The growing prevalence of deadly microbes with resistance to previously life-saving drug therapies is a dire threat to human health. Detection of low abundance pathogen sequences remains a challenge for metagenomic Next Generation Sequencing (NGS). We introduce FLASH (Finding Low Abundance Sequences by Hybridization), a next-generation CRISPR/Cas9 diagnostic method that takes advantage of the efficiency, specificity and flexibility of Cas9 to enrich for a programmed set of sequences. FLASH-NGS achieves up to 5 orders of magnitude of enrichment and sub-attomolar gene detection with minimal background. We provide an open-source software tool (FLASHit) for guide RNA design. Here we applied it to detection of antimicrobial resistance genes in respiratory fluid and dried blood spots, but FLASH-NGS is applicable to all areas that rely on multiplex PCR.

Vibrio cholerae O1 is a noninvasive enteric pathogen and serves as a model for studies of mucosal immunity. Although symptomatic V. cholerae infection induces durable protection against subsequent disease, vaccination with oral killed whole-cell V. cholerae stimulates less long-lasting protection against cholera. In this study, we demonstrated that cholera induces an early proinflammatory cellular immune response that results in priming of Th1-and Th17-type cytokine responses to ex vivo antigenic stimulation and an increase in the ratio of Th1 to Th2 CD4 ؉ T-cell responses. Comparable priming of Th1 and Th17 responses, with an increased ratio of Th1 to Th2 CD4 ؉ T-cell responses, was not observed in subjects who received two doses of the oral cholera vaccine Dukoral (a whole-cell cholera toxin B subunit containing [WC-CTB] vaccine). These findings suggest that natural V. cholerae infection induces an early, proinflammatory cellular immune response, despite the apparent lack of clinical signs of inflammation. The failure of the WC-CTB vaccine to activate equivalent, CD4؉ T-cell responses is a potential explanation for the shorter duration of protection following immunization with this vaccine. Additional studies are needed to determine whether these early T-cell-mediated events predict the subsequent duration of immunologic memory.Vibrio cholerae causes 3 million to 5 million cases of diarrhea and over 100,000 deaths annually (2). Organisms colonize the epithelial surface of the small intestine and elaborate cholera toxin (CT), an essential virulence factor for all pandemic strains of V. cholerae (30). Because V. cholerae is a prototypical noninvasive pathogen, it serves as a model for the study of mucosal immunity (17).Natural infection with V. cholerae provides greater than 90% protection against subsequent disease for at least 3 years in U.S. volunteer studies (the maximum period studied) and an average of 3 to 8 years on the basis of epidemiological studies in areas of endemicity (5,19,21). The mechanism(s) of protective immunity against cholera is not well understood but has been hypothesized to depend on anamnestic responses of memory B cells (16,25,35). Consistent with this, our group previously demonstrated that V. cholerae protein antigen-specific memory B cells remain detectable in the circulation for over a year following cholera, after circulating antibody levels have returned to baseline levels. In contrast, memory B-cell responses to lipopolysaccharide, a T-cell-independent antigen, were found to decrease more rapidly after infection (16). Such long-lasting memory B cells against V. cholerae protein antigens could play a role in mediating anamnestic responses and protection against subsequent infection.In contrast, a currently licensed cholera vaccine, Dukoral (a whole-cell CT B subunit [WC-CTB] vaccine), consists of killed V. cholerae O1 supplemented with recombinant cholera toxin B subunit (rCTB) and provides over 60% protection. However, protection wanes by 3 years after vaccination, and the vacc...

The growing prevalence of deadly microbes with resistance to previously life-saving drug therapies is a dire threat to human health. Detection of low abundance pathogen sequences remains a challenge for metagenomic Next Generation Sequencing (NGS). We introduce FLASH (Finding Low Abundance Sequences by Hybridization), a next-generation CRISPR/Cas9 diagnostic method that takes advantage of the efficiency, specificity and flexibility of Cas9 to enrich for a programmed set of sequences. FLASH-NGS achieves up to 5 orders of magnitude of enrichment and sub-attomolar gene detection with minimal background. We provide an open-source software tool (FLASHit) for guide RNA design.Here we applied it to detection of antimicrobial resistance genes in respiratory fluid and dried blood spots, but FLASH-NGS is applicable to all areas that rely on multiplex PCR.

