Secondary attack rate following on-site isolation of patients with suspected COVID-19 in multiple-bed rooms.

The implementation of isolation precautions for patients with suspected Coronavirus Disease 2019 (COVID-19) and pending test results is resource intensive. Due to the limited availability of single-bed rooms at our institution, we isolated patients with suspected COVID-19 together with patients without suspected COVID-19 on-site in multiple-bed rooms until SARS-CoV-2-test results were available. We evaluated the likelihood of SARS-CoV-2 transmission to individuals sharing the room with patients isolated on-site. This observational study was performed at the University Hospital Basel, Switzerland, from 03/20 - 11/20. Secondary attack rates were compared between patients hospitalized in multiple-bed rooms and exposed to individuals subjected to on-site isolation precautions (on-site isolation group), and patients exposed to individuals initially not identified as having COVID-19, and not placed under isolation precautions until the diagnosis was suspected (control group). Transmission events were confirmed by whole-genome sequencing. Among 1,218 patients with suspected COVID-19, 67 (5.5%) tested positive for SARS-CoV-2. Of these, 21 were isolated on-site potentially exposing 27 patients sharing the same room. Median contact time was 12 h (interquartile range 7-18 h). SARS-CoV-2 transmission was identified in none of the patients in the on-site isolation group vs. 10/63 (15.9%) in the control group (p = 0.03). Isolation on-site of suspected COVID-19-patients in multiple-bed rooms avoided single-room occupancy and subsequent in-hospital relocation for many patients without confirmed SARS-CoV-2-infection. The absence of secondary transmission among the exposed patients in the on-site isolation group allows for assessment of the risk/benefit ratio of this strategy given the limitation of a small sample size.

Iodide based electrochemical gold quantification method for lateral flow assays.

The lateral flow assay (LFA) is an ideal technology for at-home medical diagnostic tests due to its ease of use, cost-effectiveness, and rapid results. Despite these advantages, only few LFAs, such as the pregnancy and COVID-19 tests, have been translated from the laboratory to the homes of patients. To date, the medical applicability of LFAs is limited by the fact that they only provide yes/no answers unless combined with optical readers that are too expensive for at-home applications. Furthermore, LFAs are unable to compete with the state-of-the-art technologies in centralized laboratories in terms of detection limits. To address those shortcomings, we have developed an electrochemical readout procedure to enable quantitative and sensitive LFAs. This technique is based on a voltage-triggered in-situ dissolution of gold nanoparticles, the conventional label used to visualize target-specific signals on the test line in LFAs. Following the dissolution, the amount of gold is measured by electroplating onto an electrode and subsequent electrochemical quantification of the deposited gold. The measured current has a low noise, which achieves superior detection limits compared to optical techniques where background light scattering is limiting the readout performance. In addition, the hardware for the readout was developed to demonstrate translatability towards low-cost electronics.

Self-reported neurocognitive complaints in the Swiss HIV Cohort Study: a viral genome-wide association study.

People with HIV may report neurocognitive complaints, with or without associated neurocognitive impairment, varying between individuals and populations. While the HIV genome could play a major role, large systematic viral genome-wide screens to date are lacking. The Swiss HIV Cohort Study biannually enquires neurocognitive complaints. We quantified broad-sense heritability estimates using partial 'pol' sequences from the Swiss HIV Cohort Study resistance database and performed a viral near full-length genome-wide association study for the longitudinal area under the curve of neurocognitive complaints. We performed all analysis (i) restricted to HIV Subtype B and (ii) including all HIV subtypes. From 8547 people with HIV with neurocognitive complaints, we obtained 6966 partial 'pol' sequences and 2334 near full-length HIV sequences. Broad-sense heritability estimates for presence of memory loss complaints ranged between 1% and 17% (Subtype B restricted 1-22%) and increased with the stringency of the phylogenetic distance thresholds. The genome-wide association study revealed one amino acid (Env L641E), after adjusting for multiple testing, positively associated with memory loss complaints (P = 4.3 * 10-6). Other identified mutations, while insignificant after adjusting for multiple testing, were reported in other smaller studies (Tat T64N, Env *291S). We present the first HIV genome-wide association study analysis of neurocognitive complaints and report a first estimate for the heritability of neurocognitive complaints through HIV. Moreover, we could identify one mutation significantly associated with the presence of memory loss complaints. Our findings indicate that neurocognitive complaints are polygenetic and highlight advantages of a whole genome approach for pathogenicity determination.

