SARS-CoV-2 genome variations and evolution patterns in Egypt: a multi-center study.

A serious global public health emergency emerged late November 2019 in Wuhan City, China, by a new highly pathogenic virus, SARS-CoV-2. The virus evolution spread has been tracked by three developing databases: GISAID, Nextstrain and PANGO to understand its circulating variants. In this study, 110 diagnosed positive COVID-19 patient's samples, were collected from Kasr Al-Aini Hospital and the Children Cancer Hospital Egypt 57357 between May 2020 and January 2021, with clinical severity ranging from mild to severe. The viral genomes were sequenced by next generation sequencing, and phylogenetic analysis was performed to understand viral transmission dynamics. According to Nextstrain clades, most of our sequenced samples belonged to clades 20A and 20D, which in addition to clade 20B were present from the beginning of sample collection in May 2020. Clades 19A and 19B, on the other hand, appeared in the mid and late 2020 respectively, followed by the disappearance of clade 20B at the end of 2020. We identified a relatively high prevalence of the D614G spike protein variant and novel patterns of mutations associated together and with different clades. We also identified four mutations, spike H49Y, ORF3a H78Y, ORF8 E64stop and nucleocapsid E378V, associated with higher disease severity. Altogether, our study contributes genetic, phylogenetic, and clinical correlation data about the spread of the SARS-CoV-2 pandemic in Egypt.

Urethral instillation of chlorhexidine gel is an effective method of sterilisation.

Objective: To examine the effectiveness of preoperative urethral sterilisation with chlorhexidine gel in rendering the urethra as sterile as the skin of the genital area, with the skin sterilised as per the International Society for Sexual Medicine guidelines for penile prosthesis implantation. Patients and methods: A total of 111 male patients undergoing sterile andrological surgical procedures were divided into a control group (N = 61) and a chlorhexidine gel group (N = 50). Patients in the chlorhexidine group received urethral instillation with 6 mL of chlorhexidine preoperatively and on table. Patients from both groups received on-table skin preparation using povidone iodine and chlorhexidine povidone iodine. At the end of surgery, swabs were obtained from urethra and the penile skin. Skin and urethral swabs were compared for bacterial colonisation by culture and sensitivity. Results: Of the 111 patients, 16 had urethral colonisation and 10 had skin contamination, and they were all in the control group. The most common organism detected in both the urethral and skin samples was coagulase-negative Staphylococcus aureus. Urethral colonisation was significantly greater in the control group compared to the chlorhexidine group, at 16/61 vs 0/50 (P = 0.001). Similarly, skin colonisation was significantly greater in the control group compared to the chlorhexidine group, at 10/61 vs 0/50, (P = 0.002). Conclusion: Chlorhexidine gel is a powerful sterilising agent that will render the urethra sterile.

Genomic characterization of SARS-CoV-2 in Egypt.

Introduction: The novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread throughout the globe, causing a pandemic. In Egypt over 115,000 individuals were infected so far. Objective: In the present study, the objective is to perform a complete genome sequence of SAR-CoV2 isolated from Egyptian coronavirus disease (COVID-19) patients. Methods: Nasopharyngeal swabs were collected from 61 COVID-19 patients who attended at National Cancer Institute, Kasr Al-Aini Hospital and the army hospital. Viral RNA was extracted and whole genomic sequencing was conducted using Next Generation Sequencing. Results: In all cases, the sequenced virus has at least 99% identity to the reference Wuhan 1. The sequence analysis showed 204 distinct genome variations including 114 missense mutations, 72 synonymous mutations, 1 disruptive in-frame deletion, 7 downstream gene mutations, 6 upstream gene mutations, 3 frame-shift deletions, and 1 in-frame deletion. The most dominant clades were G/GH/GR/O and the dominant type is B. Conclusion: The whole genomic sequence of SARS-CoV2 showed 204 variations in the genomes of the Egyptian isolates, where the Asp614Gly (D614G) substitution is the most common among the samples (60/61). So far, there were no strikingly variations specific to the Egyptian population, at least for this set of samples.

A Gapless, Unambiguous RNA Metagenome-Assembled Genome Sequence of a Unique SARS-CoV-2 Variant Encoding Spike S813I and ORF1a A859V Substitutions.

