Whole Blood as a Sample Matrix in Homogeneous Time-Resolved Assay-Förster Resonance Energy Transfer-Based Antibody Detection.

The protein-L-utilizing Förster resonance energy transfer (LFRET) assay enables mix-and-read antibody detection, as demonstrated for sera from patients with, e.g., severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Zika virus, and orthohantavirus infections. In this study, we compared paired serum and whole blood (WB) samples of COVID-19 patients and SARS-CoV-2 vaccine recipients. We found that LFRET also detects specific antibodies in WB samples. In 44 serum-WB pairs from patients with laboratory-confirmed COVID-19, LFRET showed a strong correlation between the sample materials. By analyzing 89 additional WB samples, totaling 133 WB samples, we found that LFRET results were moderately correlated with enzyme-linked immunosorbent assay results for samples collected 2 to 14 months after receiving COVID-19 diagnosis. However, the correlation decreased for samples >14 months after receiving a diagnosis. When comparing the WB LFRET results to neutralizing antibody titers, a strong correlation emerged for samples collected 1 to 14 months after receiving a diagnosis. This study also highlights the versatility of LFRET in detecting antibodies directly from WB samples and suggests that it could be employed for rapidly assessing antibody responses to infectious agents or vaccines.

Diagnostic accuracy of DPP Fever Panel II Asia tests for tropical fever diagnosis.

BACKGROUND: Fever is the most frequent symptom in patients seeking care in South and Southeast Asia. The introduction of rapid diagnostic tests (RDTs) for malaria continues to drive patient management and care. Malaria-negative cases are commonly treated with antibiotics without confirmation of bacteraemia. Conventional laboratory tests for differential diagnosis require skilled staff and appropriate access to healthcare facilities. In addition, introducing single-disease RDTs instead of conventional laboratory tests remains costly. To overcome some of the delivery challenges of multiple separate tests, a multiplexed RDT with the capacity to diagnose a diverse range of tropical fevers would be a cost-effective solution. In this study, a multiplex lateral flow immunoassay (DPP Fever Panel II Assay) that can detect serum immunoglobulin M (IgM) and specific microbial antigens of common fever agents in Asia (Orientia tsutsugamushi, Rickettsia typhi, Leptospira spp., Burkholderia pseudomallei, Dengue virus, Chikungunya virus, and Zika virus), was evaluated. METHODOLOGY/PRINCIPAL FINDINGS: Whole blood (WB) and serum samples from 300 patients with undefined febrile illness (UFI) recruited in Vientiane, Laos PDR were tested using the DPP Fever Panel II, which consists of an Antibody panel and Antigen panel. To compare reader performance, results were recorded using two DPP readers, DPP Micro Reader (Micro Reader 1) and DPP Micro Reader Next Generation (Micro Reader 2). WB and serum samples were run on the same fever panel and read on both micro readers in order to compare results. ROC analysis and equal variance analysis were performed to inform the diagnostic validity of the test compared against the respective reference standards of each fever agent (S1 Table). Overall better AUC values were observed in whole blood results. No significant difference in AUC performance was observed when comparing whole blood and serum sample testing, except for when testing for R. typhi IgM (p = 0.04), Leptospira IgM (p = 0.02), and Dengue IgG (p = 0.03). Linear regression depicted R2 values had ~70% agreement across WB and serum samples, except when testing for leptospirosis and Zika, where the R2 values were 0.37 and 0.47, respectively. No significant difference was observed between the performance of Micro Reader 1 and Micro Reader 2, except when testing for the following pathogens: Zika IgM, Zika IgG, and B pseudomallei CPS Ag. CONCLUSIONS/SIGNIFICANCE: These results demonstrate that the diagnostic accuracy of the DPP Fever Panel II is comparable to that of commonly used RDTs. The optimal cut-off would depend on the use of the test and the desired sensitivity and specificity. Further studies are required to authenticate the use of these cut-offs in other endemic regions. This multiplex RDT offers diagnostic benefits in areas with limited access to healthcare and has the potential to improve field testing capacities. This could improve tropical fever management and reduce the public health burden in endemic low-resource areas.

