Molekulare Aspekte und chemische Inaktivierung von Influenza H5N1-Viren aus ägyptischen Hühnerbeständen von Ausbrüchen der Jahre 2006 bis 2010

The primary objective of the current study was to identify two of the highly pathogenic avian influenza virus (HPAIV) isolates of subtype H5N1 genotypically using one step Reverse Transcriptase Polymerase Chain Reaction (RT-PCR), followed by sequence and phylogenetic analyses. A further objective was to determine in vitro the virucidal efficacy of four types of chemical disinfectants, namely Formalin, Glutaraldehyde, TH4® and Virkon®S at different concentrations and contact times on the two HPAI isolates. A/chicken/Egypt/0626/2006 (EGY06) and A/chicken/Egypt/1094/2010 (EGY10) were isolated from cloacal and tracheal swabs from broiler during HPAI H5N1 outbreaks in Egypt in 2006 and 2010. The first strain, EGY06, was isolated from a non-vaccinated flock in February 2006 in the Alexandria governorate. The second strain, EGY10, was isolated from a vaccinated flock in November 2010 in the Marsa Matrouh governorate. Classical identification of the two isolates was carried out in the Department of Poultry and Hygiene, Faculty of Veterinary Medicine, Alexandria University, Egypt. Molecular identification and genetic analyses were conducted in the Gene Analysis Unit of the National Laboratory for Veterinary Quality Control on Poultry Production (NLQP), Egypt. Using RT-PCR with specific sets of primers for H5 and N1 genes of AIV it was confirmed that the two isolates belonged to AI subtype H5N1. After molecular characterization and phylogenetic analysis of the HA and NA genes, the strain EGY06 was closely related to the 2006 predecessor Egyptian viruses of 2.2.1 clade, whereas EGY10 clustered within the classic 2.2.1/c group that commonly isolated from small-scale commercial farms and human since 2009. The efficacy of four chemical disinfectants to inactivate both isolates was carried out in accordance to the guidelines of the German Veterinary Medical Society (Deutsche Veterinärmedizinische Gesellschaft, DVG) for testing of disinfection procedures and chemical disinfectants. The experiments were performed using suspension tests without and with protein load (40% Bovine Calf Serum "BCS") as well as wood and gauze as a carriers (also loaded with BCS), at room temperature and incubation times of 15 to 120 min. The obtained results showed that the use of Glutaraldehyde, Formalin or TH4® 0.5% without protein load led to complete inactivation of the virus after 15, 30, 60 or 120 min contact time. Use of Virkon®S 0.5% with and without protein load led to survival of the virus even after 60 min. In contrast, using Formalin and TH4® (1% and 2%) with and without protein load led to complete inactivation of the virus even at the shortest contact time of 15 min. Similar results were obtained after using Glutaraldehyde 1%, while treatment of H5N1 with Glutaraldehyde 2% led to gel formation. After treatment of contaminated carriers (poplar wood and gauze) with Formalin, Glutaraldehyde and TH4® 0.5%, the virus was inactivated after 30 min. Concentration of 1% of the three disinfectants was sufficient to inactivate the two isolates within 15 min contact time, except in case of Virkon®S which required higher concentrations to give similar results. The study indicated that the four chemical disinfectants could efficiently inactivate the two tested H5N1 viruses when used at higher concentration than the manufacturers recommended. The results of the present thesis highlight the sensitivity of HPAIV H5N1 to different disinfectants, which may improve biosecurity measures on the farms and reduce the economic losses caused by HPAIV H5N1.Das primäre Ziel der aktuellen Studie war es, hoch pathogene aviäre Influenza- Viren (HPAIV) des Subtyps H5N1 genotypisch durch eine einschrittige Reverse Transkriptase- Polymerase-Kettenreaktion (RT-PCR) zu identifizieren und anschließend molekularbiologisch zu charakterisieren. Ein weiteres Ziel war, die Wirksamkeit von verschiedenen Konzentrationen und Einwirkungszeiten von vier chemischen Desinfektionsmitteln (Formalin, Glutaraldehyd, TH4® und Virkon®S) auf zwei Stämme (A/chicken/Egypt/0626/2006 "EGY06" und A/chicken/Egypt/1094/2010 "EGY10") des aviären Influenzavirus (AIV) des Subtyps H5N1 in vitro zu prüfen. Die beiden Isolate des AIV-Subtyps H5N1 wurden aus Kloaken- und Trachealtupfern von infizierten Masthühnerherden während der Ausbrüche aviärer Influenza (AI) 2006 und 2010 isoliert. Während der erste Stamm EGY06 aus einer nicht geimpften Herde im Februar 2006 im Gouvernement Alexandria isoliert wurde, wurde der zweite Stamm, EGY10, aus einer geimpften Herde im November 2010 im Gouvernement Marsa Matrouh isoliert. Die klassischen Methoden zur Identifizierung der beiden Isolate wurden in der Abteilung für Geflügel und Hygiene, Veterinärmedizinische Fakultät, Universität Alexandria, Ägypten durchgeführt. Die molekulare Identifizierung und genetische Analyse erfolgten in der Gen- Analyse-Einheit des Nationalen Labors zur Qualitätskontrolle der Geflügelproduktion (NLQP), Ägypten. Mittels RT-PCR unter Verwendung spezifischer Primersets für die H5 und N1 Gene konnte bestätigt werden, dass es sich bei beiden Isolaten um AIV des Subtyps H5N1 handelt. Der molekularen Charakterisierung und der phylogenetischen Analyse der HA und NA zufolge war der Stamm EGY06 sehr eng verwandt mit dem früher im Jahr 2006 isolierten klassischen Stamm und wurde dem Clade 2.2.1 zugeordnet. Im Gegensatz dazu wurde der Stamm EGY10 im klassischen 2.2.1/c Gruppe zugeordnet, welcher häufig von kleinen kommerziellen Farmen und menschlichen seit 2009 isoliert. Die Empfindlichkeit der Viren gegen verschiedene Desinfektionsmittel wurde auf Grundlage der Richtlinien der Deutschen Veterinärmedizinischen Gesellschaft (DVG) für die Prüfung von Desinfektionsverfahren und chemischen Desinfektionsmitteln geprüft. Die Experimente wurden mittels Suspensions-Test ohne und mit Proteinbelastung (40% Bovines Calf Serum (BCS)) sowie auf Keimträgern aus Holz und Gaze, die mit BCS belastet waren, bei Raumtemperatur und Einwirkzeiten von 15 bis 120 Min durchgeführt. Die Verwendung von Glutaraldehyd, Formalin oder TH4® in einer Konzentration von 0,5% führte ohne Proteinbelastung zu einer Inaktivierung der Viren nach allen Einwirkzeiten (15, 30, 60 und 120 Min). Die Verwendung von Virkon®S 0,5% mit und ohne Proteinbelastung führte zum Überleben des Virus sogar nach 60 Min. Demgegenüber führte die Verwendung von Formalin und TH4® in einer Konzentration von 1% und 2% mit und ohne Proteinbelastung zu einer vollständigen Inaktivierung des Virus sogar bei der kürzesten Einwirkungszeit von 15 Min. Ähnliche Ergebnisse wurden nach Verwendung von Glutaraldehyd in einer Konzentration von 1% beobachtet. Die Behandlung von H5N1 mit Glutaraldehyd in einer Konzentration von 2% führte zu einer Gelbildung. Nach der Behandlung von kontaminierten Keimträgern (Pappelholz und Gaze) mit Formalin, Glutaraldehyd und TH4® in Konzentrationen von 0,5% wurde das Virus nach 30 Min inaktiviert. Während eine Konzentration von 1% der drei Desinfektionsmittel ausreichend war, um die beiden Isolate in 15 Min Einwirkzeit zu inaktivieren, konnte dieses Ergebnis im Fall von Virkon®S nicht erreicht werden, und eine höhere Konzentration war erforderlich um ähnliche Ergebnisse zu erzielen. Die Studie zeigte, dass die vier chemischen Desinfektionsmittel, wenn die verwendeten Konzentrationen höher als die vom Hersteller empfohlenen Konzentrationen sind, beide getesteten H5N1 Viren effektiv inaktivieren. Die Ergebnisse dieser Studie bieten einen neuen Ansatz zur Verbesserung der Biosicherheitsmaßnahmen in Geflügelbeständen und können zur Reduzierung der wirtschaftlichen Verluste beitragen

