The emergence and ongoing convergent evolution of the SARS-CoV-2 N501Y lineages.

The independent emergence late in 2020 of the B.1.1.7, B.1.351, and P.1 lineages of SARS-CoV-2 prompted renewed concerns about the evolutionary capacity of this virus to overcome public health interventions and rising population immunity. Here, by examining patterns of synonymous and non-synonymous mutations that have accumulated in SARS-CoV-2 genomes since the pandemic began, we find that the emergence of these three "501Y lineages" coincided with a major global shift in the selective forces acting on various SARS-CoV-2 genes. Following their emergence, the adaptive evolution of 501Y lineage viruses has involved repeated selectively favored convergent mutations at 35 genome sites, mutations we refer to as the 501Y meta-signature. The ongoing convergence of viruses in many other lineages on this meta-signature suggests that it includes multiple mutation combinations capable of promoting the persistence of diverse SARS-CoV-2 lineages in the face of mounting host immune recognition.

Multiplex qPCR discriminates variants of concern to enhance global surveillance of SARS-CoV-2.

With the emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants that may increase transmissibility and/or cause escape from immune responses, there is an urgent need for the targeted surveillance of circulating lineages. It was found that the B.1.1.7 (also 501Y.V1) variant, first detected in the United Kingdom, could be serendipitously detected by the Thermo Fisher TaqPath COVID-19 PCR assay because a key deletion in these viruses, spike Δ69-70, would cause a "spike gene target failure" (SGTF) result. However, a SGTF result is not definitive for B.1.1.7, and this assay cannot detect other variants of concern (VOC) that lack spike Δ69-70, such as B.1.351 (also 501Y.V2), detected in South Africa, and P.1 (also 501Y.V3), recently detected in Brazil. We identified a deletion in the ORF1a gene (ORF1a Δ3675-3677) in all 3 variants, which has not yet been widely detected in other SARS-CoV-2 lineages. Using ORF1a Δ3675-3677 as the primary target and spike Δ69-70 to differentiate, we designed and validated an open-source PCR assay to detect SARS-CoV-2 VOC. Our assay can be rapidly deployed in laboratories around the world to enhance surveillance for the local emergence and spread of B.1.1.7, B.1.351, and P.1.

Acquired HIV drug resistance and virologic monitoring in a HIV hyper-endemic setting in KwaZulu-Natal Province, South Africa.

BACKGROUND: Introduction of tenofovir (TDF) plus lamivudine (3TC) and dolutegravir (DTG) in first- and second-line HIV treatment regimens in South Africa warrants characterization of acquired HIV-1 drug resistance (ADR) mutations that could impact DTG-based antiretroviral therapy (ART). In this study, we sought to determine prevalence of ADR mutations and their potential impact on susceptibility to drugs used in combination with DTG among HIV-positive adults (≥ 18 years) accessing routine care at a selected ART facility in KwaZulu-Natal, South Africa. METHODS: We enrolled adult participants in a cross-sectional study between May and September 2019. Eligible participants had a most recent documented viral load (VL) ≥ 1000 copies/mL after at least 6 months on ART. We genotyped HIV-1 reverse transcriptase and protease genes by Sanger sequencing and assessed ADR. We characterized the effect of ADR mutations on the predicted susceptibility to drugs used in combination with DTG. RESULTS: From 143 participants enrolled, we obtained sequence data for 115 (80%), and 92.2% (95% CI 85.7-96.4) had ADR. The proportion with ADR was similar for participants on first-line ART (65/70, 92.9%, 95% CI 84.1-97.6) and those on second-line ART (40/44, 90.9%, 95% CI 78.3-97.5), and was present for the single participant on third-line ART. Approximately 89% (62/70) of those on first-line ART had dual class NRTI and NNRTI resistance and only six (13.6%) of those on second-line ART had major PI mutations. Most participants (82%) with first-line viraemia maintained susceptibility to Zidovudine (AZT), and the majority of them had lost susceptibility to TDF (71%) and 3TC (84%). Approximately two in every five TDF-treated individuals had thymidine analogue mutations (TAMs). CONCLUSIONS: Susceptibility to AZT among most participants with first-line viraemia suggests that a new second-line regimen of AZT + 3TC + DTG could be effective. However, atypical occurrence of TAMs in TDF-treated individuals suggests a less effective AZT + 3TC + DTG regimen in a subpopulation of patients. As most patients with first-line viraemia had at least low-level resistance to TDF and 3TC, identifying viraemia before switch to TDF + 3TC + DTG is important to avoid DTG functional monotherapy. These findings highlight a need for close monitoring of outcomes on new standardized treatment regimens.

