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A topical collection in Diagnostics (ISSN 2075-4418). This collection belongs to the section "Diagnostic Microbiology and Infectious Disease".

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Prof. Dr. Nuno Taveira

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Instituto de Investigação do Medicamento (iMed.ULisboa), Faculdade de Farmácia, Universidade de Lisboa, 1649-003 Lisboa, Portugal
Interests: HIV; antivirals and vaccines; drug resistance; pathogenesis; antibody neutralization; virus evolution
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Dear Colleagues,

In these troubled COVID-19 times we are living in, it has become more evident than ever that rapid, deployable, and highly sensitive, specific and precise diagnostic assays can save many lives. Together with the established qPCR-based and isothermal amplification-based methods, new technologies such as those based on CRISPR-Cas13 system and metagenomics are emerging and are enabling the rapid and sensitive detection and identification of multiple viruses at the same time with an unprecedented quality, both in the lab and outside the lab (PoC assays). These molecular methods of diagnostics rely on nucleic acid extraction methods whose performance characteristics may be very diverse and need a better assessment. High quality serologic assays are also paramount for the diagnosis and management of virus diseases, and for monitoring the response to vaccination. A better understanding of the advantages and limitations of currently available molecular and serologic assays for the diagnosis, treatment management, and cure assessment of each virus disease is needed. There is no better time for this Topical Collection in diagnostic virology. I would like to invite you to submit your best review or original work on all aspects of diagnostic virology mentioned above and others that I have missed to this topical collection issue. We are particularly seeking top level contributions on the development, validation, and implementation of molecular and serologic assays for the diagnosis, prognosis, and management of diseases caused by human viruses in all settings from the lab to the house and the bed of the patient.

Prof. Dr. Nuno Taveira
Collection Editor

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Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the collection website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

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Keywords

Detecton and identification of viral pathogens
Development and validation of diagnostic tests (molecular, antigenic and serologic)
Implementation of diagnostic tests in different settings and geographies
Performance of nucleic acid extraction tests
Point of care assays
Multiplex assays
CRISPR diagnostics
Metagenomic diagnostics
Drug resistance assays
Diagnostic virology and public health

Published Papers (9 papers)

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2023

Jump to: 2022, 2021, 2020

Open AccessArticle

Use of Envelope Domain III Protein for the Detection of IgG Type Antibodies Specific to Zika Virus by Indirect ELISA

Oumar Ndiaye

Cheikh Tidiane Diagne

Silvania Da Veiga Leal

Menilita dos Santos

Maria da Luz de Lima Mendonça

Carolina Cardoso da Silva Leite

Cheikh Saad Bouh Boye

and

Diagnostics 2023, 13(3), 462; https://doi.org/10.3390/diagnostics13030462 - 26 Jan 2023

Viewed by 877

Abstract

Zika virus (ZIKV) diagnostics are crucial for proper antenatal and postnatal care and also for surveillance and serosurvey studies. Since the viremia during ZIKV infection is fleeting, serological testing is highly valuable to inform diagnosis. However, current serology tests using whole virus antigens frequently suffer from cross reactivity issues, delays, and technical complexity, especially in low and middle income countries (LMICs) and endemic countries. Here, we describe an indirect ELISA to detect specific IgG antibodies using the ZIKV envelope domain III (EDIII) protein expressed in Drosophila S2 cells as an immunogen. Using a total of 367 clinical samples, we showed that the EDIII-ELISA was able to detect IgG antibodies against ZIKV with high sensitivity of 100.0% and specificity of 94.7% when compared to plaque reduction neutralization tests (PRNTs) as the gold standard and using 0.208 as the cut-off OD value. These results show the usefulness of the recombinant envelope domain III as an alternative to standard whole virus proteins for ZIKV diagnostics as it improves the sensitivity and specificity of IgG ELISA assay when used as an immunogen. This method should, therefore, be extended to serological diagnostic techniques for other members of the flavivirus genus and for use in IgM diagnostic testing. Full article

