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Introduction: Persistent oral infections with high-risk human papillomavirus (HR-HPV) are a potential cause of most oropharyngeal cancers (OPCs). Oral HR-HPV infection and persistence are significantly higher in people living with HIV (PLWH). Most data on oral HR-HPV in PLWH come from developed countries or adult cohorts. This study aims to investigate oral HR-HPV susceptibility and persistence among children and adolescents living with HIV (CALHIV) and to understand the roles of perinatal HIV exposure, infection, antiretroviral treatment, and the oral microbiome., Methods and Analysis: This prospective cohort study is ongoing at the University of Benin Teaching Hospital (UBTH), Nigeria, involving mother-child pairs followed at 6-month intervals for 2 years. Participants include children aged 9-18 and their mothers aged 18 and above. The study targets 690 adolescents in three groups: 230 CALHIV, 230 HIV-exposed but uninfected and 230 HIV-unexposed and uninfected. Oral rinse, saliva, buccal swabs and supragingival plaque samples are collected at each visit. Blood samples are tested for HIV, Hepatitis B virus (HBV) and Hepatitis C virus (HCV), with CD4, CD8 and full blood counts performed. Oral HPV is assessed for incidence, persistence, and clearance. Statistical analyses to look for associations between cohort baseline characteristics and findings will be conducted using univariable and multivariable models for repeated data and high-dimensional microbiome data. All statistical tests will be two-sided; a p value <0.05 will indicate significance. Multiple comparisons will be adjusted using the False Discovery Rate (FDR) correction to control for Type I error., Ethics and Dissemination: The study was approved by Rutgers State University (Pro2022000949) and the UBTH (ADM/E22/A/VOL. VII/14813674). Informed consent was obtained from all parents/guardians., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2025. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ Group.)

Since late 2021, Omicron variants have dominated the epidemiological scenario as the most successful severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sublineages, driving new and breakthrough infections globally over the past two years. In this study, we investigated for the first time the host salivary response of COVID-19 patients infected with Omicron variants (BA.1, BA.2, and BA.4/5) by using an untargeted four-dimensional data-independent acquisition (4D-DIA)-based proteomics approach. We identified 137 proteins whose abundance levels differed between the COVID-19 positive and negative groups. Salivary signatures were mainly enriched in ribosomal proteins, linked to mRNAviral translation, protein synthesis and processing, immune innate, and antiapoptotic signaling. The higher abundance of 14-3-3 proteins (YWHAG, YWHAQ, YWHAE, and SFN) in saliva, first reported here, may be associated with increased infectivity and improved viral replicative fitness. We also identified seven proteins (ACTN1, H2AC2, GSN, NDKA, CD109, GGH, and PCYOX) that yielded comprehension into Omicron infection and performed outstandingly in screening patients with COVID-19 in a hospital setting. This panel also presented an enhanced anti-COVID-19 and anti-inflammatory signature, providing insights into disease severity, supported by comparisons with other proteome data sets. The salivary signature provided valuable insights into the host's response to SARS-CoV-2 Omicron infection, shedding light on the pathophysiology of COVID-19, particularly in cases associated with mild disease. It also underscores the potential clinical applications of saliva for disease screening in hospital settings. Data are available via ProteomeXchange with the identifier PXD054133.

The COVID-19 (SARS-CoV-2) pandemic has led to a significant mortality globally and persistent health challenges in many survivors. Early accurate diagnosis, surveillance, identification of cohorts, and prophylaxis are considered essential measures to reduce the spread of infectious viral pathogens such as SARS-CoV-2. A reliable, fast, high-throughput screening method that can detect viral particles and identify the pathogenic virus in infected individuals could help to reduce the spread of the next viral threat through quick knowledge and implementation of appropriate prevention strategies. Since respiratory viruses are typically present in nasal and oral secretions, saliva is a good target for testing for viral infections. Saliva testing has slowly gained popularity in the diagnostics based on biomarkers and other constituents ranging from organic compounds (e.g., food additives), peptides, and even microorganisms. Polymerase chain reaction (PCR) remains the gold standard for sensitive detection of SARS-CoV-2 infection in biological samples. However, while PCR testing for COVID is sensitive and widely used by hospitals, the method has a false-negative rate of 15-20% and is kit-based necessitating the development of alternative methods of detection that provide higher accuracy. This paper describes the use of a SpecID Mass Spectrometer that can detect the presence of viral particles in saliva at very low levels (<500 virions/0.5 ml). The main goal of this study was to demonstrate that our previously developed, portable, mass spectrometry based method, SpecID, could also be sued for detecting viruses in saliva, including but not limited to SARS-CoV-2; the SpecID method has the potential to provide a reliable solution that overcomes some of the challenges with molecular testing like PCR., Competing Interests: The authors are inventors of the technology highlighted in this article. The technology has a granted patent, US-8704169-B2., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Background: Efforts are underway to support the development of novel mucosal coronavirus disease 2019 (COVID-19) vaccines. However, there is limited consensus about the complementary role of mucosal immunity in disease progression and how to evaluate immunogenicity of mucosal vaccines. This study investigated the role of oral mucosal antibody responses in viral clearance and COVID-19 symptom duration., Methods: Participants with polymerase chain reaction (PCR)-confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection provided oral fluid for testing with SARS-CoV-2 antibody multiplex assays, nasal swabs for reverse-transcription PCR, and symptom information at up to 8 follow-ups from April 2020 to February 2022., Results: High and moderate oral fluid anti-spike (S) secretory IgA (SIgA) postinfection was associated with significantly faster viral clearance and symptom resolution across age groups with effect sizes equivalent to prior COVID-19 vaccine immunity at the time of infection. Those with high and moderate anti-S SIgA cleared the virus 14 (95% confidence interval [CI], 10-18) days and recovered 9-10 (95% CI, 6-14) days earlier. Delayed and higher anti-S IgG was associated with significantly longer time to clearance and recovery. Experiencing symptoms >4 weeks was associated with lower anti-receptor-binding domain SIgA 15-30 days after infection onset (P < .001)., Conclusions: Robust mucosal SIgA early postinfection appears to support faster clearance of SARS-CoV-2 and recovery from COVID-19 symptoms. This research underscores the importance of harmonizing mucosal immune response assays to evaluate new mucosal vaccines., Competing Interests: Potential conflicts of interest . The authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (Published by Oxford University Press on behalf of Infectious Diseases Society of America 2024.)

The CRISPR-based detection methods have been widely applied, yet they remain limited by the non-universal nature of one-pot diagnostic approaches. Here, we report a universal one-pot fluorescent method for the detection of epidemic pathogens, delivering results within 15-20 min. This method uses heparin sodium to precisely tunes the cis-cleavage capability of Cas12 via interference with the Cas12a-crRNA binding process, thereby generating significant fluorescence due to the accumulation of isothermal amplification products. Additionally, this universal assay accommodates both classic and suboptimal PAMs, as well as various Cas12a subtypes such as LbCas12a, AsCas12a, and AapCas12b. Such a robust method demonstrates sensitivity and specificity exceeding 95% in the detection of monkeypox pseudovirus, influenza A virus, and SARS-CoV-2 from saliva or wastewater samples, when compared with qPCR or RT-qPCR. Moreover, the cost of heparin sodium per thousand uses is $0.01 to $0.04 only. Collectively, this universal and fast one-pot approach based on heparin sodium offers potential possibilities for point-of-care testing., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Background: Birth cohort screening has been implemented in some countries to identify the potentially 'missed population' of people with undiagnosed chronic hepatitis C virus (HCV) who may not be found through targeted approaches., Aim: To determine uptake of HCV antibody testing using an oral swab screening method, the overall yield, whether those testing positive had risk markers in their primary care record, and the cost per case detected., Design and Setting: This was a pilot screening study set in general practices in the Southwest of England, Yorkshire and Humber, and South London., Method: Participants consenting were sent an oral swab kit in the post and saliva samples were tested for antibodies to HCV., Results: In total, 16 436/98 396 (16.7%) patients consented and were sent an oral swab kit. Of these, 12 216 (12.4%) returned a kit, with 31 participants (yield 0.03%) testing positive for HCV antibodies. Of those positive, 14/35 (45%) had a risk marker for HCV on their primary care record. Two (yield 0.002%) were confirmed RNA positive and referred for treatment, both had HCV risk markers. The cost per case was £16 000 per HCV antibody detected and £247 997 per chronic HCV detected., Conclusion: Wide-scale screening could be delivered and identify people infected with HCV, however, most of these individuals could have been detected through lower-cost targeted screening. The yield and cost per case found in patients were substantially worse than model estimates and targeted screening studies. Birth cohort screening should not be rolled out in primary care in England., (© The Authors.)

Precise and rapid disease detection is critical for controlling infectious diseases like COVID-19. Current technologies struggle to simultaneously identify viral RNAs and host immune antibodies due to limited integration of sample preparation and detection. Here, we present acoustofluidic integrated molecular diagnostics (AIMDx) on a chip, a platform enabling high-speed, sensitive detection of viral immunoglobulins [immunoglobulin A (IgA), IgG, and IgM] and nucleic acids. AIMDx uses acoustic vortexes and Gor'kov potential wells at a 1/10,000 subwavelength scale for concurrent isolation of viruses and antibodies while excluding cells, bacteria, and large (>200 nanometers) vesicles from saliva samples. The chip facilitates on-chip viral RNA enrichment, lysis in 2 minutes, and detection via transcription loop-mediated isothermal amplification, alongside electrochemical sensing of antibodies, including mucin-masked IgA. AIMDx achieved nearly 100% recovery of viruses and antibodies, a 32-fold RNA detection improvement, and an immunity marker sensitivity of 15.6 picograms per milliliter. This breakthrough provides a transformative tool for multiplex diagnostics, enhancing early infectious disease detection.

