GENOMIC SURVEILLANCE OF SARS-CoV-2 REVEALS SEVERITY OF DELTA VARIANT IN CAMEROON

Background: At the global level, the dynamics of the COVID-19 pandemic have been driven by several epidemiological waves, determined by the emergence of new SARS-CoV-2 variants from the original viral lineage from Wuhan, China. While the SARS-CoV-2 dynamic has been described globally, there is a lack of data from Sub-Saharan African. Method(s): A laboratory-based survey was conducted in Cameroon, from March 1, 2020 to March 30, 2022, through an assessment of the evolutionary patterns of SARS-CoV-2 lineages across the four COVID-19 waves in the country. Data on full-length sequencing from all four sequencing laboratories were consecutively entered into the GISAID platform. These data were downloaded, and the molecular phylogeny of the SARS-CoV-2 sequences was performed using Nexstrain. The Mann-Whitney U test was used to calculate the correlation between the duration of each outbreak and the number of confirmed cases and between hospitalised cases and CFR, with a p value < 0.05 considered statistically significant. Result(s): A total of 3,881 samples were successfully processed, of which 38.9% (n=1,509) using PCR mutation assay, 41.5% (n=1,612) using targeted sequencing, and 19.6% (n=760) using whole-genome sequencing. The mean age of the study population was 36 years (min-max: 2-86), and 45% were within the age range 26-45. Regarding gender distribution, 50.9% were male, and 49.1% were female. Phylogenetic analysis of the 760 whole-genome sequences generated from March 2020 to March 2022 revealed that the greater proportion of SARS-CoV-2 circulating in Cameroon belonged to the viral sub-lineages of the original strain from Wuhan (74%), 15% Delta variant, 6% Omicron variant, 3% Alpha variant and 2% Beta variant.The pandemic was driven by SARS-CoV-2 lineages of origin in Wave 1 (16 weeks, 2.3% CFR), the Alpha and Beta variants in Wave 2 (21 weeks, 1.6% CFR), Delta variants in Wave 3 (11 weeks, 2.0% CFR), and Omicron variants in Wave 4 (8 weeks, 0.73% CFR), with a declining trend over time (p=0.01208). Conclusion(s): In a nutshell, the SARS-CoV-2 epidemic in Cameroon appears to have been driven by the SARS-CoV-2 lineage of origin in Wave 1, the cointroduction of the Alpha and Beta variants in Wave 2, the Delta variant in Wave 3, and the Omicron variant in Wave 4, with an overall declining trend in the wave duration, confirmed cases and hospitalisations over time.The SARS-CoV-2 lineage of origin and the Delta variant appeared to be the drivers of COVID-19 severity in Cameroon.

Genetic diversity of Mycobacterium tuberculosis isolates from northwest of Iran during COVID-19 era

Background: Tuberculosis (TB) is considered one of the most infectious diseases in the world. In this study, we intended to examine the epidemiology of tuberculosis by MIRU-VNTR to define the changes that occur in the transmission of tuberculosis in the region during the COVID-19 era. A total of 120 Mycobacterium tuberculosis isolates were collected from sputum samples of patients referred to East Azerbaijan Center TB from December 2020 to August 2021. Demographic information such as age, sex, place of birth, previous TB history, and relevant medical data was collected. The proportion method was performed for drug susceptibility testing, and the PCR-based MIRU-VNTR method was applied to identify molecular epidemiology relationships. Result(s): The isolates were collected from 78 male (65%) and 39 female (32.5%) Iranian patients and 3 (2.5%) Azerbaijani patients. Ninety-three distinct patterns were identified including 15 clustered patterns and 36 unique patterns. The largest cluster was composed of seven isolates. Furthermore, one cluster with 5 members, four clusters with 3 members, and nine clusters with 2 members. In MIRU-VNTR typing, 75 clusters belonged to the Tabriz region and just 3 to the Republic of Azerbaijan. All isolates were sensitive to rifampin, isoniazid, and ethambutol. Conclusion(s): Results of the current study showed COVID-19 pandemic had a direct effect on the transmission and diagnosis of tuberculosis. Less diagnosis and less clustering can indicate public controls and hygiene, and the use of masks had a direct effect on the transmission and diagnosis of tuberculosis. However, misidentification and less focus on other respiratory infections are expected during the pandemic. Studies on the co-infection of COVID-19 and tuberculosis and the role of mask and sanitization against TB are strongly recommended. Copyright © 2023, The Author(s).