Comparative evaluation of Anyplex II HPV28 and Allplex HPV28 molecular assays for human papillomavirus detection and genotyping in anogenital cancer screening.

Persistent infection with high-risk human papillomavirus (HPV) is recognized as the main cause for the development of anogenital cancers. This study prospectively evaluated the diagnostic performance of the novel Allplex-HPV28 assay with the Anyplex-II-HPV28 to detect and genotype HPV in 234 consecutive swabs and 32 biopsies of the anogenital tract from 265 patients with atypical findings in cytomorphological screening. Agreement in HPV-DNA detection between the Anyplex-II and Allplex-HPV28 assays was 99%. There was a notable diversity in the HPV-virome, with the most prevalent high-risk HPV types being 16, 53, 66, and 68. The agreement rates for detecting these genotypes exceeded 93% between the Anyplex-II and Allplex-HPV28 assays. Discrepancies in test results were solely noted for Anyplex-II-HPV28 results with a low signal intensity of "+", and for Allplex-HPV28 results with cycle thresholds of ≥36. The semi-quantitative analysis of HPV-DNA loads showed significant agreement between the Anyplex-II-HPV28 and Allplex-HPV28 assays (p < 0.001). Furthermore, HPV-DNA detection rates and mean HPV-DNA loads significantly correlated with the grade of abnormal changes identified in cytopathological assessment, being highest in cases of HSIL, condyloma accuminatum, and squamous cell carcinoma. Overall agreement rates for detecting specific HPV-types among the Anyplex-II and Allplex-HPV28 assays exceeded 99.5% in cases of atypical squamous cells, condyloma accuminatum, and squamous cell carcinoma. The novel Allplex-HPV28 assay shows good diagnostic performance in detecting and genotyping HPV commonly associated with anogenital cancers. Consequently, this assay could offer substantial potential for incorporation into future molecular screening programs for anogenital cancers in clinical settings.

Emergence of acquired dolutegravir resistance in treatment-experienced people with HIV in Lesotho.

BACKGROUND: Since 2019, the World Health Organization has recommended dolutegravir-based antiretroviral therapy (ART) as the preferred regimen for HIV management. Large-scale programmatic transitioning to dolutegravir-based ART was subsequently implemented across Africa, often in the absence of recent viral load testing and without access to genotypic resistance testing (GRT) in case of viremia. METHODS: This study assessed for emerging dolutegravir resistance in the routine care Viral Load Cohort North-East Lesotho (VICONEL). We included pediatric and adult participants who changed from non-nucleoside transcriptase inhibitor- (NNRTI-) to dolutegravir-based ART and had at least one viral load assessment before and after the change. We sequenced available samples of participants fulfilling the additional virological criteria of having two viraemic episodes while taking dolutegravir, thereof at least one viral load ≥500 copies/mL taken ≥18 months after changing to dolutegravir. RESULTS: Among 15'349 participants, 157 (1.0%) met the virological criteria and GRT was successful for 85 (0.6%). Among these 85, eight (9.4%) had dolutegravir resistance, with two (2.4%) and six (7.1%) predicted to have intermediate and high-level dolutegravir resistance, respectively. One participant had two, two had one, and five had zero active drugs in their regimen. A GRT from before the change to dolutegravir is available for five of these eight participants: four had zero and one had one active drug in their NNRTI-based regimen. CONCLUSIONS: Nine percent of people with persistent or recurring HIV viremia ≥18 months after changing to dolutegravir-based ART had dolutegravir resistance. Detection and management of emerging dolutegravir resistance must be addressed across Africa.

Structural implications of BK polyomavirus sequence variations in the major viral capsid protein Vp1 and large T-antigen: a computational study.