SARS-CoV-2 is the viral pathogen responsible for COVID-19. Although morbidity and mortality frequently occur as a result of lung disease, the gastrointestinal (GI) tract is recognized as a primary location for SARS-CoV-2. Connections and interactions between the microbiome of the gut and respiratory system have been linked with viral infections via what has been referred to as the 'gut-lung axis' with potential aerodigestive communication in health and disease. This research explored the relationship between the microbiomes of the upper respiratory and GI tracts in patients with COVID-19 and examined Extraesophageal reflux (EOR), a mechanism which could contribute to dysregulated communication between the GI and respiratory tract (as identified in COVID-19). 97 patients with a laboratory diagnosis of COVID-19 infection, and 50 age-matched controls were recruited and stool, saliva and sputum were obtained from each participant. ELISA Pepsin tests and Reflux Symptom Index scores (RSI) were conducted for EOR assessment. DNA sequencing of the V4 region of the 16 S rRNA gene was performed for microbiome analysis. No differences were observed between the fecal microbiome's alpha and Shannon diversity indices; however, a distinct microbial composition was observed in COVID-19 patients (when compared to the controls). The respiratory microbiota from individuals with COVID-19 demonstrated a statistically significant reduction in Shannon diversity and bacterial richness alongside an overall reduction in the prevalence of organisms from a typical healthy respiratory microbiome. Furthermore, the bacterial richness of the stool and sputum samples was significantly lower among COVID-19 patients admitted to ICU. A significantly higher RSI score and salivary pepsin level were detected among those with COVID-19. The data indicates that COVID-19 is associated with a dysregulation of both the gut and lung microbiome with a more marked perturbation in the lung, particularly among COVID-19 patients who had been admitted to the ICU. The presence of increased RSI scores, combined with elevated levels of Pepsin, suggests that increased micro-aspiration may occur, which is consistent with of under-recognized interactions between the GI and lung microbiomes in COVID-19 patients and requires additional study. Such studies would benefit from the insights provided by biological samples which reflect the continuum of the aerodigestive tract., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Background: Blood and oral fluid-based HIV self-tests are important for reaching the undiagnosed living with HIV. The study objectives were to evaluate the oral fluid-based OraQuick® HIV Self-Test (HIV-ST) performance in comparison to laboratory reference testing; determine if laypersons can correctly perform the HIV-ST; document if intended users can successfully interpret pre-made contrived positive, negative, and invalid results; and document if intended users can understand the key messages in the product labeling., Methods: This prospective study enrolled consenting adult intended users of HIV self-testing from six community health centres in four Canadian provinces between June 2022 and January 2024. Positive and negative agreement was determined by comparing the results of the HIV self-tests with the results of the laboratory-based "gold standard" Abbott Alinity HIV Antigen/Antibody Combo test. Descriptive statistics were used to summarize usability self-test procedure steps., Results: Overall, 951 participants were recruited and consented with 911 available for all analyses. With respect to sociodemographics: 84% of participants were between 18-45 years of age, 73% had at least a college education, 48% were Cis-male, 45% were employed; and 26% identified as White, 23% as African, Caribbean or Black, 5% as Indigenous [First Nations, Métis or Inuit], 33% as Asian, and 6% as LatinX. Primary efficacy analysis on the 911 who completed HIV-ST revealed a single confirmed positive participant and a negative percent agreement of 100% (880/880, 95% CI: 99.9-100%) with the comparator method. For usability determination, the average success rate for "critical" steps for completing the test was 94.1%. Approximately 97% of participants found the instructions easy to follow and 98% of participants reported they would use the test again. Of the 465 participants who interpreted the strong positive, weak positive, negative, and invalid pre-made contrived results, the average of correct interpretations ranged from 59-97% CONCLUSIONS: A licensed oral fluid-based HIV self-test in Canada can present an accurate, easy-to-use, and less invasive alternative to blood-based HIV testing. The addition of an oral-fluid self-test along with the current licensed blood-based HIV self-test could help reach the undiagnosed with HIV in Canada and positively impact HIV testing rates overall by offering individuals a choice of self-testing devices., Competing Interests: Declarations. Ethics approval and consent to participate: Research Ethics Boards (REB) in Ontario, Quebec, British Columbia and Manitoba approved this study at participating recruitment sites including St. Michael’s Hospital REB, University of Toronto REB, Veritas IRB British Columbia, and Quebec, as well as the University of Manitoba HREB. This study was conducted under Health Canada-approved Investigational Testing Authorization (ITA), application ITA# 336702, issued on 11 May 2022. All individual participants provided informed consent prior to study enrollment. The respective REBs approved anonymous participation was approved allowing participants to read the informed consent form with a signature and attestation by the study observer indicating that they received the participant’s verbal consent to participate in the study. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Measuring virus in biofluids is complicated by confounding biomolecules coisolated with viral nucleic acids. To address this, we developed an affinity-based microfluidic device for specific capture of intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Our approach used an engineered angiotensin-converting enzyme 2 to capture intact virus from plasma and other complex biofluids. Our device leverages a staggered herringbone pattern, nanoparticle surface coating, and processing conditions to achieve detection of as few as 3 viral copies per milliliter. We further validated our microfluidic assay on 103 plasma, 36 saliva, and 29 stool samples collected from unique patients with COVID-19, showing SARS-CoV-2 detection in 72% of plasma samples. Longitudinal monitoring in the plasma revealed our device's capacity for ultrasensitive detection of active viral infections over time. Our technology can be adapted to target other viruses using relevant cell entry molecules for affinity capture. This versatility underscores the potential for widespread application in viral load monitoring and disease management.

Biosensors are transforming point-of-care diagnostics by simplifying the detection process and enabling rapid, accurate testing. This study introduces a novel, reusable biosensor designed for direct viral RNA detection from unfiltered saliva, targeting SARS-CoV-2. Unlike conventional methods requiring filtration, our biosensor leverages a unique electrode design that prevents interference from saliva debris, allowing precise measurements. The biosensor is based on electrochemical principles, employing oligonucleotide probes immobilized on a hydrophobic-coated electrode, which prevents air bubbles and salt crystal formation. During validation, the biosensor demonstrated a sensitivity and specificity of 100%, accurately identifying SARS-CoV-2 in saliva samples without false positives or negatives. Cross-validation with RT-qPCR, the gold standard for COVID-19 diagnostics, confirmed the reliability of our device. The biosensor's performance was tested on 60 participants, yielding 12 true positive results and 48 true negatives, aligning perfectly with RT-qPCR outcomes. This reusable, easy-to-use biosensor offers significant potential for point-of-care applications in various healthcare settings, providing a fast, efficient, and cost-effective method for detecting viral infections such as COVID-19. Its robust design, minimal sample preparation requirements, and multiple-use capability mark a significant advancement in biosensing technology.

