Molecular surveillance of influenza A virus in Saudi Arabia: whole-genome sequencing and metagenomic approaches.

Outbreaks of influenza A viruses are generally seasonal and cause annual epidemics worldwide. Due to their frequent reassortment and evolution, annual surveillance is of paramount importance to guide vaccine strategies. The aim of this study was to explore the molecular epidemiology of influenza A virus and nasopharyngeal microbiota composition in infected patients in Saudi Arabia. A total of 103 nasopharyngeal samples from 2015 and 12 samples from 2022 were collected from patients positive for influenza A. Sequencing of influenza A as well as metatranscriptomic analysis of the nasopharyngeal microbiota was conducted using Oxford Nanopore sequencing. Phylogenetic analysis of hemagglutinin, neuraminidase segments, and concatenated influenza A genomes was performed using MEGA7. Whole-genome sequencing analysis revealed changing clades of influenza A virus: from 6B.1 in 2015 to 5a.2a in 2022. One sample containing the antiviral resistance-mediating mutation S247N toward oseltamivir and zanamivir was found. Phylogenetic analysis showed the clustering of influenza A strains with the corresponding vaccine strains in each period, thus suggesting vaccine effectiveness. Principal component analysis and alpha diversity revealed the absence of a relationship between hospital admission status, age, or gender of infected patients and the nasopharyngeal microbial composition, except for the infecting clade 5a.2a. The opportunistic pathogens Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis were the most common species detected. The molecular epidemiology appears to be changing in Saudi Arabia after the COVID-19 pandemic. Antiviral resistance should be carefully monitored in future studies. In addition, the disease severity of patients as well as the composition of the nasopharyngeal microbiota in patients infected with different clades should also be assessed.IMPORTANCEIn this work, we have found that the clade of influenza A virus circulating in Riyadh, KSA, has changed over the last few years from 6B.1 to 5a.2a. Influenza strains clustered with the corresponding vaccine strains in our population, thus emphasizing vaccine effectiveness. Metatranscriptomic analysis showed no correlation between the nasopharyngeal microbiome and the clinical and/or demographic characteristics of infected patients. This is except for the 5a.2a strains isolated post-COVID-19 pandemic. The influenza virus is among the continuously evolving viruses that can cause severe respiratory infections. Continuous surveillance of its molecular diversity and the monitoring of anti-viral-resistant strains are thus of vital importance. Furthermore, exploring potential microbial markers and/or dysbiosis of the nasopharyngeal microbiota during infection could assist in the better management of patients in severe cases.

Metagenomic next-generation sequencing of nasopharyngeal microbiota in COVID-19 patients with different disease severities.

Throughout the COVID-19 pandemic, extensive research has been conducted on SARS-COV-2 to elucidate its genome, prognosis, and possible treatments. However, few looked at the microbial markers that could be explored in infected patients and that could predict possible disease severity. The aim of this study is to compare the nasopharyngeal microbiota of healthy subjects, moderate, under medication, and recovered SARS-COV-2 patients. In 2020, 38 nasopharyngeal swabs were collected from 6 healthy subjects, 14 moderates, 10 under medication and 8 recovered SARS-COV-2 patients at the Prince Mohammed Bin Abdulaziz Hospital Riyadh. Metatranscriptomic sequencing was performed using Minion Oxford nanopore sequencing. No significant difference in alpha as well as beta diversity was observed among all four categories. Nevertheless, we have found that Streptococcus spp including Streptococcus pneumoniae and Streptococcus thermophilus were among the top 15 most abundant species detected in COVID-19 patients but not in healthy subjects. The genus Staphylococcus was found to be associated with COVID-19 patients compared to healthy subjects. Furthermore, the abundance of Leptotrichia was significantly higher in healthy subjects compared to recovered patients. Corynebacterium on the other hand, was associated with under-medication patients. Taken together, our study revealed no differences in the overall microbial composition between healthy subjects and COVID-19 patients. Significant differences were seen only at specific taxonomic level. Future studies should explore the nasopharyngeal microbiota between controls and COVID-19 patients while controlling for confounders including age, gender, and comorbidities; since these latter could affect the results and accordingly the interpretation.IMPORTANCEIn this work, no significant difference in the microbial diversity was seen between healthy subjects and COVID-19 patients. Changes in specific taxa including Leptotrichia, Staphylococcus, and Corynebacterium were only observed. Leptotrichia was significantly higher in healthy subjects, whereas Staphylococcus and Corynebacterium were mostly associated with COVID-19, and specifically with under-medication SARS-COV-2 patients, respectively. Although the COVID-19 pandemic has ended, the SARS-COV-2 virus is continuously evolving and the emergence of new variants causing more severe disease should be always kept in mind. Microbial markers in SARS-COV-2 infected patients can be useful in the early suspicion of the disease, predicting clinical outcomes, framing hospital and intensive care unit admission as well as, risk stratification. Data on which microbial marker to tackle is still controversial and more work is needed, hence the importance of this study.