BK polyomavirus (BKPyV) is a double-stranded DNA virus causing nephropathy, hemorrhagic cystitis, and urothelial cancer in transplant patients. The BKPyV-encoded capsid protein Vp1 and large T-antigen (LTag) are key targets of neutralizing antibodies and cytotoxic T-cells, respectively. Our single-center data suggested that variability in Vp1 and LTag may contribute to failing BKPyV-specific immune control and impact vaccine design. We, therefore, analyzed all available entries in GenBank (1516 VP1; 742 LTAG) and explored potential structural effects using computational approaches. BKPyV-genotype (gt)1 was found in 71.18% of entries, followed by BKPyV-gt4 (19.26%), BKPyV-gt2 (8.11%), and BKPyV-gt3 (1.45%), but rates differed according to country and specimen type. Vp1-mutations matched a serotype different than the assigned one or were serotype-independent in 43%, 18% affected more than one amino acid. Notable Vp1-mutations altered antibody-binding domains, interactions with sialic acid receptors, or were predicted to change conformation. LTag-sequences were more conserved, with only 16 mutations detectable in more than one entry and without significant effects on LTag-structure or interaction domains. However, LTag changes were predicted to affect HLA-class I presentation of immunodominant 9mers to cytotoxic T-cells. These global data strengthen single center observations and specifically our earlier findings revealing mutant 9mer epitopes conferring immune escape from HLA-I cytotoxic T cells. We conclude that variability of BKPyV-Vp1 and LTag may have important implications for diagnostic assays assessing BKPyV-specific immune control and for vaccine design. IMPORTANCE: Type and rate of amino acid variations in BKPyV may provide important insights into BKPyV diversity in human populations and an important step toward defining determinants of BKPyV-specific immunity needed to protect vulnerable patients from BKPyV diseases. Our analysis of BKPyV sequences obtained from human specimens reveals an unexpectedly high genetic variability for this double-stranded DNA virus that strongly relies on host cell DNA replication machinery with its proof reading and error correction mechanisms. BKPyV variability and immune escape should be taken into account when designing further approaches to antivirals, monoclonal antibodies, and vaccines for patients at risk of BKPyV diseases.

How much should we sequence? An analysis of the Swiss SARS-CoV-2 surveillance effort.

During the SARS-CoV-2 pandemic, many countries directed substantial resources toward genomic surveillance to detect and track viral variants. There is a debate over how much sequencing effort is necessary in national surveillance programs for SARS-CoV-2 and future pandemic threats. We aimed to investigate the effect of reduced sequencing on surveillance outcomes in a large genomic data set from Switzerland, comprising more than 143k sequences. We employed a uniform downsampling strategy using 100 iterations each to investigate the effects of fewer available sequences on the surveillance outcomes: (i) first detection of variants of concern (VOCs), (ii) speed of introduction of VOCs, (iii) diversity of lineages, (iv) first cluster detection of VOCs, (v) density of active clusters, and (vi) geographic spread of clusters. The impact of downsampling on VOC detection is disparate for the three VOC lineages, but many outcomes including introduction and cluster detection could be recapitulated even with only 35% of the original sequencing effort. The effect on the observed speed of introduction and first detection of clusters was more sensitive to reduced sequencing effort for some VOCs, in particular Omicron and Delta, respectively. A genomic surveillance program needs a balance between societal benefits and costs. While the overall national dynamics of the pandemic could be recapitulated by a reduced sequencing effort, the effect is strongly lineage-dependent-something that is unknown at the time of sequencing-and comes at the cost of accuracy, in particular for tracking the emergence of potential VOCs.IMPORTANCESwitzerland had one of the most comprehensive genomic surveillance systems during the COVID-19 pandemic. Such programs need to strike a balance between societal benefits and program costs. Our study aims to answer the question: How would surveillance outcomes have changed had we sequenced less? We find that some outcomes but also certain viral lineages are more affected than others by sequencing less. However, sequencing to around a third of the original effort still captured many important outcomes for the variants of concern such as their first detection but affected more strongly other measures like the detection of first transmission clusters for some lineages. Our work highlights the importance of setting predefined targets for a national genomic surveillance program based on which sequencing effort should be determined. Additionally, the use of a centralized surveillance platform facilitates aggregating data on a national level for rapid public health responses as well as post-analyses.

Immune response to the recombinant herpes zoster vaccine in people living with HIV over 50 years of age compared to non-HIV age-/gender-matched controls (SHINGR'HIV): a multicenter, international, non-randomized clinical trial study protocol.

