Estimating cutoff values for diagnostic tests to achieve target specificity using extreme value theory.

BACKGROUND: Rapidly developing tests for emerging diseases is critical for early disease monitoring. In the early stages of an epidemic, when low prevalences are expected, high specificity tests are desired to avoid numerous false positives. Selecting a cutoff to classify positive and negative test results that has the desired operating characteristics, such as specificity, is challenging for new tests because of limited validation data with known disease status. While there is ample statistical literature on estimating quantiles of a distribution, there is limited evidence on estimating extreme quantiles from limited validation data and the resulting test characteristics in the disease testing context. METHODS: We propose using extreme value theory to select a cutoff with predetermined specificity by fitting a Pareto distribution to the upper tail of the negative controls. We compared this method to five previously proposed cutoff selection methods in a data analysis and simulation study. We analyzed COVID-19 enzyme linked immunosorbent assay antibody test results from long-term care facilities and skilled nursing staff in Colorado between May and December of 2020. RESULTS: We found the extreme value approach had minimal bias when targeting a specificity of 0.995. Using the empirical quantile of the negative controls performed well when targeting a specificity of 0.95. The higher target specificity is preferred for overall test accuracy when prevalence is low, whereas the lower target specificity is preferred when prevalence is higher and resulted in less variable prevalence estimation. DISCUSSION: While commonly used, the normal based methods showed considerable bias compared to the empirical and extreme value theory-based methods. CONCLUSIONS: When determining disease testing cutoffs from small training data samples, we recommend using the extreme value based-methods when targeting a high specificity and the empirical quantile when targeting a lower specificity.

Electrochemical Immunoassay for the Detection of SARS-CoV-2 Nucleocapsid Protein in Nasopharyngeal Samples.

Point-of-care (POC) methods currently available for detecting SARS-CoV-2 infections still lack accuracy. Here, we report the development of a highly sensitive electrochemical immunoassay capable of quantitatively detecting the presence of the SARS-CoV-2 virus in patient nasopharyngeal samples using stencil-printed carbon electrodes (SPCEs) functionalized with capture antibodies targeting the SARS-CoV-2 nucleocapsid protein (N protein). Samples are added to the electrode surface, followed by horseradish peroxidase (HRP)-conjugated detection antibodies also targeting the SARS-CoV-2 N protein. The concentration of the virus in samples is quantified using chronoamperometry in the presence of 3,3'5,5'-tetramethylbenzidine. Limits of detection equivalent to less than 50 plaque forming units/mL (PFU/mL) were determined with virus sample volumes of 20 µL. No cross-reactivity was detected with the influenza virus and other coronavirus N proteins. Patient nasopharyngeal samples were tested as part of a proof-of-concept clinical study where samples were also tested using the gold-standard real-time quantitative polymerase chain reaction (RT-qPCR) method. Preliminary results from a data set of 22 samples demonstrated a clinical specificity of 100% (n = 9 negative samples according to RT-qPCR) and a clinical sensitivity of 70% for samples with RT-PCR cycle threshold (Ct) values under 30 (n = 10) and 100% for samples with Ct values under 25 (n = 5), which complies with the World Health Organization (WHO) criteria for POC COVID-19 diagnostic tests. Our functionalized SPCEs were also validated against standard plaque assays, and very good agreement was found between both methods (R2 = 0.9993, n = 6), suggesting that our assay could be used to assess patient infectivity. The assay currently takes 70 min from sampling to results.

Towards a method for cryopreservation of mosquito vectors of human pathogens.