We evaluated the diagnostic performance of Rapiim SARS-CoV-2-H (Rapiim-H)-a point-of-care qualitative antigen test-using nasopharyngeal swabs (NPS) and saliva samples and compared its results with those from antigen quantification and nucleic acid amplification tests. NPS and saliva were collected from patients with confirmed and suspected coronavirus disease (COVID-19). In total, 142 NPS and saliva samples were collected. In symptomatic cases, in which the first NPS sample was collected within 10 days of disease onset, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for COVID-19 diagnosis were 91.7 %, 88.6 %, 93.2 %, and 86.1 %, respectively. A similar analysis was performed on saliva samples, and the sensitivity, specificity, PPV, and NPV were 50.8 %, 91.4 %, 91.2 %, and 51.6 %, respectively. The Rapiim-H test using NPS demonstrated approximately 90 % sensitivity and specificity, particularly within the first 10 days after disease onset., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Kei Yamamoto reports financial support was provided by Canon Medical Systems Corporation. Hideaki Kato reports a relationship with Shionogi and Co Ltd that includes: funding grants. Hideaki Kato reports a relationship with Asahi Kasei Pharma Corporation that includes: funding grants. Kei Yamamoto reports a relationship with FUJIREBIO Inc that includes: funding grants. Kei Yamamoto reports a relationship with Mizuho Medy Co Ltd that includes: funding grants. Kei Yamamoto reports a relationship with VisGene Co. Ltd. that includes: funding grants. Kei Yamamoto reports a relationship with Sanyo Chemical Industries Ltd that includes: funding grants. Kei Yamamoto reports a relationship with CarbGeM Inc. that includes: funding grants. Kei Yamamoto reports a relationship with Sysmex Corp that includes: funding grants. Kei Yamamoto reports a relationship with Toyobo Co Ltd that includes: funding grants. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: When diagnosing viral infections in humans and animals, the presence of virus in a sample in trace amounts that are below the analytical sensitivity of the detection system may cause false negative results and inaccurate diagnosis. We previously reported the development of a simple virion concentration technique using 12 ml large-volume samples that can dramatically improve diagnostic sensitivity by increasing analytical sensitivity by 100-fold over conventional methods. The present study was conducted to further improve the simplicity and versatility of this method. We constructed a simple and highly sensitive method for the detection of SARS-CoV-2 in human saliva after concentration using a magnetic nanoparticle conjugated with polyethylene glycol (PEG)., Results: Performance of the method was evaluated by comparing a combination of automated nucleic acid extraction and RT-qPCR or triplex RT-LAM detection in a spiked sample of 20 ml saliva collected from healthy humans. The method theoretically achieved 300-fold concentration of spiked SARS-CoV-2 in saliva, enabling 10- to 1000-fold higher analytical sensitivity for detection compared to conventional RNA extraction methods., Conclusions: This newly developed method allows for easy and reliable concentration of the virion in less than 60 min, improving the analytical sensitivity of the SARS-CoV-2 test. Further, the method allows for easy and reliable enrichment of the virus in less than 60 min, improving the analytical sensitivity of the SARS-CoV-2 test. This method is easily used for highly sensitive virus detection from a variety of human oral fluid samples and may also be applied to rapid and labor-saving screening tests by pooling a large number of samples., Competing Interests: Declaration of Competing Interest The Pegcsion kit was provided free of charge to the corresponding author (WY) by JNC Corporation. The authors declare no other competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Vector competence studies in mosquitoes present valuable opportunities to explore arboviral transmission and virus-vector interactions. However, oral infection studies in mosquitoes can be challenging. An alternative approach is to infect mosquitoes during their aquatic larval stage, resulting in the emergence of infected adults. To investigate the potential of this method, Culex pipiens biotype molestus larvae were infected with Usutu virus (USUV, Orthoflavivirus usutuense). For this purpose, larvae were exposed to USUV-infected mammalian and mosquito cell cultures for 24 h before being reared to adults. Subsequent analysis via RT-qPCR revealed that the Culex larvae successfully acquired USUV from the infected cells and exhibited high susceptibility to infection. Immediately after emergence, 32.10 % (26/81) of male and 41.03 % (16/39) of female mosquitoes tested positive for USUV RNA. Notably, females that were incubated for 15 days post-emergence demonstrated even higher infection rates, reaching 100.00 % (23/23). In addition, viral RNA and infectious particles were detected in some saliva samples, indicating the potential for transmission. This experimental infection of mosquito larvae thus offers the opportunity to produce infected adult mosquitoes for studies enhancing our understanding of virus-vector interactions, co-infections, and transmission routes. Such research contributes to better public health strategies addressing arboviral diseases., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Usutu virus (USUV) is an emerging flavivirus, closely related to West Nile virus (WNV), that has spread into Europe from Africa. Since Culex tarsalis Coquillett is an important vector for WNV transmission in the United States, we tested the ability of USUV to replicate in and be transmitted by these mosquitoes. Materials and Methods: USUV was used to infect 3-4 day-old Cx. tarsalis with 5.6 to 7.5 log 10 pfu/ml in goose bloodmeals. Saliva, heads, and bodies were collected on day 13 or 14 and analyzed by RT-qPCR for detection for USUV vRNA. Blotting paper punches were also collected daily to assess viral transmissibility. Results: The low and high dose blood meal resulted in 0% and 19.6% of the mosquitoes having established infections, respectively. All of the high dose had a dissemination of USUV RNA to the heads and none of the filter papers had detectable USUV RNA, but five of the capillary saliva collections were positive, representing 45.5% of the infected mosquitoes. Conclusions: Limited infection of Cx. tarsalis was observed when exposed to bloodmeals with greater than 107 pfu/mL of USUV, indicating this vector is not likely to have a key role in transmission of the virus.

Virion-mediated outbreaks are imminent and despite rapid responses, continue to cause adverse symptoms and death. Therefore, tunable, sensitive, high-throughput assays are needed to help diagnose future virion-mediated outbreaks. Herein, it is developed a tunable in situ assay to selectively enrich virions and extracellular vesicles (EVs) and simultaneously detect antigens and nucleic acids at a single-particle resolution. The Biochip Antigen and RNA Assay (BARA) enhanced sensitivities compared to quantitative reverse-transcription polymerase chain reaction (qRT-PCR), enabling the detection of virions in asymptomatic patients, genetic mutations in single virions, and enabling the continued long-term expression of viral RNA in the EV-enriched subpopulation in the plasma of patients with post-acute sequelae of the coronavirus disease of 2019 (COVID-19). BARA revealed highly accurate diagnoses of COVID-19 by simultaneously detecting the spike glycoprotein and nucleocapsid-encoding RNA in saliva and nasopharyngeal swab samples. Altogether, the single-particle detection of antigens and viral RNA provides a tunable framework for the diagnosis, monitoring, and mutation screening of current and future outbreaks., (© 2024 The Author(s). Advanced Healthcare Materials published by Wiley‐VCH GmbH.)

Introduction: Dengue cases in the Americas in 2024 have reached record highs, especially in Brazil. However, surveillance remains suboptimal and new methods are needed to monitor Dengue Virus (DENV) spread. To assess whether wastewater-based epidemiology would be a useful tool, we investigated the presence of DENV RNA in dengue patients' urine and oral fluid from an endemic area to inform how shedding in these fluids occurs and provide insight for wastewater surveillance., Methods: We examined how often DENV RNA is detected in urine and oral fluid from dengue patients confirmed by serum RT-qPCR, NS1 ELISA or IgM seroconversion in Salvador, Brazil., Results: Of 88 confirmed cases, 9.1 % were positive for DENV RNA in urine (7/88) or oral fluid (1/88). Of 53 serum RT-qPCR-positive patients, 6 (11.3 %) showed detectable DENV RNA in acute- or convalescent-phase urine. Patients with RT-qPCR-positive urine had a lower frequency of DENV IgG in acute-phase serum (a proxy for secondary infection) (57 % vs. 74 %) and a lower median serum RT-qPCR cycle threshold than those with negative urine (21.8 vs. 23.9)., Conclusion: The low presence of DENV RNA in urine suggests that additional research is needed to evaluate whether using wastewater-based epidemiology to monitor DENV transmission is possible., Competing Interests: Conflicts of interest The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results., (Copyright © 2024 Sociedade Brasileira de Infectologia. Published by Elsevier España, S.L.U. All rights reserved.)

Congenital cytomegalovirus (CMV) infection is the leading infectious cause of childhood disability, in particular sensorineural hearing loss (SNHL). Timeliness of diagnosis is crucial, since the presence of CMV in any compartment (eg, blood, urine, or saliva) after age 21 days can mean postnatal acquisition of infection, particularly in breastfed infants. Given these issues, there is considerable interest in implementation of screening programmes-either universal screening (where all newborns are tested) or targeted screening. Targeted screening is typically based on the outcome of a newborn hearing screen, and can be influenced in some strategies by findings of other signs suggestive of congenital CMV. Universal screening is likely to have the greatest overall benefit. Early identification of congenital CMV allows for interventions such as antiviral therapy (when indicated) and enables anticipatory audiological monitoring that facilitates timely detection of delayed-onset SNHL. However, there are debates about the effectiveness of screening programmes. Most infants with congenital CMV are unaffected and do not appear to be at risk for adverse neurodevelopment outcomes, except for SNHL. Screening can, therefore, raise unwarranted concern among parents and clinicians in these cases. The best clinical sample for diagnostic testing is unclear. PCR testing of saliva is sensitive but has a risk of yielding false-positive results in infants without congenital CMV. Resolving the technological issues has improved the sensitivity of dried blood spot (DBS) PCR but the technique remains suboptimum. An advantage to DBS PCR testing is that an infrastructure exists to add this test to existing newborn screening programmes. In this Review, the advantages and disadvantages of congenital CMV screening are discussed, along with high-priority areas for future research that will inform and direct this rapidly evolving field., Competing Interests: Declaration of interests MRS reports grant support to the University of Minnesota, but no personal honoraria, from Moderna. MRS reports advisory board consultancy work for GSK vaccines. DB-G reports consulting fees from Moderna and honoraria for lectures and educational activities from MSD and Medscape., (Copyright © 2024 Elsevier Ltd. All rights reserved, including those for text and data mining, AI training, and similar technologies.)

