A 16S Next Generation Sequencing Based Molecular and Bioinformatics Pipeline to Identify Processed Meat Products Contamination and Mislabelling.

Processed meat is a target in meat adulteration for economic gain. This study demonstrates a molecular and bioinformatics diagnostic pipeline, utilizing the mitochondrial 16S ribosomal RNA (rRNA) gene, to determine processed meat product mislabeling through Next-Generation Sequencing. Nine pure meat samples were collected and artificially mixed at different ratios to verify the specificity and sensitivity of the pipeline. Processed meat products (n = 155), namely, minced meat, biltong, burger patties, and sausages, were collected across South Africa. Sequencing was performed using the Illumina MiSeq sequencing platform. Each sample had paired-end reads with a length of ±300 bp. Quality control and filtering was performed using BBDuk (version 37.90a). Each sample had an average of 134,000 reads aligned to the mitochondrial genomes using BBMap v37.90. All species in the artificial DNA mixtures were detected. Processed meat samples had reads that mapped to the Bos (90% and above) genus, with traces of reads mapping to Sus and Ovis (2-5%) genus. Sausage samples showed the highest level of contamination with 46% of the samples having mixtures of beef, pork, or mutton in one sample. This method can be used to authenticate meat products, investigate, and manage any form of mislabeling.

Transmission dynamics of SARS-CoV-2 within-host diversity in two major hospital outbreaks in South Africa.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes acute, highly transmissible respiratory infection in humans and a wide range of animal species. Its rapid global spread has resulted in a major public health emergency, necessitating commensurately rapid research to improve control strategies. In particular, the ability to effectively retrace transmission chains in outbreaks remains a major challenge, partly due to our limited understanding of the virus' underlying evolutionary dynamics within and between hosts. We used high-throughput sequencing whole-genome data coupled with bottleneck analysis to retrace the pathways of viral transmission in two nosocomial outbreaks that were previously characterised by epidemiological and phylogenetic methods. Additionally, we assessed the mutational landscape, selection pressures, and diversity at the within-host level for both outbreaks. Our findings show evidence of within-host selection and transmission of variants between samples. Both bottleneck and diversity analyses highlight within-host and consensus-level variants shared by putative source-recipient pairs in both outbreaks, suggesting that certain within-host variants in these outbreaks may have been transmitted upon infection rather than arising de novo independently within multiple hosts. Overall, our findings demonstrate the utility of combining within-host diversity and bottleneck estimations for elucidating transmission events in SARS-CoV-2 outbreaks, provide insight into the maintenance of viral genetic diversity, provide a list of candidate targets of positive selection for further investigation, and demonstrate that within-host variants can be transferred between patients. Together these results will help in developing strategies to understand the nature of transmission events and curtail the spread of SARS-CoV-2.

Early transmission of SARS-CoV-2 in South Africa: An epidemiological and phylogenetic report.

OBJECTIVES: The Network for Genomic Surveillance in South Africa (NGS-SA) was formed to investigate the introduction and understand the early transmission dynamics of the SARS-CoV-2 epidemic in South-Africa. DESIGN: This paper presents the first results from this group, which is a molecular epidemiological study of the first 21 SARS-CoV-2 whole genomes sampled in the first port of entry - KwaZulu-Natal (KZN) - during the first month of the epidemic. By combining this with calculations of the effective reproduction number (R), it aimed to shed light on the patterns of infections in South Africa. RESULTS: Two of the largest provinces - Gauteng and KZN - had a slow growth rate for the number of detected cases, while the epidemic spread faster in the Western Cape and Eastern Cape. The estimates of transmission potential suggested a decrease towards R = 1 since the first cases and deaths, but a subsequent estimated R average of 1.39 between 6-18 May 2020. It was also demonstrated that early transmission in KZN was associated with multiple international introductions and dominated by lineages B1 and B. Evidence for locally acquired infections in a hospital in Durban within the first month of the epidemic was also provided. CONCLUSION: The COVID-19 pandemic in South Africa was very heterogeneous in its spatial dimension, with many distinct introductions of SARS-CoV2 in KZN and evidence of nosocomial transmission, which inflated early mortality in KZN. The epidemic at the local level was still developing and NGS-SA aimed to clarify the dynamics in South Africa and devise the most effective measures as the outbreak evolved.