Side effects of COVID-19 vaccines in the middle eastern population.

The COVID-19 pandemic has caused severe worldwide health concerns since its first description as the SARS-COV-2 virus in December 2019. The wide dissemination of this virus, together with the lack of treatment, prompted vaccine development within a short period of time to elicit a protective immunity against COVID-19. Due to their rapid development, potential subsequent side effects of COVID-19 vaccines were overlooked, which might lead to many health concerns. This is especially true for patients at a greater risk of harm from COVID-19, such as pregnant women, children, and patients with pre-existing chronic diseases. In this review, we provide a summary of common to rare side effects of administrated COVID-19 vaccines in a Middle Eastern population. We have found that the distinction between side effects from COVID-19 vaccines in terms of frequency and severity is attributed to the differences in study populations, gender, and age. Pain at the injection site, fever, headache, fatigue, and muscle pain were the most common reported side effects. Vaccinated subjects with previous COVID-19 infection exhibited an equivalent neutralizing response after just one dose compared to two doses of vaccine. Consequently, individuals who experienced more side effects had significantly higher antibody levels. This indicates that having better immunity correlates with higher antibody levels, leading to a higher frequency of vaccine side effects. Individuals with underlying comorbidities, particularly having known allergies and with illnesses such as diabetes and cancer, might be more prone to post-vaccination side effects. Studies of a high-risk population in Middle Eastern countries are limited. Future studies should be considered to determine long-term side effects, side effects after booster doses, and side effect differences in cases of heterologous and homologous vaccination for better understanding and proper handling of high-risk populations and patients who experience these side effects.

Prevalence of Common Respiratory Viruses in Children at a Tertiary Care Hospital in Saudi Arabia.

Acute lower respiratory viral infections are significant contributors to morbidity/mortality in children less than 2 years of age. In this retrospective study conducted at a major hospital, between 2017 and 2019, it was observed that rhinovirus, respiratory-syncytial-virus and influenza virus are the most frequently detected with the seasonal distribution. Mortality rate was significantly higher in children less than 12 months and significantly increased in males and in those requiring intubation.

Antibacterial effect of a diet pill supplement on the human intestinal bacteria.

INTRODUCTION: Many people worldwide attempt to lose weight or adopt strategies to control it. Some have resorted to the consumption of commercialized diet pills to achieve this goal. Multiple brands exist without clearly indicating their mechanism of action or adverse effects on human health. This study aims to determine the antibacterial effect of commercial diet pills on members of the intestinal microbiota. METHODOLOGY: Commercialized diet pills were bought from a pharmacy in the North of Lebanon. Broth microdilution test was performed to determine the Minimum Inhibitory Concentrations (MICs) of the aqueous suspension against forty-two isolates distributed into four Enterobacterales species. MIC of the digested form was determined against six different strains. GC-MS analysis was performed to elucidate the components of the diet pill compared to the manufacturer's list. RESULTS: Broth microdilution results revealed that MICs of the diet pill aqueous suspension ranged from 3.9x103-9.76x102 µg/mL for Escherichia coli, Enterobacter spp., and Proteus spp. For Klebsiella species, MIC of carbapenem-resistant isolates reached 1.95x103 µg/mL. The digested form had a significantly lower antibacterial effect compared to the aqueous suspension. GC-MS analysis results corresponded with the list of ingredients provided by the manufacturer. CONCLUSIONS: The results showed significant antibacterial activity of a commercial diet pill on different members of the human intestinal microbiota regardless of their resistance profile. Further work is needed to elucidate the antibacterial effect of the digested components to accurately understand their effect on the intestinal microflora and thus on human health.