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2022

Jump to: 2023, 2021, 2020

Open AccessArticle

Highly Specific and Rapid Detection of Hepatitis C Virus Using RT-LAMP-Coupled CRISPR–Cas12 Assay

Nang Kham-Kjing

Nicole Ngo-Giang-Huong

Khajornsak Tragoolpua

Woottichai Khamduang

and

Sayamon Hongjaisee

Diagnostics 2022, 12(7), 1524; https://doi.org/10.3390/diagnostics12071524 - 23 Jun 2022

Cited by 9 | Viewed by 2228

Abstract

Hepatitis C virus (HCV) infection can be cured with pan-genotypic direct-acting antiviral agents. However, identifying individuals with current hepatitis C remains a major challenge, especially in resource-limited settings where access to or availability of molecular tests is still limited. The goal of this study was to develop and validate a molecular assay for the rapid detection of HCV RNA in resource-limited settings. It is based on a combination of reverse transcription loop-mediated isothermal amplification (RT-LAMP) with the clustered regularly interspaced short palindromic repeats–CRISPR-associated protein 12a (CRISPR–Cas12a) cleavage assay that allows the recognition of specific HCV nucleic acid sequences. Amplified products after the cleavage reactions can be visualized on lateral flow strips or measured with a fluorescence detector. When tested on clinical samples from individuals infected with HCV, HIV, or HBV, or from healthy donors, the RT-LAMP-coupled CRISPR–Cas12 assay yielded 96% sensitivity, 100% specificity, and 97% agreement as compared to the reference method (Roche COBAS AmpliPrep/COBAS TaqMan HCV Test). This assay could detect HCV RNA concentrations as low as 10 ng/µL (an estimated 2.38 Log₁₀ IU/mL). Therefore, this sensitive and specific assay may represent an affordable and reliable point-of-care test for the identification of individuals with active hepatitis C in low-resource settings. Full article

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Open AccessArticle

Unusual N Gene Dropout and Ct Value Shift in Commercial Multiplex PCR Assays Caused by Mutated SARS-CoV-2 Strain

and

Konstantina Gartzonika

Diagnostics 2022, 12(4), 973; https://doi.org/10.3390/diagnostics12040973 - 13 Apr 2022

Cited by 4 | Viewed by 1452

Abstract

Several SARS-CoV-2 variants have emerged and early detection for monitoring their prevalence is crucial. Many identification strategies have been implemented in cases where sequencing data for confirmation is pending or not available. The presence of B.1.1.318 among prevalent variants was indicated by an unusual amplification pattern in various RT-qPCR commercial assays. Positive samples for SARS-CoV-2, as determined using the Allplex SARS-CoV-2 Assay, the Viasure SARS-CoV-2 Real Time Detection Kit and the GeneFinder COVID-19 Plus RealAmp Kit, presented a delay or failure in the amplification of the N gene, which was further investigated. Whole-genome sequencing was used for variant characterization. The differences between the mean Ct values for amplification of the N gene vs. other genes were calculated for each detection system and found to be at least 14 cycles. Sequencing by WGS revealed that all the N gene dropout samples contained the B.1.1.318 variant. All the isolates harbored three non-synonymous mutations in the N gene, which resulted in four amino acid changes (R203K, G204R, A208G, Met234I). Although caution should be taken when the identification of SARS-CoV-2 variants is based on viral gene amplification failure, such patterns could serve as a basis for rapid and cost-effective screening, functioning as indicators of community circulation of specific variants, requiring subsequent verification via sequencing. Full article

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2021

Jump to: 2023, 2022, 2020

Open AccessArticle

A Reliable Indirect ELISA Protocol for Detection of Human Antibodies Directed to SARS-CoV-2 NP Protein

and

Diagnostics 2021, 11(5), 825; https://doi.org/10.3390/diagnostics11050825 - 02 May 2021