Objectives It is currently thought that most—but not all—individuals infected with SARS-CoV-2 develop symptoms, but that the infectious period starts on average two days before the first overt symptoms appear. It is estimated that pre- and asymptomatic individuals are responsible for more than half of all transmissions. By detecting infected individuals before they have overt symptoms, wearable devices could potentially and significantly reduce the proportion of transmissions by pre-symptomatic individuals. Using laboratory-confirmed SARS-CoV-2 infections (detected via serology tests [to determine if there are antibodies against the SARS-CoV-2 in the blood] or SARS-CoV-2 infection tests such as polymerase chain reaction [PCR] or antigen tests) as the gold standard, we will determine the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for the following two algorithms to detect first time SARS-CoV-2 infection including early or asymptomatic infection: the algorithm using Ava bracelet data when coupled with self-reported Daily Symptom Diary data (Wearable + Symptom Data Algo; experimental condition) the algorithm using self-reported Daily Symptom Diary data alone (Symptom Only Algo; control condition) In addition, we will determine which of the two algorithms has superior performance characteristics for detecting SARS-CoV-2 infection including early or asymptomatic infection as confirmed by SARS-CoV-2 virus testing. Trial design The trial is a randomized, single-blinded, two-period, two-sequence crossover trial. All subjects will participate in an initial Learning Phase (varying from 2 weeks to 3 months depending on enrolment date), followed by two contiguous 3-month test phases, Period 1 and Period 2. Each subject will undergo the experimental condition (the Wearable + Symptom Data Algo) in one of these periods and the control condition (Symptom Only Algo) in the other period. The order will be randomly assigned, resulting in subjects being allocated 1:1 to either Sequence 1 (experimental condition first) or Sequence 2 (control condition first). Based on demographics, medical history and/or profession, each subject will be stratified at baseline into a high-risk and normal-risk group within each sequence. Participants The trial will be conducted in the Netherlands. A target of 20,000 subjects will be enrolled. Based on demographics, medical history and/or profession, each subject will be stratified at baseline into a high-risk and normal-risk group within each sequence. This results in approximately 6,500 normal-risk individuals and 3,500 high-risk individuals per sequence. Subjects will be recruited from previously studied cohorts as well as via public campaigns and social media. All data for this study will be collected remotely through the Ava COVID-RED app, the Ava bracelet, surveys in the COVID-RED web portal, and self-sampling serology and PCR kits. During recruitment, subjects will be invited to visit the COVID-RED web portal (www.covid-red.eu). After successfully completing the enrolment questionnaire, meeting eligibility criteria and indicating interest in joining the study, subjects will receive the subject information sheet and informed consent form. Subjects can enrol in COVID-RED if they comply with the following inclusion and exclusion criteria. Inclusion criteria: Resident of the Netherlands At least 18 years old Informed consent provided (electronic) Willing to adhere to the study procedures described in the protocol Must have a smartphone that runs at least Android 8.0 or iOS 13.0 operating systems and is active for the duration of the study (in the case of a change of mobile number, study team should be notified) Be able to read, understand and write Dutch Exclusion criteria: Previous positive SARS-CoV-2 test result (confirmed either through PCR/antigen or antibody tests; self-reported) Previously received a vaccine developed specifically for COVID-19 or in possession of an appointment for vaccination in the near future (self-reported) Current suspected (e.g., waiting for test result) COVID-19 infection or symptoms of a COVID-19 infection (self-reported) Participating in any other COVID-19 clinical drug, vaccine, or medical device trial (self-reported) Electronic implanted device (such as a pacemaker; self-reported) Pregnant at time of informed consent (self-reported) Suffering from cholinergic urticaria (per the Ava bracelet’s User Manual; self-reported) Staff involved in the management or conduct of this study Intervention and comparator All subjects will be instructed to complete the Daily Symptom Diary in the Ava COVID-RED app daily, wear their Ava bracelet each night and synchronise it with the app each day for the entire period of study participation. Provided with wearable sensor and/or self-reported symptom data within the last 24 hours, the Ava COVID-RED app’s underlying algorithms will provide subjects with a real-time indicator of their overall health and well-being. Subjects will see one of three messages, notifying them that: no seeming deviations in symptoms and/or physiological parameters have been detected; some changes in symptoms and/or physiological parameters have been detected and they should self-isolate; or alerting them that deviations in their symptoms and/or physiological parameters could be suggestive of a potential COVID-19 infection and to seek additional testing. We will assess intraperson performance of the algorithms in the experimental condition (Wearable + Symptom Data Algo) and control conditions (Symptom Only Algo). Main outcomes The trial will evaluate the use and performance of the Ava COVID-RED app and Ava bracelet, which uses sensors to measure breathing rate, pulse rate, skin temperature, and heart rate variability for the purpose of early and asymptomatic detection and monitoring of SARS-CoV-2 in general and high-risk populations. Using laboratory-confirmed SARS-CoV-2 infections (detected via serology tests, PCR tests and/or antigen tests) as the gold standard, we will determine the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for each of the following two algorithms to detect first-time SARS-CoV-2 infection including early or asymptomatic infection: the algorithm using Ava Bracelet data when coupled with the self-reported Daily Symptom Diary data, and the algorithm using self-reported Daily Symptom Diary data alone. In addition, we will determine which of the two algorithms has superior performance characteristics for detecting SARS-CoV-2 infection including early or asymptomatic infection as confirmed by SARS-CoV-2 virus testing. The protocol contains an additional seventeen secondary outcomes which address infection incidence rates, health resource utilization, symptoms reported by SARS-CoV-2 infected participants, and the rate of breakthrough and asymptomatic SARS-CoV-2 infections among individuals vaccinated against COVID-19. PCR or antigen testing will occur when the subject receives a notification from the algorithm to seek additional testing. Subjects will be advised to get tested via the national testing programme, and report the testing result in the Ava COVID-RED app and a survey. If they cannot obtain a test via the national testing programme, they will receive a nasal swab self-sampling kit at home, and the sample will be tested by PCR in a trial-affiliated laboratory. In addition, all subjects will be asked to take a capillary blood sample at home at baseline (Month 0), and at the end of the Learning Phase (Month 3), Period 1 (Month 6) and Period 2 (Month 9). These samples will be used for SARS-CoV-2-specific antibody testing in a trial-affiliated laboratory, differentiating between antibodies resulting from a natural infection and antibodies resulting from COVID-19 vaccination (as vaccination will gradually be rolled out during the trial period). Baseline samples will only be analysed if the sample collected at the end of the Learning Phase is positive, and samples collected at the end of Period 1 will only be analysed if the sample collected at the end of Period 2 is positive. When subjects obtain a positive PCR/antigen or serology test result during the study, they will continue to be in the study but will be moved into a so-called “COVID-positive” mode in the Ava COVID-RED app. This means that they will no longer receive recommendations from the algorithms but can still contribute and track symptom and bracelet data. The primary analysis of the main objective will be executed using data collected in Period 2 (Month 6 through 9). Within this period, serology tests (before and after Period 2) and PCR/antigen tests (taken based on recommendations by the algorithms) will be used to determine if a subject was infected with SARS-CoV-2 or not. Within this same time period, it will be determined if the algorithms gave any recommendations for testing. The agreement between these quantities will be used to evaluate the performance of the algorithms and how these compare between the study conditions. Randomisation All eligible subjects will be randomized using a stratified block randomization approach with an allocation ratio of 1:1 to one of two sequences (experimental condition followed by control condition or control condition followed by experimental condition). Based on demographics, medical history and/or profession, each subject will be stratified at baseline into a high-risk and normal-risk group within each sequence, resulting in equal numbers of high-risk and normal-risk individuals between the sequences. Blinding (masking) In this study, subjects will be blinded as to study condition and randomization sequence. Relevant study staff and the device manufacturer will be aware of the assigned sequence. The subject will wear the Ava bracelet and complete the Daily Symptom Diary in the Ava COVID-RED app for the full duration of the study, and they will not know if the feedback they receive about their potential infection status will only be based on data they entered in the Daily Symptom Diary within the Ava COVID-RED app or based on both the data from the Daily Symptom Diary and the Ava bracelet. Numbers to be randomised (sample size) 20,000 subjects will be recruited and randomized 1:1 to either Sequence 1 (experimental condition followed by control condition) or Sequence 2 (control condition followed by experimental condition), taking into account their risk level. This results in approximately 6,500 normal-risk and 3,500 high-risk individuals per sequence. Trial Status Protocol version: 1.2, dated January 22nd, 2021 Start of recruitment: February 22nd, 2021 End of recruitment (estimated): April 2021 End of follow-up (estimated): December 2021 Trial registration The trial has been registered at the Netherlands Trial Register on the 18th of February, 2021 with number NL9320 (https://www.trialregister.nl/trial/9320) Full protocol The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol.

