Choice of immunoassay influences population seroprevalence estimates. Post hoc adjustments for assay performance could improve comparability of estimates across studies and enable pooled analyses. We assessed post hoc adjustment methods using data from 2021 to 2023 SARS-CoV-2 serosurveillance studies in Alberta, Canada: one that tested 124 008 blood donations using Roche immunoassays (SARS-CoV-2 nucleocapsid total antibody and anti-SARS-CoV-2 S) and another that tested 214 780 patient samples using Abbott immunoassays (SARS-CoV-2 IgG and anti-SARS-CoV-2 S). Comparing datasets, seropositivity for antibodies against nucleocapsid (anti-N) diverged after May 2022 due to differential loss of sensitivity as a function of time since infection. The commonly used Rogan-Gladen adjustment did not reduce this divergence. Regression-based adjustments using the assays' semiquantitative results produced more similar estimates of anti-N seroprevalence and rolling incidence proportion (proportion of individuals infected in recent months). Seropositivity for antibodies targeting SARS-CoV-2 spike protein was similar without adjustment, and concordance was not improved when applying an alternative, functional threshold. These findings suggest that assay performance substantially impacted population inferences from SARS-CoV-2 serosurveillance studies in the Omicron period. Unlike methods that ignore time-varying assay sensitivity, regression-based methods using the semiquantitative assay resulted in increased concordance in estimated anti-N seropositivity and rolling incidence between cohorts using different assays.

Abstract Background Hepatitis A virus (HAV) incident infection in Canada is rarely diagnosed (i.e. incidence of 3.6-10 cases/100,000 persons) (PMID: 18159360). Infections generally relate to imported contaminated food-products, or travelers returning from endemic countries. Due to its fecal-oral spread, possible underdiagnosis, and often-cryptic presentation, HAV is an ideal candidate for wastewater (WW)-based surveillance, a tool increasingly utilized to monitor infectious diseases globally.Figure 1.Longitudinal monitoring of HAV RNA wastewater abundance across eight Alberta municipalities over a 4-month period. Methods 24-hour composite WW was collected weekly from eight geographically disparate, and socioeconomically diverse municipal WW treatment plants in Alberta from August to December 2023. After short-term cold storage, WW was centrifuged, and RNA from the raw pellet was extracted using Qiagen’s RNeasy PowerFecal pro. HAV levels were quantified by RT-qPCR of the vp1 gene. 2021 Canadian census data was used to define population demographics for each participating site. Results HAV was detected in 18/117 (15.4%) WW samples and 5/8 (62.5%) municipalities over the 4-month period (Figure 1). RNA abundance in HAV positive WW samples was a median of 3.4 copies/mL (IQR 0.44 - 6.97). Larger population size (p=0.007), and greater density (p=0.001) were associated with increased likelihood of HAV WW detection, whereas social and economic demographics of populations within sewershed catchments did not associate with likelihood of HAV detection (Table 1). Conclusion HAV RNA is rarely detected in the wastewater of Alberta. Detection was more frequently observed in larger municipalities, which is consistent with non-endemic, imported disease. WW surveillance can potentially be adapted to monitor HAV in the context of outbreaks to reduce secondary transmission, and safeguard public health. Disclosures Mark Swain, MD MSc, Abbott: Advisor/Consultant|Advanz: Advisor/Consultant|Gilead, BMS, CymaBay, Intercept, Genfit, Pfizer, Novartis, Astra Zeneca, GSK, Celgene, Novo Nordisk, Axcella Health Inc., Merck, Galectin Therapeutics: Grant/Research Support|GSK: Advisor/Consultant|Ipsen: Advisor/Consultant|Novo Nordisk: Advisor/Consultant Carla Coffin, MD MSc, Altimmune: Grant/Research Support|Gilead: Grant/Research Support|GSK: Grant/Research Support|Janssen: Grant/Research Support Steven J. Drews, PhD FCCM D(ABMM), Abbott: Grant/Research Support|Danaher: Honoraria|Roche: Advisor/Consultant|Roche: Grant/Research Support

BACKGROUND:We sought to examine the prevalence of West Nile Virus (WNV) nucleic acid test (NAT) positivity over time in Canadian blood donors, examine the level of false-positive NAT screening, and understand the effectiveness of travel-related testing outside of WNV seasonality in Canada. STUDY DESIGN AND METHODS:All blood donations in Canada (except Québec) between 2018 and 2023 were tested by the Roche Cobas WNV-NAT assay during the summer-fall season by Canadian Blood Services. Testing for recent travel outside Canada was performed in the winter-spring season. All 2023 WNV-NAT-positives were confirmed by two independent WNV-specific polymerase chain reaction (PCRs). RESULTS:While WNV positivity varied by location and time, no increasing trend of positivity was observed over the 6-year study period. No WNV-NAT-positive donors with a history of recent travel outside of Canada in the winter-spring season were identified by donor screening (2018-2023). In total, 2,454,102 samples were tested during summer months and 267,988 in winter months. In 2023, WNV-specific PCR confirmed 10 of 13 (76.9%) screening WNV-NATs. Recent Japanese Encephalitis Virus (JEV) vaccination was identified as a potential false positive for initial WNV screening results. DISCUSSION:Between 2018 and 2023, WNV-NAT positivity amongst Canadian blood donors remained stable, with no evidence of an increasing positivity trend. The utility of off-season travel-related testing may be limited, as no donors were identified during the six-year study timeframe. Finally, confirmation testing using WNV-specific PCR is recommended to identify true cases and to limit misidentification of other genetically similar viruses, such as JEV.