Mosquito-borne diseases are responsible for millions of human deaths every year, posing a massive burden on global public health. Mosquitoes transmit a variety of bacteria, parasites and viruses. Mosquito control efforts such as insecticide spraying can reduce mosquito populations, but they must be sustained in order to have long term impacts, can result in the evolution of insecticide resistance, are costly, and can have adverse human and environmental effects. Technological advances have allowed genetic manipulation of mosquitoes, including generation of those that are still susceptible to insecticides, which has greatly increased the number of mosquito strains and lines available to the scientific research community. This generates an associated challenge, because rearing and maintaining unique mosquito lines requires time, money and facilities, and long-term maintenance can lead to adaptation to specific laboratory conditions, resulting in mosquito lines that are distinct from their wild-type counterparts. Additionally, continuous rearing of transgenic lines can lead to loss of genetic markers, genes and/or phenotypes. Cryopreservation of valuable mosquito lines could help circumvent these limitations and allow researchers to reduce the cost of rearing multiple lines simultaneously, maintain low passage number transgenic mosquitoes, and bank lines not currently being used. Additionally, mosquito cryopreservation could allow researchers to access the same mosquito lines, limiting the impact of unique laboratory or field conditions. Successful cryopreservation of mosquitoes would expand the field of mosquito research and could ultimately lead to advances that would reduce the burden of mosquito-borne diseases, possibly through rear-and-release strategies to overcome mosquito insecticide resistance. Cryopreservation techniques have been developed for some insect groups, including but not limited to fruit flies, silkworms and other moth species, and honeybees. Recent advances within the cryopreservation field, along with success with other insects suggest that cryopreservation of mosquitoes may be a feasible method for preserving valuable scientific and public health resources. In this review, we will provide an overview of basic mosquito biology, the current state of and advances within insect cryopreservation, and a proposed approach toward cryopreservation of Anopheles stephensi mosquitoes.

Human dengue virus serotype 2 neutralizing antibodies target two distinct quaternary epitopes.

Dengue virus (DENV) infection causes dengue fever, dengue hemorrhagic fever and dengue shock syndrome. It is estimated that a third of the world's population is at risk for infection, with an estimated 390 million infections annually. Dengue virus serotype 2 (DENV2) causes severe epidemics, and the leading tetravalent dengue vaccine has lower efficacy against DENV2 compared to the other 3 serotypes. In natural DENV2 infections, strongly neutralizing type-specific antibodies provide protection against subsequent DENV2 infection. While the epitopes of some human DENV2 type-specific antibodies have been mapped, it is not known if these are representative of the polyclonal antibody response. Using structure-guided immunogen design and reverse genetics, we generated a panel of recombinant viruses containing amino acid alterations and epitope transplants between different serotypes. Using this panel of recombinant viruses in binding, competition, and neutralization assays, we have finely mapped the epitopes of three human DENV2 type-specific monoclonal antibodies, finding shared and distinct epitope regions. Additionally, we used these recombinant viruses and polyclonal sera to dissect the epitope-specific responses following primary DENV2 natural infection and monovalent vaccination. Our results demonstrate that antibodies raised following DENV2 infection or vaccination circulate as separate populations that neutralize by occupying domain III and domain I quaternary epitopes. The fraction of neutralizing antibodies directed to different epitopes differs between individuals. The identification of these epitopes could potentially be harnessed to evaluate epitope-specific antibody responses as correlates of protective immunity, potentially improving vaccine design.

Analyzing the Human Serum Antibody Responses to a Live Attenuated Tetravalent Dengue Vaccine Candidate.

Background: Dengue virus serotypes 1-4 (DENV-1-4) are the most common vector-borne viral pathogens of humans and the etiological agents of dengue fever and dengue hemorrhagic syndrome. A live-attenuated tetravalent dengue vaccine (TDV) developed by Takeda Vaccines has recently progressed to phase 3 safety and efficacy evaluation. Methods: We analyzed the qualitative features of the neutralizing antibody (nAb) response induced in naive and DENV-immune individuals after TDV administration. Using DENV-specific human monoclonal antibodies (mAbs) and recombinant DENV displaying different serotype-specific Ab epitopes, we mapped the specificity of TDV-induced nAbs against DENV-1-3. Results: Nearly all subjects had high levels of DENV-2-specific nAbs directed to epitopes centered on domain III of the envelope protein. In some individuals, the vaccine induced nAbs that tracked with a DENV-1-specific neutralizing epitope centered on domain I of the envelope protein. The vaccine induced binding Abs directed to a DENV-3 type-specific neutralizing epitope, but findings of mapping of DENV-3 type-specific nAbs were inconclusive. Conclusion: Here we provide qualitative measures of the magnitude and epitope specificity of the nAb responses to TDV. This information will be useful for understanding the performance of TDV in clinical trials and for identifying correlates of protective immunity.

The Molecular Specificity of the Human Antibody Response to Dengue Virus Infections.