Background: Ozonated water is expected to be an effective disinfectant for SARS-CoV-2 present on environmental fomites; however, ozone is consumed by organic substances, resulting in attenuation of its effect. SARS-CoV-2 present in saliva can contaminate environmental surfaces; therefore, it is essential to understand the effect of organic substances in saliva on the disinfectant properties of ozonated water., Aim: To assess organic factors in saliva and the extent to which they diminish the effect of ozonated water on SARS-CoV-2., Methods: Ozonated water was exposed to salivary organic factors and residual ozone concentrations were measured. SARS-CoV-2 was exposed to a salivary factor and virus inactivation by ozonated water was measured., Findings: Amylase and mucin consumed ozone in a concentration-dependent manner. Urea did not. Ozonated water appeared to inactivate SARS-CoV-2 within 30 s. The amount of inactivated SARS-CoV-2 decreased as the protein concentration increased. Virus inactivation was stronger by 1.5 mg/L ozonated water than by 0.5 mg/L ozonated water., Conclusion: This study suggests that the salivary amylase and mucin decay ozone in a concentration-dependent manner, thereby attenuating the disinfection properties of ozonated water for SARS-CoV-2. An increase of the initial amount of ozone can ameliorate the disinfection effect of ozonated water on SARS-CoV-2. Ozone consumption should be taken into consideration for virus infection control. These results provide fundamental information about the effect of ozonated water when used to decontaminate surfaces harbouring SARS-CoV-2 in saliva., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

At the beginning of the COVID-19 pandemic, diagnostic testing was not accessible for mildly ill or asymptomatic individuals. Military operational circumstances exclude the usage of reference laboratory tests. For that reason, at the beginning of the pandemic alternative test methods were needed in order to gain insight into the SARS-CoV-2 status of military personnel. The objectives of this study are to assess whether SARS-CoV-2 antibody rapid lateral flow assay (LFA) in combination with semi-autonomous SARS-CoV-2 antisense and sense genomic RNA fluorescence in situ hybridization (FISH) could establish disease status in military personnel in a fieldable setting, and to assess how this combination performed and to determine which type of sample performed best. A proof of concept sub-study regarding the SARS-CoV-2 application of the fieldable Biotrack-MED® FISH analyzer, a semi-autonomous multi-sample filter cytometer, preceded this observational prospective cohort pilot study. Dutch military personnel were included in the 26 June 2020-11 May 2021 period. Blood, nasopharyngeal and oropharyngeal swabs and saliva were tested at days 0 and 14. SPSS version 25 descriptive statistics and Cohen's kappa assessed agreement between test methods. Both the sensitivity and specificity of the field tests were calculated with ELISA and PCR as reference. Saliva appeared to be the preferred sample type for FISH, where blood was not useful. FISH analysis and LFA results had a concordance of 42% for testing negative, 30% for recovered from infection, 22% for ongoing-and 58% for acute infection in a reference laboratory lab result outcome (RT-PCR or ELISA respectively). The LFA results on serum and full blood corresponded with the ELISA-obtained results (kappa of 0.61 and 0.63 respectively at day 0 and 0.81 and 0.77 respectively at day 14). LFA (full blood-serum), FISH and RT-PCR on saliva did not reach the 90% sensitivity level advised by WHO, with 64-54, 38 and 71% at day 0 and 80-79, 53 and 24% at day 14 respectively., Competing Interests: GJ is cofounder of the compagny that developed the used automated FISH analyser. MW and JR are working at the Biotrack laboratory. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2024 Wijnberg et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Rheumatoid arthritis (RA), an autoimmune disease with complex pathogenesis, is characterized by an immune imbalance reflected, e.g., in the disturbed cytokines' profile. Various viruses and bacteria can cause the upregulation of pro-inflammatory cytokines influencing RA development. In particular, oral cavity dysbiosis, observed in multiple chronic diseases including periodontitis, may be linked to RA. The cytokine profile (IL-1β, IP-10, IL-29, GM-CSF, IFN-α2, IFN-β, TGF-β1, MPC-1, TNF-α, IFN-γ, IL-6, IL-10, IL-17A, IL-12p70, IL-2, and IL-4) of RA patients' saliva was evaluated using flow cytometry and benchmarked with their levels in saliva of healthy controls and patients with other rheumatic diseases. The levels of IL-1β, IP-10, IL-2, and IL-4 were significantly elevated in RA patients' saliva compared to other studied groups. To define the potential role of the most suspicious microbial agents (Epstein-Barr Virus (EBV), Cytomegalovirus, Parvovirus B19, Porphyromonas gingivalis , and Segatella copri ) for RA pathogenesis, the amounts of their DNA in the saliva of patients with RA were assessed in all the groups mentioned above. The EBV and P. gingivalis DNA levels measured by qRT-PCR were significantly higher in RA patients' saliva than in other groups, indicating either the important role of these agents in RA pathogenesis or the higher susceptibility of RA patients for those infectious factors. The comprehension of the association of specific cytokine profiles in RA and the occurrence of specific viral and/or bacterial infections can be a key to a better understanding of RA pathogenesis. These results illustrate the complexity of the immunological profile of RA, show the high diagnostic potential of saliva, and provide insight into how various infections can contribute to RA development.

We developed a 4-parameter clinical assay using Electric Field Induced Release and Measurement (EFIRM) technology to simultaneously assess SARS-CoV-2 RNA (vRNA), nucleocapsid antigen, host binding (BAb) and neutralizing antibody (NAb) levels from a drop of saliva with performance that equals or surpasses current EUA-approved tests. The vRNA and antigen assays achieved lower limit of detection (LOD) of 100 copies/reaction and 3.5 TCID₅₀/mL, respectively. The vRNA assay differentiated between acutely infected (n = 10) and infection-naïve patients (n = 33) with an AUC of 0.9818, sensitivity of 90%, and specificity of 100%. The antigen assay similarly differentiated these patient populations with an AUC of 1.000. The BAb assay detected BAbs with an LOD of 39 pg/mL and distinguished acutely infected (n = 35), vaccinated with prior infection (n = 13), and vaccinated infection-naïve patients (n = 13) from pre-pandemic (n = 81) with AUC of 0.9481, 1.000, and 0.9962, respectively. The NAb assay detected NAbs with a LOD of 31.6 Unit/mL and differentiated between COVID-19 recovered or vaccinated patients (n = 31) and pre-pandemic controls (n = 60) with an AUC 0.923, sensitivity of 87.10%, and specificity of 86.67%. Our combo assay represents a significant technological advancement to simultaneously address SARS-CoV-2 infection and immunity, and it lays the foundation for tackling potential future pandemics., Competing Interests: Declarations. Competing interests: O.Y. is the scientific advisory board for CytoDyn (stock options and cash) and board of directors for Applied Medical (stock and cash). D.W. is a consultant for AIONCO/Avellino and has stock options in AIONCO/Avellino. Other authors declared no conflict of interests., (© 2024. The Author(s).)

Geography is a well-known factor shaping genetic variation in human populations. However, the potential role played by cultural variables remains much understudied. This study investigates the impact of socio-cultural variables on genomic similarity and the saliva microbiome, using data from populations in Lesotho and Namibia. Geographic distance within Lesotho increases genetic differentiation, while shared clan affiliation surprisingly increases it. In Namibia, ethnicity is the predominant factor influencing genetic affinity. Saliva metagenomic data shows a negative correlation between age and alpha diversity, with notable differences in host-interacting taxa and viral load. These findings highlight the role of geography in shaping genetic affinity even at small scales and the complex influences of cultural factors. The saliva microbiome appears primarily affected by unrecorded individual behaviors rather than geographic or cultural variables. At population-level these oral microbiomes reveal insights into some dietary habits, oral health, and also the communal viral load, which appears to have greater incidence in Lesotho possibly related to the long-term effects of the HIV epidemic in the country.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants are a continuous threat to human life. An urgent need remains for simple and fast tests that reliably detect active infections with SARS-CoV-2 and its variants in the early stage of infection. Here we introduce a simple and rapid activity-based diagnostic (ABDx) test that identifies SARS-CoV-2 infections by measuring the activity of a viral enzyme, Papain-Like protease (PLpro). The test system consists of a peptide that fluoresces when cleaved by SARS PLpro that is active in crude, unprocessed lysates from human tongue scrapes and saliva. Test results are obtained in 30 minutes or less using widely available fluorescence plate readers, or a battery-operated portable instrument for on-site testing. Proof-of-concept was obtained in a study on clinical specimens collected from patients with COVID-19 like symptoms who tested positive (n = 10) or negative (n = 10) with LIAT RT-PCR using nasal mid turbinate swabs. When saliva from these patients was tested with in-house endpoint RT-PCR, 17 were positive and only 5 specimens were negative, of which 2 became positive when tested 5 days later. PLpro activity correlated in 17 of these cases (3 out of 3 negatives and 14 out of 16 positives, with one invalid specimen). Despite the small number of samples, the agreement was significant (p value = 0.01). Two false negatives were detected, one from a sample with a late Ct value of 35 in diagnostic RT-PCR, indicating that an active infection was no longer present. The PLpro assay is easily scalable and expected to detect all viable SARS-CoV-2 variants, making it attractive as a screening and surveillance tool. Additionally, we show feasibility of the platform as a new homogeneous phenotypic assay for rapid screening of SARS-CoV-2 antiviral drugs and neutralizing antibodies., Competing Interests: Mesa Photonics holds two utility patents covering diagnostics of SARS-CoV-2 infections and antiviral screening based on PLpro activity: US11603552B2, Method for pathogen identification, Inventors: Frauke Henrike Rininsland, Daniel James Kane, Application granted: 2023-03-14 and US11851698B2, Method for pathogen identification, Continuation of US11603552B2, Inventors: Frauke Henrike Rininsland, Daniel James Kane, Application granted: 2023-12-26. The other authors declare no conflict of interest. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2024 Jimenez-Campos et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Establishing effective mitigation strategies to reduce the spread of influenza virus requires an improved understanding of the mechanisms of transmission. We evaluated the use of a controlled human infection model using an H3N2 seasonal influenza virus to study critical aspects of transmission, including symptom progression and the dynamics of virus shedding. Eight volunteers were challenged with influenza A/Perth/16/2009 (H3N2) virus between July and September 2022 at Emory University Hospital. Viral shedding in the nasopharynx, saliva, stool, urine, and respiratory aerosols was monitored over the quarantine period, and symptoms were tracked until day 15. In addition, environmental swabs were collected from participant rooms to examine fomite contamination, and participant sera were collected to assess seroconversion by hemagglutination inhibition or microneutralization assays. Among the eight participants, influenza virus infection was confirmed in six (75%). Infectious virus or viral RNA was found in multiple physiological compartments, fecal samples, aerosol particles, and on surfaces in the immediate environment. Illness was moderate, with upper respiratory symptoms dominating. In participants with the highest viral loads, antibody titers rose by day 15 post-inoculation, while in participants with low or undetectable viral loads, there was little or no increase in functional antibody titers. These data demonstrate the safety and utility of the human infection model to study features critical to influenza virus transmission dynamics in a controlled manner and will inform the design of future challenge studies focused on modeling and limiting transmission.CLINICAL TRIALSThis study is registered with ClinicalTrials.gov as NCT05332899., Importance: We use a controlled human infection model to assess respiratory and aerosol shedding kinetics to expand our knowledge of influenza infection dynamics and help inform future studies aimed at understanding human-to-human transmission., Competing Interests: N.G.R. receives consulting fees from Krog and participates on the advisory boards of Moderna, Sanofi, Seqirus, and Pfizer. M.F.E. recieves consulting fees from Medtronic and Boston Scientific. L.C.M. receives consulting fees from MITRE.