BACKGROUND AND OBJECTIVES:Seasonal vaccinations reduce donor illness and appointment cancellations and ensure plasma products have antibodies to vaccine-directed strains. We aimed to describe donor influenza and COVID-19 vaccination history and compare this with the general population. MATERIALS AND METHODS:Two online donor surveys were carried out in 2021 and 2024. Donors were asked about demographics, influenza (2019/2020, 2020/2021 and 2023/2024 seasons) and COVID-19 (ever and 2023/2024 season) vaccination and reasons for vaccination choices. General population vaccination statistics were extracted from public reports. Percentages of donors receiving vaccination were calculated with 95% confidence intervals. Multiple logistic regression models were fitted with demographics as independent variables. RESULTS:In survey 1, 4582 (30.4% response rate) donors completed a questionnaire; in survey 2, 6376 (21% response rate). More donors under age 65 received the influenza vaccine compared with the general population under age 65 (58% vs. 30% in 2019/2020, 63% vs. 28% in 2023/2024, p < 0.0001) and aged 65+ (81% vs. 70% in 2019/2020, 90% vs. 73% in 2023/2024, p < 0.0001). Fewer donors and the general population received the COVID-19 vaccine in 2023/2024 (under 65 45% vs. 39%; 65+ 76% vs. 67%, p < 0.0001). Most said they were vaccinated to prevent infection and protect others. CONCLUSION:Seasonal vaccination rates are higher in older donors, consistent with public health recommendations. Blood donors are more likely to be vaccinated against seasonal influenza than the general population, but post-pandemic uptake of the COVID-19 booster vaccine was low, more similar to the general population.

BACKGROUND:Blood donors represent a unique population. Pre-donation screening questions, donor self-deferral, and temporary deferral and re-testing of repeat reactive donors result in lower prevalence of infectious disease compared to the general population. Prevalence directly affects the clinical performance of assay systems apart from the analytical sensitivity and specificity of the assay. STUDY DESIGN AND METHODS:It is important that blood operators understand what situations frequently elicit false reactive and false nonreactive cases, and how to mitigate the effect of these. RESULTS:False reactive transmissible disease assays are seen more frequently in low-prevalence populations due to a reduced positive predictive value, whereas false nonreactives may be derived from failures in quality control (control failures, temperature and humidity deviations), pathophysiology in the donor, or how specimens are processed and tested (e.g., pooling). Reactive screening results require confirmation testing to differentiate between true infection and false reactive interpretation, and result in either temporary or permanent donor deferral (for false reactive or confirmed screens respectively). Complete validation or verification of laboratory assays can mitigate these donor impacts and help laboratorians understand how assays function in their local environments. DISCUSSION:Here we describe how blood donor screening assays are characterized prior to use, explore possible contributors to false reactive and nonreactive results, and the importance of tracking and monitoring infectious disease positivity rates. Two case examples: one for serology and one for molecular testing demonstrate how common errors in laboratory testing can be mitigated.

INTRODUCTION:Donors are deferred if they are on antiretroviral medications (ARV) as post-exposure or pre-exposure prophylaxis (PEP or PrEP) for human immunodeficiency virus (HIV). We assessed donor compliance by measuring ARV levels in selected anonymized donor samples collected from September 22, 2022 to December 31, 2024, almost all after the introduction of sexual risk behavior screening. METHODS:EDTA plasma samples collected at the time of donation (retention samples) were retrieved, frozen, and shipped for measurement of tenofovir and emtricitabine. Samples were from randomly selected first-time male donors in large urban areas (n = 520), syphilis (n = 133), or HIV (n = 6) confirmed positive donors, and donors deferred for PEP or PrEP use on a previous donation attempt who returned to successfully donate (n = 225 tests, 115 unique donors). We compare our results to international studies. RESULTS:All samples from first-time male donors, HIV-positive donors, syphilis-positive female donors, and female donors previously deferred for PEP or PrEP were negative. Three of 110 male syphilis-positive donors (2.7%) and 14 of 85 male donors previously deferred for PEP or PrEP (16.5%) were positive for ARV. Eleven of these donors were tested multiple times, and 10 were positive more than once. CONCLUSION:Results on syphilis-positive male donors were similar to findings in England and the Netherlands. Noncompliance with criteria for ARV use was high in male donors previously deferred for PEP/PrEP. Messaging regarding recipient risk is particularly difficult in this group, since it is at odds with the reduction in individual HIV risk.

Background and ObjectivesDespite screening procedures, a few blood donors confirm positive for transfusion-transmissible infections and are deferred. Effective notification of laboratory results is essential to ensure that donors are advised of confirmed results and to seek medical care. Here we report results from post-notification interviews of Canadian Blood Services donors.Materials and MethodsOver 17 years, 2006-2022, all donors with confirmed positive results for hepatitis B virus (HBV), hepatitis C virus (HCV), human T-cell lymphotropic virus (HTLV) and syphilis were notified by registered mail of their result and advised to see a physician. In a separate communication, all donors were later invited to participate in a scripted interview asking whether they tested positive for an infection; if yes, which one, what their reaction was, whether they consulted a physician and whether public health contacted them. Frequencies of responses were calculated.ResultsOf 2654 donors with confirmed positive test results, 876 (33%) participated; 90% said they were informed of a positive test result. Of these, about a quarter did not know for which infection they were positive. Most were surprised, and some were sad or disappointed. Most saw a physician after notification (77%). About two-thirds with HBV or HCV said they were contacted by public health, slightly fewer (58%) with syphilis, 27% of those with HTLV.ConclusionMost donors recalled being notified and were aware of their positive test, but details of the infection were sometimes not understood or recalled, and not all donors consulted a physician about the infection.