Design, development, and validation of multi-epitope proteins for serological diagnosis of Zika virus infections and discrimination from dengue virus seropositivity.

Zika virus (ZIKV), an arbovirus from the Flaviviridae family, is the causative agent of Zika fever, a mild and frequent oligosymptomatic disease in humans. Nonetheless, on rare occasions, ZIKV infection can be associated with Guillain-Barré Syndrome (GBS), and severe congenital complications, such as microcephaly. The oligosymptomatic disease, however, presents symptoms that are quite similar to those observed in infections caused by other frequent co-circulating arboviruses, including dengue virus (DENV). Moreover, the antigenic similarity between ZIKV and DENV, and even with other members of the Flaviviridae family, complicates serological testing due to the high cross-reactivity of antibodies. Here, we designed, produced in a prokaryotic expression system, and purified three multiepitope proteins (ZIKV-1, ZIKV-2, and ZIKV-3) for differential diagnosis of Zika. The proteins were evaluated as antigens in ELISA tests for the detection of anti-ZIKV IgG using ZIKV- and DENV-positive human sera. The recombinant proteins were able to bind and detect anti-ZIKV antibodies without cross-reactivity with DENV-positive sera and showed no reactivity with Chikungunya virus (CHIKV)- positive sera. ZIKV-1, ZIKV-2, and ZIKV-3 proteins presented 81.6%, 95%, and 66% sensitivity and 97%, 96%, and 84% specificity, respectively. Our results demonstrate the potential of the designed and expressed antigens in the development of specific diagnostic tests for the detection of IgG antibodies against ZIKV, especially in regions with the circulation of multiple arboviruses.

Ophthalmologic Findings in a Premature Infant leading to a Zika Diagnosis during the COVID-19 Pandemic: A Case Report.

We report on the first case of congenital Zika syndrome to be identified during the COVID-19 pandemic in Puerto Rico. The Zika virus (ZIKV) infection was first seen in Puerto Rico in December 2015. It is a flavivirus with vertical transmission, spreading from infected mothers to their fetuses and having a broad spectrum of clinical manifestations, of which microcephaly is the most worrisome. In Puerto Rico, routine ZIKV screening during pregnancy was implemented in October 2016. However, this practice has become less frequent over time. Nevertheless, the transmission of ZIKV continues, so it is important to ensure routine ZIKV screening in endemic regions, such as Puerto Rico.

Multiplex Serology for Sensitive and Specific Flavivirus IgG Detection: Addition of Envelope Protein Domain III to NS1 Increases Sensitivity for Tick-Borne Encephalitis Virus IgG Detection.

Tick-borne encephalitis is a vaccine-preventable disease of concern for public health in large parts of Europe, with EU notification rates increasing since 2018. It is caused by the orthoflavivirus tick-borne encephalitis virus (TBEV) and a diagnosis of infection is mainly based on serology due to its short viremic phase, often before symptom onset. The interpretation of TBEV serology is hampered by a history of orthoflavivirus vaccination and by previous infections with related orthoflaviviruses. Here, we sought to improve TBEV sero-diagnostics using an antigen combination of in-house expressed NS1 and EDIII in a multiplex, low-specimen-volume set-up for the detection of immune responses to TBEV and other clinically important orthoflaviviruses (i.e., West Nile virus, dengue virus, Japanese encephalitis virus, Usutu virus and Zika virus). We show that the combined use of NS1 and EDIII results in both a specific and sensitive test for the detection of TBEV IgG for patient diagnostics, vaccination responses and in seroprevalence studies. This novel approach potentially allows for a low volume-based, simultaneous analysis of IgG responses to a range of orthoflaviviruses with overlapping geographic circulations and clinical manifestations.

Quantitative detection of chikungunya, Zika, and dengue viruses by one-step real-time PCR in different cell substrates.