Dengue viruses (DENV) are mosquito-borne positive sense RNA viruses in the family Flaviviridae. The four serotypes of DENV (DENV1, DENV2, DENV3, DENV4) are widely distributed and it is estimated over a third of the world's population is at risk of infection [4]. While the majority of infections are asymptomatic, DENV infection can cause a spectrum of disease, from mild flu-like symptoms, to the more severe DENV hemorrhagic fever and shock syndrome [24]. Over the past 20 years, there have been intense efforts to develop a tetravalent live-attenuated DENV vaccine [36]. The process of vaccine development has been largely empirical, because effective live attenuated vaccines have been developed for other flaviviruses like yellow fever and Japanese encephalitis viruses. However, recent results from phase III live attenuated DENV vaccine efficacy trials are mixed with evidence for efficacy in some populations but not others [20]. In light of unexpected results from DENV vaccine trials, in this chapter we will review recent discoveries about the human antibody response to natural DENV infection and discuss the relevance of this work to understanding vaccine performance.

CD-loop Extension in Zika Virus Envelope Protein Key for Stability and Pathogenesis.

With severe disease manifestations including microcephaly, congenital malformation, and Guillain-Barré syndrome, Zika virus (ZIKV) remains a persistent global public health threat. Despite antigenic similarities with dengue viruses, structural studies have suggested the extended CD-loop and hydrogen-bonding interaction network within the ZIKV envelope protein contribute to stability differences between the viral families. This enhanced stability may lead to the augmented infection, disease manifestation, and persistence in body fluids seen following ZIKV infection. To examine the role of these motifs in infection, we generated a series of ZIKV recombinant viruses that disrupted the hydrogen-bonding network (350A, 351A, and 350A/351A) or the CD-loop extension (Δ346). Our results demonstrate a key role for the ZIKV extended CD-loop in cell-type-dependent replication, virion stability, and in vivo pathogenesis. Importantly, the Δ346 mutant maintains similar antigenicity to wild-type virus, opening the possibility for its use as a live-attenuated vaccine platform for ZIKV and other clinically relevant flaviviruses.

Release of Cell-free MicroRNA Tumor Biomarkers into the Blood Circulation with Pulsed Focused Ultrasound: A Noninvasive, Anatomically Localized, Molecular Liquid Biopsy.

Purpose To compare the abilities of three pulsed focused ultrasound regimes (that cause tissue liquefaction, permeabilization, or mild heating) to release tumor-derived microRNA into the circulation in vivo and to evaluate release dynamics. Materials and Methods All rat experiments were approved by the University of Washington Institutional Animal Care and Use Committee. Reverse-transcription quantitative polymerase chain reaction array profiling was used to identify candidate microRNA biomarkers in a rat solid tumor cell line. Rats subcutaneously grafted with these cells were randomly assigned among three pulsed focused ultrasound treatment groups: (a) local tissue liquefaction via boiling histotripsy, (b) tissue permeabilization via inertial cavitation, and (c) mild (<10°C) heating of tissue, as well as a sham-treated control group. Blood specimens were drawn immediately prior to treatment and serially over 24 hours afterward. Plasma microRNA was quantified with reverse-transcription quantitative polymerase chain reaction, and statistical significance was determined with one-way analysis of variance (Kruskal-Wallis and Friedman tests), followed by the Dunn multiple-comparisons test. Results After tissue liquefaction and cavitation treatments (but not mild heating), plasma quantities of candidate biomarkers increased significantly (P value range, <.0001 to .04) relative to sham-treated controls. A threefold to 32-fold increase occurred within 15 minutes after initiation of pulsed focused ultrasound tumor treatment, and these increases persisted for 3 hours. Histologic examination confirmed complete liquefaction of the targeted tumor area with boiling histotripsy, in addition to areas of petechial hemorrhage and tissue disruption by means of cavitation-based treatment. Conclusion Mechanical tumor tissue disruption with pulsed focused ultrasound-induced bubble activity significantly increases the plasma abundance of tumor-derived microRNA rapidly after treatment. © RSNA, 2016 Online supplemental material is available for this article.

Quantitative and stoichiometric analysis of the microRNA content of exosomes.