West Nile virus (WNV) is a mosquito-borne zoonotic flavivirus which often causes asymptomatic infection in humans but may develop into a deadly neuroinvasive disease. In this study, we aimed to investigate variables potentially associated with human WNV infection using human and mosquito WNV surveillance and monitoring datasets, established over 20 years, from 2003 to 2022, across the province of Ontario, Canada. We combined climatic and geographic data, mosquito surveillance data (n = 3010 sites), blood donation arboviral detection testing data in the human population, and demographic and socio-economic data from Canadian population censuses. We hypothesized that spatio-temporal indices related to mosquito vector habitat and phenology, in addition to human demographic and socio-economic factors, were associated with WNV infection in the human population. Our results show that high habitat suitability of the main WNV vector in southerly locations of this region, Cx. pipiens/restuans (IRR = 2.0), and variables related to lower income (IRR = 2.8), and shelter infrastructure spending (IRR = 0.7), were key risk factors associated with WNV infection among blood donors from 2003 to 2022 across Ontario (R2 = 0.67). These results may inform points of entry for practical intervention aimed at reducing risk of mosquito-borne pathogens in Canada.

ObjectivesIn high-income countries hepatitis E virus (HEV) is an uncommonly diagnosed porcine-derived zoonoses. After identifying disproportionate chronic HEV infections in persons with cystic fibrosis (pwCF) postlung transplant, we sought to understand its epidemiology and potential drivers.DesignAll pwCF post-transplant attending our regional CF centre were screened for HEV. HEV prevalence was compared against non-transplanted pwCF and with all persons screened for suspected HEV infection from 2016 to 2022 in Alberta, Canada. Those with chronic HEV infection underwent genomic sequencing and phylogenetic analysis. Owing to their swine derivation, independently sourced pancreatic enzyme replacement therapy (PERT) capsules were screened for HEV.ResultsHEV seropositivity was similar between transplanted and non-transplanted pwCF (6/29 (21%) vs 16/83 (19%); p=0.89). Relative to all other Albertans investigated for HEV as a cause of hepatitis (n=115/1079, 10.7%), pwCF had a twofold higher seropositivity relative risk and this was four times higher than the Canadian average. Only three chronic HEV infection cases were identified in all of Alberta, all in CF lung transplant recipients (n=3/29, 10.3%). Phylogenetics confirmed cases were unrelated porcine-derived HEV genotype 3a. Ninety-one per cent of pwCF were taking PERT (median 8760 capsules/person/year). HEV RNA was detected by RT-qPCR in 44% (47/107) of PERT capsules, and sequences clustered with chronic HEV cases.ConclusionPwCF had disproportionate rates of HEV seropositivity, regardless of transplant status. Chronic HEV infection was evident only in CF transplant recipients. HEV may represent a significant risk for pwCF, particularly post-transplant. Studies to assess HEV incidence and prevalence in pwCF, and potential role of PERT are required.

BACKGROUND AND OBJECTIVES:Until recently, gay, bisexual and other men who have sex with men (MSM) were deferred from donating blood for 3-12 months since the last male-to-male sexual contact. This MSM deferral has been discontinued by several high-income countries (HIC) that now perform gender-neutral donor selection.MATERIALS AND METHODS:An international symposium (held on 20-04-2023) gathered experts from seven HICs to (1) discuss how this paradigm shift might affect the mitigation strategies for transfusion-transmitted infections and (2) address the challenges related to gender-neutral donor selection.RESULTS:Most countries employed a similar approach for implementing a gender-neutral donor selection policy: key stakeholders were consulted; the transition was bridged by time-limited deferrals; donor compliance was monitored; and questions or remarks on anal sex and the number and/or type of sexual partners were often added. Many countries have now adopted a gender-neutral approach in which questions on pre- and post-exposure prophylaxis for human immunodeficiency virus (HIV) have been added (or retained, when already in place). Other countries used mitigation strategies, such as plasma quarantine or pathogen reduction technologies for plasma and/or platelets.CONCLUSION:The experience with gender-neutral donor selection has been largely positive among the countries covered herein and seems to be acceptable to stakeholders, donors and staff. The post-implementation surveillance data collected so far appear reassuring with regards to safety, although longer observation periods are necessary. The putative risks associated with HIV antiretrovirals should be further investigated.

Hepatitis B is transmitted sexually, by blood contact, and vertically from mother to child. Chronic hepatitis B is often seen in immigrants from higher-prevalence countries and their Canadian-born children. We assessed the relationship between hepatitis B and social determinants of health. Included were 1,539,869 first-time Canadian blood donors from April 2005 to December 2022. All donations were tested for hepatitis B markers. Logistic regression was fit with chronic hepatitis B as the dependent variable and age, sex, year, and ethnocultural composition and material deprivation quintiles as independent variables. Chronic hepatitis B prevalence was 47.5/100,000 (95% CI 41.5–53.5, years 2017–2022). Chronic hepatitis B prevalence was elevated in males, older age groups, and those living in more materially deprived and higher ethnocultural neighbourhoods. Of 212,518 donors from 2020 to 2022 with race/ethnicity data, chronic hepatitis B prevalence was highest in East Asians. The findings are consistent with infections in immigrants, acquired in their country of origin, in their Canadian-born children and in those with other risks. As blood donors are a low-risk population unaware of their infection and unlikely to seek testing, our results highlight the ongoing public health challenges of diagnosing chronic hepatitis B and treating it when appropriate.

