Emerging Oncogenic Viruses in Head and Neck Cancers from Romanian Patients

(1) Background: Head and neck squamous cell carcinomas (HNSCCs) are some of the most frequent malignancies globally. Oncogenic viruses MCPyV, EBV and HPVs are recognized to be related to HNSCCs and skin cancers. There are no data from Romania regarding the involvement of herpes viruses and polyomaviruses in these types of cancer. We aim to evaluate the association of oncogenic viruses from Papillomaviridae, Herpesviridae, and Polyomaviridae families in HNSCCs and skin cancers. (2) Methods: A total of 26 fresh tumors (6/26 women) were tested for 67 viral agents using a multiplex PCR genotyping assay. (3) Results: A total of 23/26 (88.5%) samples were positive for one or more viruses. All the tested tumors were negative for any HPV (alpha or beta types). In total, we detected as positive samples: 16 (61.63%) EBV1, 12 (46.15%) HHV7, 8 (30.76%) MCV, 6 (23.07%) CMV and HHV6, 2 (7.69%) HHV8, 1 (3.8%) HPyV6 and EBV2. (4) Conclusions: We detected HPV-negative cases that are HPyV and HHV positive. In these fractions of HPV-negative HNSCCs cases, other oncogenic viruses may be involved, such as EBV1, MCV or CMV. Additional research is required for clarifying the natural history of these viruses in HNSCCs, as virus detection would have a decisive impact on diagnostic and decisional algorithms.

Real-Time SARS-CoV-2 Genotyping by High-Throughput Multiplex PCR Reveals the Epidemiology of the Variants of Concern in Qatar

Complementing whole genome sequencing strategies with high-throughput multiplex RT-qPCR genotyping allows for more comprehensive and real-time tracking of SARS-CoV-2 variants of concern. During the second and third waves of COVID-19 in Qatar, PCR genotyping, combined with Sanger sequencing of un-typeable samples, was employed to describe the epidemiology of the Alpha, Beta and Delta variants. A total of 9792 nasopharyngeal PCR-positive samples collected between April-June 2021 were successfully genotyped, revealing the importation and transmission dynamics of these three variants in Qatar.

Assessment of single nucleotide polymorphism RS2596542 of MICA gene among nasopharyngeal cancer patients

MICA is an antigen that is commonly expressed on cell surface of tumoRS. MICA had a low expression level in normal cells but increased in pathological/cancer cells. The goal of the study was to determine the association of SNP RS2596542 with the risk of nasopharyngeal cancer (NPC). 144 biopsy samples diagnosed with NPC and 135 blood samples from healthy people were collected. Genotypes and alleles of RS2596542 MICA were determined by Realtime PCR genotyping. The genotype rate of MICA at RS2596542 in patients with NPC was 34.72% CC; 44.44% CT and 20.84% TT. The genotype of TT and T allele in the disease group was significantly higher than the control group (p=0.01*; OR=2.47; 95%CI: 1.22-4.97) and (p=0.01*; OR=1.51; 95%CI: 1.07-2.13). The T-allele at SNP RS2596542 of the MICA gene increases the risk of NPC and suggests that this could be used as a biomarker for NPC screening in Viet Nam. Keywords: MICA, RS2596542, nasopharyngeal cancer, NPC, Single Nucleotide Polymorphism, SNPs, Realtime PCR

A prospective diagnostic study to measure the accuracy of detection of SARS-CoV-2 Variants Of Concern (VOC) utilising a novel RT-PCR GENotyping algorithm in an In silico Evaluation (VOC-GENIE)

Background SARS-CoV-2 variants of concern (VOCs) have been associated with higher rate of transmission, and evasion of immunisation and antibody therapeutics. Variant sequencing is widely utilized in the UK. However, only 0.5% (~650k) of the 133 million cumulative positive cases worldwide were sequenced (in GISAID) on 08 April 2021 with 97% from Europe and North America and only ~0.25% (~320k) were variant sequences. This may be due to the lack of availability, high cost, infrastructure and expert staff required for sequencing. Public health decisions based on a non-randomised sample of 0.5% of the population may be insufficiently powered, and subject to sampling bias and systematic error. In addition, sequencing is rarely available in situ in a clinically relevant timeframe and thus, is not currently compatible with diagnosis and treatment patient care pathways. Therefore, we investigated an alternative approach using polymerase chain reaction (PCR) genotyping to detect the key single nucleotide polymorphisms (SNPs) associated with increased transmission and immune evasion in SARS-CoV-2 variants. Methods We investigated the utility of SARS-CoV-2 SNP detection with a panel of PCR-genotyping assays in a large data set of 640,482 SARS-CoV-2 high quality, full length sequences using a prospective in silico trial design and explored the potential impact of rapid in situ variant testing on the COVID-19 diagnosis and treatment patient pathway. Results Five SNPs were selected by screening the published literature for a reported association with increased transmission and / or immune evasion. 344881 sequences contained one or more of the five SNPs. This algorithm of SNPs was found to be able to identify the four variants of concern (VOCs) and sequences containing the E484K and L452R escape mutations. Interpretation The in silico analysis suggest that the key mutations and variants of SARS-CoV-2 may be reliably detected using a focused algorithm of biologically relevant SNPs. This highlights the potential for rapid in situ PCR genotyping to compliment or replace sequencing or to be utilized instead of sequences in settings where sequencing is not feasible, accessible or affordable. Rapid detection of variants with in situ PCR genotyping may facilitate a more effective COVID-19 diagnosis and treatment patient pathway.

