Rate of response to initial antiretroviral therapy according to level of pre-existing HIV-1 drug resistance detected by next-generation sequencing in the strategic timing of antiretroviral treatment (START) study.

OBJECTIVES: The main objective of this analysis was to evaluate the impact of pre-existing drug resistance by next-generation sequencing (NGS) on the risk of treatment failure (TF) of first-line regimens in participants enrolled in the START study. METHODS: Stored plasma from participants with entry HIV RNA >1000 copies/mL were analysed using NGS (llumina MiSeq). Pre-existing drug resistance was defined using the mutations considered by the Stanford HIV Drug Resistance Database (HIVDB v8.6) to calculate the genotypic susceptibility score (GSS, estimating the number of active drugs) for the first-line regimen at the detection threshold windows of >20%, >5%, and >2% of the viral population. Survival analysis was conducted to evaluate the association between the GSS and risk of TF (viral load >200 copies/mL plus treatment change). RESULTS: Baseline NGS data were available for 1380 antiretroviral therapy (ART)-naïve participants enrolled over 2009-2013. First-line ART included a non-nucleoside reverse transcriptase inhibitor (NNRTI) in 976 (71%), a boosted protease inhibitor in 297 (22%), or an integrase strand transfer inhibitor in 107 (8%). The proportions of participants with GSS <3 were 7% for >20%, 10% for >5%, and 17% for the >2% thresholds, respectively. The adjusted hazard ratio of TF associated with a GSS of 0-2.75 versus 3 in the subset of participants with mutations detected at the >2% threshold was 1.66 (95% confidence interval 1.01-2.74; p = 0.05) and 2.32 (95% confidence interval 1.32-4.09; p = 0.003) after restricting the analysis to participants who started an NNRTI-based regimen. CONCLUSIONS: Up to 17% of participants initiated ART with a GSS <3 on the basis of NGS data. Minority variants were predictive of TF, especially for participants starting NNRTI-based regimens.

SARS-CoV-2 Variants BQ.1 and XBB.1.5 in Wastewater of Aircraft Flying from China to Denmark, 2023.

We analyzed wastewater samples from 14 aircraft arriving in Denmark directly from China during January 9-February 12, 2023. Wastewater from 11 aircraft was SARS-CoV-2-positive by PCR; 6 predominantly contained BQ.1 and XBB.1 subvariants. Wastewater-based surveillance can contribute to public health monitoring of SARS-CoV-2 and other emerging infectious agents.

First cases of SARS-CoV-2 BA.2.86 in Denmark, 2023.

We describe 10 cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant BA.2.86 detected in Denmark, including molecular characteristics and results from wastewater surveillance that indicate that the variant is circulating in the country at a low level. This new variant with many spike gene mutations was classified as a variant under monitoring by the World Health Organization on 17 August 2023. Further global monitoring of COVID-19, BA.2.86 and other SARS-CoV-2 variants is highly warranted.

Associations of functional human leucocyte antigen class I groups with HIV viral load in a heterogeneous cohort.

OBJECTIVE: Human leucocyte antigen (HLA) class I alleles are the main host genetic factors involved in controlling HIV-1 viral load (VL). Nevertheless, HLA diversity has proven a significant challenge in association studies. We assessed how accounting for binding affinities of HLA class I alleles to HIV-1 peptides facilitate association testing of HLA with HIV-1 VL in a heterogeneous cohort. DESIGN: Cohort from the Strategic Timing of AntiRetroviral Treatment (START) study. METHODS: We imputed HLA class I alleles from host genetic data (2546 HIV+ participants) and sampled immunopeptidomes from 2079 host-paired viral genomes (targeted amplicon sequencing). We predicted HLA class I binding affinities to HIV-1 and unspecific peptides, grouping alleles into functional clusters through consensus clustering. These functional HLA class I clusters were used to test associations with HIV VL. RESULTS: We identified four clades totaling 30 HLA alleles accounting for 11.4% variability in VL. We highlight HLA-B∗57:01 and B∗57:03 as functionally similar but yet overrepresented in distinct ethnic groups, showing when combined a protective association with HIV+ VL (log, ß -0.25; adj. P-value < 0.05). We further demonstrate only a slight power reduction when using unspecific immunopeptidomes, facilitating the use of the inferred functional HLA groups in other studies. CONCLUSION: The outlined computational approach provides a robust and efficient way to incorporate HLA function and peptide diversity, aiding clinical association studies in heterogeneous cohorts. To facilitate access to the proposed methods and results we provide an interactive application for exploring data.

Deep-sequencing of viral genomes from a large and diverse cohort of treatment-naive HIV-infected persons shows associations between intrahost genetic diversity and viral load.