The novel severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) is causing an unprecedented pandemic, threatening planetary health, society, and economy. Genomic surveillance continues to be a critical effort toward tracking the virus and containing its spread, and more genomes from diverse geographical areas and different time points are needed to provide an appropriate representation of the virus evolution. In this study, we report the successful assembly of one single gapless, unambiguous contiguous sequence representing the complete viral genome from a nasopharyngeal swab of an infected health care worker in Cairo, Egypt. The sequence has all typical features of SARS-CoV-2 genomes, with no protein-disrupting mutations. However, three mutations are worth highlighting and future tracking: a synonymous mutation causing a rare spike S813I variation and two less frequent ones leading to an A41V variation in NSP3, encoded by ORF1a (ORF1a A895V), and a Q677H variation in the spike protein. Both affected proteins, S and NSP3, are relevant to vaccine and drug development. Although the genome, named CU_S3, belongs to the prevalent global genotype, marked by the D614G spike variation, the combined variations in the spike proteins and ORF1a do not co-occur in any of the 197,000 genomes reported to date. Future studies will assess the biological, pathogenic, and epidemiological implications of this set of genetic variations. This line of research is needed to inform vaccine and therapeutic innovation to stem the COVID-19 pandemic.

Molecular Characterization of Carbapenem/Colistin-Resistant Acinetobacter baumannii Clinical Isolates from Egypt by Whole-Genome Sequencing.

PURPOSE: The rise of carbapenem-resistant A. baumannii (CRAB) is considered a public health problem limiting the treatment options. Our current work studied the emergence and mechanisms of colistin-resistance among CRAB isolates in Egypt. MATERIALS AND METHODS: Seventeen clinically recovered A. baumannii were identified and screened for their antimicrobial susceptibilities using VITEK-2 system. Colistin susceptibility was evaluated using broth microdilution, and characterization of carbapenem/colistin resistance determinants was performed using whole-genome sequencing (Illumina MiSeq). RESULTS: About 52.9% (9/17) were colistin-resistant. PCR results revealed that all isolates carried bla OXA-51-like genes, bla OXA-23-like was detected in 82.3% (14/17) and bla NDM in 23.5% (4/17). Two isolates harboured bla GES-35 and bla OXA-23. Furthermore, genome analysis of seven isolates revealed six belonged to international clone 2 (IC2) while the remaining isolate was a singleton (ST158), representing a clone circulating in Mediterranean/Middle Eastern countries. CONCLUSION: The emergence and high incidence of colistin-resistance among CRAB clinical isolates in Egypt are alarming because it further limits therapy options and requires prudent antimicrobial stewardship and stringent infection control measures. Whole-genome sequence analyses suggest that the resistance to colistin was associated with multiple mutations in the pmrCAB genes. The high incidence of the high-risk lineage IC2 harbouring bla OXA-23-like as well as bla NDM is also of concern.

Genomic Characterization of Methicillin-Resistant Staphylococcus aureus (MRSA) by High-Throughput Sequencing in a Tertiary Care Hospital.

Methicillin-resistant Staphylococcus aureus (MRSA) strains are associated with serious complications and poor clinical outcome. In Egypt, they contribute to more than 70% of S. aureus healthcare-associated infections. This study combined whole-genome sequencing, bioinformatics, and statistical analyses to identify the phylogeny, resistome, virulome and potential genotype-phenotype-clinical correlation among 18 clinical isolates of MRSA in a tertiary hospital in Cairo, Egypt. The ST1535-V MRSA clone was the most frequently isolated (16.6%), followed by ST5-VI, ST1-V and ST239-III (11.1% each). SCCmec V, VI, IV and III types were detected at frequencies of 50%, 16.6%, 11.1% and 11.1%, respectively. None of the tested virulence genes were detected in all isolates, but they ranged in distribution from 1/18 to 17/18. The Panton-Valentine leukocidin (PVL)-encoding genes were detected in only four isolates and were enriched in isolates causing non-severe cases. Phylogenetic analysis revealed relatedness between three ST1535-Vs, two ST5-VIs, two ST239-IIIs and two ST1-Vs; however, only the two genetically related ST1-V isolates were epidemiologically linked. While disease outcome and source of infection had no correlation with a particular genotypic pattern, the sequence type was the most correlated factor with phylogeny and genotypic patterns, and a few genes were associated with non-severe cases.