Emergence of Colistin and Carbapenem Resistance in Extended-Spectrum β-Lactamase Producing Klebsiella pneumoniae Isolated from Chickens and Humans in Egypt

This study investigated the frequency of carbapenem and colistin resistance in ESBL-producing K. pneumoniae (ESBLK) isolates recovered from chickens and their environment, contact farm workers and hospitalized patients in Egypt. Further, the phenotypic and genotypic relationships between the community and hospital-acquired K. pneumoniae isolates in the same geographical area were investigated. From 272 total samples, 37 (13.6%) K. pneumoniae isolates were identified, of which 20 (54.1%) were hypervirulent. All isolates (100%) were multidrug-resistant (MDR) with multiple antibiotic resistance (MAR) indices ranging from 0.19 to 0.94. Colistin-resistant isolates (18.9%) displayed colistin MIC values >2 μg/mL, all harbored the mcr-1 gene. All isolates from patients (13/90, 14.4%), workers (5/22, 22.7%), chickens (9/100, 9%) and the environment (10/60, 16.7%) harbored a single or multiple β-lactamase genes, blaSHV, blaTEM, blaCTX-M1 and blaOXA-1, often in combination with carbapenemase genes (blaVIM, blaNDM-1 or blaIMP; 45.9%), the mcr-1 gene (18.9%) or both (13.5%). Enterobacterial repetitive intergenic consensus (ERIC)–PCR genotyping revealed 24 distinct ERIC types (ETs) with a discrimination index of 0.961. Six ETs showed clusters of identical isolates from chicken and human sources. The increased frequency and genetic relatedness of ESBLK and carbapenemase-producing K. pneumoniae (CPK) from chickens and humans pose a public health threat that urge more prudent use of antimicrobials in chicken farms to avoid the propagation and expansion of both ESBLK and CPK from the chicken sources to humans.

ARID5B rs10821936 and rs10994982 gene polymorphism and susceptibility to juvenile systemic lupus erythematosus and lupus nephritis

Background The prevalence of SLE and the spectrum of clinical manifestations vary widely in different races and geographical populations. Objective To investigate the possible role of ARID5B rs10821936 and rs10994982 polymorphism as a risk factor for the development of SLE in children (jSLE) and to evaluate their role in relation to clinical manifestations especially lupus nephritis (LN). Methods DNA extraction and Real-time PCR genotyping of ARID5B rs10821936 and rs10994982 were done for 104 jSLE and 282 healthy controls. Results The C allele and C containing genotypes (CC, CT and CC+CT) of ARID5B rs10821936 were higher in children with SLE (p = 0.009, OR = 1.56, 0.037, OR = 2.35, 0.016, OR = 1.81 and 0.008 OR = 1.88 respectively). ARID5B rs10994982 alleles, genotypes and haplotypes are not associated with jSLE (p > 0.05). The ARID5B rs10821936 and rs10994982 genotypes showed non-significant associations with LN, proliferative versus non proliferative and biopsy grades (p > 0.05). Conclusion ARID5B rs10821936 SNP may be a susceptibility risk factor for juvenile SLE in the studied cohort of Egyptian children.

Antibiotic Resistance and Genotypes of Nosocomial Strains of Acinetobacter baumannii in Kazakhstan

The aim of this study was to determine the prevalence of A. baumannii antibiotic-resistant strains in Kazakhstan and to characterize genotypes related to epidemic “high-risk” clones. Two hundred and twenty four A. baumannii isolates from four cities of Kazakhstan in 2011–2019 were studied. Antibiotic susceptibility testing was performed by using broth microdilutions method according to EUCAST (v 11.0) recommendations. The presence of blaOXA-23-like, blaOXA-24/40-like,blaOXA-58-like,blaVIM,blaIMP, and blaNDM genes was determined by PCR. Genotyping was performed using high-throughput real-time PCR detection of 21 SNPs at 10 chromosomal loci used in existing MLST schemes. Resistance rates to imipenem, meropenem, amikacin, gentamicin, and ciprofloxacin were 81.3%, 78.6%, 79.9%, 65.2%, and 89.3%, respectively. No colistin resistant isolates were detected. The values of the MIC 50% and the MIC 90% of tigecycline were 0.125 mg/L, only four isolates (1.8%) had the ECOFF value >0.5 mg/L. The presence of acquired carbapenemase genes was found in 82.2% strains, including blaOXA-23-like (78.6%) or blaOXA-58-like (3.6%) genes. The spreading of carbapenem resistant A. baumannii strains in Kazakhstan was associated with epidemic “high-risk” clonal groups, predominantly, CG208(92)OXF/CG2PAS (80.8%) and less often CG231(109)OXF/CG1PAS (1.8%).

A versatile platform for locus-scale genome rewriting and verification

Routine rewriting of loci associated with human traits and diseases would facilitate their functional analysis. However, existing DNA integration approaches are limited in terms of scalability and portability across genomic loci and cellular contexts. We describe Big-IN, a versatile platform for targeted integration of large DNAs into mammalian cells. CRISPR/Cas9-mediated targeting of a landing pad enables subsequent recombinase-mediated delivery of variant payloads and efficient positive/negative selection for correct clones in mammalian stem cells. We demonstrate integration of constructs up to 143 kb, and an approach for one-step scarless delivery. We developed a staged pipeline combining PCR genotyping and targeted capture sequencing for economical and comprehensive verification of engineered stem cells. Our approach should enable combinatorial interrogation of genomic functional elements and systematic locus-scale analysis of genome function.