Cited by 7 | Viewed by 4383

Abstract

A few months ago, the availability of a reliable and cost-effective testing capacity for COVID-19 was a concern for many countries. With the emergence and circulation of new SARS-CoV-2 variants, another layer of challenge can be added for COVID-19 testing at both molecular and serological levels. This is particularly important for the available tests principally designed to target the S gene/protein where multiple mutations have been reported. Herein, the SARS-CoV-2 NP recombinant protein was utilized to develop a simple and reliable COVID-19 NP human IgG ELISA. The optimized protocol was validated against a micro-neutralization (MN) assay, in-house S-based ELISA, and commercial chemiluminescence immunoassay (CLIA). The developed assay provides 100% sensitivity, 98.9% specificity, 98.9% agreement, and high overall accuracy with an area under curve equal to 0.9998 ± 0.0002 with a 95% confidence interval of 0.99 to 1.00. The optical density values of positive samples significantly correlated with their corresponding MN titers. The assay specifically detects IgG antibodies to the SARS-CoV-2 NP protein and does not cross-detect IgG to the viral S protein. Moreover, it does not cross-react with antibodies related to other coronaviruses (e.g., the Middle East respiratory syndrome coronavirus or human coronavirus HKU1). The availability of this reliable COVID-19 NP IgG ELISA protocol is highly valuable for its diagnostic and epidemiological applications. Full article

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Open AccessArticle

Detection and Differentiation of SARS-CoV-2, Influenza, and Respiratory Syncytial Viruses by CRISPR

Huifen Zhou

Jen-Hui Tsou

Molangur Chinthalapally

Hongjie Liu

and

Feng Jiang

Diagnostics 2021, 11(5), 823; https://doi.org/10.3390/diagnostics11050823 - 01 May 2021

Cited by 6 | Viewed by 2459

Abstract

SARS-CoV-2, influenza, and respiratory syncytial viruses (RSVs) cause acute respiratory infections with similar symptoms. Since the treatments and outcomes of these infections are different, the early detection and accurate differentiation of the viruses are clinically important for the prevention and treatment of the diseases. We previously demonstrated that clustered regularly interspaced short palindromic repeats (CRISPR) could rapidly and precisely detect SARS-CoV-2. The objective of this study was to develop CRISPR as a test for simultaneously detecting and accurately distinguishing the viruses. The CRISPR assay with an RNA guide against each virus was performed in the reference standards of SARS-CoV-2, influenza A and B, and RSV. The CRISPR assay had a limit of detection of 1–100 copies/µL for specifically detecting SARS-CoV-2, influenza A and B, and RSV without cross-reaction with other respiratory viruses. The validation of the test in nasopharyngeal specimens showed that it had a 90–100% sensitivity and 100% specificity for the detection of SARS-CoV-2, influenza A and B, and RSV. The CRISPR assay could potentially be used for sensitive detection and specific differentiation of the respiratory viruses. Full article

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Open AccessArticle

Risk Score for Predicting In-Hospital Mortality in COVID-19 (RIM Score)

Alejandro López-Escobar

Rodrigo Madurga

José María Castellano

Sara Velázquez

Rafael Suárez del Villar

and

Santiago Ruiz de Aguiar

Diagnostics 2021, 11(4), 596; https://doi.org/10.3390/diagnostics11040596 - 26 Mar 2021