Serotypes and Antimicrobial Susceptibility of Nasopharyngeal Isolates of Streptococcus pneumoniae from Children Less Than 5 Years Old in Egypt.

PURPOSE: Streptococcus pneumoniae (S. pneumoniae) is the etiology of severe and life-threatening infections in children less than 5 years old. Though pneumococcal conjugate vaccines (PCVs) are effective in the prevention of pneumococcal infections, yet they are not included in the National Immunization Program in Egypt pending the identification of pathogenic serotypes. As S. pneumoniae colonization of the pharynx predisposes to pneumonia and invasive pneumococcal disease (IPD) caused by the colonizing serotypes, identification of the nasopharyngeal (NP) serotypes can be a surrogate to the invasive serotypes. In this study, we aimed to 1. Identify the serotypes and antimicrobial susceptibility testing (AST) of Streptococcus pneumoniae colonizing the nasopharynx of Egyptian children younger than 5 years in two successive winter seasons. 2. Correlate the identified serotypes with vaccine coverage of the 13-valent conjugate pneumococcal vaccines (PCV13). 3. Compare the serotypes and AST of S. pneumoniae from NP to those of IPD that were routinely identified in our clinical laboratory during the study period. MATERIALS AND METHODS: The study was conducted in two successive winter seasons (December 2015-March 2016; December 2016-March 2017). We enrolled 334 children, aged 6 months to 5 years, attending the outpatient general clinics of Cairo University Children Hospital, excluding those with fever, signs of infection, history of antibiotic intake or hospitalization in the preceding month. We tested NP swabs for S. pneumoniae by culture and real-time PCR. Serotyping was performed by sequential multiplex PCR for all positive samples. AST was done to S. pneumoniae isolates by Vitek-2™ (BioMérieux, Marcy-L'Etoile, France). We included routinely detected S. pneumoniae from sterile body sites during the study period, and identified their serotypes and AST. RESULTS: PCR was positive for pneumococci in 217 out of 334 pharyngeal swabs (65%), including 186 typable samples. The most common serotypes were serotypes 1, 6ABC, 19 F, 5 and 18ABC. By culture, we isolated only 110 out of 334 pharyngeal swabs (32.9%). The theoretical coverage of the PCV13 vaccine for the detected serotypes was 77.4%. The AST of NP isolates revealed low susceptibility rates to all antimicrobials except for vancomycin, linezolid, levofloxacin and clindamycin. During the study period, we identified 40 IPD; 21 identified by PCR and 19 by culture. The commonest pneumococcal serotypes were 1, 18ABC, 6ABC and 5. The PCV13 coverage was 75%. By Vitek-2, the isolates showed 100%, 100%, 94.7%, 89.5%, 84.2%, 84.2% and 78.9% susceptibility to vancomycin, linezolid, clindamycin, levofloxacin, penicillin, cefotaxim and erythromycin, respectively. CONCLUSION: Based on the serotype vaccine coverage and the emerging antimicrobial resistance of S. pneumoniae, PCVs will be valuable to Egyptian children.

Characterization and mutational analysis of haemagglutinin and neuraminidase of H3N2 and H1N1pdm09 human influenza A viruses in Egypt.

Seasonal influenza viruses constitute a major global concern. Currently, H3N2 and H1N1pdm09 are the commonly circulating influenza A viruses. The haemagglutin and neuraminidase genes of influenza A(H3N2) and A(H1N1)pdm09 viruses from Egyptian paediatric patients with respiratory distress were sequenced. Mutational analysis of all published sequences from Egypt was evolutionary tracked for both HA and NA genes. Phylogenetic analysis of H3N2 HA showed that the Egyptian strains belong to 3C2 subclade while Egyptian A(H1N1)pdm09 strains belong to 6B1 subclade. Some Egyptian A(H1N1)pdm09, 2013-2014, strains form a new subclade; 6B3. High score of mutations were recorded in HA of H1N1pdm09 but higher was recorded in H3N2 strains. These findings confirmed a high mutation rate of influenza A subtypes specially H3N2 strains.

Reliability of phenotypic methods for detection of colistin resistance among carbapenem-resistant Acinetobacter baumannii clinical isolates from Egypt.