Exosomes have been proposed as vehicles for microRNA (miRNA) -based intercellular communication and a source of miRNA biomarkers in bodily fluids. Although exosome preparations contain miRNAs, a quantitative analysis of their abundance and stoichiometry is lacking. In the course of studying cancer-associated extracellular miRNAs in patient blood samples, we found that exosome fractions contained a small minority of the miRNA content of plasma. This low yield prompted us to perform a more quantitative assessment of the relationship between miRNAs and exosomes using a stoichiometric approach. We quantified both the number of exosomes and the number of miRNA molecules in replicate samples that were isolated from five diverse sources (i.e., plasma, seminal fluid, dendritic cells, mast cells, and ovarian cancer cells). Regardless of the source, on average, there was far less than one molecule of a given miRNA per exosome, even for the most abundant miRNAs in exosome preparations (mean ± SD across six exosome sources: 0.00825 ± 0.02 miRNA molecules/exosome). Thus, if miRNAs were distributed homogenously across the exosome population, on average, over 100 exosomes would need to be examined to observe one copy of a given abundant miRNA. This stoichiometry of miRNAs and exosomes suggests that most individual exosomes in standard preparations do not carry biologically significant numbers of miRNAs and are, therefore, individually unlikely to be functional as vehicles for miRNA-based communication. We propose revised models to reconcile the exosome-mediated, miRNA-based intercellular communication hypothesis with the observed stoichiometry of miRNAs associated with exosomes.

Absolute quantification by droplet digital PCR versus analog real-time PCR.

Nanoliter-sized droplet technology paired with digital PCR (ddPCR) holds promise for highly precise, absolute nucleic acid quantification. Our comparison of microRNA quantification by ddPCR and real-time PCR revealed greater precision (coefficients of variation decreased 37-86%) and improved day-to-day reproducibility (by a factor of seven) of ddPCR but with comparable sensitivity. When we applied ddPCR to serum microRNA biomarker analysis, this translated to superior diagnostic performance for identifying individuals with cancer.

Systematic design and functional analysis of artificial microRNAs.

Unlike short interfering RNAs (siRNAs), which are commonly designed to repress a single messenger RNA (mRNA) target through perfect base pairing, microRNAs (miRNAs) are endogenous small RNAs that have evolved to concurrently repress multiple mRNA targets through imperfect complementarity. MicroRNA target recognition is primarily determined by pairing of the miRNA seed sequence (nucleotides 2-8) to complementary match sites in each mRNA target. Whereas siRNA technology is well established for single target knockdown, the design of artificial miRNAs for multi-target repression is largely unexplored. We designed and functionally analysed over 200 artificial miRNAs for simultaneous repression of pyruvate carboxylase and glutaminase by selecting all seed matches shared by their 3' untranslated regions. Although we identified multiple miRNAs that repressed endogenous protein expression of both genes, seed-based artificial miRNA design was highly inefficient, as the majority of miRNAs with even perfect seed matches did not repress either target. Moreover, commonly used target prediction programs did not substantially discriminate effective artificial miRNAs from ineffective ones, indicating that current algorithms do not fully capture the features important for artificial miRNA targeting and are not yet sufficient for designing artificial miRNAs. Our analysis suggests that additional factors are strong determinants of the efficacy of miRNA-mediated target repression and remain to be discovered.

Strain-Dependent Assessment of Powassan Virus Transmission to Ixodes scapularis Ticks.

Powassan virus (POWV) is an emerging tick-borne encephalitic virus in Lyme disease-endemic sites in North America. Due to range expansion and local intensification of blacklegged tick vector (Ixodes scapularis) populations in the northeastern and upper midwestern U.S., human encephalitis cases are increasingly being reported. A better understanding of the transmission cycle between POWV and ticks is required in order to better predict and understand their public health burden. Recent phylogeographic analyses of POWV have identified geographical structuring, with well-defined northeastern and midwestern clades of the lineage II subtype. The extent that geographic and genetically defined sublineages differ in their ability to infect and be transmitted by blacklegged ticks is unclear. Accordingly, we determined whether there are strain-dependent differences in the transmission of POWV to ticks at multiple life stages. Five recent, low-passage POWV isolates were used to measure aspects of vector competence, using viremic and artificial infection methods. Infection rates in experimental ticks remained consistent between all five isolates tested, resulting in a 12-20% infection rate and some differences in viral load. We confirm that these differences are likely not due to differences in host viremia. Our results demonstrate that blacklegged ticks are susceptible to, and capable of transmitting, all tested strains and suggest that the tick-virus association is stable across diverse viral genotypes.