Human cytomegalovirus (CMV) causes significant disease in immunocompromised patients and serious birth defects if acquired in utero. Available CMV antivirals target the viral DNA polymerase, have significant toxicities, and suffer from resistance. New drugs targeting different pathways would be beneficial. The anthraquinone emodin is proposed to inhibit herpes simplex virus by blocking the viral nuclease. Emodin and related anthraquinones are also reported to inhibit CMV. In the present study, emodin reduced CMV infectious yield with an EC50 of 4.9 μM but was cytotoxic at concentrations only two-fold higher. Related anthraquinones acid blue 40 and alizarin violet R inhibited CMV at only high concentrations (238–265 μM) that were also cytotoxic. However, atanyl blue PRL inhibited infectious yield of CMV with an EC50 of 6.3 μM, significantly below its 50% cytotoxic concentration of 216 μM. Atanyl blue PRL reduced CMV infectivity and inhibited spread. When added up to one h after infection, it dramatically reduced CMV immediate early protein expression and blocked viral DNA synthesis. However, it had no antiviral activity when added 24 h after infection. Interestingly, atanyl blue PRL inhibited nuclease activities of purified CMV UL98 protein with IC50 of 4.5 and 9.3 μM. These results indicate that atanyl blue PRL targets very early post-entry events in CMV replication and suggest it may act through inhibition of UL98, making it a novel CMV inhibitor. This compound may provide valuable insights into molecular events that occur at the earliest times post-infection and serve as a lead structure for antiviral development.