Chikungunya (CHIKV), Zika (ZIKV), and dengue viruses (DENV) are vector-borne pathogens that cause emerging and re-emerging epidemics throughout tropical and subtropical countries. The symptomatology is similar among these viruses and frequently co-circulates in the same areas, making the diagnosis arduous. Although there are different methods for detecting and quantifying pathogens, real-time reverse transcription-polymerase chain reaction (real-time RT-qPCR) has become a leading technique for detecting viruses. However, the currently developed assays frequently involve probes and high-cost reagents, limiting access in low-income countries. Therefore, this study aims to design and evaluate a quantitative one-step RT-qPCR assay to detect CHIKV, ZIKV, and DENV with high specificity, reproducibility, and low cost in multiple cell substrates. We established a DNA intercalating green dye-based RT-qPCR test that targets nsP1 of CHIKV, and NS5 gene of ZIKV, and DENV for the amplification reaction. The assay exhibited a high specificity confirmed by the melting curve analysis. No cross-reactivity was observed between the three viruses or unspecific amplification of host RNA. The sensitivity of the reaction was evaluated for each virus assay, getting a limit of detection of one RNA copy per virus. Standard curves were constructed, obtaining a reaction efficiency of ~ 100%, a correlation coefficient (R2) of ~ 0.97, and a slope of -3.3. The coefficient of variation (CV) ranged from 0.02 to 1.43. In addition, the method was optimized for viral quantification and tested in Vero, BHK-21, C6/36, LULO, and the Aedes cell lines. Thus, the DNA intercalating green dye-based RT-qPCR assay was a highly specific, sensitive, reproducible, and effective method for detecting and quantifying CHIKV, ZIKV, and DENV in different cell substrates that could also be applied in clinical samples.

Noninvasive detection of Zika virus in mosquito excreta sampled from wild mosquito populations in French Guiana.

Arboviruses can be difficult to detect in the field due to relatively low prevalence in mosquito populations. The discovery that infected mosquitoes can release viruses in both their saliva and excreta gave rise to low-cost methods for the detection of arboviruses during entomological surveillance. We implemented both saliva and excreta-based entomological surveillance during the emergence of Zika virus (ZIKV) in French Guiana in 2016 by trapping mosquitoes around households of symptomatic cases with confirmed ZIKV infection. ZIKV was detected in mosquito excreta and not in mosquito saliva in 1 trap collection out of 85 (1.2%). One female Ae. aegypti L. (Diptera: Culicidae) was found with a ZIKV systemic infection in the corresponding trap. The lag time between symptom onset in a ZIKV-infected individual living near the trap site and ZIKV detection in this mosquito was 1 wk. These results highlight the potential of detection in excreta from trapped mosquitoes as a sensitive and cost-effective method to non invasively detect arbovirus circulation.

Detection of arboviruses in Aedes aegypti through transovarian analysis: A study in Goiânia, Goiás.

BACKGROUND: Arboviral diseases are a group of infectious diseases caused by viruses transmitted by arthropods, mainly mosquitoes. These diseases, such as those caused by the dengue (DENV), Zika (ZIKV), chikungunya (CHIKV), and yellow fever (YFV) viruses, have a significant impact worldwide. In this context, entomological surveillance plays a crucial role in the control and prevention of arboviruses by providing essential information on the presence, distribution, and activity of vector mosquitoes. Based on entomological surveillance, transovarian transmission provides information regarding the maintenance and dissemination of arboviruses. The objective of this study was to detect these arboviruses in Goiânia, Goiás, and analyze the occurrence of transovarian transmission. METHODS: Aedes aegypti eggs were collected from different regions of Goiânia and cultivated under controlled laboratory conditions until the emergence of adult mosquitoes. Adult females were grouped into pools containing their heads and thoraxes. These pools were subsequently evaluated using reverse-transcription quantitative polymerase chain reaction (RT-qPCR) assay. RESULTS: A total of 157 pools (N=1570) were analyzed, with two pools testing positive for CHIKV and one pool testing positive for ZIKV, indicating that the offspring resulting from transovarian transmission are potentially infectious. CONCLUSIONS: In summary, the demonstration of the vertical transmission mechanisms of CHIKV and ZIKV in A. aegypti serves as an alert to health authorities, as these diseases are still underreported, and their primary urban vector has likely acquired this capacity, contributing to the dissemination of these infections.