BACKGROUND: Infection with human immunodeficiency virus type 1 (HIV) typically results from transmission of a small and genetically uniform viral population. Following transmission, the virus population becomes more diverse because of recombination and acquired mutations through genetic drift and selection. Viral intrahost genetic diversity remains a major obstacle to the cure of HIV; however, the association between intrahost diversity and disease progression markers has not been investigated in large and diverse cohorts for which the majority of the genome has been deep-sequenced. Viral load (VL) is a key progression marker and understanding of its relationship to viral intrahost genetic diversity could help design future strategies for HIV monitoring and treatment. METHODS: We analysed deep-sequenced viral genomes from 2,650 treatment-naive HIV-infected persons to measure the intrahost genetic diversity of 2,447 genomic codon positions as calculated by Shannon entropy. We tested for associations between VL and amino acid (AA) entropy accounting for sex, age, race, duration of infection, and HIV population structure. RESULTS: We confirmed that the intrahost genetic diversity is highest in the env gene. Furthermore, we showed that mean Shannon entropy is significantly associated with VL, especially in infections of >24 months duration. We identified 16 significant associations between VL (p-value<2.0x10-5) and Shannon entropy at AA positions which in our association analysis explained 13% of the variance in VL. Finally, equivalent analysis based on variation in HIV consensus sequences explained only 2% of VL variance. CONCLUSIONS: Our results elucidate that viral intrahost genetic diversity is associated with VL and could be used as a better disease progression marker than HIV consensus sequence variants, especially in infections of longer duration. We emphasize that viral intrahost diversity should be considered when studying viral genomes and infection outcomes. TRIAL REGISTRATION: Samples included in this study were derived from participants who consented in the clinical trial, START (NCT00867048) (23), run by the International Network for Strategic Initiatives in Global HIV Trials (INSIGHT). All the participant sites are listed here: http://www.insight-trials.org/start/my_phpscript/participating.php?by=site.

Introduction and transmission of SARS-CoV-2 lineage B.1.1.7, Alpha variant, in Denmark.

BACKGROUND: In early 2021, the SARS-CoV-2 lineage B.1.1.7 (Alpha variant) became dominant across large parts of the world. In Denmark, comprehensive and real-time test, contact-tracing, and sequencing efforts were applied to sustain epidemic control. Here, we use these data to investigate the transmissibility, introduction, and onward transmission of B.1.1.7 in Denmark. METHODS: We analyzed a comprehensive set of 60,178 SARS-CoV-2 genomes generated from high-throughput sequencing by the Danish COVID-19 Genome Consortium, representing 34% of all positive cases in the period 14 November 2020 to 7 February 2021. We calculated the transmissibility of B.1.1.7 relative to other lineages using Poisson regression. Including all 1976 high-quality B.1.1.7 genomes collected in the study period, we constructed a time-scaled phylogeny, which was coupled with detailed travel history and register data to outline the introduction and onward transmission of B.1.1.7 in Denmark. RESULTS: In a period with unchanged restrictions, we estimated an increased B.1.1.7 transmissibility of 58% (95% CI: [56%, 60%]) relative to other lineages. Epidemiological and phylogenetic analyses revealed that 37% of B.1.1.7 cases were related to the initial introduction in November 2020. The relative number of cases directly linked to introductions varied between 10 and 50% throughout the study period. CONCLUSIONS: Our findings corroborate early estimates of increased transmissibility of B.1.1.7. Both substantial early expansion when B.1.1.7 was still unmonitored and continuous foreign introductions contributed considerably to case numbers. Finally, our study highlights the benefit of balanced travel restrictions and self-isolation procedures coupled with comprehensive surveillance efforts, to sustain epidemic control in the face of emerging variants.

Increased transmission of SARS-CoV-2 in Denmark during UEFA European championships.

Denmark hosted four games during the 2020 UEFA European championships (EC2020). After declining positive SARS-CoV-2 test rates in Denmark, a rise occurred during and after the tournament, concomitant with the replacement of the dominant Alpha lineage (B.1.1.7) by the Delta lineage (B.1.617.2), increasing vaccination rates and cessation of several restrictions. A cohort study including 33 227 cases was conducted from 30 May to 25 July 2021, 14 days before and after the EC2020. Included was a nested cohort with event information from big-screen events and matches at the Danish national stadium, Parken (DNSP) in Copenhagen, held from 12 June to 28 June 2021. Information from whole-genome sequencing, contact tracing and Danish registries was collected. Case-case connections were used to establish transmission trees. Cases infected on match days were compared to cases not infected on match days as a reference. The crude incidence rate ratio (IRR) of transmissions was 1.55, corresponding to 584 (1.76%) cases attributable to EC2020 celebrations. The IRR adjusted for covariates was lower (IRR 1.41) but still significant, and also pointed to a reduced number of transmissions from fully vaccinated cases (IRR 0.59). These data support the hypothesis that the EC2020 celebrations contributed to the rise of cases in Denmark in the early summer of 2021.