INTRODUCTION: Acinetobacter baumannii is a challenging pathogen responsible for serious nosocomial infections. Colistin resistance in carbapenem-resistant A. baumannii strains is a critical health problem as it limits the available therapeutic options. The current work aimed to study the reliability of several phenotypic methods for the detection of colistin resistance among carbapenem-resistant A. baumannii isolates in Egypt. METHODS: A total of 22 carbapenem-resistant A. baumannii isolates were recovered. Colistin minimum inhibitory concentrations (MICs) were determined using broth microdilution (BMD) and compared to agar dilution (AD), automated system (VITEK-2) and gradient test (E-test) and were analyzed by statistical methods. RESULTS: Phenotypic testing showed that nine of 22 isolates (40.9%) were colistin-resistant by BMD and seven of them were also resistant by AD, with the categorical agreement (CA) of 72.7% and essential agreement (EA) of 90.9%. Colistin MIC results ranged from 1-8 µg/mL and 1-32 µg/mL by both AD and BMD respectively. Detection of colistin resistance by gradient test and automated system showed high very major error (VME) rates (40.9%) compared to BMD with a lack of CA between them. AD gave moderate agreement with BMD by 90.9% EA, 72.7% CA and only 9.1% VME. CONCLUSIONS: In delineating colistin breakpoints BMD followed by AD method are defined as the only reliable phenotypic methods for colistin resistance evaluation. More rapid and reliable tests, other than BMD and AD, are required for the convenient detection of colistin resistance in the routine clinical microbiology laboratory daily workflow.

Respiratory Viruses and Atypical Bacteria Co-Infection in Children with Acute Respiratory Infection.

BACKGROUND: Acute respiratory infections (ARI) are one of the prevalent pediatric diseases. Coinfections of respiratory viruses and atypical bacterial respiratory pathogens are common. AIM: This study aimed to determine the prevalence of co-infection between respiratory pathogens including viruses, bacteria and atypical bacteria in a sample of Egyptian children presenting with symptoms of acute respiratory tract infection. METHODS: This one-year prospective cohort study conducted in Abo El Rish Pediatric Hospital, Cairo University over one year included children presenting with symptoms of acute respiratory infection. Enrolled children were subjected to nasopharyngeal swabs or throat swabs and then processed to detect viral, bacterial and atypical bacterial causative agents by culture), retrotranscription polymerase, Monoplex polymerase chain reaction (PCR) and Multiplex PCR. RESULTS: Viral etiological agents were detected in 20 cases (20.8%), while 76 patients (79.2%) had no definite viral aetiology. The most abundant virus detected was Rhinovirus in 36 (27.3%), followed by 21 (15.9%) were positive for RSV, 12 (9.1%) were positive for HMPV, 6 (4.5%) were positive for adenovirus and 3 (2.3%) were positive for influenza B. For Atypical bacterial causes Mycoplasma were positive for 9 (6.8%) cases and one case was positive for Bordetella parapertussis. Viral and atypical bacteria Co infection were detected in 14 (10.6%) of cases. CONCLUSION: These results suggest that coinfection with bacteria or atypical bacteria in children with acute respiratory tract infection is common and this co-infection can induce serious illness. The multiplex reverse-transcriptase polymerase chain reaction should become an essential tool for epidemiological studies and can fill the gap between clinical presentation and definitive diagnosis.

Sequence analysis of the G gene of hRSVA ON1 genotype from Egyptian children with acute respiratory tract infections.

Human respiratory syncytial virus causes severe lower respiratory tract infection in neonates and children. Genotype ON1, with duplication of 72-nt in the G gene, was first detected in Canada and then recorded in other countries. In the current study, we describe the first detection of the ON1 genotype among children in Egypt in 2014/2015. Sequence analysis of the full-attachment G gene revealed that the majority of the strains examined were related to the ON1 genotype and only one sample related to N1 genotype. The Egyptian ON1 strains showed unique non-silent mutations in addition to variable mutations near the antigenic sites in comparison to the original ON1 ancestor strain. Continuous surveillance of hRSV regionally and globally is needed to understand the evolutionary mechanisms and strategies adopted by hRSV and their inducers for better adaption to the host.