BACKGROUND:Hepatitis B core antibody (anti-HBc) screening has been implemented in many blood establishments to help prevent transmission of hepatitis B virus (HBV), including from donors with occult HBV infection (OBI). We review HBV screening algorithms across blood establishments globally and their potential effectiveness in reducing transmission risk. MATERIALS AND METHODS:A questionnaire on HBV screening and follow-up strategies was distributed to members of the International Society of Blood Transfusion working party on transfusion-transmitted infectious diseases. Screening data from 2022 were assimilated and analyzed. RESULTS:A total of 30 unique responses were received from 25 countries. Sixteen respondents screened all donations for anti-HBc, with 14 also screening all donations for HBV DNA. Anti-HBc prevalence was 0.42% in all blood donors and 1.19% in new donors in low-endemic countries; however, only 44% of respondents performed additional anti-HBc testing to exclude false reactivity. 0.68% of anti-HBc positive, HBsAg-negative donors had detectable HBV DNA. Ten respondents did universal HBV DNA screening without anti-HBc, whereas four respondents did not screen for either. Deferral strategies for anti-HBc positive donors were highly variable. One transfusion-transmission from an anti-HBc negative donor was reported. DISCUSSION:Anti-HBc screening identifies donors with OBI but also results in the unnecessary deferral of a significant number of donors with resolved HBV infection and donors with false-reactive anti-HBc results. Whilst confirmation of anti-HBc results could be improved to reduce donor deferral, transmission risks associated with anti-HBc negative OBI donors must be considered. In high-endemic areas, highly sensitive HBV DNA testing is required to identify infectious donors.

Background: SARS-CoV-2 seroprevalence monitors cumulative infection rates irrespective of case testing protocols. We aimed to describe Nova Scotia blood donor seroprevalence in relation to public health policy and reported data over the course of the COVID-19 pandemic (May 2020 to August 2022). Methods: Monthly random Nova Scotia blood donation samples (24,258 in total) were tested for SARS-CoV-2 infection antibodies (anti-nucleocapsid) from May 2020 to August 2022, and vaccination antibodies (anti-spike) from January 2021 to August 2022. Multivariable logistic regression for infection antibodies and vaccination antibodies separately with month, age, sex, and racialization identified independent predictors. The provincial nucleic acid amplification test (NAAT)-positive case rate over the pandemic was calculated from publicly available data. Results: Anti-N seroprevalence was 3.8% in January 2022, increasing to 50.8% in August 2022. The general population COVID-19 case rate was 3.5% in January 2022, increasing to 12.5% in August 2022. The percentage of NAAT-positive samples in public health laboratories increased from 1% in November 2021 to a peak of 30.7% in April 2022 with decreasing numbers of tests performed. Higher proportions of younger donors as well as Black, Indigenous, and racialized blood donors were more likely to have infection antibodies ( p < 0.01). Vaccination antibodies increased to 100% over 2021, initially in older donors (60+ years), and followed by progressively younger age groups. Conclusions: SARS-CoV-2 infection rates were relatively low in Nova Scotia until the more contagious Omicron variant dominated, after which about half of Nova Scotia donors had been infected despite most adults being vaccinated (although severity was much lower in vaccinated individuals). Most COVID-19 cases were detected by NAAT until Omicron arrived. When NAAT testing priorities focused on high-risk individuals, infection rates were better reflected by seroprevalence.

Estimate HTLV-1/2 (human T-cell lymphotropic viruses) prevalence in Canadian blood donors and the association of demographic variables with infection and their corresponding risk factors. First-time blood donors in all Canadian provinces (except Quebec) from 1990 to 2022 were included. Blood samples were tested for HTLV-1/2 by enzyme-linked immunoassay, confirmed by Western blot. Multivariable logistic regression with year, age group, sex, region, neighbourhood material deprivation, and ethnocultural composition indices predicted HTLV-1/2. Since 2005, all HTLV-1/2-positive donors (cases) were invited to participate in a risk factor interview, and 4 non-positive donors (controls per case) were matched for age, sex, and region. Case–control predictors of HTLV-1/2 were analyzed using logistic regression. There were 3,085,554 first-time donors from 1990 to 2022. HTLV-1/2 prevalence remained low (12 per 100,000 in 2022, 95

BACKGROUND AND OBJECTIVES:There is a growing infectious syphilis outbreak in Western Canada. Although blood donors are screened for syphilis risks, some blood donors will still be confirmed test-positive for syphilis. This study compares the characteristics of confirmed test-positive syphilis donations in both Western Canada and Eastern Canada, November 2022-August 2023. MATERIALS AND METHODS:Donors were defined as Western or Eastern Canadian. Blood donations were tested for syphilis using the PK-TP assay (Beckman Coulter PK7300 Automated Microplate System). Confirmatory Treponema pallidum particle agglutination (TPPA) and rapid plasma reagin (RPR) assays were performed by one of two reference laboratories. An RPR titre ≥1:8 was used as a proxy for possible infectious syphilis. RESULTS:Rates of laboratory-confirmed syphilis were higher in Western (n = 43, 13.4/100,000 donations) versus Eastern donors (n = 19, 4.7/100,000 donations; Fisher's exact test, two-sided, p ≤ 0.0001). Most syphilis confirmations were in first-time donors (Western Canada n = 31/43, 72.1%, Eastern Canada 12/19, 63.2%). CONCLUSION:Although rates of laboratory-confirmed syphilis were higher in Western versus Eastern donors, Western donors did not have higher rates of infectious syphilis. Further studies might assess whether donors with laboratory-confirmed syphilis understood pre-donation screening questions or were completely unaware of a past infection.