Molecular epidemiology of the SARS-CoV-2 variant Omicron BA.2 sub-lineage in Denmark, 29 November 2021 to 2 January 2022.

Following emergence of the SARS-CoV-2 variant Omicron in November 2021, the dominant BA.1 sub-lineage was replaced by the BA.2 sub-lineage in Denmark. We analysed the first 2,623 BA.2 cases from 29 November 2021 to 2 January 2022. No epidemiological or clinical differences were found between individuals infected with BA.1 versus BA.2. Phylogenetic analyses showed a geographic east-to-west transmission of BA.2 from the Capital Region with clusters expanding after the Christmas holidays. Mutational analysis shows distinct differences between BA.1 and BA.2.

Neutralisation of the SARS-CoV-2 Delta variant sub-lineages AY.4.2 and B.1.617.2 with the mutation E484K by Comirnaty (BNT162b2 mRNA) vaccine-elicited sera, Denmark, 1 to 26 November 2021.

Several factors may account for the recent increased spread of the SARS-CoV-2 Delta sub-lineage AY.4.2 in the United Kingdom, Romania, Poland, and Denmark. We evaluated the sensitivity of AY.4.2 to neutralisation by sera from 30 Comirnaty (BNT162b2 mRNA) vaccine recipients in Denmark in November 2021. AY.4.2 neutralisation was comparable to other circulating Delta lineages or sub-lineages. Conversely, the less prevalent B.1.617.2 with E484K showed a significant more than 4-fold reduction in neutralisation that warrants surveillance of strains with the acquired E484K mutation.

Development and Dynamics of Cytomegalovirus UL97 Ganciclovir Resistance Mutations in Transplant Recipients Detected by Next-Generation Sequencing.

BACKGROUND: (Val)ganciclovir resistance mutations in CMV UL97 (UL97-GCV-R) complicate anti-CMV therapy in recipients of solid organ and hematopoietic stem cell transplants, but comprehensive data on prevalence, emergence, and outcome are scarce. METHODS: Using next-generation sequencing (NGS; Illumina MiSeq platform), we analyzed UL97-GCV-R in patients with available plasma samples and refractory CMV replication/DNAemia (n = 87) containing viral loads ≥910 IU/mL. Twenty-one patients with CMV DNAemia resolving under antiviral therapy were analyzed as controls. Detected mutations were considered induced and of potential clinical significance if they increased by ≥10% compared with the first detected frequency or if they had a maximum frequency ≥25%. RESULTS: Nineteen of 87 (21.8%) with refractory CMV replication had ≥1 UL97-GCV-R detected by NGS, in comparison to 0/21 of the controls (P = .02). One-third of the recipients had 2 or more induced UL97-GCV-R mutations. The most frequently induced mutations affected codons 595 (42% [8/19]), 594 (32% [6/19]), and 603 (32% [6/19]). C592G was present in all episodes of both cases and controls at frequencies <15%, but never induced. UL97-GCV-R tended to be more frequent in donor/recipient CMV immunoglobulin G mismatch or following failure to complete primary prophylaxis, and many developed invasive CMV disease. CONCLUSIONS: UL97-GCV-R is common among transplant patients with refractory CMV replication. Early testing by NGS allows for identification of major mutations at codons 595, 594, and 603 and excludes a major role of C592G in ganciclovir resistance. Large prospective studies on UL97-GCV-R are warranted.

Phylogenetic analysis of HIV-1 shows frequent cross-country transmission and local population expansions.

Understanding of pandemics depends on the characterization of pathogen collections from well-defined and demographically diverse cohorts. Since its emergence in Congo almost a century ago, Human Immunodeficiency Virus Type 1 (HIV-1) has geographically spread and genetically diversified into distinct viral subtypes. Phylogenetic analysis can be used to reconstruct the ancestry of the virus to better understand the origin and distribution of subtypes. We sequenced two 3.6-kb amplicons of HIV-1 genomes from 3,197 participants in a clinical trial with consistent and uniform sampling at sites across 35 countries and analyzed our data with another 2,632 genomes that comprehensively reflect the HIV-1 genetic diversity. We used maximum likelihood phylogenetic analysis coupled with geographical information to infer the state of ancestors. The majority of our sequenced genomes (n = 2,501) were either pure subtypes (A-D, F, and G) or CRF01_AE. The diversity and distribution of subtypes across geographical regions differed; USA showed the most homogenous subtype population, whereas African samples were most diverse. We delineated transmission of the four most prevalent subtypes in our dataset (A, B, C, and CRF01_AE), and our results suggest both continuous and frequent transmission of HIV-1 over country borders, as well as single transmission events being the seed of endemic population expansions. Overall, we show that coupling of genetic and geographical information of HIV-1 can be used to understand the origin and spread of pandemic pathogens.