BACKGROUND: The burden of herpes zoster (shingles) virus and associated complications, such as post-herpetic neuralgia, is higher in older adults and has a significant impact on quality of life. The incidence of herpes zoster and post-herpetic neuralgia is increased in people living with HIV (PLWH) compared to an age-matched general population, including PLWH on long-term antiretroviral therapy (ART) with no detectable viremia and normal CD4 counts. PLWH - even on effective ART may- exhibit sustained immune dysfunction, as well as defects in cells involved in the response to vaccines. In the context of herpes zoster, it is therefore important to assess the immune response to varicella zoster virus vaccination in older PLWH and to determine whether it significantly differs to that of HIV-uninfected healthy adults or younger PLWH. We aim at bridging these knowledge gaps by conducting a multicentric, international, non-randomised clinical study (SHINGR'HIV) with prospective data collection after vaccination with an adjuvant recombinant zoster vaccine (RZV) in two distinct populations: in PLWH on long-term ART (> 10 years) over 50 years of and age/gender matched controls. METHODS: We will recruit participants from two large established HIV cohorts in Switzerland and in France in addition to age-/gender-matched HIV-uninfected controls. Participants will receive two doses of RZV two months apart. In depth-evaluation of the humoral, cellular, and innate immune responses and safety profile of the RZV will be performed to address the combined effect of aging and potential immune deficiencies due to chronic HIV infection. The primary study outcome will compare the geometric mean titer (GMT) of gE-specific total IgG measured 1 month after the second dose of RZV between different age groups of PLWH and between PLWH and age-/gender-matched HIV-uninfected controls. DISCUSSION: The SHINGR'HIV trial will provide robust data on the immunogenicity and safety profile of RZV in older PLWH to support vaccination guidelines in this population. TRIAL REGISTRATION: ClinicalTrials.gov NCT05575830. Registered on 12 October 2022. Eu Clinical Trial Register (EUCT number 2023-504482-23-00).

Impact of Swabbing Location, Self-Swabbing, and Food Intake on SARS-CoV-2 RNA Detection.

This study compared SARS-CoV-2 RNA loads at different anatomical sites, and the impact of self-swabbing and food intake. Adult symptomatic patients with SARS-CoV-2 or non-SARS-CoV-2 respiratory tract infection were included between 2021 and 2022. Patients performed a nasal and buccal swab before a professionally collected nasopharyngeal/oropharyngeal swab (NOPS). Buccal swabs were collected fasting and after breakfast in a subgroup of patients. SARS-CoV-2 RNA loads were determined by nucleic acid testing. Swabbing convenience was evaluated using a survey. The median age of 199 patients was 54 years (interquartile range 38-68); 42% were female and 52% tested positive for SARS-CoV-2. The majority of patients (70%) were hospitalized. The mean SARS-CoV-2 RNA load was 6.6 log10 copies/mL (standard deviation (SD), ±1.5), 5.6 log10 copies/mL (SD ± 1.9), and 3.4 log10 copies/mL (SD ± 1.9) in the professionally collected NOPS, and self-collected nasal and buccal swabs, respectively (p < 0.0001). Sensitivity was 96.1% (95% CI 90.4-98.9) and 75.3% (95% CI 63.9-81.8) for the nasal and buccal swabs, respectively. After food intake, SARS-CoV-2 RNA load decreased (p = 0.0006). Buccal swabbing was the preferred sampling procedure for the patients. In conclusion, NOPS yielded the highest SARS-CoV-2 RNA loads. Nasal self-swabbing emerged as a reliable alternative in contrast to buccal swabs. If buccal swabs are used, they should be performed before food intake.

Deciphering factors linked with reduced SARS-CoV-2 susceptibility in the Swiss HIV Cohort Study.