This edition of the Journal of Thrombosis and Haemostasis includes a paper by Singh et al. [1Singh A. Ghosh R. Asuru T.R. Prajapat S.K. Joshi G. Gaur K.K. et al.Inhibition of cellular activation induced by platelet factor 4 via the CXCR3 pathway ameliorates Japanese encephalitis and dengue viral infections.J Thromb Haemost. 2024; 22: 818-833Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar] entitled "Inhibition of platelet factor 4: CXCR3 pathway prevents JEV and DENV infection." In this article, the authors provide extensive evidence that both dengue virus (DENV) and Japanese encephalitis virus (JEV) infections of mice and cell cultures could be attenuated by a deficiency of the chemokine platelet factor 4 (PF4; chemokine [C-X-C motif] ligand 4) or inhibition of the PF4 via the C-X-C motif chemokine receptor 3 (CXCR3) pathway. The authors also undertook extensive experiments to begin to map out how PF4 impacts antiviral cytokine production (eg, interferon [IFN]-α and IFN-β) and autophagy flux in DENV and JEV infection models. Finally, the authors collected blood from DENV-infected patients during acute and convalescent phases and noted that patients had higher levels of PF4 and nonstructural protein (NS)+ monocytes in the acute phase compared to the convalescent phase. In this editorial, I will outline why this work mapping out the role that PF4 and CXCR3 play in DENV and JEV infections and disease is so important. Both DENV (Orthoflavivirus denguei) and JEV (Orthoflavivirus japonicum) are separate species within the genus Orthoflavivirus and family Flaviviridae. Humans can be infected with 4 serotypes (DENV-1,2,3,4) by mosquito vectors, which are primarily Aedes aegypti and Aedes albopictus. Serotypes may vary between regions and within regions over time, and in some regions, all 4 serotypes may circulate, with 1 being dominant [2Norshidah H. Vignesh R. Lai N.S. Updates on Dengue vaccine and antiviral: where are we heading?.Molecules. 2021; 26: 6768Crossref PubMed Scopus (23) Google Scholar]. In 2023, the World Health Organization (WHO) estimated that half of the world's population is at risk of DENV infection, and it is estimated that 100 to 400 million infections occur each year in tropical/subtropical regions and urban/semiurban settings. Most cases are asymptomatic or misdiagnosed. Between 2000 and 2019, the number of dengue cases reported to the WHO increased from 505 430 cases to 5.2 million in 2019 [3World Health OrganizationDengue and severe dengue.https://www.who.int/news-room/fact-sheets/detail/dengue-and-severe-dengueGoogle Scholar]. The epidemiology of DENV is expected to change in the face of climate change, and there are already signals for autochthonous DENV transmission in Western Europe [4Zatta M. Brichler S. Vindrios W. Melica G. Gallien S. Autochthonous Dengue outbreak, Paris region, France, September-October 2023.Emerg Infect Dis. 2023; 29: 2538-2540Crossref PubMed Scopus (3) Google Scholar]. Three main presentations may occur with infection: Dengue fever, dengue hemorrhagic fever, and dengue shock syndrome, and these are described elsewhere [5Heymann D.L. Control of communicable diseases manual.21st ed. APHA Press, Washington, DC2022Google Scholar]. Currently, there are no specific antiviral agents for DENV infection. In the United States, Dengvaxia dengue vaccine (tetravalent live-attenuated dengue vaccine; Sanofi Pasteur Inc.) is approved in specific populations and has a good vaccine effectiveness profile. JEV transmission to humans is via the bite of infected Culex mosquitoes (mainly Culex tritaeniorhynchus), and a transmission cycle is maintained between birds, humans, mosquitoes, and pigs, usually in rural and periurban environments. Most individuals at risk for JEV infection live in 2 WHO regions (Southeast Asia and Western Pacific), where the population is estimated to be over 3 billion people. In 2011, the WHO estimated that there were 67 900 clinical JEV cases every year [6Campbell G.L. Hills S.L. Fischer M. Jacobson J.A. Hoke C.H. Hombach J.M. Marfin A.A. Solomon T. Tsai T.F. Tsu V.D. Ginsburg A.S. Estimated global incidence of Japanese encephalitis: a systematic review.Bull World Health Organ. 2011; 89: 766-774ECrossref PubMed Scopus (730) Google Scholar]. Most JEV infections are usually asymptomatic or mild (eg, fever and headache) or without apparent symptoms. However, 1 in 250 infections involve severe disease symptoms, and in those patients, the case fatality rate can be as high as 30%. There is no specific antiviral treatment for JEV; however, due to the role of inflammation in poor clinical outcomes, antiviral agents that also dampen inflammation may be candidates for study. Although current vaccines are highly effective and immunogenic, there are several challenges to JEV vaccine programs, including maintaining JEV vaccine coverage, maintaining a stable supply, and poor surveillance/vaccination data [7Vannice K.S. Hills S.L. Schwartz L.M. Barrett A.D. Heffelfinger J. Hombach J. Letson G.W. Solomon T. Marfin A.A. Japanese encephalitis vaccination experts panel. The future of Japanese encephalitis vaccination: expert recommendations for achieving and maintaining optimal JE control.NPJ Vaccines. 2021; 6: 82Crossref PubMed Scopus (32) Google Scholar]. In the last sentence of the conclusion of their article, Singh et al. state "Our studies suggest that the PF4-CXCR3 axis is a potential target to develop a treatment regimen against the flaviviruses, JEV and DENV." Modulating the PF4-CXCR3 axis to manage DENV and JEV infection would be a very different approach from the directly acting antiviral agents used to treat a distant relative, hepatitis C virus (species Hepacivirus C, genus Hepacivirus, family Flaviviridae) [8Martinello M. Naggie S. Rockstroh J.K. Matthews G.V. Direct-acting antiviral therapy for treatment of acute and recent hepatitis C virus infection: a narrative review.Clin Infect Dis. 2023; 77: S238-S244Crossref PubMed Scopus (8) Google Scholar]. Controlling CXCR3-mediated disease processes has been a proposed approach for other disease processes. Although CXCR3 is potentially druggable, there have been no successful CXCR3 antagonist clinical trials to date. One agent AMG487 (N-[(1R)-1-[3-(4-ethoxyphenyl)-3,4-dihydro-4-oxopyrido[2,3-d]pyrimidin-2-yl]ethyl]-N-(3-pyridinylmethyl)-4-(trifluoromethoxy)-benzeneacetamide; CAS number 473719-41-4) progressed to phase II clinical trials focused on psoriasis but was withdrawn due to a lack of efficacy [9National Center for Advancing Translational SciencesInxight drugs: AMG-487.https://drugs.ncats.io/substances?q=%22AMG-487%20METABOLITE%20M3%22&facet=Substance%20Form%2FPrincipal%20FormGoogle Scholar]. A review by Pease [10Pease J.E. Designing small molecule CXCR3 antagonists.Expert Opin Drug Discov. 