) of 83 isolates by using DDS method with a distance of 25 and 22-20 mm between antibiotic disks, respectively. Agar screening plates demonstrated 16 (19.3%) ESBL positive isolates and was more sensitive compared to DDS method with 25 mm distance between disks. However, agar screening method gave a successful presumptive bacterial identification in only 10 of 16 ESBL positive isolates. In conclusion, the potential of the new agar screening test in direct identification of ESBL production in clinical samples should be evaluated.

Evaluation of the Helicobacter pylori stool antigen test (HpSA) for the detection of Helicobacter pylori infection and comparison with other methods.

BACKGROUND/AIMS: Several invasive and non-invasive methods are available for the detection of H. pylori infection. The accuracy of anti-H. pylori antibodies in serum is low. There is a need for a quick, inexpensive and reliable non-invasive test to detect H. pylori. The aim of this study was to evaluate the enzyme immunoassay for the detection of H. pylori antigen in stool in the Turkish population and compare it to other methods. METHODOLOGY: 50 patients who were admitted to Hacettepe University Department of Internal Medicine, Division of Gastroenterology with the symptom of dyspepsia for whom the indication of upper gastrointestinal endoscopy was present were included in the study. With their permission stool samples were taken. The patients were evaluated with histology, culture, serology, rapid urease test and HpSA (Helicobacter pylori Stool Antigen test). Forty-one patients had gastritis and biopsies were taken from those. RESULTS: Excluding HpSA if three of the rest of four methods were positive, patients were accepted as H. pylori positive. Nineteen patients were positive for H. pylori, 22 were negative. HpSA was positive in 16 of 19. The sensitivity and specificity of the methods were as follows: histology 100% sensitive, and 86% specific, culture 63% and 100%, HpIgG 58% and 73%, rapid urease test 89% and 82%, respectively. The results were as 84% and 82% for HpSA. Comparing with the 'Gold Standard' histology using McNemar's test Kappa results were as 0.610, 0.181, 0.610, 0.708 for culture, HpIgG, Rapid Urease Test and HpSA, respectively. CONCLUSIONS: HpSA is a cheap, effective method for the diagnosis of H. pylori infection in the Turkish population.

The cagA status of Helicobacter pylori isolates from dyspeptic children in Turkey.

There are inconsistent reports regarding cytotoxin-associated gene A (cagA) status of Helicobacter pylori isolates and the severity of the mucosal lesions in children. The aim of this study was to determine the prevalence of cagA(+) strains and to evaluate its correlation with clinic and endoscopic findings. We examined 45 H. pylori strains that were grown on brain-heart infusion agar supplemented with 7% horse blood. Following 72 h of incubation colonies were harvested and bacterial DNA was extracted. Polymerase chain reaction primers F1 and B1 were used to amplify a 348-bp internal fragment of cagA. The prevalence of cagA in Turkish pediatric patients was 55.6%. No association was found between cagA status and the severity of gastro-duodenal lesions.

Viral studies in the cerebrospinal fluid in subacute sclerosing panencephalitis.

OBJECTIVES: The pathogenesis of subacute sclerosing panencephalitis (SSPE), and particularly, the cause of measles virus (MV) reactivation following a latent period after primary measles infection is unknown. The hypothesis of other viruses contributing to the pathogenesis of SSPE by affecting the in vivo state of MV was investigated. METHODS: We examined the cerebrospinal fluid of SSPE patients (n=43) for DNA or RNA and antibodies against HSV type 1 and 2, EBV, CMV, VZV, Hepatitis B, Hepatitis C, JC virus, human herpesvirus (HHV)-6, HHV-7, HHV-8, HTLV-1, and HTLV-2. We compared the findings with those of patients with other neurological disorders (n=39). RESULTS: CMV DNA and HSV type 1 IgG were found more frequently in SSPE patients. Other positive results were at similar incidence in SSPE and control groups. The clinical features of SSPE cases with and without positive viral tests did not differ from each other. CONCLUSION: These data do not support a specific role for these agents in SSPE, but imply that the passage of some viruses to the CNS and local antibody synthesis may be facilitated by inflammation. The persistence or reactivation of MV in SSPE may be related to other factors pertaining to the host or environment.

[Evaluation of western blot methods for serologic diagnosis of Helicobacter pylori infections]. / Helicobacter pylori enfeksiyonlarinin serolojik tanisinda western blot yönteminin degerlendirilmesi.

Recent studies have demonstrated that immunoblot method is more sensitive and specific than enzyme immunoassays (EIA) to diagnose Helicobacter pylori infections, and it also permits the detection of antibody profiles against different bacterial antigens including the virulence factors. The aim of this study was to evaluate the diagnostic value of Western Blot (WB) method in the serological diagnosis of H. pylori infections. For this purpose, H. pylori IgG and IgA antibodies have been searched qualitatively by a commercial WB test (Euroimmun GmbH, Germany), in the sera of 23 patients (9 female, 14 male) ages between 6-70 years old (13 children, 10 adults), who were diagnosed as H. pylori infection with the positive results of at least three of the conventional methods (culture and polymerase chain reaction of endoscopic biopsy specimens, 13C urea breath test, serum IgG-EIA methods). All of the patients (100%) were found positive by WB-IgG, and 9 (39%) were positive by WB-IgA tests. H.pylori IgG and IgA antibodies showed reactivity with the different combinations of p120 (CagA), p95 (VacA), p29 (UreA) and p66 (UreB) antigens. The number of positive samples for CagA-IgG, VacA-IgG, UreA-IgG, CagA-IgA and UreA-IgA were 19, 17, 21, 8 and 4, respectively, while there were no positive sample for VacA-IgA. The antigenic distribution did not show any difference between age and sex of the patients. In our study the sensitivities of WB-IgG and WB-IgA methods were estimated as 100% and 39%, respectively, which were in accordance with the studies in literature. As a result, WB-IgG method appears to be useful and reliable for the detection of antibody profiles to H. pylori antigens and virulence factors, rather than routine and screening purposes, however the diagnostic value of WB-IgA method is low. It was thought that the results of our preliminary study should be confirmed by other studies including larger patient populations and control groups, in our country.