An overview of the public health challenges in diagnosing and controlling human foodborne pathogens

Pathogens found in food are believed to be the leading cause of foodborne illnesses; and they are considered a serious problem with global ramifications. During the last few decades, a lot of attention has been paid to determining the microorganisms that cause foodborne illnesses and developing new methods to identify them. Foodborne pathogen identification technologies have evolved rapidly over the last few decades, with the newer technologies focusing on immunoassays, genome-wide approaches, biosensors, and mass spectrometry as the primary methods of identification. Bacteriophages (phages), probiotics and prebiotics were known to have the ability to combat bacterial diseases since the turn of the 20th century. A primary focus of phage use was the development of medical therapies; however, its use quickly expanded to other applications in biotechnology and industry. A similar argument can be made with regards to the food safety industry, as diseases directly endanger the health of customers. Recently, a lot of attention has been paid to bacteriophages, probiotics and prebiotics most likely due to the exhaustion of traditional antibiotics. Reviewing a variety of current quick identification techniques is the purpose of this study. Using these techniques, we are able to quickly identify foodborne pathogenic bacteria, which forms the basis for future research advances. A review of recent studies on the use of phages, probiotics and prebiotics as a means of combating significant foodborne diseases is also presented. Furthermore, we discussed the advantages of using phages as well as the challenges they face, especially given their prevalent application in food safety

Burnout among surgeons before and during the SARS-CoV-2 pandemic: an international survey

Background: SARS-CoV-2 pandemic has had many significant impacts within the surgical realm, and surgeons have been obligated to reconsider almost every aspect of daily clinical practice. Methods: This is a cross-sectional study reported in compliance with the CHERRIES guidelines and conducted through an online platform from June 14th to July 15th, 2020. The primary outcome was the burden of burnout during the pandemic indicated by the validated Shirom-Melamed Burnout Measure. Results: Nine hundred fifty-four surgeons completed the survey. The median length of practice was 10 years; 78.2% included were male with a median age of 37 years old, 39.5% were consultants, 68.9% were general surgeons, and 55.7% were affiliated with an academic institution. Overall, there was a significant increase in the mean burnout score during the pandemic; longer years of practice and older age were significantly associated with less burnout. There were significant reductions in the median number of outpatient visits, operated cases, on-call hours, emergency visits, and research work, so, 48.2% of respondents felt that the training resources were insufficient. The majority (81.3%) of respondents reported that their hospitals were included in the management of COVID-19, 66.5% felt their roles had been minimized; 41% were asked to assist in non-surgical medical practices, and 37.6% of respondents were included in COVID-19 management. Conclusions: There was a significant burnout among trainees. Almost all aspects of clinical and research activities were affected with a significant reduction in the volume of research, outpatient clinic visits, surgical procedures, on-call hours, and emergency cases hindering the training. Trial registration: The study was registered on clicaltrials.gov "NCT04433286" on 16/06/2020

Mass Spectrometry Technology and qPCR for Detection of Enterococcus faecalis in Diabetic Foot Patients