Increased Prevalence of EBV Infection in Nasopharyngeal Carcinoma Patients: A Six-Year Cross-Sectional Study.

Epstein Barr Virus (EBV) is implicated in the carcinogenesis of nasopharyngeal carcinoma (NPC) and currently associated with at least 1% of global cancers. The differential prognosis analysis of NPC in EBV genotypes remains to be elucidated. Medical, radiological, pathological, and laboratory reports of 146 NPC patients were collected retrospectively over a 6-year period between 2015 and 2020. From the pathology archives, DNA was extracted from tumor blocks and used for EBV nuclear antigen 3C (EBNA-3C) genotyping by nested polymerase chain reaction (PCR). We found a high prevalence of 96% of EBV infection in NPC patients with a predominance of genotype I detected in 73% of NPC samples. Histopathological examination showed that most of the NPC patients were in the advanced stages of cancer: stage III (38.4%) or stage IV-B (37.7%). Only keratinized squamous cell carcinoma was significantly higher in EBV negative NPC patients compared with those who were EBV positive (OR = 0.01, 95%CI = (0.004-0.32; p = 0.009)), whereas the majority of patients (91.8%) had undifferentiated, non-keratinizing squamous cell carcinoma, followed by differentiated, non-keratinizing squamous cell carcinoma (7.5%). Although NPC had metastasized to 16% of other body sites, it was not associated with EBV infection, except for lung metastasis. A statistically significant reverse association was observed between EBV infection and lung metastasis (OR = 0.07, 95%CI = (0.01-0.51; p = 0.008)). Although 13% of NPC patients died, the overall survival (OS) mean time was 5.59 years. Given the high prevalence of EBV-associated NPC in our population, Saudi could be considered as an area with a high incidence of EBV-associated NPC with a predominance of EBV genotype I. A future multi-center study with a larger sample size is needed to assess the true burden of EBV-associated NPC in Saudi Arabia.

Prognosis of COVID-19 in the middle eastern population, knowns and unknowns.

Since its emergence in China in 2019, the SARS-CoV-2 virus has affected all countries worldwide. The virus is easily transmitted from one person to another via infected aerosols or contaminated surfaces. Unlike its counterparts, the prognosis of COVID-19 ranges from asymptomatic to critical disease or death. Several factors play a role in determining the severity of the disease in infected patients. Among others, is the pre-existence of an underlying medical condition such as diabetes, cancer, and others. Furthermore, although children are less prone to the severe form of the COVID-19 disease, they require attention due to the report of many atypical presentations of the infection, post-asymptomatic exposure. In the Middle East, little is known about the prognosis of the SARS-CoV-2 infection in high-risk categories, notably patients with diabetes, cancer, and pregnant women. The aim of this review is to summarize the current knowledge about this group of population in the middle eastern region as well as to highlight the gap in the literature. We have found that the majority of the papers were from the Gulf countries. Although, few studies were conducted; high-risk patients appear to have an increased risk of morbidity and mortality from COVID-19 compared to their counterparts. Higher levels of inflammatory markers, C-reactive protein, erythrocyte sedimentation rate, D-dimer, and ferritin levels were also observed. Children are often asymptomatic or present with atypical presentations. More studies should be conducted to determine the clinical biomarkers of COVID-19 in high-risk categories to help in patient risk stratification and management in the middle eastern population.

Dissemination of Multidrug-Resistant and mcr-1 Gram-Negative Bacilli in Broilers, Farm Workers, and the Surrounding Environment in Lebanon.

Poultry are currently regarded as reservoirs from which multidrug resistance can be readily transferred to the surrounding ecosystem. The aim of this study was to explore the prevalence of extended spectrum beta-lactamases (ESBLs) and ampC and mcr-1 Gram-negative bacilli in chicken, farmers, and environment in Lebanon. In May 2017, we revisited the farm where the first mcr-1 was detected in 2015 in Lebanon. Overall 200 chicken fecal swabs, 6 farmers' fecal samples, and 41 environmental samples were collected. Real time (RT)-PCR was performed for beta-lactamases and mcr genes' screening using newly designed primers/probes. Multilocus sequence typing (MLST) was also performed. ESBL/ampCs were found in 118 samples from chicken, 4 from workers, and all environmental ones. mcr-1 was detected in all chicken and farmers' samples from which 314 and 7 strains were isolated, respectively. Three mcr-1 Escherichia coli strains were isolated from litter and feed. Compared to 2015, the prevalence of ESBL/ampC producers, TEM and CTX-M genes increased significantly in 2017. Main spectra profiles dendrogram of isolated E. coli strains in 2015/2017 and MLST revealed the presence of different clones and sequence types. The evolution of resistance appears to be multi-clonal and related to the diffusion of plasmids carrying ESBL and mcr-1 genes. More work is needed to quantify the magnitude of this emerging problem in Lebanon.