Breeding habitats, bionomics and phylogenetic analysis of Aedes aegypti and first detection of Culiseta longiareolata, and Ae. hirsutus in Somali Region, eastern Ethiopia.

INTRODUCTION: Arboviral diseases, such as dengue, chikungunya, yellow fever, and Zika, are caused by viruses that are transmitted to humans through mosquito bites. However, the status of arbovirus vectors in eastern Ethiopia is unknown. The aim of this study was to investigate distribution, breeding habitat, bionomics and phylogenetic relationship of Aedes aegypti mosquito species in Somali Regional State, Eastern Ethiopia. METHODS: Entomological surveys were conducted in four sites including Jigjiga, Degehabur, Kebridehar and Godey in 2018 (October to December) to study the distribution of Ae. aegypti and with a follow-up collection in 2020 (July-December). In addition, an investigation into the seasonality and bionomics of Ae. aegypti was conducted in 2021 (January-April) in Kebridehar town. Adult mosquitoes were collected from indoor and outdoor locations using CDC light traps (LTs), pyrethrum spray collection (PSCs), and aspirators. Larvae and pupae were also collected from a total of 169 water-holding containers using a dipper between October and November 2020 (rainy season) in Kebridehar town. The species identification of wild caught and reared adults was conducted using a taxonomic key. In addition, species identification using mitochondrial and nuclear genes maximum likelihood-based phylogenetic analysis was performed. RESULTS: In the 2018 collection, Ae. aegypti was found in all study sites (Jigjiga, Degahabour, Kebridehar and Godey). In the 2020-2021 collection, a total of 470 (Female = 341, Male = 129) wild caught adult Ae. aegypti mosquitoes were collected, mostly during the rainy season with the highest frequency in November (n = 177) while the lowest abundance was in the dry season (n = 14) for both February and March. The majority of Ae. aegypt were caught using PSC (n = 365) followed by CDC LT (n = 102) and least were collected by aspirator from an animal shelter (n = 3). Aedes aegypti larval density was highest in tires (0.97 larvae per dip) followed by cemented cisterns (0.73 larvae per dip) and the Relative Breeding Index (RBI) was 0.87 and Container Index (CI) was 0.56. Genetic analysis of ITS2 and COI revealed one and 18 haplotypes, respectively and phylogenetic analysis confirmed species identification. The 2022 collection revealed no Ae. aegpti, but two previously uncharacterized species to that region. Phylogenetic analysis of these two species revealed their identities as Ae. hirsutus and Culiseta longiareolata. CONCLUSION: Data from our study indicate that, Ae. aegypti is present both during the wet and dry seasons due to the availability of breeding habitats, including water containers like cemented cisterns, tires, barrels, and plastic containers. This study emphasizes the necessity of establishing a national entomological surveillance program for Aedes in Somali region.

Detection of anti-premembrane antibody as a specific marker of four flavivirus serocomplexes and its application to serosurveillance in endemic regions.

In the past few decades, several emerging/re-emerging mosquito-borne flaviviruses have resulted in disease outbreaks of public health concern in the tropics and subtropics. Due to cross-reactivities of antibodies recognizing the envelope protein of different flaviviruses, serosurveillance remains a challenge. Previously we reported that anti-premembrane (prM) antibody can discriminate between three flavivirus infections by Western blot analysis. In this study, we aimed to develop a serological assay that can discriminate infection or exposure with flaviviruses from four serocomplexes, including dengue (DENV), Zika (ZIKV), West Nile (WNV) and yellow fever (YFV) viruses, and explore its application for serosurveillance in flavivirus-endemic countries. We employed Western blot analysis including antigens of six flaviviruses (DENV1, 2 and 4, WNV, ZIKV and YFV) from four serocomplexes. We tested serum samples from YF-17D vaccinees, and from DENV, ZIKV and WNV panels that had been confirmed by RT-PCR or by neutralization assays. The overall sensitivity/specificity of anti-prM antibodies for DENV, ZIKV, WNV, and YFV infections/exposure were 91.7%/96.4%, 91.7%/99.2%, 88.9%/98.3%, and 91.3%/92.5%, respectively. When testing 48 samples from Brazil, we identified multiple flavivirus infections/exposure including DENV and ZIKV, DENV and YFV, and DENV, ZIKV and YFV. When testing 50 samples from the Philippines, we detected DENV, ZIKV, and DENV and ZIKV infections with a ZIKV seroprevalence rate of 10%, which was consistent with reports of low-level circulation of ZIKV in Asia. Together, these findings suggest that anti-prM antibody is a flavivirus serocomplex-specific marker and can be employed to delineate four flavivirus infections/exposure in regions where multiple flaviviruses co-circulate.