Background: Determinants of maternal-fetal cytomegalovirus (CMV) transmission and factors influencing the severity of congenital CMV (cCMV) infection are not well understood., Methods: We conducted a descriptive, multicenter study in pregnant women ≥18 years old with primary CMV infection and their newborns to explore maternal immune responses to CMV and determine potential immunologic/virologic correlates of cCMV following primary infection during pregnancy. We developed alternative approaches looking into univariate/multivariate factors associated with cCMV, including a participant clustering/stratification approach and an interpretable predictive model-based approach using trained decision trees for risk prediction (post hoc analyses)., Results: Pregnant women were grouped in 3 distinct clusters with similar baseline characteristics, particularly gestational age at diagnosis. We observed a trend for higher viral loads in urine and saliva samples from mothers of infants with cCMV versus without cCMV. When using a trained predictive-model approach that accounts for interaction effects between variables, anti-pentamer immunoglobulin G antibody concentration and viral load in saliva were identified as biomarkers jointly associated with the risk of maternal-fetal CMV transmission., Conclusions: We identified biomarkers of CMV maternal-fetal transmission. After validation in larger studies, our findings will guide the management of primary infection during pregnancy and the development of vaccines against cCMV., Clinical Trials Registration: NCT01251744., Competing Interests: Potential conflicts of interest. S. A., H. A., V. B., N. D. S., J.-M. D., I. D., M. J., C. S., A. A. P., R. A. v. d. B., and R. P. are/were employed by GSK. S. A., V. B., and R. P. hold financial equities in GSK. M. J. received personal fees from GSK. R. P. is a Moderna employee, and he owns Moderna restricted and unrestricted stock shares and stock options. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Background: Understanding antibody responses to SARS-CoV-2 vaccination is crucial for refining COVID-19 immunization strategies. Generation of mucosal immune responses, including mucosal IgA, could be of potential benefit to vaccine efficacy; however, limited evidence exists regarding the production of mucosal antibodies following the administration of current mRNA vaccines to young children., Methods: We measured the levels of antibodies against SARS-CoV-2 from a cohort of children under 5 years of age (n = 24) undergoing SARS-CoV-2 mRNA vaccination (serially collected, matched serum and saliva samples) or in a convenience sample of children under 5 years of age presenting to pediatric emergency department (nasal swabs, n = 103). Furthermore, we assessed salivary and nasal samples for the ability to induce SARS-CoV-2 spike-mediated neutrophil extracellular traps (NET) formation., Results: Longitudinal analysis of post-vaccine responses in saliva revealed the induction of SARS-CoV-2-specific IgG but not IgA. Similarly, SARS-CoV-2-specific IgA was only observed in nasal samples obtained from previously infected children with or without vaccination, but not in vaccinated children without a history of infection. In addition, oronasopharyngeal samples obtained from children with prior infection were able to trigger enhanced spike-mediated NET formation, and IgA played a key role in driving this process., Conclusions: Despite the induction of specific IgG in the oronasal mucosa, current intramuscular vaccines have limited ability to generate mucosal IgA in young children. These results confirm the independence of mucosal IgA responses from systemic humoral responses following mRNA vaccination and suggest potential future vaccination strategies for enhancing mucosal protection in this young age group., Competing Interests: Potential conflicts of interest. D. R. W. has a financial interest in Quanterix Corporation, a company that develops an ultrasensitive digital immunoassay platform; is an inventor of the Simoa technology, a founder of the company, and also serves on its Board of Directors. D. R. W.’s interests were reviewed and are managed by Brigham and Women's Hospital and Partners Healthcare in accordance with their conflict of interest policies. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Many biological markers of normal and disease states can be detected in saliva. The benefits of saliva collection for research include being non-invasive, ease of frequent sample collection, saving time, and being cost-effective. A small volume (≈1 mL) of saliva is enough for these analyses that can be collected in just a few minutes. For "dry" saliva paper matrices, additional drying times (about 30 min) may be needed, but this can be performed at room temperature without the need for freezers and specialized equipment. Together, these make saliva an ideal choice of body fluid for many clinical studies from diagnosis to monitoring measurable biological substances in hospital settings, remote, and other general locations including disaster areas. For these reasons, we have been using saliva (dry as well as wet) from astronauts participating in short- and long-duration space missions for over two decades to conduct viral, stress, and immunological studies. We have also extended the use of saliva to space analogs including bed rest, Antarctica, and closed-chamber studies. Saliva is a biomarker-rich and easily accessible body fluid that could enable larger and faster public health screenings, earlier disease detection, and improved patient outcomes. This review summarizes our lessons learned from utilizing saliva in spaceflight research and highlights the advantages and disadvantages of saliva in clinical diagnostics.

Saliva contains antimicrobial peptides considered integral components of host innate immunity, and crucial for protection against colonizing microbial species. Most notable is histatin-5 which is exclusively produced in salivary glands with uniquely potent antifungal activity against the opportunistic pathogen Candida albicans. Recently, SARS-CoV-2 was shown to replicate in salivary gland acinar cells eliciting local immune cell activation. In this study, we performed studies to investigate the implications of SARS-CoV-2 infection on salivary histatin-5 production and Candida colonization. Bulk RNA-sequencing of parotid salivary glands from COVID-19 autopsies demonstrated statistically significant decreased expression of histatin and amylase genes. In situ hybridization, coupled with immunofluorescence for co-localization of SARS-CoV-2 spike and histatin in salivary gland cells, showed that histatin was absent or minimally present in acinar cells with replicating viruses. To investigate the clinical implications of these findings, salivary histatin-5 levels and oral Candida burden in saliva samples from three independent cohorts of mild and severe COVID-19 patients and matched healthy controls were evaluated. Results revealed significantly reduced histatin-5 in SARS-CoV-2 infected subjects, concomitant with enhanced prevalence of C. albicans. Analysis of prospectively recovered samples indicated that the decrease in histatin-5 is likely reversible in mild-moderate disease as concentrations tended to increase during the post-acute phase. Importantly, salivary cytokine profiling demonstrated correlations between activation of the Th17 inflammatory pathway, changes in histatin-5 concentrations, and subsequent clearance of C. albicans in a heavily colonized subject. The importance of salivary histatin-5 in controlling the proliferation of C. albicans was demonstrated using an ex vivo assay where C. albicans was able to proliferate in COVID-19 saliva with low histatin-5, but not with high histatin-5. Taken together, the findings from this study potentially implicate SARS-CoV-2 infection of salivary glands with compromised oral innate immunity, and potential predisposition to oral candidiasis., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Plasmonic nanoparticle-based biosensors often report a colorimetric signal through the aggregation or clustering of the nanoparticles (NPs), but these mechanisms typically struggle to function in complex biofluids. Here, we report a matrix-insensitive sensor array approach to detect bacteria, fungi, and viruses whose signal is based on the dissociation of the peptide-aggregated NPs by thiolated polyethylene glycol (HS-PEG) polymers. We show that the HS-PEGs of differing sizes have varying capabilities to dissociate citrate-capped gold nanoparticle (AuNP) and silver nanoparticle (AgNP) assemblies. The dissociative abilities of the HS-PEGs were used in this sensor array to discriminate at the 90% confidence level the microorganisms Porphyromonas gingivalis , Fusobacterium nucleatum , and Candida albicans in water and saliva using linear discriminant analysis (LDA). We further demonstrate the versatility of the sensor array by detecting various subtypes of the viruses SARS-CoV-2 (beta, delta, and omicron) and influenza (H3N2) spiked in saliva samples using LDA. In the final demonstration, the sensor array design stratified healthy saliva samples from patient samples diagnosed with periodontitis as well as COVID-19.