Cited by 19 | Viewed by 2299

Abstract

Infection by SARS-CoV2 has devastating consequences on health care systems. It is a global health priority to identify patients at risk of fatal outcomes. 1955 patients admitted to HM-Hospitales from 1 March to 10 June 2020 due to COVID-19, were were divided into two groups, 1310 belonged to the training cohort and 645 to validation cohort. Four different models were generated to predict in-hospital mortality. Following variables were included: age, sex, oxygen saturation, level of C-reactive-protein, neutrophil-to-platelet-ratio (NPR), neutrophil-to-lymphocyte-ratio (NLR) and the rate of changes of both hemogram ratios (VNLR and VNPR) during the first week after admission. The accuracy of the models in predicting in-hospital mortality were evaluated using the area under the receiver-operator-characteristic curve (AUC). AUC for models including NLR and NPR performed similarly in both cohorts: NLR 0.873 (95% CI: 0.849–0.898), NPR 0.875 (95% CI: 0.851–0.899) in training cohort and NLR 0.856 (95% CI: 0.818–0.895), NPR 0.863 (95% CI: 0.826–0.901) in validation cohort. AUC was 0.885 (95% CI: 0.885–0.919) for VNLR and 0.891 (95% CI: 0.861–0.922) for VNPR in the validation cohort. According to our results, models are useful in predicting in-hospital mortality risk due to COVID-19. The RIM Score proposed is a simple, widely available tool that can help identify patients at risk of fatal outcomes. Full article

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Open AccessReview

Diagnosis of SARS-Cov-2 Infection by RT-PCR Using Specimens Other Than Naso- and Oropharyngeal Swabs: A Systematic Review and Meta-Analysis

and

Diagnostics 2021, 11(2), 363; https://doi.org/10.3390/diagnostics11020363 - 21 Feb 2021

Cited by 30 | Viewed by 3980

Abstract

The rapid and accurate testing of SARS-CoV-2 infection is still crucial to mitigate, and eventually halt, the spread of this disease. Currently, nasopharyngeal swab (NPS) and oropharyngeal swab (OPS) are the recommended standard sampling techniques, yet, these have some limitations such as the complexity of collection. Hence, several other types of specimens that are easier to obtain are being tested as alternatives to nasal/throat swabs in nucleic acid assays for SARS-CoV-2 detection. This study aims to critically appraise and compare the clinical performance of RT-PCR tests using oral saliva, deep-throat saliva/posterior oropharyngeal saliva (DTS/POS), sputum, urine, feces, and tears/conjunctival swab (CS) against standard specimens (NPS, OPS, or a combination of both). In this systematic review and meta-analysis, five databases (PubMed, Scopus, Web of Science, ClinicalTrial.gov and NIPH Clinical Trial) were searched up to the 30th of December, 2020. Case-control and cohort studies on the detection of SARS-CoV-2 were included. The methodological quality was assessed using the Quality Assessment of Diagnostic Accuracy Studies 2 (QUADAS 2). We identified 1560 entries, 33 of which (1.1%) met all required criteria and were included for the quantitative data analysis. Saliva presented the higher accuracy, 92.1% (95% CI: 70.0–98.3), with an estimated sensitivity of 83.9% (95% CI: 77.4–88.8) and specificity of 96.4% (95% CI: 89.5–98.8). DTS/POS samples had an overall accuracy of 79.7% (95% CI: 43.3–95.3), with an estimated sensitivity of 90.1% (95% CI: 83.3–96.9) and specificity of 63.1% (95% CI: 36.8–89.3). The remaining index specimens could not be adequately assessed given the lack of studies available. Our meta-analysis shows that saliva samples from the oral region provide a high sensitivity and specificity; therefore, these appear to be the best candidates for alternative specimens to NPS/OPS in SARS-CoV-2 detection, with suitable protocols for swab-free sample collection to be determined and validated in the future. The distinction between oral and extra-oral salivary samples will be crucial, since DTS/POS samples may induce a higher rate of false positives. Urine, feces, tears/CS and sputum seem unreliable for diagnosis. Saliva testing may increase testing capacity, ultimately promoting the implementation of truly deployable COVID-19 tests, which could either work at the point-of-care (e.g. hospitals, clinics) or at outbreak control spots (e.g., schools, airports, and nursing homes). Full article