Corrigendum: Intrinsic factors driving mosquito vector competence and viral evolution: a review.

[This corrects the article DOI: 10.3389/fcimb.2023.1330600.].

The Incompetence of Mosquitoes-Can Zika Virus Be Adapted To Infect Culex tarsalis Cells?

The molecular evolutionary mechanisms underpinning virus-host interactions are increasingly recognized as key drivers of virus emergence, host specificity, and the likelihood that viruses can undergo a host shift that alters epidemiology and transmission biology. Zika virus (ZIKV) is mainly transmitted between humans by Aedes aegypti mosquitoes. However, the 2015 to 2017 outbreak stimulated discussion regarding the role of Culex spp. mosquitoes in transmission. Reports of ZIKV-infected Culex mosquitoes, in nature and under laboratory conditions, resulted in public and scientific confusion. We previously found that Puerto Rican ZIKV does not infect colonized Culex quinquefasciatus, Culex pipiens, or Culex tarsalis, but some studies suggest they may be competent ZIKV vectors. Therefore, we attempted to adapt ZIKV to Cx. tarsalis by serially passaging virus on cocultured Ae. aegypti (Aag2) and Cx. tarsalis (CT) cells to identify viral determinants of species specificity. Increasing fractions of CT cells resulted in decreased overall virus titer and no enhancement of Culex cell or mosquito infection. Next-generation sequencing of cocultured virus passages revealed synonymous and nonsynonymous variants throughout the genome that arose as CT cell fractions increased. We generated nine recombinant ZIKVs containing combinations of the variants of interest. None of these viruses showed increased infection of Culex cells or mosquitoes, demonstrating that variants associated with passaging were not specific to increased Culex infection. These results reveal the challenge of a virus adapting to a new host, even when pushed to adapt artificially. Importantly, they also demonstrate that while ZIKV may occasionally infect Culex mosquitoes, Aedes mosquitoes likely drive transmission and human risk. IMPORTANCE ZIKV is mainly transmitted between humans by Aedes mosquitoes. In nature, ZIKV-infected Culex mosquitoes have been found, and ZIKV infrequently infects Culex mosquitoes under laboratory conditions. Yet, most studies show that Culex mosquitoes are not competent vectors for ZIKV. We attempted to adapt ZIKV to Culex cells to identify viral determinants of species specificity. We sequenced ZIKV after it was passaged on a mixture of Aedes and Culex cells and found that it acquired many variants. We generated recombinant viruses containing combinations of the variants of interest to determine if any of these changes enhance infection in Culex cells or mosquitoes. Recombinant viruses did not show increased infection in Culex cells or mosquitoes, but some variants increased infection in Aedes cells, suggesting adaptation to those cells instead. These results reveal that arbovirus species specificity is complex, and that virus adaptation to a new genus of mosquito vectors likely requires multiple genetic changes.

Loss of West Nile virus genetic diversity during mosquito infection due to species-dependent population bottlenecks.

Vector competence (VC) refers to the efficiency of pathogen transmission by vectors. Each step in the infection of a mosquito vector constitutes a barrier to transmission that may impose bottlenecks on virus populations. West Nile virus (WNV) is maintained by multiple mosquito species with varying VC. However, the extent to which bottlenecks and VC are linked is poorly understood. Similarly, quantitative analyses of mosquito-imposed bottlenecks on virus populations are limited. We used molecularly barcoded WNV to quantify tissue-associated population bottlenecks in three variably competent WNV vectors. Our results confirm strong population bottlenecks during mosquito infection that are capable of dramatically reshaping virus population structure in a non-selective manner. In addition, we found that mosquitoes with differing VC uniquely shape WNV population structure: highly competent vectors are more likely to contribute to the maintenance of rare viral genotypes. These findings have important implications for arbovirus emergence and evolution.

Intrinsic factors driving mosquito vector competence and viral evolution: a review.