A comparison study of meat eaters and non-meat eaters on mind attribution and moral disengagement of animals.

This study aims to investigate how the distinction between animals that humans consume (AHCs) and pet animals influence meat eaters' and non-meat eaters' perceived mind attribution on animals and moral disengagement. Following this, a two-way mixed ANOVA with repeated measures on the type of animals being slaughtered and type of eaters was conducted. For meat-eaters, perceived mental capacity ratings for AHCs were lower than pet animals. For non-meat eaters, the difference between these animals was negligible. In addition, meat eaters had higher levels of moral disengagement in comparison to non-meat eaters. Further analysis showed that meat eaters who reported lower perceived mental capacities of AHCs appeared to feel such animals were more edible and were less likely to perceive killing them for food as morally wrong. Moral disengagement was also negatively associated with mental capacity of AHCs as food, suggesting that there was a higher moral disengagement among meat eaters who tend to view AHCs as lacking in mental capacities.

Development assistance for health: past trends, associations, and the future of international financial flows for health.

BACKGROUND: Disbursements of development assistance for health (DAH) have risen substantially during the past several decades. More recently, the international community's attention has turned to other international challenges, introducing uncertainty about the future of disbursements for DAH. METHODS: We collected audited budget statements, annual reports, and project-level records from the main international agencies that disbursed DAH from 1990 to the end of 2015. We standardised and combined records to provide a comprehensive set of annual disbursements. We tracked each dollar of DAH back to the source and forward to the recipient. We removed transfers between agencies to avoid double-counting and adjusted for inflation. We classified assistance into nine primary health focus areas: HIV/AIDS, tuberculosis, malaria, maternal health, newborn and child health, other infectious diseases, non-communicable diseases, Ebola, and sector-wide approaches and health system strengthening. For our statistical analysis, we grouped these health focus areas into two categories: MDG-related focus areas (HIV/AIDS, tuberculosis, malaria, child and newborn health, and maternal health) and non-MDG-related focus areas (other infectious diseases, non-communicable diseases, sector-wide approaches, and other). We used linear regression to test for structural shifts in disbursement patterns at the onset of the Millennium Development Goals (MDGs; ie, from 2000) and the global financial crisis (impact estimated to occur in 2010). We built on past trends and associations with an ensemble model to estimate DAH through the end of 2040. FINDINGS: In 2015, US$36·4 billion of DAH was disbursed, marking the fifth consecutive year of little change in the amount of resources provided by global health development partners. Between 2000 and 2009, DAH increased at 11·3% per year, whereas between 2010 and 2015, annual growth was just 1·2%. In 2015, 29·7% of DAH was for HIV/AIDS, 17·9% was for child and newborn health, and 9·8% was for maternal health. Linear regression identifies three distinct periods of growth in DAH. Between 2000 and 2009, MDG-related DAH increased by $290·4 million (95% uncertainty interval [UI] 174·3 million to 406·5 million) per year. These increases were significantly greater than were increases in non-MDG DAH during the same period (p=0·009), and were also significantly greater than increases in the previous period (p<0·0001). Between 2000 and 2009, growth in DAH was highest for HIV/AIDS, malaria, and tuberculosis. Since 2010, DAH for maternal health and newborn and child health has continued to climb, although DAH for HIV/AIDS and most other health focus areas has remained flat or decreased. Our estimates of future DAH based on past trends and associations present a wide range of potential futures, although our mean estimate of $64·1 billion (95% UI $30·4 billion to $161·8 billion) shows an increase between now and 2040, although with a large uncertainty interval. INTERPRETATION: Our results provide evidence of two substantial shifts in DAH growth during the past 26 years. DAH disbursements increased faster in the first decade of the 2000s than in the 1990s, but DAH associated with the MDGs increased the most out of all focus areas. Since 2010, limited growth has characterised DAH and we expect this pattern to persist. Despite the fact that DAH is still growing, albeit minimally, DAH is shifting among the major health focus areas, with relatively little growth for HIV/AIDS, malaria, and tuberculosis. These changes in the growth and focus of DAH will have critical effects on health services in some low-income countries. Coordination and collaboration between donors and domestic governments is more important than ever because they have a great opportunity and responsibility to ensure robust health systems and service provision for those most in need. FUNDING: Bill & Melinda Gates Foundation.

