Reem Hassan

The overall pattern of the SARS-CoV-2 pandemic so far has been a series of waves; surges in new cases followed by declines. The appearance of novel mutations and variants underlie the rises in infections, making surveillance of SARS-CoV-2 mutations and prediction of variant evolution of utmost importance. In this study, we sequenced 320 SARS-CoV-2 viral genomes isolated from patients from the outpatient COVID-19 clinic in the Children's Cancer Hospital Egypt 57357 (CCHE 57357) and the Egypt Center for Research and Regenerative Medicine (ECRRM). The samples were collected between March and December 2021, covering the third and fourth waves of the pandemic. The third wave was found to be dominated by Nextclade 20D in our samples, with a small number of alpha variants. The delta variant was found to dominate the fourth wave samples, with the appearance of omicron variants late in 2021. Phylogenetic analysis reveals that the omicron variants are closest genetically to early pandemic variants. Mutation analysis shows SNPs, stop codon mutation gain, and deletion/insertion mutations, with distinct patterns of mutations governed by Nextclade or WHO variant. Finally, we observed a large number of highly correlated mutations, and some negatively correlated mutations, and identified a general inclination toward mutations that lead to enhanced thermodynamic stability of the spike protein. Overall, this study contributes genetic and phylogenetic data, as well as provides insights into SARS-CoV-2 viral evolution that may eventually help in the prediction of evolving mutations for better vaccine development and drug targets.

BACKGROUND: Candidemia is a pervasive problem associated with significant morbidity and mortality in health care settings. This study aimed to determine the changing distribution of Candida species and the emergence of uncommon species.

METHODS: This was a cross-sectional study performed in two Cairo University hospitals between 2019 and 2020. All Candida species isolates recovered from blood cultures of adults and pediatrics patients admitted to the hospitals were included. Candida isolates were identified by chromogenic Candida agar and Vitek2 YST identification card. Candida kefyr was confirmed by chip array.

RESULTS: Candida species were responsible for 1.6% of bloodstream infections in adults and 10.8% in pediatric patients. C. albicans was the most prevalent species representing 27.8% in adults and 48.3% in pediatrics. Non-albicans species (NAC) represented the most isolated Candida species among adults and pediatrics (72.2% and 51.6%, respectively) with the predominance of C. tropicalis (27.8% and 22.5%, respectively) followed by C. parapsilosis (16.7% and 10.8%, respectively). The uncommon Candida, which is Candida species other than C. albicans, C. parapsilosis, C. tropicalis, C. glabrata, and C. krusei, represents 16.6% and 14% of all candidemia in adults and pediatrics, respectively. Only one of each of C. lusitaniae, C. utilis, and C. kefyr were detected in adults. C. lusitaniae was the most frequently recovered uncommon Candida among pediatrics resulting in 6.4% of candidemia followed by C. famata (4.3%), C. utilis (2.2%), and C. kefyr (1.1%).

CONCLUSIONS: C. albicans is still the primary species isolated from pediatrics and adults with candidemia despite the considerable shift to the non-albicans species. C. tropicalis and C. parapsilosis are the most prevalent NAC. The increased prevalence of uncommon Candida species is alarming and necessitates a prompt stewardship program.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) is a significant healthcare-associated (HA-MRSA) pathogen due to its increased morbidity and mortality rates. There is a paucity of data regarding MRSA clones circulating in the Middle East in the literature, especially from Egypt. We aimed to identify the pattern of resistance and virulence in the propagating clones using NGS technologies for the whole genome sequence.

METHODS: From an 18-month surveillance program for MRSA-positive patients, 18 MRSA isolates from surgical healthcare associated infections were selected. The Vitek2 system was used to assess antimicrobial susceptibility. The whole genome sequencing was performed using the NovaSeq6000. The reads were mapped to the reference genome (Staphylococcus_aureus_ATCC_BAA_1680), used for variant calling, screened for virulence/resistance genes, and typed using multi-locus sequence typing and spa typing. Correlation between demographic and clinical data and molecular findings were performed.

RESULTS: All the MRSA isolates were highly resistant to tetracycline followed by gentamicin (61%) and highly susceptible to trimethoprim/sulfamethoxazole. Most of the isolates showed a high virulence profile. ST239 was the predominant sequence type (6/18), while t037 was the predominant spa type (7/18). Five isolates shared the same ST239 and spa t037. ST1535, an emerging MRSA strain, was the second most prevalent in our study. One isolate showed a unique pattern of a high abundance of resistance and virulence genes.