Detection of carbapenemase-producers: Evaluating the performance of the carbapenem inactivation method and Carba NP test versus multiplex PCR.

OBJECTIVES: With a worrisome surge of carbapenem-resistant bacterial isolates, the diagnostic arsenal has become in dire need of affordable and timely assays to detect the rapidly transmissible carbapenemases. Employing multiplex PCR as a reference method, the purpose of the present study was to compare the performance of the carbapenem inactivation method (CIM) and the Carba NP test in the detection of carbapenemase-producers. METHODS: A panel of 203 Gram-negative bacterial isolates screened for carbapenem resistance were subjected to the CIM and Carba NP test. The results were compared with multiplex PCR targeting various carbapenemase genes. RESULTS: According to multiplex PCR, 92 (45.3%) of 203 isolates were found to harbour one or more carbapenemase genes, with blaNDM and blaKPC being the most commonly encountered. The sensitivity and specificity of the CIM were 95.7% and 95.5% respectively, whilst those of the Carba NP test were 75.0% and 99.1%, respectively. Both methods were found to be rapid and reliable in the detection of carbapenemases and showed a high agreement with multiplex PCR. CONCLUSIONS: As the list of carbapenemase genes continues to expand, the reliability of PCR has become doubtful; hence, the CIM and Carba NP test could offer promising alternatives, with the CIM being of a lower cost and less labour intensive.

Prevalence and risk factors of MRSA, ESBL and MDR bacterial colonization upon admission to an Egyptian medical ICU.

INTRODUCTION: Bacterial colonization of the skin and mucous membranes of intensive care unit (ICU) patients with virulent organisms such as methicillin-resistant Staphylococcus aureus (MRSA), extended-spectrum beta-lactamase (ESBL) producers, and multidrug-resistant Gram-negative bacteria (MDR-GNB) frequently results in life-threatening infections. Universal screening of ICU patients upon admission has been suggested. The aim of the current study was to evaluate the prevalence and pattern of MRSA, ESBL, and MDR-GNB colonization in patients upon admission to an Egyptian medical ICU, along with the related demographic and clinical risk factors. METHODOLOGY: Throat, axillary, and groin swabs were obtained from all study participants in addition to rectal swabs from consenting patients. These swabs were screened for MRSA, ESBL, and MDR-GNB. RESULTS: Of the patients included in the study, 33%, 13%, and 63% were colonized with ESBL, MDR-GNB, and MRSA organisms, respectively. Those suffering from a more severe disease with a simplified acute physiology score II (SAPS II) > 29 demonstrated higher levels of MDR-GNB colonization upon admission, while MDR-GNB or ESBL colonization upon admission was associated with higher ICU mortality. CONCLUSIONS: Colonization of ICU patients with superbugs upon admission has an impact on outcome and mortality. In this Egyptian example, colonization rates were higher than in other literature reports, demonstrating the need for routine screening and decolonization, if applicable.

Respiratory syncytial virus and human metapneumovirus in severe lower respiratory tract infections in children under two.

INTRODUCTION: Viruses are the most important causative agents of acute lower respiratory tract infections (ALRTIs), ranked as the second leading cause of death and the primary cause of hospitalization in children. Respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) are among the commonest viral causes of severe ALRTI. In this study, we aimed to study the burden of both RSV and hMPV in causing severe ALRTI in children younger than two years of age admitted to the pediatric intensive care unit (PICU). METHODOLOGY: Nasopharyngeal swabs were collected from children admitted to the PICU with a diagnosis of community-acquired ALRTI who were two years of age or younger. Real-time polymerase chain reaction (RT-PCR) was used to test for RSV and hMPV. RESULTS: A total of 127 swabs were screened for RSV and hMPV, of which 49.6% were negative for RSV and hMPV, 46.4% were positive for RSV, and 3.9% were positive for hMPV. With respect to RSV, the mean age of cases (4.01 ± 5.05) and the monthly distribution (mainly January) were the most important risk factors. There were no statistically significant differences between the RSV group and control group regarding duration of hospital stay, mechanical ventilation need or duration, and underlying chronic conditions. CONCLUSIONS: RSV is important viral cause of severe ALRTIs in children younger than two years of age during this study period; hMPV played a minor role.