BACKGROUND: Factors influencing susceptibility to SARS-CoV-2 remain to be resolved. Using data of the Swiss HIV Cohort Study (SHCS) on 6,270 people with HIV (PWH) and serologic assessment for SARS-CoV-2 and circulating-human-coronavirus (HCoV) antibodies, we investigated the association of HIV-related and general parameters with SARS-CoV-2 infection. METHODS: We analyzed SARS-CoV-2 PCR-tests, COVID-19 related hospitalizations, and deaths reported to the SHCS between January 1, 2020 and December 31, 2021. Antibodies to SARS-CoV-2 and HCoVs were determined in pre-pandemic (2019) and pandemic (2020) bio-banked plasma and compared to HIV-negative individuals. We applied logistic regression, conditional logistic regression, and Bayesian multivariate regression to identify determinants of SARS-CoV-2 infection and Ab responses to SARS-CoV-2 in PWH. RESULTS: No HIV-1-related factors were associated with SARS-CoV-2 acquisition. High pre-pandemic HCoV antibodies were associated with a lower risk of subsequent SARS-CoV-2 infection and with higher SARS-CoV-2 antibody responses upon infection. We observed a robust protective effect of smoking on SARS-CoV-2-infection risk (aOR= 0.46 [0.38,0.56], p=2.6*10-14), which occurred even in previous smokers, and was highest for heavy smokers. CONCLUSIONS: Our findings of two independent protective factors, smoking and HCoV antibodies, both affecting the respiratory environment, underscore the importance of the local immune milieu in regulating susceptibility to SARS-CoV-2.

Differential Gene Expression of SARS-CoV-2 Positive Bronchoalveolar Lavages: A Case Series.

BACKGROUND: Transcriptomic data on bronchoalveolar lavage (BAL) from COVID-19 patients are currently scarce. OBJECTIVES: This case series seeks to characterize the intra-alveolar immunopathology of COVID-19. METHOD: BALs were performed on 14 patients (5 COVID-19, of which 3 mild and 2 largely asymptomatic, 9 controls). Controls included asthma (n = 1), unremarkable BALs (n = 3), infections with respiratory syncytial virus (n = 1), influenza B (n = 1), and infections with other coronaviruses (n = 3). SARS-CoV-2 RNA load was measured by quantitative nucleic acid testing, while the detection of other pathogens was performed by immunofluorescence or multiplex NAT. RESULTS: Gene expression profiling showed 71 significantly downregulated and 5 upregulated transcripts in SARS-CoV-2-positive lavages versus controls. Downregulated transcripts included genes involved in macrophage development, polarization, and crosstalk (LGALS3, MARCO, ERG2, BTK, RAC1, CD83), and genes involved in chemokine signaling and immunometabolism (NUPR1, CEBPB, CEBPA, PECAM1, CCL18, PPARG, ALOX5, ALOX5AP). Upregulated transcripts featured genes involved in NK-T cell signaling (GZMA, GZMH, GNLY, PRF1, CD3G). Patients with mild COVID-19 showed a significant upregulation of genes involved in blood mononuclear cell/leukocyte function (G0S2, ANXA6, FCGR2B, ADORA3), coagulation (von Willebrand factor [VWF]), interferon response (IFRD1, IL12RB2), and a zinc metalloprotease elevated in asthma (CPA3) compared to asymptomatic cases. In-silico comparison of the 5 COVID-19 BAL cases to a published cohort of lethal COVID-19 showed a significant upregulation of "antigen processing and presentation" and "lysosome" pathways in lethal cases. CONCLUSIONS: These data underscore the heterogeneity of immune response in COVID-19. Further studies with a larger dataset are required to gain a better understanding of the hallmarks of SARS-CoV-2 immunological response.

Impact of bronchoalveolar lavage on the management of immunocompromised hosts.

BACKGROUND: Respiratory infections are an important cause of morbidity and mortality in immunocompromised individuals. Fiberoptic bronchoscopy with bronchoalveolar lavage (BAL) is an important tool to detect infectious agents in immunocompromised patients with low respiratory tract infections (LRTI). RESEARCH QUESTION: BAL changes the management of immunocompromised patients with suspected LRTI. STUDY DESIGN AND METHODS: Immunocompromised patients with a suspicion of LRTI underwent diagnostic BAL. The primary composite outcome consisted of pre-defined modifications in the management of the immunocompromised patients following BAL. We quantified the impact of bronchoscopy up to 30 days after the procedure. RESULTS: A total of 2666 visits from 1301 patients were included in the study and immunosuppression was classified as haematological (n = 1040; 544 patients), solid organ transplantation (n = 666; 107 patients) and other causes (n = 960; 650 patients). BAL led to a change in management in 52.36% (n = 1396) of all cases. This percentage, as well as the 30-day mortality differed significantly amongst the three groups. Age, C-reactive protein and aetiology of infection determined significantly the risk of 30-day mortality in all patients. In 1.89% (n = 50) of all cases, a combination of 2 respiratory viral agents was identified and 24.23% (n = 646) were diagnosed with a single respiratory viral agent. INTERPRETATION: BAL leads to changes in management in the majority of immunosuppressed patients. There is a high prevalence of multimicrobial infections and respiratory viral infections in immunocompromised patients with respiratory symptoms. Individual virus infection is associated with diverse risk of a negative outcome.