Diabetic foot ulcer (DFU) is one of the most serious and costly complications of diabetic patients. Enterococcus faecalis (E. faecalis) represents one of the most virulent microorganisms in diabetic foot infections (DFIs). We therefore aimed to study the frequency and precise identification of E. faecalis in DFU. Six hundred thirty specimens collected from diabetic foot patients were used in the current investigation. Biochemical identification was carried out by the Vitek® 2 system. Proteomic analysis was implemented by MALDI-TOF MS and confirmed by SYBER Green real-time polymerase chain reaction (SYBER Green qPCR). According to the results, the overall frequency of E. faecalis in patients with DFU was 8.25% (52/630). Out of 52 E. faecalis strains, 40 isolates were isolated from males and 12 from females. The results of biochemical identification revealed that 92.30% (48/52) of E. faecalis isolates were properly recognized at the species level. Whereas 100% (52/52) of E. faecalis isolates were properly recognized by MALDI-TOF MS as 44.23% (23/52), 51.92% (27/52) and 3.84% (2/52) with a score value ranging from 2.300 to 3.000, 2.000-2.299 and 1.700-1.999 Da, respectively. Seven E. faecalis virulence genes, including asa1, GelE, cylA, esp, hy1, VanA, and VanB, were detected by SYBER Green RT-PCR. In conclusion, E. faecalis was the utmost predominant single organism isolated from the DFIs. MALDI-TOF mass spectrometry is considered a fast, trustworthy and economic detection method for various significant microorganisms. E. faecalis isolates were also found to carry several virulence genes. Our findings may serve as an urgent issue for supplementary investigations of contagions caused by E. faecalis

Antibacterial effects and resistance induction of silver and gold nanoparticles against Staphylococcus aureus‐induced mastitis and the potential toxicity in rats

Abstract Staphylococcus aureus (S. aureus) is one of the prevalent mastitis‐inducing pathogens worldwide. The resistance of S. aureus to antibiotics is a common issue for dairy farms. Recently, nanoparticles (NPs) have been used for treating antibiotic‐resistant bacteria. We therefore aimed to investigate the antimicrobial effect of silver and gold NPs (AgNPs and AuNPs, respectively) and the resistance developed by S. aureus as well as the toxic effects of both NPs in rats. We used 198 S. aureus strains to determine the antibacterial effects of AgNPs and AuNPs. The microdilution method was used to establish the minimum inhibitory concentrations (MICs) of both NPs. To induce resistance, 20 S. aureus strains were passaged 10 times in broth medium with sublethal doses of NPs and an additional 10 times without NPs to examine the stability of resistance. Histopathology was performed after oral administration to the rats with the study doses of 0.25, 0.5, 1, and 2 mg/kg of NPs for 30 days. The MICs of 10‐nm AgNPs, 20‐nm AgNPs, 10‐nm AuNPs, and 20‐nm AuNPs against S. aureus were 14.70 ± 1.19 μg/ml, 9.15 ± 0.13 μg/ml, 24.06 ± 2.36 μg/ml, and 18.52 ± 1.26 μg/ml, respectively. Most strains developed strong resistance when treated with 20‐nm or 10‐nm AgNPs, whereas only two strains were resistant to 10‐nm AuNPs and three strains to 20‐nm AuNPs. No cross‐resistance between NPs and various antibiotics was identified in any of the adapted S. aureus strains. Organ histopathology revealed that 0.25, 0.5, and 1 mg/kg doses of AgNPs and AuNPs were not toxic to rat tissue. In contrast, a higher dose (2 mg/kg) of NPs impaired all organs tested. This study demonstrates the antibacterial effects of NPs. S. aureus strains develop resistance less frequently against AuNPs than AgNPs, and neither AuNPs nor AgNPs was toxic to rats at low doses

Comparison of LCD array and IS6110-PCR with conventional techniques for detection of Mycobacterium bovis isolated from Egyptian cattle and Buffaloes