Understanding the Epidemiology of Multi-Drug Resistant Gram-Negative Bacilli in the Middle East Using a One Health Approach.

In the last decade, extended-spectrum cephalosporin and carbapenem resistant Gram-negative bacilli (GNB) have been extensively reported in the literature as being disseminated in humans but also in animals and the environment. These resistant organisms often cause treatment challenges due to their wide spectrum of antibiotic resistance. With the emergence of colistin resistance in animals and its subsequent detection in humans, the situation has worsened. Several studies reported the transmission of resistant organisms from animals to humans. Studies from the middle east highlight the spread of resistant organisms in hospitals and to a lesser extent in livestock and the environment. In view of the recent socio-economical conflicts that these countries are facing in addition to the constant population mobilization; we attempt in this review to highlight the gaps of the prevalence of resistance, antibiotic consumption reports, infection control measures and other risk factors contributing in particular to the spread of resistance in these countries. In hospitals, carbapenemases producers appear to be dominant. In contrast, extended spectrum beta lactamases (ESBL) and colistin resistance are becoming a serious problem in animals. This is mainly due to the continuous use of colistin in veterinary medicine even though it is now abandoned in the human sphere. In the environment, despite the small number of reports, ESBL and carbapenemases producers were both detected. This highlights the importance of the latter as a bridge between humans and animals in the transmission chain. In this review, we note that in the majority of the Middle Eastern area, little is known about the level of antibiotic consumption especially in the community and animal farms. Furthermore, some countries are currently facing issues with immigrants, poverty and poor living conditions which has been imposed by the civil war crisis. This all greatly facilitates the dissemination of resistance in all environments. In the one health concept, this work re-emphasizes the need to have global intervention measures to avoid dissemination of antibiotic resistance in humans, animals and the environment in Middle Eastern countries.

Analysis of CAST in 9 Lebanese hospitals between 2008 and 2017.

BACKGROUND: Cumulative Antibiotic Susceptibility Testing (CAST) plays a crucial role in providing knowledge about the evolution of bacterial resistance. The preparation of such report is however prone to many errors. This study investigated the variety of mistakes detected in the CAST of 9 Lebanese hospitals. METHODS: Nine Lebanese hospitals were involved, where 21 different errors were looked for and analyzed. The total number of errors in each year was calculated and averaged according to the number of hospitals. Obtaining the average number of errors per hospital per year allowed the comparison of each hospital individually. RESULTS: The average number of errors in 2008 was 38.75 and increased to 51.5 in 2012. The average number of errors then decreased to 37.89 by 2017. The most common error between 2008-2017 was the incoherent percentages. Superimposing these results allowed to determine if hospitals in general were following the trend of average errors. Some hospitals were constantly improving, others were making a variable number of errors over the years. CONCLUSION: The percentages of errors found here are alarming, urging therefore educating microbiologists on preparing CAST correctly. Future studies should aim to study the physician's level of knowledge on the proper utilization of CAST.

Acinetobacter spp in a Third World Country with Socio-economic and Immigrants Challenges.