Under-5 mortality in 2851 Chinese counties, 1996-2012: a subnational assessment of achieving MDG 4 goals in China.

BACKGROUND: In the past two decades, the under-5 mortality rate in China has fallen substantially, but progress with regards to the Millennium Development Goal (MDG) 4 at the subnational level has not been quantified. We aimed to estimate under-5 mortality rates in mainland China for the years 1970 to 2012. METHODS: We estimated the under-5 mortality rate for 31 provinces in mainland China between 1970 and 2013 with data from censuses, surveys, surveillance sites, and disease surveillance points. We estimated under-5 mortality rates for 2851 counties in China from 1996 to 2012 with the reported child mortality numbers from the Annual Report System on Maternal and Child Health. We used a small area mortality estimation model, spatiotemporal smoothing, and Gaussian process regression to synthesise data and generate consistent provincial and county-level estimates. We compared progress at the county level with what was expected on the basis of income and educational attainment using an econometric model. We computed Gini coefficients to study the inequality of under-5 mortality rates across counties. FINDINGS: In 2012, the lowest provincial level under-5 mortality rate in China was about five per 1000 livebirths, lower than in Canada, New Zealand, and the USA. The highest provincial level under-5 mortality rate in China was higher than that of Bangladesh. 29 provinces achieved a decrease in under-5 mortality rates twice as fast as the MDG 4 target rate; only two provinces will not achieve MDG 4 by 2015. Although some counties in China have under-5 mortality rates similar to those in the most developed nations in 2012, some have similar rates to those recorded in Burkina Faso and Cameroon. Despite wide differences, the inter-county Gini coefficient has been decreasing. Improvement in maternal education and the economic boom have contributed to the fall in child mortality; more than 60% of the counties in China had rates of decline in under-5 mortality rates significantly faster than expected. Fast reduction in under-5 mortality rates have been recorded not only in the Han population, the dominant ethnic majority in China, but also in the minority populations. All top ten minority groups in terms of population sizes have experienced annual reductions in under-5 mortality rates faster than the MDG 4 target at 4.4%. INTERPRETATION: The reduction of under-5 mortality rates in China at the country, provincial, and county level is an extraordinary success story. Reductions of under-5 mortality rates faster than 8.8% (twice MDG 4 pace) are possible. Extremely rapid declines seem to be related to public policy in addition to socioeconomic progress. Lessons from successful counties should prove valuable for China to intensify efforts for those with unacceptably high under-5 mortality rates. FUNDING: National "Twelfth Five-Year" Plan for Science and Technology Support, National Health and Family Planning Commission of The People's Republic of China, Program for Changjiang Scholars and Innovative Research Team in University, the National Institute on Aging, and the Bill & Melinda Gates Foundation.

Tracking development assistance and government health expenditures for 35 malaria-eliminating countries: 1990-2017.