2017; 12: 159-168Crossref PubMed Scopus (16) Google Scholar] highlights potential problems in moving CXCR3 antagonist through clinical trials and emphasizes that researchers undertaking discovery and development work need to prove "beyond reasonable doubt that the target is critical for disease pathogenesis and that effective blockade will be therapeutic." Thus, the complex interactions mapped out by Singh et al. are key in determining if the interaction between PF4 and CXCR3 in the context of DENV and JEV is a hopeful target for future therapeutics discovery and development. DENV infections can activate platelets and lead to the release of α-granules containing PF4. The role of JEV in platelet activation remains less clear, and treatment of platelets with increasing doses of JEV did not significantly activate platelets in one set of experiments [11Ojha A. Nandi D. Batra H. Singhal R. Annarapu G.K. Bhattacharyya S. Seth T. Dar L. Medigeshi G.R. Vrati S. Vikram N.K. Guchhait P. Platelet activation determines the severity of thrombocytopenia in dengue infection.Sci Rep. 2017; 741697Crossref Scopus (118) Google Scholar] (Table). In a prior series of experiments, Ojha et al. [12Ojha A. Bhasym A. Mukherjee S. Annarapu G.K. Bhakuni T. Akbar I. Seth T. Vikram N.K. Vrati S. Basu A. Bhattacharyya S. Guchhait P. Platelet factor 4 promotes rapid replication and propagation of Dengue and Japanese encephalitis viruses.EBioMedicine. 2019; 39: 332-347Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar] showed that PF4 could enhance the in vivo and in vitro propagation of both DENV and JEV. This most recent work further elucidates the relationship between PF4-CXCR3 and the role of this interaction in DENV and JEV infection. It also prompts us to think about future studies that could be undertaken (Table).TableKey elements raised in this study and how they might support further work targeting PF4-CXCR3 and downstream processes as druggable targets in DENV and JEV infections and disease.Key element identified by Singh et al.Why this is importantWhat else can be doneA receptor-ligand interaction is key: an interaction between PF4 and CXCR3 enhances DENV and JEV propagation in infection models.Provides further context to prior research on the role of PF4-CXCR3 in flavivirus infections; describes virus-specific protocols and biomarkers for future PF4-CXCR3 studies in cell lines and mouse models infected with flaviviruses (eg, cell/protein staining, nucleic acid testing, and mouse viability); describes potential controls (eg, AMG487) for other small-molecule studies.Understand the role of PF4 in JEV infection; generate a more detailed understanding of CXCR3 and PF4 binding kinetics in the context of flavivirus infection; study other pathways that might synergize with PF-CXCR3 in flavivirus infection; screen for small molecules or other antibodies that could block PF4-CXCR3 interactions.Lysosome degradation: The interaction between PF4 and CXCR3 blocks lysosome degradation and promotes DENV and JEV replication.Fulfills guideline recommendations for autophagy studies; provides protocols for lysosome degradation of flaviviruses; identifies controls (eg, AM487) for other small-molecule inhibitors of lysosome degradation of flaviviruses; identifies PF4-mediated targets in mTOR-p38-ULK1 axis that might block phagosome-lysosome fusion and prevent DENV degradation.Compare/contrast role of the mTOR-p38-ULK1 axis important in JEV infection; clarify crosstalk with proinflammatory/anti-inflammatory signal transduction pathways; use small-molecule inhibitors to block ULK1, p38, and mTOR serine/threonine kinase activity.IFN-α/β expression blockage: PF4 and CXCR3 interactions lead to reduced IFN-α/β expression in parallel to reduced autophagosome-lysosome fusion in DENV and JEV infection models.Identifies potential crosstalk pathways between cytokine expression and autophagy pathways.Can these signaling pathways be targeted with small-molecule inhibitors? Can these linkages between autophagy and cytokine pathways be clarified?DENV, dengue virus; IFN, interferon; JEV, Japanese encephalitis virus. Open table in a new tab DENV, dengue virus; IFN, interferon; JEV, Japanese encephalitis virus. It is still not clear where PF4 is binding and activating CXCR3 to promote DENV and JEV replication (Table). CXCR3 is a transmembrane G protein–coupled receptor with an N-terminus extracellular domain, 3 extracellular loop (ECL) structures, 3 intracellular loop structures, and an intracellular C-terminal domain that is widely expressed on human cells (eg, activated CD8+ T cells, B cells, dendritic cells, endothelial cells, macrophages, mast cells, natural killer cells, and Th1 cells). In humans, the CXCR3 gene is transcribed into 3 splice variants: CXCR3A, CXCR3B, and CXCR3-alt. Monomers of PF4 (PF4 is a tetramer in solution) likely bind to the ECL2 and N-terminal domain of CXCR3A and CXCR3B [13Liu Y.H. Chuang C.H. Lee Y.Z. Lee E.T. Lo C.L. Wu C.Y. Huang L.K. Bikfalvi A. Sue S.C. Structural properties of CXCL4L1 and its recognition of the CXCR3 N-terminus.Biochemistry. 2023; 62: 722-734Crossref PubMed Scopus (1) Google Scholar]. What happens next is unclear in this model of infection, but CXCR3 binding to other agonists (eg, CXCL9, CXCL10, and CXCL11) uses a multistep approach to ligand binding and receptor activation [14Xanthou G. Williams T.J. Pease J.E. Molecular characterization of the chemokine receptor CXCR3: evidence for the involvement of distinct extracellular domains in a multi-step model of ligand binding and receptor activation.Eur J Immunol. 