INTRODUCTION: In the last decade, Acinetobacter species have taken a major public health concern. This is mainly due the increased resistance to a wide range of antibiotics causing treatment challenges. In view of the constant population mobilization and the economic crisis that Lebanon is currently facing, it becomes a necessity to re-evaluate the real threat of Acinetobacter spp and its implication in the one health. METHODOLOGY: This review was conducted through the analysis of 45 research papers and reports pertaining to Acinetobacter spp performed in Lebanon. More than 82% of the papers consulted were published in international journals and more than 70 percent of them had received impact factor. RESULTS: An in depth description of the involvement of this organism in human infection and its role as potential pathogen or simple colonizer was performed. In addition, the different aspects of resistance, mostly to carbapenems and colistin was studied and summarized. While in animals and environment, susceptible strains were mostly isolated, OXA-23/OXA-24 were predominant in humans. Recently, NDM-1 producing Acinetobacter spp was detected in a Syrian refugee which then was reported in Lebanese patients. The bacterial identification procedures are non-systematic and not always reliable in the Lebanese studies presenting sometimes discrepancies an inconsistency. CONCLUSION: Acinetobacter is commonly isolated Lebanon. In view of the spread of resistance among these isolated and their dissemination, Infection control measures attempting to control the spread of this genus in and outside hospitals are lacking and thus require more attention and stewardship activities.

Prevalence of Extended-Spectrum Beta-Lactamase-Producing Gram-Negative Bacilli and Emergence of mcr-1 Colistin Resistance Gene in Lebanese Swine Farms.

Livestock are considered reservoirs of multidrug-resistant organisms that can be transferred to humans through direct/indirect routes. Once transmitted, these organisms can be responsible for infections with therapeutic challenges. The aim of this study was to determine the prevalence of extended-spectrum cephalosporin and colistin-resistant Gram-negative bacilli in Lebanese swine farms. In May 2017, 114 fecal samples were collected from swine farms in south Lebanon. Separate media supplemented with cefotaxime, ertapenem, and colistin were used for the screening of resistant organisms. Double-disk synergy test and ampC disk test were performed to detect extended-spectrum beta-lactamase (ESBL) and ampC producers, respectively. Detection of beta-lactamase and mcr genes was performed using real time PCR. Of 114 fecal samples, 76 showed growth on the medium with cefotaxime. In total, 111 strains were isolated with 94.5% being Escherichia coli. Phenotypic tests showed that 98, 6, and 7 strains were ESBL, ampC, and ESBL/ampC producers, respectively. CTX-M and CMY were the main beta-lactamase genes detected. On the medium with colistin, 19 samples showed growth. In total, 23 colistin-resistant E. coli strains harboring the mcr-1 gene were isolated. This is the first study in Lebanon determining multidrug resistance epidemiology in pigs. The prevalence of ESBLs is high and the emergence of colistin resistance is alarming.

Colistin- and Carbapenem-Resistant Klebsiella pneumoniae Clinical_Isolates: Algeria.

This study investigates the molecular mechanisms of colistin and carbapenem resistance in Klebsiella pneumoniae ST101 strains. The three K. pneumoniae carried blaCTX-M-15, blaTEM-183, and blaSHV-106 genes and two coharbored blaOXA-48. As for colistin resistance, the isolates had amino acid substitutions in PmrA/B and a truncated mgrB gene in one isolate.

Prevalence and Emergence of Extended-Spectrum Cephalosporin-, Carbapenem- and Colistin-Resistant Gram Negative Bacteria of Animal Origin in the Mediterranean Basin.

In recent years, extended ESBL and carbapenemase producing Gram negative bacteria have become widespread in hospitals, community settings and the environment. This has been triggered by the few therapeutic options left when infections with these multi-drug resistant organisms occur. The emergence of resistance to colistin, the last therapeutic option against carbapenem-resistant bacteria, worsened the situation. Recently, animals were regarded as potent antimicrobial reservoir and a possible source of infection to humans. Enteric Gram negative bacteria in animals can be easily transmitted to humans by direct contact or indirectly through the handling and consumption of undercooked/uncooked animal products. In the Mediterranean basin, little is known about the current overall epidemiology of multi-drug resistant bacteria in livestock, companion, and domestic animals. This review describes the current epidemiology of ESBL, carbapenemase producers and colistin resistant bacteria of animal origin in this region of the world. The CTX-M group 1 seems to prevail in animals in this area, followed by SHV-12 and CTX-M group 9. The dissemination of carbapenemase producers and colistin resistance remains low. Isolated multi-drug resistant bacteria were often co-resistant to non-beta-lactam antibiotics, frequently used in veterinary medicine as treatment, growth promoters, prophylaxis and in human medicine for therapeutic purposes. Antibiotics used in veterinary medicine in this area include mainly tetracycline, aminoglycosides, fluoroquinolones, and polymyxins. Indeed, it appears that the emergence of ESBL and carbapenemase producers in animals is not related to the use of beta-lactam antibiotics but is, rather, due to the co-selective pressure applied by the over usage of non-beta-lactams. The level of antibiotic consumption in animals should be, therefore, re-considered in the Mediterranean area especially in North Africa and western Asia where no accurate data are available about the level of antibiotic consumption in animals.