BACKGROUND: Donor financing for malaria has declined since 2010 and this trend is projected to continue for the foreseeable future. These reductions have a significant impact on lower burden countries actively pursuing elimination, which are usually a lesser priority for donors. While domestic spending on malaria has been growing, it varies substantially in speed and magnitude across countries. A clear understanding of spending patterns and trends in donor and domestic financing is needed to uncover critical investment gaps and opportunities. METHODS: Building on the Institute for Health Metrics and Evaluation's annual Financing Global Health research, data were collected from organizations that channel development assistance for health to the 35 countries actively pursuing malaria elimination. Where possible, development assistance for health (DAH) was categorized by spend on malaria intervention. A diverse set of data points were used to estimate government health budgets expenditure on malaria, including World Malaria Reports and government reports when available. Projections were done using regression analyses taking recipient country averages and earmarked funding into account. RESULTS: Since 2010, DAH for malaria has been declining for the 35 countries actively pursuing malaria elimination (from $176 million in 2010 to $62 million in 2013). The Global Fund is the largest external financier for malaria, providing 96% of the total external funding for malaria in 2013, with vector control interventions being the highest cost driver in all regions. Government expenditure on malaria, while increasing, has not kept pace with diminishing DAH or rising national GDP rates, leading to a potential gap in service delivery needed to attain elimination. CONCLUSION: Despite past gains, total financing available for malaria in elimination settings is declining. Health financing trends suggest that substantive policy interventions will be needed to ensure that malaria elimination is adequately financed and that available financing is effectively targeted to interventions that provide the best value for money.

A post-partum single-dose TDF/FTC tail does not prevent the selection of NNRTI resistance in women receiving pre-partum ZDV and intrapartum single-dose nevirapine to prevent mother-to- child HIV-1 transmission.

Although the rates of vertical transmission of HIV in the developing world have improved to around 3% in countries like South Africa, resistance to antiretrovirals (ARV) used in Prevention of Mother-to-Child transmission (pMTCT) strategies may thwart such outcomes and affect the efficacy of future ARV regimens in mothers and children. This study conducted in Durban, South Africa, between 2010 and 2013 found a high rate of nevirapine (NVP) resistance among women receiving Zidovudine (AZT) from 14 weeks gestation, single dose nevirapine (sd NVP) at the onset of labor and a single dose of coformulated Tenofovir/Emtricitabine (TDF/FTC) postpartum. Using Sanger sequencing, high and intermediate levels of nevirapine (NVP) resistance were detected in 15/44 (34%) and in 1/44 (2%) of women tested, respectively. Most subjects selected the K103N mutation (22% (10/45) of all patients and 66% (10/15) of those with high-level NVP resistance). Such rate of NVP resistance is comparable to studies where only sd NVP was used. In conclusion, a post-partum single-dose TDF/FTC tail does not prevent the selection of NNRTI resistance in women receiving pre-partum ZDV and intrapartum sd NVP to prevent mother-to-child HIV-1 transmission.

Sources and Focus of Health Development Assistance, 1990-2014.

IMPORTANCE: The governments of high-income countries and private organizations provide billions of dollars to developing countries for health. This type of development assistance can have a critical role in ensuring that life-saving health interventions reach populations in need. OBJECTIVES: To identify the amount of development assistance that countries and organizations provided for health and to determine the health areas that received these funds. EVIDENCE REVIEW: Budget, revenue, and expenditure data on the primary agencies and organizations (n = 38) that provided resources to developing countries (n = 146-183, depending on the year) for health from 1990 through 2014 were collected. For each channel (the international agency or organization that directed the resources toward the implementing institution or government), the source and recipient of the development assistance were determined and redundant accounting of the same dollar, which occurs when channels transfer funds among each other, was removed. This research derived the flow of resources from source to intermediary channel to recipient. Development assistance for health (DAH) was divided into 11 mutually exclusive health focus areas, such that every dollar of development assistance was assigned only 1 health focus area. FINDINGS: Since 1990, $458.0 billion of development assistance has been provided to maintain or improve health in developing countries. The largest source of funding was the US government, which provided $143.1 billion between 1990 and 2014, including $12.4 billion in 2014. Of resources that originated with the US government, 70.6% were provided through US government agencies, and 41.0% were allocated for human immunodeficiency virus (HIV)/AIDS. The second largest source of development assistance for health was private philanthropic donors, including the Bill and Melinda Gates Foundation and other private foundations, which provided $69.9 billion between 1990 and 2014, including $6.2 billion in 2014. These resources were provided primarily through private foundations and nongovernmental organizations and were allocated for a diverse set of health focus areas. Since 1990, 28.0% of all DAH was allocated for maternal health and newborn and child health; 23.2% for HIV/AIDS, 4.3% for malaria, 2.8% for tuberculosis, and 1.5% for noncommunicable diseases. Between 2000 and 2010, DAH increased 11.3% annually. However, since 2010, total DAH has not increased as substantially. CONCLUSIONS AND RELEVANCE: Funding for health in developing countries has increased substantially since 1990, with a focus on HIV/AIDS, maternal health, and newborn and child health. Funding from the US government has played a substantial role in this expansion. Funding for noncommunicable diseases has been limited. Understanding how funding patterns have changed across time and the priorities of sources of international funding across distinct channels, recipients, and health focus areas may help identify where funding gaps persist and where cost-effective interventions could save lives.