PCR Amplification and Sequencing Analysis of Full-Length Turkey Coronavirus Spike Gene

Turkey coronaviral enteritis caused by turkey coronavirus (TCoV) continues to infect turkey flocks, resulting in significant economic loss. Determining and understanding genetic relationships among different TCoV isolates or strains is important for controlling the disease. Using two-step RT-PCR assays that amplify the full length of TCoV spike (S) gene, TCoV isolates can be sequenced, analyzed, and genotyped. Described in this chapter is the protocol on PCR amplification and sequencing analysis of full-length TCoV S gene. Such protocol is useful in molecular epidemiology for establishing an effective strategy to control the transmission of TCoV among turkey flocks. Copyright © Springer Science+Business Media New York 2016

Biocomputational Analysis of Spike Proteins to Identify Evolutionary Divergence of Beta- CoVs with Focusing on SARS-CoV2

Spike (S) proteins covering the outer surface of the Coronaviruses are the major hotspots of evolution and are also responsible for attaching the virus to the angiotensin-converting enzyme (ACE2) receptor on the host cells. In this study, we unveiled the evolutionary relics of the S-protein sequences of different beta Coronaviruses, namely, Severe Acute Respiratory Syndrome (SARS-CoV), SARS-CoV-2, and Middle East Respiratory Syndrome (MERSCoV). The present study aims at exploring the sequence divergence of the spike protein (S-protein) of nCov2 viruses to illuminate the evolutionary process. The nucleotide sequences of S-proteins of nCov2 viruses, namely, SARS, MERS and SARS-CoV2, representing different continents of the world, were downloaded from the NCBI Nucleotide databases. The conserved regions have been depicted through multiple sequence alignment (Clustal Omega, Jalview) and the molecular phylogeny has been studied. (using MEGA 7). Comparative analysis of the pairwise distance and selection pressure indicated that the SARS-CoV and SARS-CoV-2 are close to each other, however, distantly related to MERS-CoV and the SARS-CoV2 could have evolved from SARS (Severe Acute Respiratory Syndrome).

Outbreak of Ralstonia pickettii Bacteremia Caused by Contaminated Hydromorphone in a Universitary Hospital in Bogota, Colombia

Background. Ralstonia pickettii are aerobic non fermenter gram negative bacilli isolated in water and soil. It is related to nosocomial infection outbreaks and considered an opportunistic pathogen. There have been outbreaks reports due to contaminated water systems and sterile drug solutions which mainly occurs during manufacturing. We present the report of an outbreak of R. pickettii bacteremia secondary to a contamination of hydromorphone vials. Methods. In February 2021 an outbreak of R. pickettii bacteremia was identified. All isolates were from blood cultures with slow growth, thus indicating the culturing of liquid inputs, intravenous administration solutions and commonly used drugs among patients including hydromorphone. Mass spectrometry (MALDI-TOF) was used for the identification and automated microdilution to determine sensitivity to antimicrobials of the isolates and clonality analysis of genetic relationships was carried out using the DICE coefficient, UPGMA algorithm Results. During the outbreak, 19 patients with R. pickettii bacteremia were identified The global attack rate was 1,9%. 11/19 (58%) were women and 13/19 (68%) of the isolations were from inward patients and 6/19 (32%) were from intensive care unit. Factors that could contribute to the appearance of the outbreak were underlying pathology, 2 patients with a diagnosis of diabetes mellitus, 10 patients with a diagnosis of arterial hypertension, 5 patients with obesity, 6 patients with heart disease, additionally 7 patients with a diagnosis of SARS COV 2 and 6 patients with the use of corticosteroids. The global attack rate was 1,9% and mortality was 31.5% (6 patients). R. pickettii was identified from two batches of hydromorphone by MALDI-TOF and the clonality study concluded that the isolates analyzed, were clonal with a 100% similarity. The associated mortality rate was 5/29 (26.3%). Conclusion. We confirmed an outbreak of R. pickettii due to the contamination of two hydromorphone badges in Colombia. It is crucial to acknowledge the importance of infection control and surveillance during the COVID-19 pandemic as well as maintaining adequate quality control of medication production in order to avoid presenting this kind of outbreaks.

Temporal landscape of mutational frequencies in SARS-CoV-2 genomes of Bangladesh: possible implications from the ongoing outbreak in Bangladesh.