2003; 33: 2927-2936Crossref PubMed Scopus (78) Google Scholar]. One question arises as to whether PF4 can synergize with other pathways to impact DENV and JEV infections (Table). Another study has shown that PF4, in the absence of DENV or JEV, can synergize with toll-like receptor 8 (TLR8) to signal through receptor-interacting serine/threonine-protein kinase to activate interleukin-1 expression in monocytes [15Yang C. Bachu M. Du Y. Brauner C. Yuan R. Ah Kioon M.D. Chesi G. Barrat F.J. Ivashkiv L.B. CXCL4 synergizes with TLR8 for TBK1-IRF5 activation, epigenomic remodeling and inflammatory response in human monocytes.Nat Commun. 2022; 13: 3426Crossref PubMed Scopus (13) Google Scholar]. This is intriguing as TLR8 can also interact with flavivirus RNA. Where AMG487 disrupts PF4-CXCR3 interactions and promotes DENV and JEV replication is also unclear (Table). For other studies, we understand that AMG487 can prevent binding of CXCL10 and CXCL11 to CXCR3 [16Johnson M. Li A.R. Liu J. Fu Z. Zhu L. Miao S. Wang X. Xu Q. Huang A. Marcus A. Xu F. Ebsworth K. Sablan E. Danao J. Kumer J. Dairaghi D. Lawrence C. Sullivan T. Tonn G. Schall T. et al.Discovery and optimization of a series of quinazolinone-derived antagonists of CXCR3.Bioorg Med Chem Lett. 2007; 17: 3339-3343Crossref PubMed Scopus (81) Google Scholar]. Binding of CXCL10 and CXCL11 is thought to occur at the ECL1 region and the N-terminal region of CXCR3. CXCL10 binding is also thought to occur at the Asn22 residue near the N-terminal region of CXCR3 [17Satarkar D. Patra C. Evolution, expression and functional analysis of CXCR3 in neuronal and cardiovascular diseases: a narrative review.Front Cell Dev Biol. 2022; 10882017Crossref PubMed Scopus (16) Google Scholar]. Therefore, it is possible that AMG487 could reduce PF4 binding to a region between the N-terminus and ECL2 regions of CXCR4. In their work, Singh et al. thoroughly followed the principles of guidelines developed for using and interpreting autophagy assays. These guidelines, although not mentioned in the study, are an excellent resource and provide a glossary of molecules involved in autophagy. The role of DENV in subverting autophagy in a variety of cell lines was described in 2008 [18Lee Y.R. Lei H.Y. Liu M.T. Wang J.R. Chen S.H. Jiang-Shieh Y.F. Lin Y.S. Yeh T.M. Liu C.C. Liu H.S. Autophagic machinery activated by dengue virus enhances virus replication.Virology. 2008; 374: 240-248Crossref PubMed Scopus (301) Google Scholar]. In the current study by Singh et al., PF4 treatment of DENV-infected cells increased phosphorylation of phosphorylated (P)-unc-51 like autophagy activating kinase (ULK) 1, and P-p38 (Table). ULK1 has 3 major domains: a kinase domain, a serine proline-rich domain, and a C-terminal domain. The interaction of ULK1 with p38 is complex and may activate ULK1 or be a substrate of ULK1. The serine proline-rich domain of ULK interacts with microtubule-associated protein 1 light chain 3/LC3 (LC3). Conjugation of cytosolic LC3-I to phosphatidylethanolamine allows for generation of LC3-II associated phagosomes, which promote phagosome maturation and processing of the engulfed cargo. PF4 treatment of DENV-infected cells also increased phosphorylation of mechanistic target of rapamycin (mTOR) kinase. In mammals, mTOR interacts with binding partners to form 2 complexes, mTORC1 and mTORC2, with TORC1 directly regulating autophagy [19Klionsky D.J. Abdel-Aziz A.K. Abdelfatah S. Abdellatif M. Abdoli A. Abel S. Abeliovich H. Abildgaard M.H. Abudu Y.P. Acevedo-Arozena A. Adamopoulos I.E. Adeli K. Adolph T.E. Adornetto A. Aflaki E. Agam G. Agarwal A. Aggarwal B.B. Agnello M. Agostinis P. et al.Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition).Autophagy. 2021; 17: 1-382Crossref PubMed Scopus (1323) Google Scholar]. The association of ULK-1, mTOR, and p38 serine/threonine kinase activity in preventing lysosome-phagosome fusion in models of DENV infection provides a rationale for identifying these kinases in inhibitor studies [20Blázquez A.B. Saiz J.C. Potential for protein kinase pharmacological regulation in Flaviviridae infections.Int J Mol Sci. 2020; 21: 9524Crossref PubMed Scopus (8) Google Scholar]. Involvement of serine/threonine kinases leads to questions as to whether there could be further crosstalk between the autophagy process and other proinflammatory or anti-inflammatory pathways in DENV infection. It is also unclear whether JEV would interact with this process in the same manner as DENV (Table). DENV can evade the innate immune response by blocking type I IFN [21Castillo Ramirez J.A. Urcuqui-Inchima S. Dengue virus control of type I IFN responses: a history of manipulation and control.J Interferon Cytokine Res. 2015; 35: 421-430Crossref PubMed Scopus (58) Google Scholar]. In a prior study, members of the study team noted that treatment of infected monocytes with PF4 increased P-p38, decreased P-STAT2, and decreased interferon regulatory factor 9 (IRF9) and IFN-α production. Both anti-PF4 and AMG487 reversed the effect of PF4 by decreasing P-p38, increasing P-STAT2, and increasing IRF9 and IFN-α expression [12Ojha A. Bhasym A. Mukherjee S. Annarapu G.K. Bhakuni T. Akbar I. Seth T. Vikram N.K. Vrati S. Basu A. Bhattacharyya S. Guchhait P. Platelet factor 4 promotes rapid replication and propagation of Dengue and Japanese encephalitis viruses.EBioMedicine. 2019; 39: 332-347Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar]. In this study, Singh et al. showed that IRF3/7/9 and IFN-α/β expression in DENV-2 and JEV infection models occurred in parallel to decreased autophagosome-lysosome fusion. This leads to an interesting question as to whether there is crosstalk between signal transduction pathways and whether multiple pathways involved in DENV and JEV pathophysiology could be blocked by small molecules. DENV and JEV are viruses of global importance that can lead to substantial burdens on health systems in affected countries. Although there are vaccines for both viruses, there are no specific treatments for either DENV or JEV. Infection models such as those used by Singh et al. identify how host immune components or processes, such as autophagy, the mTOR-p38-ULK1 axis, and antiviral cytokine production, can be subverted during DENV and JEV infections. The work also provides further evidence on how the PF4-CXCR3 axis might act as a target for new antiviral agents or therapies. One question arises as to whether PF4 can synergize with other signaling pathways (eg, TLR8 and receptor-interacting serine/threonine-protein kinase) to blunt antiviral IFN response and sustain DENV and JEV infections.