CONCLUSION: WGS elucidated the resistance and virulence profiles of MRSA isolated from clinical samples of HAI patients with high-resolution tracking of clones predominant in our healthcare facility.

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Patients with COVID-19 are at risk of developing secondary complications such as invasive pulmonary aspergillosis and mucormycosis. This is a retrospective study including all cancer children diagnosed with COVID-19-associated pulmonary fungal infection (CAPFI) during the period 2020-2021. A total of 200 patients were diagnosed with COVID-19, out of which 21 (10%) patients were diagnosed with CAPFI, 19 patients (90%) with COVID-aspergillosis (CAPA), and 2 (10%) patients with COVID-mucormycosis (CAM). Patients with CAPFI were classified using the "2020 ECMM/ISHAM consensus criteria"; proven in 2 (10%) patients, probable in 12 (57%), and possible in 7 (33%) patients. Although the hematological malignancy patients were already on antifungal prophylaxis, breakthrough fungal infection was reported in 16/21 (75%), 14 (65%) patients had CAPA while on echinocandin prophylaxis, while 2 (10%) patients had CAM while on voriconazole prophylaxis. Overall mortality was reported in 8 patients (38%) while CAPFI-attributable mortality was reported in 4 patients (20%). In conclusion, clinicians caring for pediatric cancer patients with COVID-19 should consider invasive pulmonary fungal infection, even if they are on antifungal prophylaxis, especially with worsening of the clinical chest condition. A better understanding of risk factors for adverse outcomes may improve clinical management in these patients.

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.

BACKGROUND: The growing emergence of microbial resistance to antibiotics represents a worldwide challenge. Antimicrobial photodynamic inactivation (aPDI) has been introduced as an alternative technique, especially when combined with nanotechnology. Therefore, this study was designed to investigate the therapeutic merits of combined aPDI and nanoemulsion in infections caused by resistant bacterial strains.

METHODS: Cationic zinc (II) phthalocyanine nanoemulsions (ZnPc-NE) were prepared using isopropyl myristate (IPM) as oil phase, egg phosphatidylcholine (egg PC) as emulsifier, and N-cetyl-N,N,N-trimethyl ammonium bromide (CTAB). Nanoemulsions were characterized for particle size, polydispersity, zeta potential, viscosity, and skin deposition. The in-vitro aPDI was investigated on human resistant pathogens; gram-positive methicillin-resistant Staphylococcus aureus (MRSA) and gram-negative Multidrug-resistant strain of Escherichia coli (MDR E. coli), under different experimental conditions. In addition, in-vivo model of abrasion wound infected by MDR E. coli was induced in rats to investigate the therapeutic potential of the selected formulation.

RESULTS: It was evident that the selected ZnPc formulation (20 % IPM, 2 % egg PC and 0.5 % CTAB) displayed a particle size of 209.9 nm, zeta potential +73.1 mV, and 23.66 % deposition of ZnPc in skin layers. Furthermore, the selected formulation combined with light achieved almost 100 % eradication of the two bacterial strains, with superior bacterial load reduction and wound healing propertiesin-vivo, compared to either the nanoemulsion formulation or laser alone.

CONCLUSION: ZnPc nanoemulsion improved antimicrobial photodynamic therapy in inactivating resistant bacterial infections and provided a promising therapeutic means of treating serious infections, and hence could be applied in diseases caused by other bacterial strains.