The human oral cavity and upper airway serves as an early barrier and reservoir in the transmission of SARS-CoV-2. Saliva in this microenvironment may serve as a key host factor that can modulate susceptibility to infection and eventual infection of the lower respiratory tract. We sought to analyze the content and composition of heparan sulfate, a glycosaminoglycan identified as an important co-receptor for viral entry, and whether there is any correlation with SARS-CoV-2 infection. We enlisted 98 participants stratified by age, gender, race, and COVID-19 history. Notably, the concentration of heparan sulfate in saliva increased with age, and its composition showed a wide range of variability within each age group independently of age. Heparan sulfate concentration and composition did not differ significantly with gender, ethnicity or race. Compared to patients with no COVID-19 history, patients with previous infection had a similar salivary heparan sulfate concentration, but significant increases in overall sulfation were noted. Moreover, in a subset of participants, for which data was available pre- and post- infection, significant elevation in N-sulfoglucosamine in heparan sulfate was observed post- COVID-19. Examination of salivary bacterial 16S rRNA, showed a significant reduction in species predicted to possess heparan sulfate-modifying capacity among participants >60 years old, which correlates with the increase in heparan sulfate content in older individuals. These findings demonstrate a surprisingly wide variation in heparan sulfate content and composition in saliva across the sampled population and confirm other findings showing variation in content and composition of glycosaminoglycans in blood and urine., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Aim: To assess the feasibility and acceptability of massive hepatitis C virus (HCV) testing in point of care on the street using quick tests, determine the characteristics of the population included, and the prevalence of HCV infection in this population., Methods: Cross-sectional community-based study including adult men who have sex with men (MSM) who attended the three most important LGTB+ events in Sitges (Catalonia, Spain) in 2022. Points of care were set up on tents on the street and attendees were offered voluntary anti-HCV antibody self-testing. Participants were informed of the study, provided consent, completed the test for identification of risk practices (TIRP), and took the test with the OraQuick® HCV test on a saliva sample (sensitivity: 97.8% [95% confidence interval (CI), 93.2-99.4%] and specificity: 100% [95% CI, 98.4-100%]; gold standard: IgG antibody test for HCV by immunoassay [serum]); participants with positive results were offered HCV virus testing with the Xpert HCV Fingerstick® on a blood drop., Results: A total of 1249 adults participated in the large-scale screening, of which 1197 (95.8%) were identified as MSM. The screening time was 39 participants/h. Four (0.32%) participants had positive anti-HCV results, all with undetectable HCV RNA levels. Participants' median (IQR) age was 44 (35, 54) years; most were Europeans, and 13% reported being unaware of their serological HCV status. The mean (SD) TIRP score was 1.40 (1.44) ( n  = 1062), with 67.41% reporting some risk, and the self-perceived sexually transmitted disease score was 3.0 (2.82) ( n  = 969)., Conclusion: The point-of-care strategy on the street using a quick oral self-test at massive MSM events is feasible, well-accepted, and quick, and may be a useful strategy to reach other populations at risk of HCV infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Albertos, Majo, Esteban, Colom and Buti.)

Musca domestica salivary gland hypertrophy virus (MdSGHV) affects house flies by enlarging salivary glands, impeding ovary development in females, and mating behavior in both males and females. It is not known if this virus impacts the quantity of saliva produced by house flies. This study aimed to establish baseline saliva quantities in healthy M. domestica across sexes and ages and examine how MdSGHV infection influences saliva output in 5-day-old males. Results reveal that healthy female Musca domestica produce more saliva on average than males and that saliva production among both sexes decreases with age. A comparison of infected, PBS-injected, and healthy flies shows significantly higher saliva quantities in infected individuals, suggesting MdSGHV enhances saliva production to improve transmission. These findings provide insights into MdSGHV transmission dynamics, which may provide for a better understanding of how other vector-borne diseases like Zika and Dengue virus interact in the infected salivary glands of the host vector.

Zoonotic avian influenza viruses have continued to infect people on occasion. During treatment, antiviral resistant viruses have occasionally emerged, highlighting the need for a novel strategy for treating human illness. After pancreatin treatment, edible bird's nest (EBN), swiftlet saliva consumed for health purposes, possesses anti-avian viral activity by inhibiting receptor-binding hemagglutinin (HA) activity. Glycan analysis revealed an abundance of α2,3Neu5Ac decoy receptors in pancreatin-treated EBN. Fucosylated tri-α2,3Neu5Ac tri-antennary N-glycans (N-35) and di-α2,3Neu5Ac core 2 O-glycans (O-15) are predominant, accounting for 53.46% and 44.66% of total N- and O-glycan amounts, respectively. Isobologram analysis revealed that the treated EBN had a strong synergistic effect with either oseltamivir carboxylate or zanamivir, a competitive inhibitor of receptor-destroying neuraminidases (NAs), against the avian H5N1 virus. Taken together, EBN has the potential to be developed as a food-derived avian viral trap to prevent and decrease avian virus infection as well as in combination with a viral releasing-NA inhibitor to increase therapeutic potency, reduce toxicity, delay resistance development, and potentially prevent pandemic onset., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Japanese encephalitis virus (JEV) is transmitted by Culex species of mosquitoes. In 2022, JEV belonging to a previously unrecognized lineage of genotype IV (GIV) caused a major outbreak of JE in South-eastern Australia, resulting in human cases and affecting piggeries. Cx. annulirostris has previously been implicated as the major vector of JEV in northern Australia where the virus has circulated since its first detection in 1995. Here, we showed that experimental infection of a laboratory colony of Australian Cx. annulirostris with the isolate JEV NSW/22 resulted in a 100% mosquito infection rate, with 87% of mosquito saliva samples testing positive by RT-qPCR at 14 days post-infection. Immunohistochemistry confirmed the presence of a replicating virus in the mosquito midgut and dissemination throughout the body, including the salivary glands. Our results also showed evidence of transovarial transmission of this virus; however, transstadial transmission from the eggs to the adult stage was not found. Comparison with an Indonesian isolate of GIV JEV and previous Australian isolates belonging to genotypes I and II showed that infection with JEV NSW/22 resulted in higher viral titres in the early stage of infection and higher proportions of mosquitoes with JEV-positive saliva, indicating a greater transmission potential compared to other isolates. This study provides compelling experimental evidence that Australian Cx. annulirostris is a highly efficient vector for the 2022 Australian JEV GIV outbreak strain.

DNA has shown great biocompatibility, programmable mechanical properties, and precise structural addressability at the nanometer scale, rendering it a material for constructing versatile nanorobots for biomedical applications. Here, we present the design principle, synthesis, and characterization of a DNA nanorobotic hand, called DNA NanoGripper, that contains a palm and four bendable fingers as inspired by naturally evolved human hands, bird claws, and bacteriophages. Each NanoGripper finger consists of three phalanges connected by three rotatable joints that are bendable in response to the binding of other entities. NanoGripper functions are enabled and driven by the interactions between moieties attached to the fingers and their binding partners. We demonstrate that the NanoGripper can be engineered to effectively interact with and capture nanometer-scale objects, including gold nanoparticles, gold NanoUrchins, and SARS-CoV-2 virions. With multiple DNA aptamer nanoswitches programmed to generate a fluorescent signal that is enhanced on a photonic crystal platform, the NanoGripper functions as a highly sensitive biosensor that selectively detects intact SARS-CoV-2 virions in human saliva with a limit of detection of ~100 copies per milliliter, providing a sensitivity equal to that of reverse transcription quantitative polymerase chain reaction (RT-qPCR). Quantified by flow cytometry assays, we demonstrated that the NanoGripper-aptamer complex can effectively block viral entry into the host cells, suggesting its potential for inhibiting virus infections. The design, synthesis, and characterization of a sophisticated nanomachine that can be tailored for specific applications highlight a promising pathway toward feasible and efficient solutions to the detection and potential inhibition of virus infections.

Many aptamers have been generated by systematic evolution of ligands by exponential enrichment (SELEX) to recognize spike proteins of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2&ek), some of which have been engineered into dimeric and trimeric versions for enhanced affinity for diagnostic applications. However, no studies have been conducted to compare the utilities of monomeric, dimeric and trimeric aptamers in diagnostic assays with real clinical samples to answer the question of what levels of affinity an aptamer must have for accurate clinical diagnostics. Herein, we carried out a comparative study with two monomeric aptamers MSA1 and MSA5, one dimeric aptamer and two homotrimeric aptamers constructed with MSA1 and MSA5, with affinity varying by 1000-fold. Using a colorimetric sandwich assay to analyze 48 human saliva samples, we found that the trimeric aptamer assay (K d ≈10 pM) can identify the SARS-CoV-2 infection much more accurately than the dimeric aptamer assay (K d ≈100 pM) and monomeric aptamer assay (K d ≈10,000 pM). Based on the experimental data, we theoretically predict the levels of affinity an aptamer needs to possess to achieve 80-100 % sensitivity and 100 % specificity. The findings from this study highlight the need for deriving very high affinity aptamers to enable highly accurate detection of viral infection for future pandemics., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

The COVID-19 pandemic has highlighted the importance of point-of-care (POC) pathogen detection. Accurate and accessible diagnostic techniques for virus identification are crucial for controlling the spread of diseases and have profound implications for our communities and global health. Reagentless surface-enhanced Raman scattering (SERS) sensors offer a promising solution for POC testing due to their capability to function without complex processing steps. However, their application in this space is limited by the fact that these solution-based assays are challenging to administer, transport and store. To overcome these limitations, we employed a freeze-drying (lyophilization) process on reagentless SERS sensors and investigated their improved stability and shelf-life. We explored this mechanism using different concentrations of cryoprotectants. Lyophilized sensors were then tested in a mix-and-detect fashion by adding to the dry sensors a drop of the sample, consisting of saliva spiked with target DNA oligonucleotides relative to different SARS-CoV-2 variants. In addition, we further uncovered how lyophilization benefits sensors with a DNA-catalysis mechanism. In summary, our findings indicate that lyophilization substantially enhances the practicality and usability of reagentless SERS sensors, contributing to the translation of this powerful diagnostic tool to POC testing in remote areas with limited resources.