No abstract

Human cytomegalovirus encodes an alkaline nuclease, UL98, that is highly conserved among herpesviruses and has both endonuclease (endo) and exonuclease (exo) activities. This protein is thought to be important for viral replication and therefore represents a potential target for antiviral development; however, little is known about its structure or role in viral replication. Comparative structural modelling was used to build a model of UL98 based on the known structure of shutoff and exonuclease protein from Kaposi's sarcoma-associated herpesvirus. The model predicts that UL98 residues D254, E278 and K280 represent the critical aspartic acid, glutamic acid and lysine active-site residues, respectively, while R164 and S252 correspond to residues proposed to bind the 59 phosphate of the DNA substrate. UL98 with an amino-terminal hexahistidine tag was expressed in Escherichia coli, purified by affinity chromatography and confirmed to have exo and endo activities. Amino acid substitutions D254A, E278A, K280A and S252A virtually eliminated exo and endo activities, whereas R164A retained full endo activity but only 10 % of the exo activity compared with the wild-type enzyme. A mutant virus lacking UL98 was viable but severely attenuated for replication, while one expressing UL98(R164A) replicated normally. These results confirm the utility of the model in representing the active-site region of UL98 and suggest a mechanism for the differentiation of endonuclease and exonuclease activities. These findings could facilitate the exploration of the roles of alkaline nucleases in herpesvirus replication and the rational design of inhibitors that target their enzymic activities.

Background & Aims: We investigated the dynamics of serum HBV pre-genomic RNA (pgRNA) and hepatitis B core-related antigen (HBcrAg) in patients receiving nucleos(t)ide analogues (NAs) and their predictability for favourable suppression of serum hepatitis B surface antigen (HBsAg).2 Methods: Serum viral biomarkers were measured at baseline, weeks 4, 12, 24, 36 and 48 of treatment. Patients were followed up thereafter and the serum HBsAg level was measured at end of follow-up (EOFU). Favourable HBsAg response (FHR) was defined as ≤100 IU/mL or HBsAg seroclearance upon EOFU.Results: Twenty-eight HBeAg-positive and 36 HBeAg-negative patients (median 38.2 years old, 71.9% male) were recruited with median follow-up duration of 17.1 (IQR 12.8-18.2) years. For the entire cohort, 22/64 (34.4%, of which 8 achieved HBsAg seroclearance) achieved FHR. For HBeAg-positive patients, serum HBV pgRNA decline at week 4 was significantly greater for patients with FHR compared to non-FHR patients (5.49 vs 4.32 log copies/mL, respectively, p=0.016). The AUROC for week 4 HBV pgRNA reduction to predict FHR in HBeAg-positive patients was 0.825 (95% CI 0.661-0.989). For HBeAg-negative patients, instead of increase in serum HBcrAg from baseline in non-FHR patients, FHR patients had median reduction in HBcrAg at week 4 (increment of 1.75 vs reduction of 2.98 log U/mL; p=0.023). The AUROC for week 4 change of HBcrAg to predict FHR in HBeAg-negative patients was 0.789 (95% CI 0.596-0.982). Conclusions:Early on-treatment changes of serum HBV pgRNA and HBcrAg at 4 weeks predict HBsAg seroclearance or ≤100 IU/mL in NA-treated CHB patients upon long-term FU.