High level of HIV-2 false positivity in KwaZulu-Natal province: a region of South Africa with a very high HIV-1 subtype C prevalence.

Human immunodeficiency virus 2 (HIV-2) is found predominantly in West Africa. It is not unlikely, however, that HIV-2 may also be found in South Africa, due to the influx of immigrants into this country. It is important to distinguish between HIV-1 and HIV-2 since the clinical courses and treatment responses of these viruses are different. Routine serological methods for diagnosing HIV do not differentiate between HIV-1 and -2 infections, while rapid tests, viral load quantification and PCR are HIV-type--specific. The objective of this study was to describe the seroprevalence and molecular epidemiology of HIV-2 in KwaZulu-Natal, one of the regions with the highest HIV prevalence in the world and home of the two largest harbors in South Africa. HIV-1 positive samples were screened for antibodies against HIV-2, using a rapid test. The confirmation of HIV-2 positive samples was done by PCR. Of the 2,123 samples screened, 319 (15%) were identified as positive by the rapid test. None of these samples were confirmed positive by PCR. To explore this discrepancy in the results, a subset (n = 52) of the rapid HIV-2 positive samples was subjected to Western blotting. Thirty-seven (71%) of these were positive, yielding an overall HIV-2 seroprevalence of 10.6%. Three out of 28 (10.7%) Western blot positive samples were positive by a Pepti-LAV assay. This discrepancy between serological and molecular confirmation may be attributed to non-specific or cross-reacting antibodies. The use of rapid tests and Western blots for HIV-2 diagnosis in South Africa should be interpreted with caution.

Exploiting benzilic acid as a modular template: controlling photoreactivity and solid to liquid transition during photodimerization.

A well-known molecule, benzilic acid, is used as a [2+2] photodimerization template by using third-generation crystal engineering principles. This template utilizes orthogonality and non-covalent interactions in an optimized way and is shown to be effective in tuning the photoreactivity of styryl pyridine derivatives. The photo-induced crystal-to-liquid transformation was observed during photodimerization. This phenomenon is explained based on slip plane and energy framework analysis.

Molecular Epidemiology and Diversity of SARS-CoV-2 in Ethiopia, 2020-2022.

Ethiopia is the second most populous country in Africa and the sixth most affected by COVID-19 on the continent. Despite having experienced five infection waves, >499,000 cases, and ~7500 COVID-19-related deaths as of January 2023, there is still no detailed genomic epidemiological report on the introduction and spread of SARS-CoV-2 in Ethiopia. In this study, we reconstructed and elucidated the COVID-19 epidemic dynamics. Specifically, we investigated the introduction, local transmission, ongoing evolution, and spread of SARS-CoV-2 during the first four infection waves using 353 high-quality near-whole genomes sampled in Ethiopia. Our results show that whereas viral introductions seeded the first wave, subsequent waves were seeded by local transmission. The B.1.480 lineage emerged in the first wave and notably remained in circulation even after the emergence of the Alpha variant. The B.1.480 was outcompeted by the Delta variant. Notably, Ethiopia's lack of local sequencing capacity was further limited by sporadic, uneven, and insufficient sampling that limited the incorporation of genomic epidemiology in the epidemic public health response in Ethiopia. These results highlight Ethiopia's role in SARS-CoV-2 dissemination and the urgent need for balanced, near-real-time genomic sequencing.

