ETIDIOH - NEW

  Highlights •  A Nipah virus real-time RT-PCR was developed for this study and display dynamic amplification, with sensitive (limit of detection 3.7-4.2 copies/µL) and specific detection. •  The assay was adapted for use on a portable, battery-powered real-time thermocycler . •  When paired with instrument-free RNA extraction , Nipah virus RNA was rapidly detected from contrived whole blood and nasopharyngeal swabs without electricity. •  The combined of Extract & Store and the Palm PCR S1e device offers a viable solution for field-based molecular detection of Nipah virus. Abstract Background Nipah virus (NiV) is a highly pathogenic, zoonotic paramyxovirus with significant public health implications due to high associated mortality and potential for human-to-human transmission. Current diagnostic testing options for NiV are limited and require extensive laboratory infrastructure. Objective Develop a field-deployable testing workflow for timely NiV detection...

  Abstract Background :  Genetic drift and host-associated adaptation in influenza A viruses threaten the long-term reliability of RT-qPCR-based diagnostics , particularly when nucleotide mismatches arise within primer and probe binding regions . Conventional assay evaluations often emphasize sequence conservation but rarely assess functional mismatch tolerance across divergent subtypes and hosts.  Methods :  We performed an in silico evaluation of a matrix (M) gene–targeted RT-qPCR assay by aligning primer and probe binding regions against 22 H1N1 isolates and representative H3N2 and H5N1 reference strains, including recent zoonotic isolates from avian and bovine hosts . Nucleotide mismatches were identified, quantified, and mapped relative to assay components and oligonucleotide termini. Mismatch burden was summarized by subtype and assay region.  Results :  H1N1 isolates exhibited complete conservation across primer and probe regions. In contrast, H3N2 a...

  Highlights •  Concern over H5N1 bird flu testing and detection in the Netherlands is increasing. •  50 human laboratories in the Netherlands, Aruba, Bonaire , and Curacao were assessed. •  The laboratories detected animal influenza viruses with high performance. •  Few laboratories identified the animal subtype of detected influenza A viruses. •  National reference laboratory capacity to identify the animal subtype is critical. Abstract Background Since 2022, highly pathogenic H5N1 influenza A virus clade 2.3.4.4b has caused global outbreaks among wild birds and poultry , with increasing mammalian and sporadic human infections . This elevates concerns about zoonotic transmission and pandemic risk , highlighting the need for accurate detection and identification of animal influenza A viruses by human clinical diagnostic laboratories (hCDL). Methods To evaluate routine diagnostic performance , an External Quality Assessment (EQA) panel containing inactivate...

  Highlights •  Viremia in children with La Crosse Virus infection is transient; viral RNA was detected in only 3.2% of sera •  Detection of La Crosse Virus RNA in respiratory samples is slightly higher at 21.7% and may reflect the temporal distribution of the virus after infection •  NAAT has limited utility in routine diagnosis of La Crosse Virus encephalitis in children but may still be useful in cases with delayed seroconversion Abstract Background La Crosse virus (LACV), a member of family Peribunyaviridae, genus Orthobunyavirus , is the leading cause of neuroinvasive arboviral infection in children in the United States . Diagnosis relies on detecting specific antibodies (IgG or IgM), a 4-fold titer rise or seroconversion, in patients with compatible presentations. NAAT used for LACV detection has largely been limited to mosquito, animal models or postmortem brain tissue. There is a lack of data on the performance of NAATs in clinical specimens from living patie...

  Abstract We evaluated 2 electrochemiluminescence serologic assays to detect avian influenza H5 antibodies . Both assays identified H5 antibodies from both serum and dried blood spots and had strong specificity and minimal cross-reactivity in human and avian samples. Such assays can support populationwide serologic surveys aimed at assessing population-level immunity. Source:  Link:  https://wwwnc.cdc.gov/eid/article/32/1/25-1117_article ____

  Abstract The highly pathogenic avian influenza viruses (HPAIV) of subtype H5N1 represent a major threat to animal and public health . The current panzootic with H5 clade 2.3.4.4b has caused numerous, widespread outbreaks in various domestic and wild avian species with high mortalities, massive losses and a high frequency of spillover events to unexpected novel mammalian hosts such as dairy cows . The global H5N1 situation raises serious concerns about zoonotic risks due to effective mammal-to-mammal transmission . Therefore, it is critical to increase surveillance intensity of a broadened species range, particularly at the human-animal interface . For this purpose, reliable and cost-effective serological tools that are easy to perform and suitable for high-throughput screenings are critically needed. The newly developed double antigen ELISA format employing a luminescence-based detection technology has demonstrated to comply with such prerequisites. The assay allowed the detectio...