COVID-19 can range from a mild to severe acute respiratory syndrome and also could result in multisystemic damage. Additionally, many people develop post-acute symptoms associated with immune and metabolic disturbances in response to viral infection, requiring longitudinal and multisystem studies to understand the complexity of COVID-19 pathophysiology. Here, we conducted a 1 H Nuclear Magnetic Resonance metabolomics in saliva of symptomatic subjects presenting mild and moderate respiratory symptoms to investigate prospective changes in the metabolism induced after acute-phase SARS-CoV-2 infection. Saliva from 119 donors presenting non-COVID and COVID-19 respiratory symptoms were evaluated in the acute phase (T1) and the post-acute phase (T2). We found two clusters of metabolite fluctuation in the COVID-19 group. Cluster 1, metabolites such as glucose, (CH 3 ) 3 choline-related metabolites, 2-hydroxybutyrate, BCAA, and taurine increased in T2 relative to T1, and in cluster 2, acetate, creatine/creatinine, phenylalanine, histidine, and lysine decreased in T2 relative to T1. Metabolic fluctuations in the COVID-19 group were associated with overweight/obesity, vaccination status, higher viral load, and viral clearance of the respiratory tract. Our data unveil metabolic signatures associated with the transition to the post-acute phase of SARS-CoV-2 infection that may reflect tissue damage, inflammatory process, and activation of tissue repair cascade. Thus, they contribute to describing alterations in host metabolism that may be associated with prolonged symptoms of COVID-19.

Saliva has emerged as a promising diagnostic fluid for viral infections, enabling the direct analysis of viral genetic material and the detection of infection markers such as proteins, metabolites, microRNAs, and immunoglobulins. This comprehensive review aimed to explore the use of saliva as a diagnostic tool for viral infections, emphasizing its advantages and limitations. Saliva stands out due to its simplicity and safety in collection, along with the convenience of self-collection without the need for healthcare supervision, while potentially being comparable to urine and blood in terms of effectiveness. Herein, we highlighted the significant potential of saliva in assessing viral loads and diagnosing viral infections, such as herpesviruses, HPV, PyV, TTV, SARS-CoV-2, and MPXV. The detection of viral shedding in saliva underscores its utility in early diagnosis, the monitoring of infection progression, and evaluating treatment responses. The non-invasive nature of saliva collection makes it an appealing alternative to more invasive methods, promoting better patient compliance and facilitating large-scale screening and surveillance. As such, we further highlight current evidence on the use of saliva as a prognostic tool. Although a significant amount of data is already available, further investigations are warranted to more comprehensively assess the added benefit from the utilization of salivary biomarkers in the clinics. Salivary biomarkers show great promise for the early detection and prevention of viral infection complications, potentially improving disease management and control at the population level. Integrating these non-invasive tools into routine clinical practice could enhance personalized healthcare strategies and patient outcomes. Future studies should focus on establishing standardization protocols, validating the accuracy of salivary diagnostics, and expanding clinical research to enhance the diagnostic and monitoring capabilities of salivary biomarkers.

Omics approaches were extensively applied during the coronavirus disease 2019 (COVID-19) pandemic to understand the disease, identify biomarkers with diagnostic and prognostic value, and discover new molecular targets for medications. COVID-19 continues to challenge the healthcare system as the virus mutates, becoming more transmissible or adept at evading the immune system, causing resurgent epidemic waves over the last few years. In this study, we used saliva from volunteers who were negative and positive for COVID-19 when Omicron and its variants became dominant. We applied a direct solid-phase extraction approach followed by non-target metabolomics analysis to identify potential salivary signatures of hospital-recruited volunteers to establish a model for COVID-19 screening. Our model, which aimed to differentiate COVID-19-positive individuals from controls in a hospital setting, was based on 39 compounds and achieved high sensitivity (85%/100%), specificity (82%/84%), and accuracy (84%/92%) in training and validation sets, respectively. The salivary diagnostic signatures were mainly composed of amino acids and lipids and were related to a heightened innate immune antiviral response and an attenuated inflammatory profile. The higher abundance of thyrotropin-releasing hormone in the COVID-19 positive group highlighted the endocrine imbalance in low-severity disease, as first reported here, underscoring the need for further studies in this area.

Evaluating SARS-CoV-2 viral loads in nasopharyngeal (NP) and saliva samples, factors affecting viral loads, and the performance of rapid antigen testing (RAT) have not been comprehensively conducted during SARS-CoV-2 Omicron epidemic. This prospective study included outpatients enrolled during Omicron variant period in Japan. Paired NP swab and saliva samples were collected to measure viral loads by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The correlation between viral loads and clinical symptoms was examined. The performance of an immunochromatography-based RAT kit was also assessed. A total of 153 patients tested within 3 days of symptom onset were included. The mean viral load was 5.60 log 10 copies/test and 3.65 log 10 copies/test in NP and saliva samples, respectively, resulting in a significant difference ( P < 0.0001). Fever over 37°C (axillary temperature) and total number of symptoms other than fever were identified as independent factors positively correlated with the viral loads in both NP and saliva samples. RAT sensitivity using NP and saliva samples was 92% and 68%, respectively, using positive RT-qPCR results as the reference. The sensitivity of RAT using NP and saliva samples was significantly higher in patients with fever ≥37°C and/or at least one symptom than in those with fever <37°C and/or no symptoms (97% vs 83% in NP swabs; 80% vs 50% in saliva). Distinct symptoms, including fever ≥37°C, may reflect high Omicron variant viral loads. Rapid antigen testing, not only using nasopharyngeal swabs but also using saliva, would be useful for COVID-19 diagnosis as point-of-care testing, particularly for symptomatic patients., Importance: We examined nasopharyngeal and salivary viral loads using samples collected from outpatients with SARS-CoV-2 infection during the Omicron epidemic in Japan and explored the outpatient factors correlated with viral loads. In addition, we evaluated the performance of an authorized rapid antigen testing (RAT) kit using nasopharyngeal and saliva samples with RT-PCR testing as the reference. Intriguingly, a correlation between fever and other symptoms and SARS-CoV-2 viral loads in nasopharyngeal and saliva samples was observed based on one COVID-19 outpatient visit. RAT sensitivity was influenced by viral loads. Nevertheless, nasopharyngeal RAT is considered useful for SARS-CoV-2 point-of-care diagnosis. In patients with distinct symptoms, including high-grade fever, salivary RAT could be a practical diagnostic tool because of the higher estimated viral loads. After the Omicron epidemic, outpatients with mild COVID-19 have become the main focus of diagnosis and treatment. Our study provides valuable information regarding the point-of-care diagnosis of these patients., Competing Interests: The authors declare no conflict of interest.

With greater population density, the likelihood of viral outbreaks achieving pandemic status is increasing. However, current viral screening techniques use specific reagents, and as viruses mutate, test accuracy decreases. Here, we present the first real-time, reagent-free, portable analysis platform for viral detection in liquid saliva, using COVID-19 as a proof-of-concept. We show that vibrational molecular spectroscopy and machine learning (ML) detect biomolecular changes consistent with the presence of viral infection. Saliva samples were collected from 470 individuals, including 65 that were infected with COVID-19 (28 from hospitalized patients and 37 from a walk-in testing clinic) and 251 that had a negative polymerase chain reaction (PCR) test. A further 154 were collected from healthy volunteers. Saliva measurements were achieved in 6 minutes or less and led to machine learning models predicting COVID-19 infection with sensitivity and specificity reaching 90%, depending on volunteer symptoms and disease severity. Machine learning models were based on linear support vector machines (SVM). This platform could be deployed to manage future pandemics using the same hardware but using a tunable machine learning model that could be rapidly updated as new viral strains emerge.