High Resolution analysis of Transmission Dynamics of Sars-Cov-2 in Two Major Hospital Outbreaks in South Africa Leveraging Intrahost Diversity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes acute, highly transmissible respiratory infection in both humans and wide range of animal species. Its rapid spread globally and devasting effects have resulted into a major public health emergency prompting the need for methodological interventions to understand and control its spread. In particular, The ability to effectively retrace its transmission pathways in outbreaks remains a major challenge. This is further exacerbated by our limited understanding of its underlying evolutionary mechanism. Using NGS whole-genome data, we determined whether inter- and intra-host diversity coupled with bottleneck analysis can retrace the pathway of viral transmission in two epidemiologically well characterised nosocomial outbreaks in healthcare settings supported by phylogenetic analysis. Additionally, we assessed the mutational landscape, selection pressure and diversity of the identified variants. Our findings showed evidence of intrahost variant transmission and evolution of SARS-CoV-2 after infection These observations were consistent with the results from the bottleneck analysis suggesting that certain intrahost variants in this study could have been transmitted to recipients. In both outbreaks, we observed iSNVs and SNVs shared by putative source-recipients pairs. Majority of the observed iSNVs were positioned in the S and ORF1ab region. AG, CT and TC nucleotide changes were enriched across SARS-COV-2 genome. Moreover, SARS-COV-2 genome had limited diversity in some loci while being highly conserved in others. Overall, Our findings show that the synergistic effect of combining withinhost diversity and bottleneck estimations greatly enhances resolution of transmission events in Sars-Cov-2 outbreaks. They also provide insight into the genome diversity suggesting purifying selection may be involved in the transmission. Together these results will help in developing strategies to elucidate transmission events and curtail the spread of Sars-Cov-2.

Impact of pretreatment low-abundance HIV-1 drug-resistant variants on virological failure among HIV-1/TB-co-infected individuals.

OBJECTIVES: To determine the impact of pretreatment low-abundance HIV-1 drug-resistant variants (LA-DRVs) on virological failure (VF) among HIV-1/TB-co-infected individuals treated with NNRTI first-line ART. METHODS: We conducted a case-control study of 170 adults with HIV-1/TB co-infection. Cases had at least one viral load (VL) ≥1000 RNA copies/mL after ≥6 months on NNRTI-based ART, and controls had sustained VLs <1000 copies/mL. We sequenced plasma viruses by Sanger and MiSeq next-generation sequencing (NGS). We assessed drug resistance mutations (DRMs) using the Stanford drug resistance database, and analysed NGS data for DRMs at ≥20%, 10%, 5% and 2% thresholds. We assessed the effect of pretreatment drug resistance (PDR) on VF. RESULTS: We analysed sequences from 45 cases and 125 controls. Overall prevalence of PDR detected at a ≥20% threshold was 4.7% (8/170) and was higher in cases than in controls (8.9% versus 3.2%), P = 0.210. Participants with PDR at ≥20% had almost 4-fold higher odds of VF (adjusted OR 3.7, 95% CI 0.8-18.3) compared with those without, P = 0.104. PDR prevalence increased to 18.2% (31/170) when LA-DRVs at ≥2% were included. Participants with pretreatment LA-DRVs only had 1.6-fold higher odds of VF (adjusted OR 1.6, 95% CI 0.6-4.3) compared with those without, P = 0.398. CONCLUSIONS: Pretreatment DRMs and LA-DRVs increased the odds of developing VF on NNRTI-based ART, although without statistical significance. NGS increased detection of DRMs but provided no additional benefit in identifying participants at risk of VF at lower thresholds. More studies assessing mutation thresholds predictive of VF are required to inform use of NGS in treatment decisions.

Whole Genome Sequencing of SARS-CoV-2: Adapting Illumina Protocols for Quick and Accurate Outbreak Investigation during a Pandemic.

The COVID-19 pandemic has spread very fast around the world. A few days after the first detected case in South Africa, an infection started in a large hospital outbreak in Durban, KwaZulu-Natal (KZN). Phylogenetic analysis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes can be used to trace the path of transmission within a hospital. It can also identify the source of the outbreak and provide lessons to improve infection prevention and control strategies. This manuscript outlines the obstacles encountered in order to genotype SARS-CoV-2 in near-real time during an urgent outbreak investigation. This included problems with the length of the original genotyping protocol, unavailability of reagents, and sample degradation and storage. Despite this, three different library preparation methods for Illumina sequencing were set up, and the hands-on library preparation time was decreased from twelve to three hours, which enabled the outbreak investigation to be completed in just a few weeks. Furthermore, the new protocols increased the success rate of sequencing whole viral genomes. A simple bioinformatics workflow for the assembly of high-quality genomes in near-real time was also fine-tuned. In order to allow other laboratories to learn from our experience, all of the library preparation and bioinformatics protocols are publicly available at protocols.io and distributed to other laboratories of the Network for Genomics Surveillance in South Africa (NGS-SA) consortium.

Early transmission of SARS-CoV-2 in South Africa: An epidemiological and phylogenetic report.