Development of a quantitative NS1 antigen enzyme-linked immunosorbent assay (ELISA) for Zika virus detection using a novel virus-specific mAb.

Viruses from the Flaviviridae family, such as Dengue virus (DENV), Yellow fever virus (YFV), and Zika virus (ZIKV) are notorious global public health problems. ZIKV emergence in Polynesia and the Americas from 2013 to 2016 raised concerns as new distinguishing features set it apart from previous outbreaks, including its association with neurological complications and heightened disease severity. Virus detection is impaired as cross-reactivity to other closely related orthoflaviviruses is common among commercially available diagnostic kits. While non-structural protein 1 (NS1) has been used as an early marker of DENV and West Nile virus (WNV) infection, little is known about NS1 expression during ZIKV infection. In the present work, we developed a NS1 capture ELISA using a novel ZIKV-specific monoclonal antibody to study NS1 expression dynamics in vitro in mosquito and human cell lines. While detectable in culture supernatants, higher concentrations of NS1 were predominantly cell-associated. To our knowledge, this is the first report of NS1 detection in human cells despite viral clearance over time. Tests with human samples need to be conducted to validate the applicability of NS1 detection for diagnosis, but overall, the tools developed in this work are promising for specific detection of acute ZIKV infection.

ICP-MS and fluorescence dual-mode detection of ZIKV-RNA based on quantum dot labeling with hybridization chain reaction.

The detection of Zika virus (ZIKV) is of great significance to human life and health. Herein, we presented an ICP-MS and fluorescent dual-mode sensor for quantitative analysis of Zika virus RNA fragments (ZIKV-RNA), which employed quantum dots (QDs) as signal tags and combined with hybridization chain reaction (HCR). The dual-mode sensor realized cross-checking of the analysis results and improved the assay accuracy. Firstly, the single-stranded DNA (ssDNA) was anchored on the surface of magnetic beads (MBs). Afterward, HCR was conducted with probe DNA-CdSe quantum dots conjugates (pDNA-QDs) and link DNA (lDNA), producing the MBs-ssDNA-[pDNA-QDs-lDNA]n conjugates. In the presence of target ZIKV-RNA, a strand displacement reaction occurred, leading to the dissociation of the [pDNA-QDs-lDNA]n labels from the conjugates into the solution. Finally, the signal intensity was detected using ICP-MS and fluorescence analysis, with achieved limits of detection of 131 pM and 152 pM, respectively. The inter-assay RSD values of fluorescence and ICP-MS were 3.94 % and 4.26 % at 10 nM level, respectively, showing that the method had good precision. This method showed high selectivity and was applied to the analysis of biological fluids. There was no significant difference between the results of ICP-MS modes and fluorescence mode. This method offers a new strategy for sensitivity analysis of ZIKV-RNA and exhibits promise in clinical applications for diagnosis.

Molecular detection of dengue and chikungunya viruses in surveillance of wild-caught Aedes mosquitoes in Punjab, North India.