Background: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants demonstrate predilection for different regions of the respiratory tract. While saliva-based reverse transcription-polymerase chain reaction (RT-PCR) testing is a convenient, cost-effective alternative to nasopharyngeal swabs (NPS), few studies to date have investigated whether saliva sensitivity differs across variants of concern., Methods: SARS-CoV-2 RT-PCR was performed on paired NPS and saliva specimens collected from individuals with acute coronavirus disease 2019 (COVID-19) symptoms or exposure to a COVID-19 household contact. Viral genome sequencing of NPS specimens and Los Angeles County surveillance data were used to determine the variant of infection. Saliva sensitivity was calculated using NPS-positive RT-PCR as the reference standard. Factors contributing to the likelihood of saliva SARS-CoV-2 RT-PCR positivity were evaluated with univariate and multivariable analyses., Results: Between June 2020 and December 2022, 548 saliva samples paired with SARS-CoV-2 positive NPS samples were tested by RT-PCR. Overall, saliva sensitivity for SARS-CoV-2 detection was 61.7% (95% CI, 57.6%-65.7%). Sensitivity was highest with Delta infection (79.6%) compared to pre-Delta (58.5%) and Omicron (61.5%) (P = 0.003 and 0.01, respectively). Saliva sensitivity was higher in symptomatic individuals across all variants compared to asymptomatic cases [pre-Delta 80.6% vs 48.3% (P < 0.001), Delta 100% vs 72.5% (P = 0.03), Omicron 78.7% vs 51.2% (P < 0.001)]. Infection with Delta, symptoms, and high NPS viral load were independently associated with 2.99-, 3.45-, and 4.0-fold higher odds of SARS-CoV-2 detection by saliva-based RT-PCR (P = 0.004, <0.001, and <0.001), respectively., Conclusions: As new variants emerge, evaluating saliva-based testing approaches may be crucial to ensure effective virus detection., (© Association for Diagnostics & Laboratory Medicine 2024. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Head and neck squamous cell carcinomas (HNSCCs) are linked to tobacco smoking, opium use, and human papillomavirus (HPV) infection. However, little is known about the association of HPV infection with risk factors of HNSCCs, including opium and tobacco use. This cross-sectional analysis of a national multi-center case-control study in Iran included 498 HNSCC cases and 242 controls. We investigated the association of opium and tobacco use with α- (n = 21), β- (n = 46), and γ-HPV (n = 52) types in saliva samples using type-specific bead-based multiplex genotyping assays (TS-MPG). We found that α-HPV positivity was significantly associated with tobacco smoking (OR = 10.35; 95% CI = 1.15, 93; p = .03), but not with opium use (OR = 1.06; 95% CI = 0.41, 2.76; p = .89). Additionally, tobacco smoking correlated with an elevated risk of β-species 2 HPV infection (OR = 1.28; 95% CI = 1.04, 1.58; p = .020). Conversely, opium use showed a positive association with γ-species 12 HPV infection (OR = 5.67; 95% CI = 1.43, 22.44; p = .013). These findings indicate that tobacco and opium use may influence the risk of HPV infection in different ways depending on the HPV genus and species. Further studies are needed to replicate these findings in other populations., (© 2024 UICC.)

Home testing technology strategy is critical for early screening of disease. However, current home testing technologies often require complex processes, which limits their application. In this study, a time-resolved cascade logic gate microfluidic chip (TCLMC) was revealed to enable capillary force-based one-step operation without manual intervention or professional equipment. By analogy with logic gates in the circuit, TCLMC could automatically control the fluid flow and regulate the incubation time to optimize the immunoassay. The limit of detection of TCLMC for SARS-CoV-2 and influenza B virus (Flu B) was 134.94 and 79.17 pg mL -1 within 10 min. Additionally, this study tested saliva samples from 12 Flu B patients and 24 healthy controls to verify its clinical application. The results showed that TCLMC had high sensitivity (100%), specificity (100%), and accuracy (100%). This study provides a new one-step strategy for home testing and demonstrates its great potential in the diagnosis field., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

In the past few years, the COVID-19 pandemic, caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) seriously threatens global public health security due to its high contagiousness. It remains of vital importance to develop a rapid and sensitive assay for SARS-CoV-2. In this work, we proposed a sandwich-type assay based on poly(N-isopropylacrylamide) (PNIPAM), allowing efficient detection of the SARS-CoV-2 S1 protein in the homogeneous solution. Firstly, a direct sandwich-type assay was established with a linear range of 0.2-2 μg/mL and a limit of detection (LOD) of 0.11 μg/mL, which could realize rapid detection in about 1 h. Furthermore, the sandwich-type assay coupled with rolling circle amplification (RCA) obtained an increase in sensitivity of 5.9 × 10 4 folds with a wide linear range of 0.01 - 100 ng/mL and a LOD of 1.88 pg/mL. The average recoveries in unpretreated saliva were 90 %-113.0 %, indicating the potential of the developed method for application in practical samples. Given the high selectivity and sensitivity of the developed method, it has a significant potential for rapid and early detection of SARS-CoV-2., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

COVID-19 is an infectious disease caused by the SARS-CoV-2 virus, which rapidly spread worldwide and resulted in a pandemic. Efficient and sensitive detection techniques have been devised since the onset of the epidemic and continue to be improved at present. Due to the crucial role of the SARS-CoV-2 S1 protein in facilitating the virus's entry into cells, efforts in detection and treatment have primarily centered upon this protein. In this study, a rapid, ultrasensitive, disposable, easy-to-use, cost-effective next generation biosensor based on optimized aptamer (Optimer, OPT) was developed by using a disposable pencil graphite electrode (PGE) and applied for the impedimetric determination of SARS-CoV-2 S1 protein. The S1 protein interacted with the OPT in the solution phase and then immobilized onto the PGE surface. Subsequently, measurements using electrochemical impedance spectroscopy (EIS) were conducted in a solution containing a redox probe of 1 mM [Fe(CN) 6 ] 3-/4- . Under optimum conditions, the limit of detection (LOD) for the S1 protein in buffer medium at concentrations ranging from 10 1 to 10 6 ag/mL was calculated as 8.80 ag/mL (0.11 aM). The selectivity of the developed biosensor was studied against MERS-CoV-S1 protein (MERS) and Influenza Hemagglutinin antigen (HA). Furthermore, the application of the biosensor in artificial saliva medium is demonstrated. The LOD was also calculated in artificial saliva medium in the concentration range of 10 1 -10 5 ag/mL and calculated as 2.01 ag/mL (0.025 aM). This medium was also used to assess the selectivity of optimized-aptamer based biosensor., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The COVID-19 pandemic caught the world unprepared. Large-scale testing efforts were urgently needed, and diagnostic strategies had to rapidly evolve in response to unprecedented worldwide demand. However, the rollout of diagnostic testing and screening for SARS-CoV-2 was often impeded by logistical challenges, including regulatory delays, workforce shortages, laboratory bottlenecks, and supply chain disruptions. Recognizing these hurdles early on, we developed a testing approach that supported frequent, repeat testing, particularly as communities reopened. We hypothesized and experimentally demonstrated that saliva was a suitable specimen for the detection of SARS-CoV-2. This finding was advanced into the development of open-source, extraction-free reverse transcription polymerase chain reaction protocols using readily available, "off-the-shelf" reagents and equipment for the direct detection of SARS-CoV-2 in saliva ("SalivaDirect''). Working with the US Food and Drug Administration (FDA), we established a novel regulatory framework wherein the FDA granted Emergency Use Authorization to Yale University to offer the SalivaDirect test protocol to high-complexity diagnostic laboratories (as designated by the Clinical Laboratory Improvement Amendments) with quality oversight provided by Yale. This grew into a network of more than 200 labs across the United States that, as of May 2024, resulted in over 6.5 million SARS-CoV-2 tests. By making the protocol flexible and open-source, laboratories were able to rapidly and economically scale testing using a simple, self-collected saliva specimen. Additionally, fostering a national network of laboratories enabled real-time exchanges, problem solving, and the development of community best practices. Preparing for the next pandemic, or simply the next seasonal epidemic, the SalivaDirect model of deploying a readily available, expandable solution and accompanying network provides a proven method for the successful implementation of pathogen testing in the United States and globally., Competing Interests: ALW has received consulting and/or advisory board fees from Pfizer, Merck, Diasorin, PPS Health, Co-Diagnostics, and Global Diagnostic Systems for work unrelated to this project and is the principal investigator on research grants from Merck, NIH RADx UP, and SalivaDirect, Inc. to Yale University and from Balvi.io, and Shield T3 to SalivaDirect, Inc. Authors BC, LB, and YA were employed by SalivaDirect, Inc. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Wyllie, Choate, Burke and Ali.)

Bats are known reservoirs for a wide range of pathogenic microorganisms, including viruses, bacteria, fungi, helminths, and protozoa, which can be transmitted and infect other zoonotic organisms. Various studies have utilised next-generation sequencing (NGS) to describe the pathogens associated with bats. Although most have characterised microbial communities in specific body fluids, few have analysed the composition and diversity of these microbial communities across different body fluids at the individual level. In this study, we employed two next-generation sequencing techniques: amplicon-based sequencing of the V4 hypervariable region of the 16S- and 18S-rRNA genes and viral metagenomics, to describe the prokaryotic, eukaryotic, and viral communities present in blood, faeces, and oral swab samples collected from two genera of bats (Carollia and Phyllostomus) in the department of Casanare, eastern Colombia. A total of 60 samples corresponding to the three bodily fluids were processed and analysed. The results indicated that the microbial communities across the body fluids were mainly composed of bacteria, fungi, protozoa, and various DNA and RNA viruses, showing a variability of microbial genera and species. The abundances, diversity metrics, and correlations of these microorganisms displayed patterns associated with bat genus and body fluids, suggesting that the ecological characteristics of these microbial communities may be influenced by the ecological and physiological traits of the bats. Additionally, we found similar community compositions of bacteria, some fungal genera, and viruses in the three body fluids, indicating a possible circulation of these microbes within the same bat. This could be due to microbial movement from the gut microbiota to other physiological systems or transmission via blood-feeding vectors. Furthermore, our results revealed the presence of various microbes of public health concern, including Bartonella spp., Mannheimia haemolytica, Rhodotorula spp., Piroplasmida spp., Toxoplasma gondii, Alphacoronavirus spp., and Bat circovirus. The abundance of these pathogenic microbial species across the three bodily fluids suggests potential transmission routes from bats to other organisms, which may contribute to the emergence of zoonotic disease outbreaks. These findings highlight the variability of microorganisms present within the same bat and the different pathogen-host interactions that may regulate the presence and transmission of these zoonotic microbes. Further research is required to elucidate the genomic features, ecological interactions, and biological activities of these microbial communities in bats., (© 2024. The Author(s).)