The emergence of carbapenem-resistant (CRKP) isolates in Egyptian hospitals has been reported. However, the genetic basis and analysis of the plasmids associated with carbapenem-resistant hypervirulent (CR-HvKP) in Egypt have not been presented. Therefore, we attempted to decipher the plasmid sequences that are responsible for transferring the determinants of carbapenem resistance, particularly and Out of 34 isolates collected from two tertiary hospitals in Egypt, 31 were CRKP. Whole-genome sequencing revealed that our isolates were related to 13 different sequence types (STs). The most prevalent ST was ST101, followed by ST383 and ST11. Among the CRKP isolates, one isolate named EBSI036 has been reassessed by Nanopore sequencing. Genetic environment analysis showed that EBSI036 carried 20 antibiotic resistance genes and was identified as a CR-HvKP strain: it harbored four plasmids, namely, pEBSI036-1-NDM-VIR, pEBSI036-2-KPC, pEBSI036-3, and pEBSI036-4. The two carbapenemase genes and were located on plasmids pEBSI036-1-NDM-VIR and pEBSI036-2-KPC, respectively. The IncFIB:IncHI1B hybrid plasmid pEBSI036-1-NDM-VIR also carried some virulence factors, including the regulator of the mucoid phenotype (), the regulator of mucoid phenotype 2 (), and aerobactin ( and ). Thus, we set out in this study to analyze in depth the genetic basis of the pEBSI036-1-NDM-VIR and pEBSI036-2-KPC plasmids. We report a high-risk clone ST11 KL47 serotype of a CR-HvKP strain isolated from the blood of a 60-year-old hospitalized female patient from the intensive care unit (ICU) in a tertiary care hospital in Egypt, which showed the cohabitation of a novel hybrid plasmid coharboring the and virulence genes and a -carrying plasmid. CRKP has been registered in the critical priority tier by the World Health Organization and has become a significant menace to public health. The emergence of CR-HvKP is of great concern in terms of both disease and treatment. In-depth analysis of the carbapenemase-encoding and virulence plasmids may provide insight into ongoing recombination and evolution of virulence and multidrug resistance in Thus, this study serves to alert contagious disease clinicians to the presence of hypervirulence in CRKP isolates in Egyptian hospitals.

Invasive Aspergillosis is a challenging infection that requires convenient, efficient, and cost-effective diagnostics. This study addresses the potential of infrared spectroscopy to satisfy this clinical need with the aid of machine learning. Two models, based on Partial Least Squares-Discriminant Analysis (PLS-DA), have been trained by a set of infrared spectral data of 9 Aspergillus-spiked and 7 Aspergillus-free plasma samples, and a set of 200 spectral data simulated by oversampling these 16 samples. Two further models have also been trained by the same sets but with auto-scaling performed prior to PLS-DA. These models were assessed using 45 mock samples, simulating the challenging samples of patients at risk of Invasive Aspergillosis, including the presence of drugs (9 tested) and other common pathogens (5 tested) as potential confounders. The simple model shows good prediction performance, yielding a total accuracy of 84.4%, while oversampling and autoscaling improved this accuracy to 93.3%. The results of this study have shown that infrared spectroscopy can identify Aspergillus species in blood plasma even in presence of potential confounders commonly present in blood of patients at risk of Invasive Aspergillosis.

Infection with multiple drug resistant (MDR) Escherichia coli poses a life threat to immunocompromised pediatric cancer patients. Our aim is to genotypically characterize the plasmids harbored in MDR E. coli isolates recovered from bacteremic patients of Children's Cancer Hospital in Egypt 57357 (CCHE 57357). In this study, 21 carbapenem-resistant E. coli (CRE) isolates were selected that exhibit Quinolones and Aminoglycosides resistance. Plasmid shot-gun sequencing was performed using Illumina next- generation sequencing platform. Isolates demonstrated resistant to all beta-lactams, carbapenems, aminoglycosides and quinolones. Of the 32 antimicrobial resistant genes identified that exceeded the analysis cutoff coverage, the highest represented genes were aph(6)-Id, sul2, aph(3″)-Ib, aph(3')-Ia, sul1, dfrA12, TEM-220, NDM-11. Isolates employed a wide array of resistance mechanisms including antibiotic efflux, antibiotic inactivation, antibiotic target replacements and antibiotic target alteration. Sequenced isolates displayed diverse insertion sequences, including IS26, suggesting dynamic reshuffling of the harbored plasmids. Most isolates carried plasmids originating from other bacterial species suggesting a possible horizontal gene transfer. Only two isolates showed virulence factors with iroA gene cluster which was found in only one of them. Outside the realms of nosocomial infections among patients in hospitals, our results indicate a transfer of resistant genes and plasmids across different organisms.