  Abstract Molnupiravir is an antiviral medicine that induces lethal copying errors during SARS-CoV-2 RNA replication . Molnupiravir reduced hospitalization in one pivotal trial by 50% and had variable effects on reducing viral RNA levels in three separate trials. We used mathematical models to simulate these trials and closely recapitulated their virologic outcomes . Model simulations suggested lower antiviral potency against pre-Omicron SARS-CoV-2 variants than against Omicron . We estimated that in vitro assays underestimated in vivo potency by 6- to 7-fold against Omicron variants. Our model suggested that because polymerase chain reaction detects molnupiravir mutated variants, the true reduction in non-mutated viral RNA was underestimated by approximately 0.4 log10 in the two trials conducted while Omicron variants dominated. Viral area under the curve estimates differed significantly between non-mutated and mutated viral RNA. Our results reinforce past work suggesting that in...

  Abstract Influenza A viruses (IAV) remain a persistent One Health threat, and whole-genome sequencing from wastewater offers a promising surveillance tool . However, IAV is at low abundance in wastewater , making it difficult to sequence. We benchmarked four targeted enrichment methods suited for whole-genome sequencing including custom and off-the-shelf amplicon and probe-based methods. Our custom HA tiled-amplicon panel was sensitive, fast, and cost-effective, making it suitable for monitoring low-abundance seasonal variants of known subtypes . However, its reliance on conserved and intact primer-binding sites limited primer design to fewer subtypes. A previously published universal amplicon method targeted all IAV subtypes , but it performed poorly in wastewater due to its reliance on intact genome segments. Probe-capture methods were resilient to RNA degradation and mismatches , potentially enabling broader surveillance and detection of emerging strains. However, probes were ...

  Highlights •  Most vector-borne viruses like alphaviruses are not included in routine diagnostics •  Lack of testing results in misdiagnoses and underdetection •  A new multiplexed real-time PCR assay detects all human pathogenic alphaviruses •  The new multiplex assay is more sensitive than available tests and highly specific •  The multiplex test can be applied broadly for diagnostics and molecular surveillance Abstract Background Knowledge of epidemiology, pathogenesis, and public health burden is scarce for many arthropod-borne viruses (arboviruses). Insufficient knowledge is partly due to lack of exhaustive laboratory diagnostics due to resource limitations . Among arboviruses , arthritogenic and encephalitogenic alphaviruses are globally widespread, can cause severe disease, and can co-occur regionally. Objectives We developed and validated a multiplexed real-time reverse transcription-PCR assay for the detection of all alphaviruses commonly causing...

Abstract Hendra virus (HeV) and Nipah virus (NiV) are two highly pathogenic RNA viruses with zoonotic potential, which can cause severe diseases with high mortality rates (50–100%) in humans and animals . Given this context, these viruses are classified as Biosafety Level 4 (BSL-4) pathogens , thus limiting research studies. Despite the high case fatalities , there are currently no human vaccines available for either virus , owing in part to the limitations in research and hesitancy in funding . In the absence of widespread vaccination, diagnostic tests are crucial for the rapid identification of cases and disease surveillance. This review synthesizes current knowledge on the epidemiology, transmission dynamics, and pathogenesis of NiV and HeV to contextualize a detailed assessment of the available diagnostic tools. We examined molecular and serological assays, including RT-PCR, ELISA, and LAMP , highlighting sample sources, detection windows, and performance. Diagnostic considerations...

Abstract Highly pathogenic avian influenza virus (HPAIV) of H5 and H7 subtypes has emerged as one of the most important zoonotic pathogens in the 21st century with significant economic consequences . The recent outbreak of H5N1 avian influenza (AI) in dairy cattle highlighted the importance of early detection in managing and mitigating HPAIV outbreaks. A successful high-speed diagnostic response requires rapid site and specimen access, minimal time for test protocols, and prompt communication of the diagnostic results to government officials. A new diagnostic paradigm that consists of miniaturized extractor and qPCR instruments (EZextractor and EZcycler MiniQ), designed for mobile, on-site testing has been compared with a platform of benchtop instruments (QIAGEN RNeasy and QuantStudio 5) for detecting inactivated H5 avian influenza virus (AIV) spiked in raw milk samples. Two sets of experiments were performed: 1) 15 raw milk samples , obtained from 15 different farms, diluted with phos...

Abstract Highly pathogenic avian influenza virus (HPAIV) of H5 and H7 subtypes has emerged as one of the most important zoonotic pathogens in the 21st century with significant economic consequences. The recent outbreak of H5N1 avian influenza (AI) in dairy cattle highlighted the importance of early detection in managing and mitigating HPAIV outbreaks. A successful high-speed diagnostic response requires rapid site and specimen access, minimal time for test protocols, and prompt communication of the diagnostic results to government officials. A new diagnostic paradigm that consists of miniaturized extractor and qPCR instruments (EZextractor and EZcycler MiniQ), designed for mobile, on-site testing has been compared with a platform of benchtop instruments (QIAGEN RNeasy and QuantStudio 5) for detecting inactivated H5 avian influenza virus (AIV) spiked in raw milk samples. Two sets of experiments were performed: 1) 15 raw milk samples, obtained from 15 different farms, diluted with phosph...