Background: The emergence of a novel coronavirus, SARS-CoV-2, in December 2019, progressed to become a world pandemic in a few months and reached South Africa at the beginning of March. To investigate introduction and understand the early transmission dynamics of the virus, we formed the South African Network for Genomics Surveillance of COVID (SANGS_COVID), a network of ten government and university laboratories. Here, we present the first results of this effort, which is a molecular epidemiological study of the first twenty-one SARS-CoV-2 whole genomes sampled in the first port of entry, KwaZulu-Natal (KZN), during the first month of the epidemic. By combining this with calculations of the effective reproduction number (R), we aim to shed light on the patterns of infections that define the epidemic in South Africa. Methods: R was calculated using positive cases and deaths from reports provided by the four major provinces. Molecular epidemiology investigation involved sequencing viral genomes from patients in KZN using ARCTIC protocols and assembling whole genomes using meticulous alignment methods. Phylogenetic analysis was performed using maximum likelihood (ML) and Bayesian trees, lineage classification and molecular clock calculations. Findings: The epidemic in South Africa has been very heterogeneous. Two of the largest provinces, Gauteng, home of the two large metropolis Johannesburg and Pretoria, and KwaZulu-Natal, home of the third largest city in the country Durban, had a slow growth rate on the number of detected cases. Whereas, Western Cape, home of Cape Town, and the Eastern Cape provinces the epidemic is spreading fast. Our estimates of transmission potential for South Africa suggest a decreasing transmission potential towards R=1 since the first cases and deaths have been reported. However, between 06 May and 18 May 2020, we estimate that R was on average 1.39 (1.04 - 2.15, 95% CI). We also demonstrate that early transmission in KZN, and most probably in all main regions of SA, was associated with multiple international introductions and dominated by lineages B1 and B. The study also provides evidence for locally acquired infections in a hospital in Durban within the first month of the epidemic, which inflated early mortality in KZN. Interpretation: This first report of SANGS_COVID consortium focuses on understanding the epidemic heterogeneity and introduction of SARS-CoV-2 strains in the first month of the epidemic in South Africa. The early introduction of SARS-CoV-2 in KZN included caused a localized outbreak in a hospital, provides potential explanations for the initially high death rates in the province. The current high rate of transmission of COVID-19 in the Western Cape and Eastern Cape highlights the crucial need to strength local genomic surveillance in South Africa. Funding: UKZN Flagship Program entitled: Afrocentric Precision Approach to Control Health Epidemic, by a research Flagship grant from the South African Medical Research Council (MRC-RFA-UFSP-01-2013/UKZN HIVEPI, by the the Technology Innovation Agency and the the Department of Science and Innovation and by National Human Genome Re- search Institute of the National Institutes of Health under Award Number U24HG006941. H3ABioNet is an initiative of the Human Health and Heredity in Africa Consortium (H3Africa).

Population genetic structure, linkage disequilibrium and effective population size of conserved and extensively raised village chicken populations of Southern Africa.

Extensively raised village chickens are considered a valuable source of biodiversity, with genetic variability developed over thousands of years that ought to be characterized and utilized. Surveys that can reveal a population's genetic structure and provide an insight into its demographic history will give valuable information that can be used to manage and conserve important indigenous animal genetic resources. This study reports population diversity and structure, linkage disequilibrium and effective population sizes of Southern African village chickens and conservation flocks from South Africa. DNA samples from 312 chickens from South African village and conservation flocks (n = 146), Malawi (n = 30) and Zimbabwe (n = 136) were genotyped using the Illumina iSelect chicken SNP60K BeadChip. Population genetic structure analysis distinguished the four conservation flocks from the village chicken populations. Of the four flocks, the Ovambo clustered closer to the village chickens particularly those sampled from South Africa. Clustering of the village chickens followed a geographic gradient whereby South African chickens were closer to those from Zimbabwe than to chickens from Malawi. Different conservation flocks seemed to have maintained different components of the ancestral genomes with a higher proportion of village chicken diversity found in the Ovambo population. Overall population LD averaged over chromosomes ranged from 0.03 ± 0.07 to 0.58 ± 0.41 and averaged 0.15 ± 0.16. Higher LD, ranging from 0.29 to 0.36, was observed between SNP markers that were less than 10 kb apart in the conservation flocks. LD in the conservation flocks steadily decreased to 0.15 (PK) and 0.24 (VD) at SNP marker interval of 500 kb. Genomewide LD decay in the village chickens from Malawi, Zimbabwe and South Africa followed a similar trend as the conservation flocks although the mean LD values for the investigated SNP intervals were lower. The results suggest low effective population sizes particularly in the conservation flocks. The utility and limitations of the iselect chicken SNP60K in village chicken populations is discussed.