Amplicon size and non-encapsidated DNA fragments define plasma cytomegalovirus DNA loads by automated nucleic acid testing platforms: A marker of viral cytopathology?

Management of cytomegalovirus (CMV) in transplant patients relies on measuring plasma CMV-loads using quantitative nucleic acid testing (QNAT). We prospectively compared the automated Roche-cobas®6800-CMV and Roche-CAP/CTM-CMV with laboratory-developed Basel-CMV-UL54-95bp, and Basel-CMV-UL111a-77bp. Roche-cobas®6800-CMV and Roche-CAP/CTM-CMV were qualitatively concordant in 142/150 cases (95%). In-depth comparison revealed higher CMV-loads of the laboratory-developed assay and correlated with smaller amplicon size. After calibration to the 1.WHO-approved CMV international standard, differences were reduced but remained significant. DNase-I pretreatment significantly reduced CMV-loads for both automated Roche-CAP/CTM-CMV and Roche-cobas®6800-CMV assays, whereby 90% and 95% of samples became undetectable. DNase-I pretreatment also reduced CMV-loads quantified by Basel-CMV-UL54-95bp and Basel-CMV-UL111a-77bp, but remaining detectable in 20% and 35%, respectively. Differences were largest for 110 samples with low-level CMV-DNAemia being detectable but not-quantifiable by Roche-cobas®6800-CMV, whereby the smaller amplicon sizes yielded higher viral loads for concordant positives. We conclude that non-encapsidated fragmented CMV-DNA is the major form of plasma CMV-loads also measured by fully-automated platforms. Amplicons of <150 bp and calibrators are needed for reliable and commutable QNAT-results. We hypothesize that non-encapsidated fragmented CMV-DNA results from lysis of CMV-replicating cells and represent a direct marker of viral cell damage, which contribute to delayed viral load responses despite effective antivirals.

Urine CXCL10 to Assess BK Polyomavirus Replication After Kidney Transplantation.

BACKGROUND: Urine CXCL10 is a biomarker for renal allograft inflammation induced by rejection, urinary tract infection, or BK polyomavirus (BKPyV) replication. This study aimed to compare urine CXCL10 levels in different stages of BKPyV reactivation and to investigate urine CXCL10 as a biomarker for BKPyV replication. METHODS: We included 763 urine samples (235 patients) from an interventional, randomized trial obtained in the context of regular screening for urine CXCL10 levels. All urine samples had a complete urine sediment analysis, no rejection episode noted within 30 d before urine collection, and a urine decoy cell analysis was conducted within ±3 d. RESULTS: Urine CXCL10 levels were 2.31 ng/mmol in samples without BKPyV viruria, slightly rose to 4.35 ng/mmol with BKPyV viruria, and then markedly increased to 16.42 ng/mmol when decoy cells were detectable, but still in the absence of BKPyV DNAemia ( P < 0.001). The highest urine CXCL10 values were observed in samples with BKPyV DNAemia (median 42.59 ng/mmol). The area under the curve of urine CXCL10 levels to detect ≥3 decoy cells was 0.816. At a CXCL10 cutoff of 3 ng/mmol, the negative predictive value was 97%. The area under the curve of urine CXCL10 levels to detect BKPyV DNAemia was 0.882, with a negative predictive value of 99% at a CXCL10 cutoff of 3 ng/mmol. CONCLUSIONS: Urine CXCL10 levels are already significantly elevated in BKPyV viruria (especially with decoy cell shedding) and further increase with BKPyV DNAemia. Low urine CXCL10 values can rule out the presence of ≥3 decoy cells and BKPyV DNAemia with high certainty.

The Swiss Pathogen Surveillance Platform - towards a nation-wide One Health data exchange platform for bacterial, viral and fungal genomics and associated metadata.