Genomic epidemiology of SARS-CoV-2 during the first four waves in Mozambique.

Mozambique reported the first case of coronavirus disease 2019 (COVID-19) in March 2020 and it has since spread to all provinces in the country. To investigate the introductions and spread of SARS-CoV-2 in Mozambique, 1 142 whole genome sequences sampled within Mozambique were phylogenetically analyzed against a globally representative set, reflecting the first 25 months of the epidemic. The epidemic in the country was marked by four waves of infection, the first associated with B.1 ancestral lineages, while the Beta, Delta, and Omicron Variants of Concern (VOCs) were responsible for most infections and deaths during the second, third, and fourth waves. Large-scale viral exchanges occurred during the latter three waves and were largely attributed to southern African origins. Not only did the country remain vulnerable to the introductions of new variants but these variants continued to evolve within the borders of the country. Due to the Mozambican health system already under constraint, and paucity of data in Mozambique, there is a need to continue to strengthen and support genomic surveillance in the country as VOCs and Variants of interests (VOIs) are often reported from the southern African region.

Insights into SARS-CoV-2 in Angola during the COVID-19 peak: Molecular epidemiology and genome surveillance.

Background: In Angola, COVID-19 cases have been reported in all provinces, resulting in >105,000 cases and >1900 deaths. However, no detailed genomic surveillance into the introduction and spread of the SARS-CoV-2 virus has been conducted in Angola. We aimed to investigate the emergence and epidemic progression during the peak of the COVID-19 pandemic in Angola. Methods: We generated 1210 whole-genome SARS-CoV-2 sequences, contributing West African data to the global context, that were phylogenetically compared against global strains. Virus movement events were inferred using ancestral state reconstruction. Results: The epidemic in Angola was marked by four distinct waves of infection, dominated by 12 virus lineages, including VOCs, VOIs, and the VUM C.16, which was unique to South-Western Africa and circulated for an extended period within the region. Virus exchanges occurred between Angola and its neighboring countries, and strong links with Brazil and Portugal reflected the historical and cultural ties shared between these countries. The first case likely originated from southern Africa. Conclusion: A lack of a robust genome surveillance network and strong dependence on out-of-country sequencing limit real-time data generation to achieve timely disease outbreak responses, which remains of the utmost importance to mitigate future disease outbreaks in Angola.

HIV-1 drug resistance in adults and adolescents on protease inhibitor-based antiretroviral therapy in KwaZulu-Natal Province, South Africa.

OBJECTIVES: In low-middle-income countries, increasing levels of HIV drug resistance (HIVDR) on second-line protease inhibitor (PI)-based regimens are a cause for concern given the limited drug options for third-line antiretroviral therapy (ART). We conducted a retrospective analysis of routine HIV-1 genotyping laboratory data from KwaZulu-Natal, South Africa, to describe the frequency and patterns of HIVDR mutations and their consequent impact on standardised third-line regimens. METHODS: This was a cross-sectional analysis of all HIV-1 genotypic resistance tests conducted by the National Health Laboratory Service in KwaZulu-Natal (January 2015 to December 2016) for adults and adolescents (age ≥10 years) on second-line PI-based ART with virological failure. We assigned a third-line regimen to each record based on a national treatment algorithm and calculated the genotypic susceptibility score (GSS) for that regimen. RESULTS: Of 348 samples analysed, 287 (82.5%) had at least one drug resistance mutation (DRM) and 114 (32.8%) had at least one major PI DRM. Major PI resistance was associated with longer duration on second-line ART (aOR per 6-months = 1.11, 95% CI 1.04-1.19) and older age (aOR = 1.03, 95% CI 1.01-1.05). Of 112 patients requiring third-line ART, 12 (10.7%) had a GSS of <2 for the algorithm-assigned third-line regimen. CONCLUSION: One-third of people failing second-line ART had significant PI DRMs. A subgroup of these individuals had extensive HIVDR, where the predicted activity of third-line ART was suboptimal, highlighting the need for continuous evaluation of outcomes on third-line regimens and close monitoring for emergent HIV-1 integrase inhibitor resistance.