BACKGROUND: Dengue (DENV), Zika (ZIKV) and chikungunya (CHIKV) viruses are transmitted mainly by Aedes mosquitoes and are responsible for a significant global healthcare burden. The current study aimed to detect arboviruses in the Aedes mosquitoes in close proximity of patients during the transmission season. METHODS: Both immature and adult mosquitoes were collected from in and around the patients' houses. Mosquito pools were homogenized and extracted RNA was subjected to reverse transcription polymerase chain reaction for arboviral detection. Transovarian transmission (TOT) was assessed by screening F0 adults. Mosquito positivity was correlated with the aetiological agents identified in patients. RESULTS: Of 46 pools, 19 consisted of wild Aedes, with arboviral positivity in 53% (10/19) of pools. Among wild A. aegypti pools, positivity of DENV mono-infection, CHIKV mono-infection and DENV+CHIKV co-infection was noted in four, two and three pools, respectively. One wild pool of Aedes albopictus was positive for DENV-1. Similarly, A. aegypti F0 (adult Aedes developed from immatures) pools showed 59.2% (16/27) positivity for arboviruses. F0 Aedes showed positivity in three, six and seven pools for DENV-2, CHIKV and DENV+CHIKV, respectively, suggestive of TOT. DENV serotypes and CHIKV from 24 patients' serum samples were matched with strains isolated from Aedes and correlation was observed in four instances. CONCLUSIONS: The study detected DENV and CHIKV from wild-caught Aedes and found evidence of DENV and CHIKV TOT in F0 adults.

Optimal 2D audio features estimation for a lightweight application in mosquitoes species: Ecoacoustics detection and classification purposes.

Mosquitoes are the vector of diseases that kill more than one million people per year worldwide. Surveillance systems are essential for understanding their complex ecology and behaviour. This is fundamental for predicting disease risk caused by mosquitoes and formulating effective control strategies against mosquito-borne diseases such as malaria, dengue, and Zika. Mosquito populations vary heterogeneously in urban and rural landscapes, fluctuating with seasonal and climatic trends and human activity. Several approaches provide environmental data for mosquito mapping and risk prediction. However, they rely traditionally upon labour-intensive techniques such as manual traps. This paper presents the optimal audio features for mosquito identification using ecoacoustics signals to automatically identify different mosquito species from their wingbeat sounds based on popular audio features. The audio selection method uses Density-Based Spatial Clustering of Applications with Noise (DBSCAN) and Silhouette coefficient to evaluate the clusters in the data through the optimal-combined audio features. To classify the mosquito species and distinguish them from environmental-urban noise, the method comprises the Gaussian Mixture Model (GMM) and Gibbs approach for Aedes aegypti, and Culex quinquefasciatus, using the acoustic recordings of their wingbeat signals. Finally, comparing GMM and Gibbs, the two have very similar accuracy, but the classification time is much faster for Gibbs sampling, making it a good candidate for a lightweight solution. These are essential when deploying the described models to monitor mosquito vectors in the wild with Internet of Things (IoT) technologies.

Laboratory diagnosis of CNS infections in children due to emerging and re-emerging neurotropic viruses.

Recent decades have witnessed the emergence and re-emergence of numerous medically important viruses that cause central nervous system (CNS) infections in children, e.g., Zika, West Nile, and enterovirus/parechovirus. Children with immature immune defenses and blood-brain barrier are more vulnerable to viral CNS infections and meningitis than adults. Viral invasion into the CNS causes meningitis, encephalitis, brain imaging abnormalities, and long-term neurodevelopmental sequelae. Rapid and accurate detection of neurotropic viral infections is essential for diagnosing CNS diseases and setting up an appropriate patient management plan. The addition of new molecular assays and next-generation sequencing has broadened diagnostic capabilities for identifying infectious meningitis/encephalitis. However, the expansion of test menu has led to new challenges in selecting appropriate tests and making accurate interpretation of test results. There are unmet gaps in development of rapid, sensitive and specific molecular assays for a growing list of emerging and re-emerging neurotropic viruses. Herein we will discuss the advances and challenges in the laboratory diagnosis of viral CNS infections in children. This review not only sheds light on selection and interpretation of a suitable diagnostic test for emerging/re-emerging neurotropic viruses, but also calls for more research on development and clinical utility study of novel molecular assays. IMPACT: Children with immature immune defenses and blood-brain barrier, especially neonates and infants, are more vulnerable to viral central nervous system infections and meningitis than adults. The addition of new molecular assays and next-generation sequencing has broadened diagnostic capabilities for identifying infectious meningitis and encephalitis. There are unmet gaps in the development of rapid, sensitive and specific molecular assays for a growing list of emerging and re-emerging neurotropic viruses.