The Swiss Pathogen Surveillance Platform (SPSP) is a shared secure surveillance platform between human and veterinary medicine, to also include environmental and foodborne isolates. It enables rapid and detailed transmission monitoring and outbreak surveillance of pathogens using whole genome sequencing data and associated metadata. It features controlled data access, complex dynamic queries, dedicated dashboards and automated data sharing with international repositories, providing actionable results for public health and the vision to improve societal well-being and health.

A systematic outbreak investigation of SARS-CoV-2 transmission clusters in a tertiary academic care center.

BACKGROUND: We sought to decipher transmission pathways in healthcare-associated infections with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within our hospital by epidemiological work-up and complementary whole genome sequencing (WGS). We report the findings of the four largest epidemiologic clusters of SARS-CoV-2 transmission occurring during the second wave of the pandemic from 11/2020 to 12/2020. METHODS: At the University Hospital Basel, Switzerland, systematic outbreak investigation is initiated at detection of any nosocomial case of SARS-CoV-2 infection, as confirmed by polymerase chain reaction, occurring more than five days after admission. Clusters of nosocomial infections, defined as the detection of at least two positive patients and/or healthcare workers (HCWs) within one week with an epidemiological link, were further investigated by WGS on respective strains. RESULTS: The four epidemiologic clusters included 40 patients and 60 HCWs. Sequencing data was available for 70% of all involved cases (28 patients and 42 HCWs), confirmed epidemiologically suspected in house transmission in 33 cases (47.1% of sequenced cases) and excluded transmission in the remaining 37 cases (52.9%). Among cases with identical strains, epidemiologic work-up suggested transmission mainly through a ward-based exposure (24/33, 72.7%), more commonly affecting HCWs (16/24, 66.7%) than patients (8/24, 33.3%), followed by transmission between patients (6/33, 18.2%), and among HCWs and patients (3/33, 9.1%, respectively two HCWs and one patient). CONCLUSIONS: Phylogenetic analyses revealed important insights into transmission pathways supporting less than 50% of epidemiologically suspected SARS-CoV-2 transmissions. The remainder of cases most likely reflect community-acquired infection randomly detected by outbreak investigation. Notably, most transmissions occurred between HCWs, possibly indicating lower perception of the risk of infection during contacts among HCWs.

Frequency matters: comparison of drug resistance mutation detection by Sanger and next-generation sequencing in HIV-1.

BACKGROUND: Next-generation sequencing (NGS) is gradually replacing Sanger sequencing (SS) as the primary method for HIV genotypic resistance testing. However, there are limited systematic data on comparability of these methods in a clinical setting for the presence of low-abundance drug resistance mutations (DRMs) and their dependency on the variant-calling thresholds. METHODS: To compare the HIV-DRMs detected by SS and NGS, we included participants enrolled in the Swiss HIV Cohort Study (SHCS) with SS and NGS sequences available with sample collection dates ≤7 days apart. We tested for the presence of HIV-DRMs and compared the agreement between SS and NGS at different variant-calling thresholds. RESULTS: We included 594 pairs of SS and NGS from 527 SHCS participants. Males accounted for 80.5% of the participants, 76.3% were ART naive at sample collection and 78.1% of the sequences were subtype B. Overall, we observed a good agreement (Cohen's kappa >0.80) for HIV-DRMs for variant-calling thresholds ≥5%. We observed an increase in low-abundance HIV-DRMs detected at lower thresholds [28/417 (6.7%) at 10%-25% to 293/812 (36.1%) at 1%-2% threshold]. However, such low-abundance HIV-DRMs were overrepresented in ART-naive participants and were in most cases not detected in previously sampled sequences suggesting high sequencing error for thresholds <3%. CONCLUSIONS: We found high concordance between SS and NGS but also a substantial number of low-abundance HIV-DRMs detected only by NGS at lower variant-calling thresholds. Our findings suggest that a substantial fraction of the low-abundance HIV-DRMs detected at thresholds <3% may represent sequencing errors and hence should not be overinterpreted in clinical practice.

Impact of nucleic acid extraction procedures on human papillomavirus (HPV) detection and genotyping.