Molecular Epidemiology and Trends in HIV-1 Transmitted Drug Resistance in Mozambique 1999-2018.

HIV drug resistance (HIVDR) can become a public health concern, especially in low- and middle-income countries where genotypic testing for people initiating antiretroviral therapy (ART) is not available. For first-line regimens to remain effective, levels of transmitted drug resistance (TDR) need to be monitored over time. To determine the temporal trends of TDR in Mozambique, a search for studies in PubMed and sequences in GenBank was performed. Only studies covering the pol region that described HIVDR and genetic diversity from treatment naïve patients were included. A dataset from seven published studies and one novel unpublished study conducted between 1999 and 2018 were included. The Calibrated Population Resistance tool (CPR) and REGA HIV-1 Subtyping Tool version 3 for sequences pooled by sampling year were used to determine resistance mutations and subtypes, respectively. The prevalence of HIVDR amongst treatment-naïve individuals increased over time, reaching 14.4% in 2018. The increase was most prominent for non-nucleoside reverse transcriptase inhibitors (NNRTIs), reaching 12.7% in 2018. Subtype C was predominant in all regions, but a higher genetic variability (19% non-subtype C) was observed in the north region of Mozambique. These findings confirm a higher diversity of HIV in the north of the country and an increased prevalence of NNRTI resistance among treatment naïve individuals over time.

Targeted Sanger sequencing to recover key mutations in SARS-CoV-2 variant genome assemblies produced by next-generation sequencing.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is adaptively evolving to ensure its persistence within human hosts. It is therefore necessary to continuously monitor the emergence and prevalence of novel variants that arise. Importantly, some mutations have been associated with both molecular diagnostic failures and reduced or abrogated next-generation sequencing (NGS) read coverage in some genomic regions. Such impacts are particularly problematic when they occur in genomic regions such as those that encode the spike (S) protein, which are crucial for identifying and tracking the prevalence and dissemination dynamics of concerning viral variants. Targeted Sanger sequencing presents a fast and cost-effective means to accurately extend the coverage of whole-genome sequences. We designed a custom set of primers to amplify a 401 bp segment of the receptor-binding domain (RBD) (between positions 22698 and 23098 relative to the Wuhan-Hu-1 reference). We then designed a Sanger sequencing wet-laboratory protocol. We applied the primer set and wet-laboratory protocol to sequence 222 samples that were missing positions with key mutations K417N, E484K, and N501Y due to poor coverage after NGS sequencing. Finally, we developed SeqPatcher, a Python-based computational tool to analyse the trace files yielded by Sanger sequencing to generate consensus sequences, or take preanalysed consensus sequences in fasta format, and merge them with their corresponding whole-genome assemblies. We successfully sequenced 153 samples of 222 (69 %) using Sanger sequencing and confirmed the occurrence of key beta variant mutations (K417N, E484K, N501Y) in the S genes of 142 of 153 (93 %) samples. Additionally, one sample had the Y508F mutation and four samples the S477N. Samples with RT-PCR Ct scores ranging from 13.85 to 37.47 (mean=25.70) could be Sanger sequenced efficiently. These results show that our method and pipeline can be used to improve the quality of whole-genome assemblies produced using NGS and can be used with any pairs of the most used NGS and Sanger sequencing platforms.

SARS-CoV-2 prolonged infection during advanced HIV disease evolves extensive immune escape.

Characterizing SARS-CoV-2 evolution in specific geographies may help predict properties of the variants that come from these regions. We mapped neutralization of a SARS-CoV-2 strain that evolved over 6 months from ancestral virus in a person with advanced HIV disease in South Africa; this person was infected prior to emergence of the Beta and Delta variants. We longitudinally tracked the evolved virus and tested it against self-plasma and convalescent plasma from ancestral, Beta, and Delta infections. Early virus was similar to ancestral, but it evolved a multitude of mutations found in Omicron and other variants. It showed substantial but incomplete Pfizer BNT162b2 escape, weak neutralization by self-plasma, and despite pre-dating Delta, it also showed extensive escape of Delta infection-elicited neutralization. This example is consistent with the notion that SARS-CoV-2 evolving in individual immune-compromised hosts, including those with advanced HIV disease, may gain immune escape of vaccines and enhanced escape of Delta immunity, and this has implications for vaccine breakthrough and reinfections.