Bovine tuberculosis is a chronic bacterial and major infectious disease of cattle and buffaloes caused by Mycobacterium bovis. Rapid diagnosis of bovine tuberculosis is considered one of the cornerstones for worldwide control as it permits early epidemiological and therapeutic interventions. Therefore, this study was designed to evaluate conventional techniques (tuberculin test, Ziehl Neelsen staining and culturing) in comparison with proven molecular laboratory techniques (LCD array and IS6110 PCR) for identification of Bovine tuberculosis. A total of 902 Egyptian animals (480 buffaloes and 422 cattle) were examined by tuberculin test, and the positive reactors were slaughtered. Tissue samples were collected for staining as well as culturing. Moreover, LCD array and PCR using IS6110 on DNA extracted from tissue and culture samples were carried out for molecular identification of M. bovis. According to the results, the tuberculin positive cases for cattle and buffaloes were 2.14% (9 cases) and 5.62% (27 cases), respectively. After post-mortem examination, the prevalence of tuberculin positive cases with visible lesions was 88.9% for cattle and 14.8% for buffaloes. Alternatively, these percentages were 11.1% and 85.2% for cattle and buffalo carcasses with non-visible lesions. The percentage of cattle and buffaloes showing positive culture was 88.9% and 62.9%, respectively. This percentage was 69.5% after staining with Ziehl Neelsen. In contrast, LCD array and IS6110 were 100%, confirming the isolation results. In conclusion, LCD array depending on 16S RNA and DNA hybridization with specific probes for detection of M. bovis are rapid, sensitive and labor-saving when combined with IS6110-PCR

Highlight on Multidrug Resistance of Enterococcus faecalis Recovered from Diabetic Foot Patients

Diabetic foot infections (DFIs) are a progressively serious health problem worldwide. Enterococcus faecalis (E. faecalis) is one of the most frequent bacteria in DFIs. The antibiotic resistance patterns of this bacterium remain a significant tool for monitoring infection. Therefore, our study aimed to determine the susceptibility of E. faecalis recovered from the wounds of hospitalized diabetic foot patients to various antimicrobial drugs. Fifty-two E. faecalis strains were recovered from 630 diabetic foot patients. All isolates were identified biochemically by a Vitek® 2 system and via a mass spectrometer (MALDI Biotyper). Antimicrobial sensitivity testing used Vitek 2 cards and Kirby-Bauer as the reference method. The findings indicated that the susceptibility of E. faecalis was 100% for ampicillin, ampicillin-sulbactam, benzylpenicillin, norfloxacin, and ofloxacin; 92% for nitrofurantoin, teicoplanin, and vancomycin; 87% for imipenem; 81% for kanamycin (high concentration) and tetracycline; 73% for levofloxacin; and 52% for streptomycin (high concentrations). The resistance was 100% for clindamycin and quinupristin-dalfopristin, 96% for cefuroxime, 90% for ciprofloxacin and erythromycin, 86% for trimethoprim-sulfamethoxazole, 54% for gentamicin (high concentration), and 48% for streptomycin (high concentration). All E. faecalis strains were resistant against numerous antibiotics with a multiple antibiotic resistance (MAR) index of 0.20–0.60. The mean value of MAR indices for all tested E. faecalis species was 0. 373. The high levels of antimicrobial resistance patterns to E. faecalis seen here are important because they restrict treatment possibilities and adversely affect the health of diabetic foot patients. Consequently, our findings should be carefully considered in public health and awareness programs

Role of aldose reductase C-106T polymorphism among diabetic Egyptian patients with different microvascular complications

The aldose reductase pathway proves that elevated blood glucose promotes cellular dysfunction. The polyol pathway converts excess intracellular glucose into alcohols via activity of the aldose reductase. This enzyme catalyzes the conversion of glucose to sorbitol which triggers variety of intracellular changes in the tissues. Among diabetes, activity is drastically increased in association with three main consequences inside the cells. The aim of this study was to detect the association of the C-106 T polymorphism of the aldose reductase gene and its frequency among a sample of 150 Egyptian adults with type 2 diabetic patients having diabetic microvascular. The detection of the aldose reductase C-106 T polymorphism gene was done by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The genotype distribution of the C-106 T polymorphism showed that CC genotype was statistically significantly higher among patients with retinopathy compared to nephropathy. Patients with nephropathy had significant association with the TT genotype when compared with diabetic retinopathy patients. Follow up study after the genotype detection among recently diagnosed diabetic patients in order to give a prophylactic aldose reductase inhibitors; studying the microvascular complications and its relation to the genotype polymorphisms. The study may include multiple gene polymorphisms to make the relation between the gene and the occurrence of these complications more evident.</p