Prevalence and Characterization of Multi-Drug-Resistant Gram-Negative Bacilli Isolated From Lebanese Poultry: A Nationwide Study.

Currently, antimicrobial resistance is one of the most prominent public health issues. In fact, there is increasing evidence that animals constitute a reservoir of antimicrobial resistance. In collaboration with the Lebanese Ministry of Agriculture, the aim of this study was to determine the prevalence of intestinal carriage of multi-drug-resistant Gram-negative Bacilli in poultry farms at the national level. Between August and December 2015, 981 fecal swabs were obtained from 49 poultry farms distributed across Lebanon. The swabs were subcultured on MacConkey agar supplemented with cefotaxime (2 µg/ml). Isolated strains were identified using MALDI-TOF mass spectrometry. Multilocus sequence typing analysis was performed for Escherichia coli. Phenotypic detection of extended spectrum ß-lactamases (ESBL) and AmpC production was performed using double disk synergy and the ampC disk test, respectively. ß-lactamase encoding genes blaCTX-M, blaTEM, blaSHV, blaFOX, blaMOX, blaEBC, blaACC, blaDHA, and blaCMY using PCR amplification. Out of 981 fecal swabs obtained, 203 (20.6%) showed bacterial growth on the selective medium. Of the 235 strains isolated, 217 were identified as E. coli (92%), eight as Klebsiella pneumoniae (3%), three as Proteus mirabilis (1%) and three as Enterobacter cloacae (1%). MLST analysis of E. coli isolates showed the presence of ST156, ST5470, ST354, ST155, and ST3224. The phenotypic tests revealed that 43.5, 28.5, and 20.5% of the strains were ampC, ESBL, and ampC/ESBL producers, respectively. The putative TEM gene was detected in 83% of the isolates, SHV in 20%, CTX-M in 53% and CMY ampC ß-lactamase gene in 65%. Our study showed that chicken farms in Lebanon are reservoirs of ESBL and AmpC producing Gram-negative bacilli. The level of antibiotic consumption in the Lebanese veterinary medicine should be evaluated. Future studies should focus on the risk factors associated with the acquisition of multi-drug-resistant organisms in farm animals in Lebanon.

Epidemiology of multi drug resistant organisms in pigs in Lebanon.

INTRODUCTION: Livestock are nowadays considered potent reservoirs of multi drug resistance. Enteric resistant organisms in animals can be transmitted to humans and be causative agents of infections with therapeutic challenges. The aim of this study is to determine the prevalence of multi drug resistant organisms in Lebanese swine farms. METHODOLOGY: In May 2017, 94 fecal samples were collected from pigs in the south of Lebanon. Three media supplemented with cefotaxime, ertapenem, colistin were used for the screening of ESBL, carbapenemase producers and colistin resistance respectively. MALDI-TOF was used for bacterial identification. Double disk synergy test, ampC disk test and carpa np test were used for the detection of ESBL, ampC and carbapenemase producers respectively. RT-PCR was performed for the screening of beta lactamase and mcr colistin resistance genes. RESULTS: 77/94 fecal samples, showed growth on the medium supplemented with cefotaxime. In total 111 strains were isolated: 94% were identified as E.coli, 6% other organisms such as E. fergusonii and K. pneumoniae. Phenotypic tests showed that 72% of isolated strains were ESBL producers while 28% were ampC beta lactamase producers. RT-PCR analysis revealed that blaCTX-M was present in 45% of isolated strains, blaTEM in 26% and blaSHV in 10%. In parallel, 22 colistin resistant E.coli strains and 1 K.pneumoniae carrying mcr-1 were isolated. CONCLUSIONS: This study showed the importance of swine farms as reservoirs of resistance in Lebanon. The emergence of colistin resistance in pigs is worrying. A re-evaluation of antibiotic consumption in pigs is therefore warranted.