Human Immunotypes Impose Selection on Viral Genotypes Through Viral Epitope Specificity.

BACKGROUND: Understanding the genetic interplay between human hosts and infectious pathogens is crucial for how we interpret virulence factors. Here, we tested for associations between HIV and host genetics, and interactive genetic effects on viral load (VL) in HIV-positive antiretroviral treatment-naive clinical trial participants. METHODS: HIV genomes were sequenced and the encoded amino acid (AA) variants were associated with VL, human single nucleotide polymorphisms (SNPs), and imputed HLA alleles using generalized linear models with Bonferroni correction. RESULTS: Human (388 501 SNPs) and HIV (3010 variants) genetic data were available for 2122 persons. Four HIV variants were associated with VL (P < 1.66 × 10-5). Twelve HIV variants were associated with a range of 1-512 human SNPs (P < 4.28 × 10-11). We found 46 associations between HLA alleles and HIV variants (P < 1.29 × 10-7). HIV variants and immunotypes when analyzed separately were associated with lower VL, whereas the opposite was true when analyzed in concert. Epitope binding predictions supported our observations. CONCLUSIONS: Our results show the importance of immunotype specificity on viral antigenic determinants, and the identified genetic interplay emphasizes that viral and human genetics should be studied in the context of each other.Clinical Trials Registration: NCT00867048.

Oxacillinase-181 Carbapenemase-Producing Klebsiella pneumoniae in Neonatal Intensive Care Unit, Ghana, 2017-2019.

We sequenced 29 carbapenemase-producing Klebsiella pneumoniae isolates from a neonatal intensive care unit in Ghana. Twenty-eight isolates were sequence type 17 with blaOXA-181 and differed by 0-32 single-nucleotide polymorphisms. Improved surveillance and infection control are needed to characterize and curb the spread of multidrug-resistant organisms in sub-Saharan Africa.

Transcriptional changes induced by bevacizumab combination therapy in responding and non-responding recurrent glioblastoma patients.

BACKGROUND: Bevacizumab combined with chemotherapy produces clinical durable response in 25-30% of recurrent glioblastoma patients. This group of patients has shown improved survival and quality of life. The aim of this study was to investigate changes in gene expression associated with response and resistance to bevacizumab combination therapy. METHODS: Recurrent glioblastoma patients who had biomarker-accessible tumor tissue surgically removed both before bevacizumab treatment and at time of progression were included. Patients were grouped into responders (n = 7) and non-responders (n = 14). Gene expression profiling of formalin-fixed paraffin-embedded tumor tissue was performed using RNA-sequencing. RESULTS: By comparing pretreatment samples of responders with those of non-responders no significant difference was observed. In a paired comparison analysis of pre- and posttreatment samples of non-responders 1 gene was significantly differentially expressed. In responders, this approach revealed 256 significantly differentially expressed genes (72 down- and 184 up-regulated genes at the time of progression). Genes differentially expressed in responders revealed a shift towards a more proneural and less mesenchymal phenotype at the time of progression. CONCLUSIONS: Bevacizumab combination treatment demonstrated a significant impact on the transcriptional changes in responders; but only minimal changes in non-responders. This suggests that non-responding glioblastomas progress chaotically without following distinct gene expression changes while responding tumors adaptively respond or progress by means of the same transcriptional changes. In conclusion, we hypothesize that the identified gene expression changes of responding tumors are associated to bevacizumab response or resistance mechanisms.

Identification of eight novel SDHB, SDHC, SDHD germline variants in Danish pheochromocytoma/paraganglioma patients.

BACKGROUND: Germline mutations in the succinate dehydrogenase complex genes SDHB, SDHC, and SDHD predispose to pheochromocytomas and paragangliomas. Here, we examine the SDHB, SDHC, and SDHD mutation spectrum in the Danish population by screening of 143 Danish pheochromocytoma and paraganglioma patients. METHODS: Mutational screening was performed by Sanger sequencing or next-generation sequencing. The frequencies of variants of unknown clinical significance, e.g. intronic, missense, and synonymous variants, were determined using the Exome Aggregation Consortium database, while the significance of missense mutations was predicted by in silico and loss of heterozygosity analysis when possible. RESULTS: We report 18 germline variants; nine in SDHB, six in SDHC, and three in SDHD. Of these 18 variants, eight are novel. We classify 12 variants as likely pathogenic/pathogenic, one as likely benign, and five as variants of unknown clinical significance. CONCLUSIONS: Identifying and classifying SDHB, SDHC, and SDHD variants present in the Danish population will augment the growing knowledge on variants in these genes and may support future clinical risk assessments.