Along with intrinsic evolution, adaptation to selective pressure in new environments might have resulted in the circulatory SARS-CoV-2 strains in response to the geoenvironmental conditions of a country and the demographic profile of its population. With this target, the current study traced the evolutionary route and mutational frequency of 198 Bangladesh-originated SARS-CoV-2 genomic sequences available in the GISAID platform over a period of 13 weeks as of 14 July 2020. The analyses were performed using MEGA X, Swiss Model Repository, Virus Pathogen Resource and Jalview visualization. Our analysis identified that majority of the circulating strains strikingly differ from both the reference genome and the first sequenced genome from Bangladesh. Mutations in nonspecific proteins (NSP2-3, NSP-12(RdRp), NSP-13(Helicase)), S-Spike, ORF3a, and N-Nucleocapsid protein were common in the circulating strains with varying degrees and the most unique mutations (UM) were found in NSP3 (UM-18). But no or limited changes were observed in NSP9, NSP11, Envelope protein (E) and accessory factors (NSP7a, ORF 6, ORF7b) suggesting the possible conserved functions of those proteins in SARS-CoV-2 propagation. However, along with D614G mutation, more than 20 different mutations in the Spike protein were detected basically in the S2 domain. Besides, mutations in SR-rich region of N protein and P323L in RDRP were also present. However, the mutation accumulation showed a significant association (p = 0.003) with sex and age of the COVID-19-positive cases. So, identification of these mutational accumulation patterns may greatly facilitate vaccine development deciphering the age and the sex-dependent differential susceptibility to COVID-19.

Genotype-phenotype correlation identified a novel SARS-CoV-2 variant possibly linked to severe disease.

The geographic location and heterogeneous multi-ethnic population of Dubai (United Arab Emirates; UAE) provide a unique setting to explore the global molecular epidemiology of SARS-CoV-2 and relationship between different viral strains and disease severity. We systematically selected (i.e. every 100th individual in the central Dubai COVID-19 database) 256 patients by age, sex, disease severity and month to provide a representative sample of laboratory-confirmed COVID-19 patients (nasopharyngeal swab PCR positive) during the first wave of the UAE outbreak (January to June 2020). Sociodemographic and clinical data were extracted from medical records and full SARS-CoV-2 genome sequences extracted from nasopharyngeal swabs were analysed. Older age was significantly associated with COVID-19-associated hospital admission and mortality. Overweight/obese or diabetic patients were 3-4 times more likely to be admitted to hospital and intensive care unit (ICU). Sequencing data showed multiple independent viral introductions into the UAE from Europe, Iran and Asia (29 January-18 March), and these early strains seeded significant clustering consistent with almost exclusive community-based transmission between April and June 2020. Majority of sequenced strains (N = 60, 52%) were from the European cluster consistent with the higher infectivity rates associated with the D614G mutation carried by most strains in this cluster. A total of 986 mutations were identified in 115 genomes, 272 were unique (majority were missense, n = 134) and 20/272 mutations were novel. A missense (Q271R) and synonymous (R41R) mutation in the S and N proteins, respectively, were identified in 2/27 patients with severe COVID-19 but not in patients with mild or moderate disease (0/86; p = .05, Fisher's Exact Test). Both patients were women (51-64 years) with no significant underlying health conditions. The same two mutations were identified in a healthy 37-year-old Indian man who was hospitalized in India due to COVID-19. Our findings provide evidence for continued community-based transmission of the European strains in the Dubai population and highlight new mutations that might be associated with severe disease in otherwise healthy adults.

Territorywide Study of Early Coronavirus Disease Outbreak, Hong Kong, China.

Initial cases of coronavirus disease in Hong Kong were imported from mainland China. A dramatic increase in case numbers was seen in February 2020. Most case-patients had no recent travel history, suggesting the presence of transmission chains in the local community. We collected demographic, clinical, and epidemiologic data from 50 patients, who accounted for 53.8% of total reported case-patients as of February 28, 2020. We performed whole-genome sequencing to determine phylogenetic relationship and transmission dynamics of severe acute respiratory syndrome coronavirus 2 infections. By using phylogenetic analysis, we attributed the community outbreak to 2 lineages; 1 harbored a common mutation, Orf3a-G251V, and accounted for 88.0% of the cases in our study. The estimated time to the most recent common ancestor of local coronavirus disease outbreak was December 24, 2019, with an evolutionary rate of 3.04 × 10-3 substitutions/site/year. The reproduction number was 1.84, indicating ongoing community spread.