A minimally invasive, field-applicable CRISPR/Cas biosensor to aid in the detection of Pseudogymnoascus destructans, the causative fungal agent of white-nose syndrome in bats.

The accessibility to CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein) genetic tools has given rise to applications beyond site-directed genome editing for the detection of DNA and RNA. These tools include precise diagnostic detection of human disease pathogens, such as SARS-CoV-2 and Zika virus. Despite the technology being rapid and cost-effective, the use of CRISPR/Cas tools in the surveillance of the causative agents of wildlife diseases has not been prominent. This study presents the development of a minimally invasive, field-applicable and user-friendly CRISPR/Cas-based biosensor for the detection of Pseudogymnoascus destructans (Pd), the causative fungal agent of white-nose syndrome (WNS), an infectious disease that has killed more than five million bats in North America since its discovery in 2006. The biosensor assay combines a recombinase polymerase amplification (RPA) step followed by CRISPR/Cas12a nuclease cleavage to detect Pd DNA from bat dermal swab and guano samples. The biosensor had similar detection results when compared to quantitative PCR in distinguishing Pd-positive versus negative field samples. Although bat dermal swabs could be analysed with the biosensor without nucleic acid extraction, DNA extraction was needed when screening guano samples to overcome inhibitors. This assay can be applied to help with more rapid delineation of Pd-positive sites in the field to inform management decisions. With further optimization, this technology has broad translation potential to wildlife disease-associated pathogen detection and monitoring applications.

Detection of arboviruses in Aedes aegypti through transovarian analysis: A study in Goiânia, Goiás

Abstract Background: Arboviral diseases are a group of infectious diseases caused by viruses transmitted by arthropods, mainly mosquitoes. These diseases, such as those caused by the dengue (DENV), Zika (ZIKV), chikungunya (CHIKV), and yellow fever (YFV) viruses, have a significant impact worldwide. In this context, entomological surveillance plays a crucial role in the control and prevention of arboviruses by providing essential information on the presence, distribution, and activity of vector mosquitoes. Based on entomological surveillance, transovarian transmission provides information regarding the maintenance and dissemination of arboviruses. The objective of this study was to detect these arboviruses in Goiânia, Goiás, and analyze the occurrence of transovarian transmission. Methods: Aedes aegypti eggs were collected from different regions of Goiânia and cultivated under controlled laboratory conditions until the emergence of adult mosquitoes. Adult females were grouped into pools containing their heads and thoraxes. These pools were subsequently evaluated using reverse-transcription quantitative polymerase chain reaction (RT-qPCR) assay. Results: A total of 157 pools (N=1570) were analyzed, with two pools testing positive for CHIKV and one pool testing positive for ZIKV, indicating that the offspring resulting from transovarian transmission are potentially infectious. Conclusions: In summary, the demonstration of the vertical transmission mechanisms of CHIKV and ZIKV in A. aegypti serves as an alert to health authorities, as these diseases are still underreported, and their primary urban vector has likely acquired this capacity, contributing to the dissemination of these infections.

Machine learning-driven SERS fingerprinting of disintegrated viral components for rapid detection of SARS-CoV-2 in environmental dust.