A novel primer set for improved direct gene sequencing of human bocavirus genotype-1 from clinical samples.

Human bocavirus genotype (HBoV-1) is a parvovirus associated with respiratory tract infections in children with different degrees of severity. The current study intended to improve the direct gene sequencing of the HBoV-1 using a newly developed primer set. Screening the presence of human bocavirus infection among in-patients children suffering from lower respiratory tract infections was another aim of the current study. Nasopharyngeal swab samples from in-patients children suffering from lower respiratory tract infections were examined. The real-time polymerase chain reaction was used for the initial screening as a highly sensitive method to detect the HBoV. Genotyping of real-time positive samples was attempted by direct sequencing of PCR amplicons using NP, VP1/2 and the newly developed VP/NC primers. HBoV-1 was present in 56.8% of the examined children. The newly developed primer set successfully amplified all real-time PCR positive samples, however, the other primer pairs did not reliably detect real-time PCR positive samples. The gene sequences of the detected HBoV-1 showed conserved sequences to each other with a low rate of discrepancies. The high rate of infection and the similarity between the detected strains strongly suggest nosocomial infections.

Risk factors of prolonged hospital stay in children with viral severe acute respiratory infections.

INTRODUCTION: Severe acute lower respiratory infections (SARIs) are one of the major causes of morbidity and mortality in young children, especially in developing countries. The present study focused on detection of risk factors for prolonged hospital stays among children with viral SARIs. METHODOLOGY: A sentinel surveillance study was conducted at Cairo University Hospital (CUH) between February 2010 and May 2011. Nasopharyngeal (NP) and oropharyngeal (OP) swabs were collected from all children admitted with SARIs. Viruses were identified using reverse transcription polymerase chain reaction (RT-PCR). RESULTS: Out of 1,046 children, 380 (36%) were positive for one or more viruses; these included respiratory syncytial virus (RSV) (22.9%), adenovirus (6.2%), parainfluenza viruses (PIVs1-3) (5.1%), human metapneumovirus (HMPV) (4.5%), influenza A (1.4%), and influenza B (0.6%). Viral etiology was mainly detected in children under one year of age (88.9%). Prolonged length of stay was independently associated with the presence of cyanosis and underlying chronic illness (OR 7.4, CI: 1.8-30.32 [p = 0.005], OR 2.5, CI: 1.36-4.64 [p = 0.004], respectively). Virus type did not affect the length of hospital stay (p > 0.05). Oxygen therapy was required in 91% of the patients. A total of 43 patients (11.6%) required intensive care admission. Twenty-one patients (5.5%) died, and 15 of them (71.4%) had an underlying chronic illness. CONCLUSIONS: The study demonstrated the important burden of respiratory viruses as a cause of SARI in hospitalized children in a tertiary Egyptian hospital. Cyanosis and underlying chronic illness were significantly associated with prolonged length of stay.

Morbidity and outcome of severe respiratory syncytial virus infection.

BACKGROUND: Respiratory syncytial virus (RSV) is the main cause of severe acute respiratory infection (SARI) in infants and young children. This study aimed to identify risk factors for intensive care unit (ICU) admission, prolonged length of stay (PLOS), and mortality in patients hospitalized with SARI caused by RSV. METHODS: This prospective cohort study included children hospitalized with SARI (according to the World Health Organization definition) and whose laboratory results proved RSV infection during the period from February 2010 to May 2011. RESULTS: Out of 240 enrolled patients, 24 patients (10%) were admitted to the ICU, 57 patients (24.3%) had a PLOS of >9 days and 12 patients (5%) died. The presence of cyanosis (P = 0.000; OR, 351.7) and lung consolidation (P = 0.006, OR, 9.3) were independent risk factors associated with ICU admission. The need for ICU admission (P = 0.000; OR, 6.1) and lung consolidation (P = 0.008, OR, 2.46) were independent risk factors associated with PLOS. The presence of an underlying congenital heart disease (P = 0.03, OR, 18.3), thrombocytopenia (P = 0.04, OR, 32.86) and mechanical ventilation (P = 0.000; OR, 449.4) were the only independent risk factors associated with mortality in our study. CONCLUSIONS: Early recognition of risk factors for complicated RSV disease on admission prompts early interventions and early ICU admissions for these children.