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Open AccessArticle

Multicenter Evaluation of the Cepheid Xpert^® HBV Viral Load Test

and

Diagnostics 2021, 11(2), 297; https://doi.org/10.3390/diagnostics11020297 - 12 Feb 2021

Cited by 7 | Viewed by 2313

Abstract

Accurate measurement of the hepatitis B virus (HBV) DNA is important for the management of patients with chronic HBV infection. Here, the performance of the Xpert^® HBV Viral Load test (Xpert HBV Viral Load) versus the Roche COBAS^® Ampliprep/COBAS^® TaqMan^® system (CAP/CTM HBV) HBV test v2.0 was evaluated. From September 2017 to December 2017, a total of 876 prospectively collected or archived serum or EDTA plasma specimens from subjects chronically infected with HBV were tested using the Xpert HBV Viral Load and the CAP/CTM HBV v2.0 assays. Of the 876 specimens tested, 560 were within the quantitative range of both assays. The agreement between the two methods was 90.0%. No difference in plasma or serum samples was observed. Deming regression analysis showed a good correlation of the Xpert HBV Viral Load assay with the CAP/CTM HBV v2.0 assay. The Bland–Altman analysis showed a good agreement between the results of the Xpert HBV Viral Load assay and the CAP/CTM HBV assay, with a mean difference (±1.96 standard deviation) of 0.0091 ± 0.3852 Log IU/mL. Comparing the two assays, only nineteen specimens (2.1%) had a difference greater than 1.96 times the standard deviation. The Xpert^® HBV Viral Load test is suitable for monitoring patients with HBV infection and is useful in diagnostic settings. Full article

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2020

Jump to: 2023, 2022, 2021

Open AccessArticle

Accuracy of the Zika IgM Antibody Capture Enzyme-Linked Immunosorbent Assay from the Centers for Disease Control and Prevention (CDC Zika MAC-ELISA) for Diagnosis of Zika Virus Infection

Moyra Machado Portilho

Laise de Moraes

Mariana Kikuti

Leile Camila Jacob Nascimento

Mitermayer Galvão Reis

Viviane Sampaio Boaventura

Ricardo Khouri

and

Guilherme Sousa Ribeiro

Diagnostics 2020, 10(10), 835; https://doi.org/10.3390/diagnostics10100835 - 18 Oct 2020

Cited by 3 | Viewed by 2425

Abstract

Serological diagnosis of Zika virus (ZIKV) infection is challenging because of antigenic cross-reactivity with dengue virus (DENV). This study evaluated the accuracy of the Zika IgM antibody capture enzyme-linked immunosorbent assay (CDC Zika IgM MAC-ELISA) in differentiating between ZIKV and DENV infections. To determine sensitivity, we used acute- and convalescent-phase sera from 21 patients with RT-PCR-confirmed ZIKV infection. To determine specificity, we used acute- and convalescent-phase sera from 60 RT-PCR-confirmed dengue cases and sera from 23 blood donors. During the acute-phase of the illness, the assay presented a sensitivity of 12.5% (2/16) for samples collected 0–4 days post symptoms onset (DPSO), and of 75.0% (3/4) for samples collected 5–9 DPSO. During the convalescent-phase of the illness, the test sensitivity was 90.9% (10/11), 100% (2/2), and 0% (0/2) for samples obtained 12–102, 258–260, and 722–727 DPSO, respectively. Specificity for acute- and convalescent-phase samples from RT-PCR-confirmed dengue cases was 100% and 93.2%, respectively. Specificity for blood donor samples was 100%. The assay is an accurate method for Zika serological diagnosis and proved to be reliable for use during surveillance and outbreak investigations in settings where ZIKV and DENV cocirculate. Full article

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Published Papers (9 papers)

2023

Jump to: 2022, 2021, 2020

2022

Jump to: 2023, 2021, 2020

2021

Jump to: 2023, 2022, 2020

2020

Jump to: 2023, 2022, 2021

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