Surveillance of airborne viruses in crowded indoor spaces is crucial for managing outbreaks, as highlighted by the SARS-CoV-2 pandemic. However, the rapid and on-site detection of fast-mutating viruses, such as SARS-CoV-2, in complex environmental backgrounds remains challenging. Our study introduces a machine learning (ML)-driven surface-enhanced Raman spectroscopy (SERS) approach for detecting viruses within environmental dust matrices. By decomposing intact virions into individual structural components via a Raman-background-free lysis protocol and concentrating them into nanogap SERS hotspots, we significantly enhance the SERS signal intensity and fingerprint information density from viral structural components. Utilizing Principal Component Analysis (PCA), we establish a robust connection between the SERS data of these structural components and their biological sequences, laying a solid foundation for virus detection through SERS. Furthermore, we demonstrate reliable quantitative detection of SARS-CoV-2 using identified SARS-CoV-2 peaks at concentrations down to 102 pfu/ml through Gaussian Process Regression (GPR) and a digital SERS methodology. Finally, applying a Principal Component Analysis-Linear Discriminant Analysis (PCA-LDA) algorithm, we identify SARS-CoV-2, influenza A virus, and Zika virus within an environmental dust background with over 86% accuracy. Therefore, our ML-driven SERS approach holds promise for rapid environmental virus monitoring to manage future outbreaks.

Development and pre-clinical evaluation of a Zika virus diagnostic for low resource settings.

Introduction: Zika virus (ZIKV) is a re-emerging flavivirus that poses a significant public health threat. ZIKV exhibits a wide array of non-vector borne human transmission routes, such as sexual transmission, transplacental transmission and blood transfusion. Detection and surveillance of ZIKV is considered paramount in prevention of major outbreaks. With the majority of cases reported in low-resource locations, simple, low-cost detection methods are considered highly desirable. Materials and Methods: Here we have developed a sensitive and specific ZIKV diagnostic using reverse transcription recombinase-aided amplification (RT-RAA) coupled with lateral flow detection (LFD) targeting a highly conserved region of the ZIKV NS1 gene. Results: We show our rapid, isothermal-ZIKV-diagnostic (Iso-ZIKV-Dx) can detect 500 copies of synthetic ZIKV RNA/µL in under 30 min at a constant 39°C. Using simulated urine samples, we observed that Iso-ZIKV-Dx also detects as low as 34.28 RNA copies/reaction of ZIKV (MR766 strain). Specificity testing confirmed that our test does not detect any co-circulating flaviviruses (dengue, West Nile, Japanese encephalitis, Murray Valley encephalitis and yellow fever viruses) or chikungunya virus. Sample processing results show complete inactivation of ZIKV (MR766 strain) in 5 min at room temperature using our novel viral RNA sample preparation reagent. Furthermore, lateral flow strips testing demonstrates positive diagnoses in as little as 5 min in running buffer. Discussion: Contrary to conventional RT-qPCR, our Iso-ZIKV-Dx does not require expensive machinery, specialised laboratory settings or extensively trained personnel. Pre-clinical evaluation demonstrates that our test exhibits robust, in-field capabilities without compromising sensitivity or specificity. When compared to the gold-standard RT-qPCR, our Iso-ZIKV-Dx test offers an array of applications that extend beyond diagnostics alone, including potential for surveillance and monitoring of ZIKV vector competency.

Detection and persistence of Zika virus in body fluids and associated factors: a prospective cohort study.

This study aimed to analyze the detection and duration of the Zika virus (ZIKV) in plasma, urine, saliva, sweat, rectal swabs, vaginal secretions, breast milk, and semen and to explore risk factors associated with prolonged viral persistence. A prospective cohort study of symptomatic patients and their household contacts was conducted in Brazil from July 2017 to June 2019. A total of 260 individuals (184 women and 76 men) with confirmed ZIKV infection were enrolled and followed up for 12 months. ZIKV RNA was present in all body fluid specimens and detectable for extended periods in urine, sweat, rectal swabs, and semen. The longest detection duration was found in semen, with high viral loads in the specimens. ZIKV RNA clearance was associated with several factors, including age, sex, education level, body mass index, non-purulent conjunctivitis, joint pain, and whether the participant had a history of yellow fever vaccination. The influence of each of these factors on the low or fast viral clearance varied according to the specific body fluid under investigation. Recurrent ZIKV detection events after total viral clearance were observed in the cohort. Our findings provide valuable insights into the persistence and potential recurrence of ZIKV infection, highlighting the need for continued monitoring and follow-up of individuals infected with ZIKV and for effective prevention measures to reduce the risk of transmission.