Detection of SARS-CoV-2 variants by Abbott molecular, antigen, and serological tests.

BACKGROUND: Viral diversity presents an ongoing challenge for diagnostic tests, which need to accurately detect all circulating variants. The Abbott Global Surveillance program monitors severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants and their impact on diagnostic test performance. OBJECTIVES: To evaluate the capacity of Abbott molecular, antigen, and serologic assays to detect circulating SARS-CoV-2 variants, including all current variants of concern (VOC): B.1.1.7 (alpha), B.1.351 (beta), P.1 (gamma) and B.1.617.2 (delta). STUDY DESIGN: Dilutions of variant virus cultures (B.1.1.7, B.1.351, B.1.429, B.1.526.1, B.1.526.2, B.1.617.1, B.1.617.2, P.1, R.1 and control isolate WA1) and a panel of N = 248 clinical samples from patients with sequence confirmed variant infections (B.1.1.7, B.1.351, B.1.427, B.1.429, B.1.526, B.1.526.1, B.1.526.2, P.1, P.2, R.1) were evaluated on at least one assay: Abbott ID NOW COVID-19, m2000 RealTime SARS-CoV-2, Alinity m SARS-CoV-2, and Alinity m Resp-4-Plex molecular assays; the BinaxNOW COVID-19 Ag Card and Panbio COVID-19 Ag Rapid Test Device; and the ARCHITECT/Alinity i SARS-CoV-2 IgG and AdviseDx IgM assays, Panbio COVID-19 IgG assay, and ARCHITECT/Alinity i AdviseDx SARS-CoV-2 IgG II assay. RESULTS: Consistent with in silico predictions, each molecular and antigen assay detected VOC virus cultures with equivalent sensitivity to the WA1 control strain. Notably, 100% of all tested variant patient specimens were detected by molecular assays (N = 197 m2000, N = 88 Alinity m, N = 99 ID NOW), and lateral flow assays had a sensitivity of >94% for specimens with genome equivalents (GE) per device above 4 log (85/88, Panbio; 54/57 Binax). Furthermore, Abbott antibody assays detected IgG and IgM in 94-100% of sera from immune competent B.1.1.7 patients 15-26 days after symptom onset. CONCLUSIONS: These data confirm variant detection for 11 SARS-CoV-2 assays, which is consistent with each assay target region being highly conserved. Importantly, alpha, beta, gamma, and delta VOCs were detected by molecular and antigen assays, indicating that these tests may be suitable for widescale use where VOCs predominate.

Emergence of SARS-CoV-2 Omicron lineages BA.4 and BA.5 in South Africa.

Three lineages (BA.1, BA.2 and BA.3) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant of concern predominantly drove South Africa's fourth Coronavirus Disease 2019 (COVID-19) wave. We have now identified two new lineages, BA.4 and BA.5, responsible for a fifth wave of infections. The spike proteins of BA.4 and BA.5 are identical, and similar to BA.2 except for the addition of 69-70 deletion (present in the Alpha variant and the BA.1 lineage), L452R (present in the Delta variant), F486V and the wild-type amino acid at Q493. The two lineages differ only outside of the spike region. The 69-70 deletion in spike allows these lineages to be identified by the proxy marker of S-gene target failure, on the background of variants not possessing this feature. BA.4 and BA.5 have rapidly replaced BA.2, reaching more than 50% of sequenced cases in South Africa by the first week of April 2022. Using a multinomial logistic regression model, we estimated growth advantages for BA.4 and BA.5 of 0.08 (95% confidence interval (CI): 0.08-0.09) and 0.10 (95% CI: 0.09-0.11) per day, respectively, over BA.2 in South Africa. The continued discovery of genetically diverse Omicron lineages points to the hypothesis that a discrete reservoir, such as human chronic infections and/or animal hosts, is potentially contributing to further evolution and dispersal of the virus.