Hepatocellular carcinoma (HCC) is the second most common cause of cancer-related death worldwide. The use of alpha fetoprotein (AFP) alone was not an accurate biomarker for HCC despite its high specificity. Therefore, we assessed the possible role of serum biomarkers that have been mentioned briefly in previous studies on Egyptian patients ion top of HCV. However these studies included small number of patients and did not assess the different stages of hepatocarcinogenesis. In the current study we assessed 1) the expression levels of Golgi protein 37(GP73),Midkine (MDK) and Dickkopf-1(DKK-1) proteins separately and in combination at different stages of hepatocarcinogenesis. GP73, MDK and DKK-1 proteins were assessed in 238 individuals divided into 4 groups (HCC, chronic HCV, and chronic HCV with cirrhosis and healthy subjects as a control) Serum levels of GP73, MDK, and DKK-1 were assessed in all subjects by ELISA. Serum levels of the studied markers were significantly higher in HCC compared to other groups (p < 0.001). The ROC curve analysis for the studied markers showed 1) 88.5% sensitivity, 80.6% specificity, 69% PPV, 93.5% NPV and (AUC 0.91)for MDK; 2) 93.6%, 86.9%, 77.7%, 96.5% for DKK-1. 3) 91%, 85%, 74.7%, 95% (AUC 0.96) for GP73 and 4) 74.4%, 84.4%, 69.9%, 87.1% (AUC 0.81) for AFP. Serum levels of GP73, MDK, and DKK-1 are comparable to AFP as promising predictor biomarkers for HCC patients from Egypt. A two markers panel including Gp73 and DKK-1 showed the highest specificity and sensitivity among different markers combinations.

PURPOSE: Antimicrobial resistance is a major concern that we are facing nowadays. This is due to antibiotic misuse and bacteria developing resistance to the commonly used antibiotics. This may lead to increased mortality and consumption of country resources. Implementation of an antimicrobial stewardship program [ASP] can limit the use of unnecessary antibiotics and subsequently decrease the infection rates with better patient outcome. We aimed to control antibiotic misuse, reduce infection rate, decrease drug costs, and reduce length of hospital stay in the ICU.

METHODS: We conducted a prospective study on the surgical neonatal ICU [SNICU] over a period of 6 months divided into pre-implementation phase, followed by an ASP phase, in which we applied the antibiotic guidelines approved by the ASP committee. Data were collected in the two phases and analyzed for demographics, compliance with guidelines, prescribed antibiotics, lab investigations, surgical site infection [SSI], length of stay and patient outcome.

RESULTS: Compliance to the guidelines was encountered in 86% and SSI rate decreased to 20%. Days of Therapy (DOT) per 1000 patient days showed a significant decrease in Ampicillin Sulbactam by 296 (p = 0.024), Imipenem by 220.34 (p = 0.024) and Vancomycin by 287.34 (p = 0.048). Drug cost showed a 1185.97 EGP decrease in the ASP period compared to the pre-implementation period (p = 0.714). Average LOS decreased in the ASP period by a mean difference of 2.5 (p = 0.027).

CONCLUSION: ASP implementation can control antibiotic misuse, decrease the medical care expenses and improve patient outcome.

TYPE OF STUDY: Clinical research paper.

LEVEL OF EVIDENCE: Level one.

Understanding the complex immune responses in sepsis is crucial to provide insight into the clinical syndrome. We evaluated the changes in the surface receptors of the cells of innate immunity, neutrophils and monocytes, in patients with sepsis. Since sepsis remains a clinical challenge, we aimed to assess the significance of altered receptor expression in diagnosis and prognosis. Critically ill patients with sepsis (n=31) were investigated for the expression of receptors for IgG heavy chain CD64 and CD16 on neutrophils and CD64 and the lipopolysaccharide receptor CD14 on monocytes by flow cytometry and compared to 23 patients with no sepsis. Patients with sepsis had increased expression of neutrophil CD64. Neutrophil CD64 was specific for discriminating patients with sepsis but showed weak sensitivity. When integrated in a scoring system, neutrophil CD64 in combination with C-reactive protein (CRP) and SOFA score showed a diagnostic accuracy of 0.93 for sepsis and significantly predicted increased mortality risk. While neutrophil CD16 did not discriminate for sepsis, decreased expression was associated with increased mortality risk. In contrast, monocyte CD64 and CD14 expression was unaltered in sepsis and was not associated with mortality risk. Our study demonstrates that unlike monocytes, neutrophil receptor expression is altered in patients with sepsis receiving intensive care. It is promising to apply a combination approach to diagnose sepsis especially in time-limited conditions.