Mosquitoes are responsible for the transmission of numerous viruses of global health significance. The term "vector competence" describes the intrinsic ability of an arthropod vector to transmit an infectious agent. Prior to transmission, the mosquito itself presents a complex and hostile environment through which a virus must transit to ensure propagation and transmission to the next host. Viruses imbibed in an infectious blood meal must pass in and out of the mosquito midgut, traffic through the body cavity or hemocoel, invade the salivary glands, and be expelled with the saliva when the vector takes a subsequent blood meal. Viruses encounter physical, cellular, microbial, and immunological barriers, which are influenced by the genetic background of the mosquito vector as well as environmental conditions. Collectively, these factors place significant selective pressure on the virus that impact its evolution and transmission. Here, we provide an overview of the current state of the field in understanding the mosquito-specific factors that underpin vector competence and how each of these mechanisms may influence virus evolution.

SARS-CoV-2 entry into and evolution within a skilled nursing facility.

SARS-CoV-2 belongs to the family Coronaviridae which includes multiple human pathogens that have an outsized impact on aging populations. As a novel human pathogen, SARS-CoV-2 is undergoing continuous adaptation to this new host species and there is evidence of this throughout the scientific and public literature. However, most investigations of SARS-CoV-2 evolution have focused on large-scale collections of data across diverse populations and/or living environments. Here we investigate SARS-CoV-2 evolution in epidemiologically linked individuals within a single outbreak at a skilled nursing facility beginning with initial introduction of the pathogen. The data demonstrate that SARS-CoV-2 was introduced to the facility multiple times without establishing an interfacility transmission chain, followed by a single introduction that infected many individuals within a week. This large-scale introduction by a single genotype then persisted in the facility. SARS-CoV-2 sequences were investigated at both the consensus and intra-host variation levels. Understanding the variability in SARS-CoV-2 during transmission chains will assist in understanding the spread of this disease and can ultimately inform best practices for mitigation strategies.

Intracellular Diversity of WNV within Circulating Avian Peripheral Blood Mononuclear Cells Reveals Host-Dependent Patterns of Polyinfection.

Arthropod-borne virus (arbovirus) populations exist as mutant swarms that are maintained between arthropods and vertebrates. West Nile virus (WNV) population dynamics are host-dependent. In American crows, purifying selection is weak and population diversity is high compared to American robins, which have 100- to 1000-fold lower viremia. WNV passed in robins leads to fitness gains, whereas that passed in crows does not. Therefore, we tested the hypothesis that high crow viremia allows for higher genetic diversity within individual avian peripheral blood mononuclear cells (PBMCs), reasoning that this could have produced the previously observed host-specific differences in genetic diversity and fitness. Specifically, we infected cells and birds with a molecularly barcoded WNV and sequenced viral RNA from single cells to quantify the number of WNV barcodes in each. Our results demonstrate that the richness of WNV populations within crows far exceeds that in robins. Similarly, rare WNV variants were maintained by crows more frequently than by robins. Our results suggest that increased viremia in crows relative to robins leads to the maintenance of defective genomes and less prevalent variants, presumably through complementation. Our findings further suggest that weaker purifying selection in highly susceptible crows is attributable to this higher viremia, polyinfections and complementation.

Vaccine-induced antibodies to contemporary strains of dengue virus type 4 show a mechanistic correlate of protective immunity.

The four dengue virus serotypes (DENV1-4) are mosquito-borne flaviviruses of humans. Several live-attenuated tetravalent DENV vaccines are at different stages of clinical development and approval. In children with no baseline immunity to DENVs, a leading vaccine (Dengvaxia) is efficacious against vaccine-matched DENV4 genotype II (GII) strains but not vaccine-mismatched DENV4 GI viruses. We use a panel of recombinant DENV4 viruses displaying GI or GII envelope (E) proteins to map Dengvaxia-induced neutralizing antibodies (NAbs) linked to protection. The vaccine stimulated antibodies that neutralize the DENV4 GII virus better than the GI virus. The neutralization differences map to 5 variable amino acids on the E protein located within a region targeted by DENV4 NAbs, supporting a mechanistic role for these epitope-specific NAbs in protection. In children with no baseline immunity to DENVs, levels of DENV4 serotype- and genotype-specific NAbs induced by vaccination are predictive of vaccine efficacy.