Background and Objectives: In Canada, plasma sent for fractionation is tested for both parvovirus B19 (B19V) and hepatitis A virus (HAV). This study compared positivity rates of B19 and HAV nucleic acid tests (NATs) in Canadian plasma samples for the pre-COVID-19 restriction era (2015 to end of February 2020 [Q1] 2020) and the post-COVID-19 restriction era. Materials and Methods: Pooled EDTA plasma specimens were tested within 24 months of blood draw using the Procleix Panther System (Grifols Diagnostic Solutions Inc, San Diego, CA, USA) for B19V and HAV detection. Reactive pools were resolved by individual specimen testing. Results: Between 1 January 2015, and 31 March 2022, 3,928,619 specimens from Canadian plasma donors were tested for B19V. For the same period, 3,922,954 specimens were tested for HAV. To account for a lag in specimen testing for up to 24 months, the data were divided into: (1) a pre-pandemic period (1 January 2015-31 March 2020; B19V tested n = 2,412,701, B19V NAT-positive n = 240 [0.01%], HAV tested n = 2,407,036, HAV NAT-positive n = 26 [0.001%]); (2) a two-year mixed-impact period (1 April 2020-31 March 2022; B19V tested n = 968,250, B19V NAT-positive n = 14 [0.001%], HAV tested n = 968,250, HAV NAT-positive n = 2 [0.0002%]); and (3) a pandemic-impact period (1 April 2022-31 March, 2023; B19V tested n = 597,668, B19V NAT-positive n = 3 [0.0005%], HAV tested n = 597,668, HAV NAT-positive n = 1 [0.0002%]). Conclusion: The percentage of B19V- and HAV-positive donations was significantly reduced from the pre-pandemic period to the pandemic-impact period.