Human papillomavirus (HPV) infections are often asymptomatic, but some of the >200 HPV genotypes confer a high risk for precancerous cervical lesions and cervical cancer. Current clinical management of HPV infections relies on reliable nucleic acid testing detection and genotyping. We prospectively compared nucleic acid extraction without and with prior centrifugation enrichment for detecting and genotyping HPV in cervical swabs with atypical squamous or glandular cells. Consecutive swabs were analyzed from 45 patients with atypical squamous or glandular cells. Nucleic acids were extracted in parallel using three procedures, Abbott-M2000, Roche-MagNA-Pure-96 Large-Volume Kit without (Roche-MP-large) and with prior centrifugation (Roche-MP-large/spin) and tested using Seegene-Anyplex-II HPV28. In total, 54 HPV-genotypes were detected in 45 samples, 51 by Roche-MP-large/spin, 48 by Abbott-M2000 and 42 by Roche-MP-large. The overall concordance was 80% for detecting any HPV and 74% for specific HPV-genotypes. Roche-MP-large/spin and Abbott-M2000 showed the highest concordance for HPV detection (88.9%; kappa 0.78), and genotyping (88.5%). Two and more HPV-genotypes were detected in 15 samples, often with one HPV being more abundant. Dilution series confirmed the specific detection of multiple HPV-genotypes and their relative abundance. In 285 consecutive follow-up samples extracted by Roche-MP-large/spin, the top three detected genotypes were the high-risk HPV16, HPV53, HPV56 and the low-risk HPV42, HPV54 and HPV61. Rate and breadth of HPV detection in cervical swabs depends on extraction protocols being highest after centrifugation/enrichment. As multivalent HPV-vaccine coverage increases, detecting the evolving HPV virome depends on improved extraction and broader genotype coverage.

Molecular Characterization of BK Polyomavirus Replication in Allogeneic Hematopoietic Cell Transplantation Patients.

BACKGROUND: High-level BK polyomavirus (BKPyV) replication in allogeneic hematopoietic cell transplantation (HCT) predicts failing immune control and BKPyV-associated hemorrhagic cystitis. METHODS: To identify molecular markers of BKPyV replication and disease, we scrutinized BKPyV DNA-loads in longitudinal urine and plasma pairs from 20 HCT patients using quantitative nucleic acid testing (QNAT), DNase-I treatment prior to QNAT, next-generation sequencing (NGS), and tested cell-mediated immunity. RESULTS: We found that larger QNAT amplicons led to under-quantification and false-negatives results (P < .001). DNase-I reduced urine and plasma BKPyV-loads by >90% (P < .001), indicating non-encapsidated BKPyV genomes. DNase-resistant urine BKPyV-loads remained infectious in cell culture. BKPyV genome fragmentation of ≤250 bp impaired NGS coverage of genetic variation using 1000-bp and 5000-bp amplicons. Conversely, 250-bp amplicons captured viral minority variants. We identified genotype-specific and genotype-independent changes in capsid Vp1 or T-antigen predicted to escape from antibody neutralization or cytotoxic CD8 T-cells, respectively. Genotype-specific changes in immunodominant 9mers were associated with reduced or absent CD8 T-cell responses. Thus, failure to control BKPyV replication in HCT Patients may involve insufficient genotype-specific cytotoxic CD8 T-cell responses, potentially predictable by low neutralizing antibodies as well as genotype-independent immune escape. CONCLUSIONS: Our results provide new insights for patient evaluation and for designing immune protection through neutralizing antibodies, adoptive T-cell therapy, or vaccines.

Swiss public health measures associated with reduced SARS-CoV-2 transmission using genome data.

Genome sequences from evolving infectious pathogens allow quantification of case introductions and local transmission dynamics. We sequenced 11,357 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes from Switzerland in 2020-the sixth largest effort globally. Using a representative subset of these data, we estimated viral introductions to Switzerland and their persistence over the course of 2020. We contrasted these estimates with simple null models representing the absence of certain public health measures. We show that Switzerland's border closures decoupled case introductions from incidence in neighboring countries. Under a simple model, we estimate an 86 to 98% reduction in introductions during Switzerland's strictest border closures. Furthermore, the Swiss 2020 partial lockdown roughly halved the time for sampled introductions to die out. Last, we quantified local transmission dynamics once introductions into Switzerland occurred using a phylodynamic model. We found that transmission slowed 35 to 63% upon outbreak detection in summer 2020 but not in fall. This finding may indicate successful contact tracing over summer before overburdening in fall. The study highlights the added value of genome sequencing data for understanding transmission dynamics.