Effect of web-based health education on nursing students’ knowledge, adaptive healthy measures and attitudes regarding polycystic ovary syndrome: a randomized controlled trial

Abstract Background Polycystic ovary syndrome (PCOS) is a complex endocrine disorder affecting women of reproductive age, and it has emerged as a significant global public health issue. This study aimed to investigate the effects of web-based health education on nursing students’ knowledge, adaptive healthy measures, and attitudes toward PCOS. Methods A two-group randomized controlled trial (RCT) with pre-test and immediate post-test assessments was conducted. Study participants were recruited using a simple random sampling method from the Faculty of Nursing, Mansoura University, Egypt. A questionnaire consisting of six sections was developed to collect data, which was analyzed with the SPSS 23.0 using Student’s t-test, Pearson’s correlation test, and chi-square test analysis of variance. Results The analysis revealed a significant increase in knowledge scores post-intervention, with the web-based learning groups (32.2 ± 10.5) outperforming the traditional learning group (22.1 ± 10.2), with (p < 0.05). Similarly, there was a notable improvement in adaptive healthy measures scores post-intervention, with the web-based learning group (8.9 ± 2.4) showing better results than the traditional group (6.5 ± 2.9), with (p < 0.05). In terms of attitudes toward PCOS, the web-based group (18.2 ± 4.9) displayed a significant improvement compared to the traditional group (11.7 ± 5.2), with (p < 0.05). Conclusions The findings suggest that web-based learning is more effective than traditional methods in enhancing nursing students’ knowledge, adaptive healthy measures, and attitudes toward PCOS. Trial Registration : This study was registered by Clinical Trials.gov Identifier: (NCT06192381|| https://www.clinicaltrials.gov/ ) on 5-1-2024

Proteomic Analysis and Molecular Characterization of Airborne Bioaerosols in Indoor and Outdoor Environment in Al-Qassim Region, Saudi Arabia

There are various sources of microbial air pollution which are seems to be a serious public health problem all over the world. For prevention and control of air pollution caused by airborne bacteria, rapid, sensitive and reliable detection techniques are required. Therefore, our study focused on using MALDI Biotyper (MBT) for rapid recognition of various microbial air pollutants. Five hundred air samples were collected from three localities, including Qassim University (150 samples), Al-Qassim hospitals (250 samples) and poultry slaughter houses (100 samples). All air samples were collected by impactor air sampler from the indoor and outdoor environment. All samples were cultivated on nutrient and blood agar media for two days and a total of 129 isolates were purified for proteomic analysis using MALDI Biotyper (MBT) then confirmed by quantitative polymerase chain reaction (qPCR). One hundred and nineteen (92.25%) isolates were identified by MBT at the species level with a log (score) value ≥2. 000 whereas; 10 (7.75%) isolates were detected at the genus level with score values ranged from 1.7000 to 1.999. The MBT was able to identify 93 (72.10%) gram-positive and 36 (27.90%) gram-negative bacterial isolates. The most common genera were Staphylococcus (n = 43, 33.33%), Escherichia (n = 16, 12.40%), Enterococcus (n = 15, 11.63%) and Bacillus (n = 15, 11.63%). Staphylococcus aureus and Escherichia coli were the most frequently identified species (n = 16, 12.40% for each). In general, we detected 53 (41.10%) various bacterial species in Al-Qassim hospitals, 41 (31.79%) in poultry slaughter houses and 35 (27.13%) in Qassim University. Throughout Al-Qassim region, the air was tainted by numerous environmental microorganisms, and the MBT was positively adjusted for their fast and accurate identification