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2.
Orv Hetil ; 162(52): 2071-2078, 2021 12 26.
Article in Hungarian | MEDLINE | ID: covidwho-1599870

ABSTRACT

Összefoglaló. Bár a SARS-CoV-2-pandémia próbára tette a diagnosztikus kapacitásokat, számos hasznos tapasztalattal is szolgált, melyek alacsonyabb mintaszám mellett nem lettek volna levonhatók. Míg korábban a PCR-vizsgálatok jellemzoen diagnosztikus, illetve kvantitatív követési célokat szolgáltak, a járvány során többségbe kerültek a szuro- és (kezdetben) a felszabadító vizsgálatok. Jól követheto volt, hogy a tesztek piacra juttatásának eroltetett üteme sokszor nem tette lehetové a teljesen kiforrott koncepciók létrehozását. Tekintettel arra, hogy a molekuláris diagnosztika során nem teljes vírusgenomokat, hanem célszakaszokat mutatunk ki, amelyek aránya a fertozés egyes szakaszaiban nem feltétlenül állandó, egyre valószínubb, hogy nem azonos célgének a legmegfelelobbek diagnosztikus, szuro- és felszabadító vizsgálatokhoz. A nagy mennyiségu, aspecifikusan végzett vizsgálat még kiváló fajlagosság mellett is a pozitív prediktív érték csökkenéséhez vezethet, amennyiben a fertozés tényleges prevalenciája a vizsgálati csoportban alacsony. Munkánkban megkíséreljük irodalmi és saját adatok felhasználásával összefoglalni az elmúlt két év fontosabb diagnosztikus tapasztalatait a teljesség igénye nélkül. Orv Hetil. 2021; 162(52): 2071-2078. Summary. Although the SARS-CoV-2 pandemic has been a great challenge for the diagnostic capacities, it also proved to be a unique source of experience. While previously PCR tests had overwhelmingly been used for targeted diagnostic and quantitative follow-up testing, screening and (initially) release tests became far more prevalent during the pandemic. It was well to be seen that the forced pace of bringing tests to market often gave way to not fully mature concepts. The PCR method is based on the detection of sequences, the proportions of which are likely to alter throughout the course of the disease. It is becoming increasingly clear that different target genes might be the best suitable for diagnostic, screening and release testing. Even with specific assays, an unprecedentedly high number of tests might result in the inflation of the positive predictive value, when the true prevalence of the infection remains very low among the tested individuals. Here we try to summarize some of the potentially most relevant diagnostic conclusions of the pandemic so far according to our own data and the literature. Orv Hetil. 2021; 162(52): 2071-2078.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Pandemics , Pathology, Molecular
3.
Anal Chem ; 93(50): 16804-16812, 2021 12 21.
Article in English | MEDLINE | ID: covidwho-1565689

ABSTRACT

To effectively control the spread of new infectious diseases, there is a need for highly sensitive diagnostic methods to detect viral nucleic acids rapidly. This study outlines a universal and simple detection strategy that uses magnetic nanoparticles (MNPs) and a novel MagR-MazE fusion protein for molecular diagnostics to facilitate sensitive detection. This study has engineered a novel MNP conjugate that can be generated easily, without using many chemical reagents. The technique is a nucleic acid detection method, using MagR-MazE fusion protein-conjugated MNPs, where the results can be visualized with the naked eye, regardless of the oligonucleotide sequences of the target in the lateral flow assay. This method could sensitively detect polymerase chain reaction (PCR) products of 16S ribosomal RNA (rRNA) and the 2019-nCoV-N-positive control gene in 5 min. It shows a low limit of detection (LoD) of 0.013 ng/µL for dsDNA. It is simpler and more rapid, sensitive, and versatile than other techniques, making it suitable for point-of-care testing. The proposed detection system and MNP conjugation strategy using a fusion protein can be widely applied to various fields requiring rapid on-site diagnosis.


Subject(s)
COVID-19 , Magnetite Nanoparticles , Humans , Pathology, Molecular , Polymerase Chain Reaction , SARS-CoV-2
4.
Eur Rev Med Pharmacol Sci ; 25(22): 7162-7184, 2021 Nov.
Article in English | MEDLINE | ID: covidwho-1552083

ABSTRACT

The last two decades have witnessed the emergence of three deadly coronaviruses (CoVs) in humans: severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). There are still no reliable and efficient therapeutics to manage the devastating consequences of these CoVs. Of these, SARS-CoV-2, the cause of the currently ongoing coronavirus disease 2019 (COVID-19) pandemic, has posed great global health concerns. The COVID-19 pandemic has resulted in an unprecedented crisis with devastating socio-economic and health impacts worldwide. This highlights the fact that CoVs continue to evolve and have the genetic flexibility to become highly pathogenic in humans and other mammals. SARS-CoV-2 carries a high genetic homology to the previously identified CoV (SARS-CoV), and the immunological and pathogenic characteristics of SARS-CoV-2, SARS-CoV, and MERS contain key similarities and differences that can guide therapy and management. This review presents salient and updated information on comparative pathology, molecular pathogenicity, immunological features, and genetic characterization of SARS-CoV, MERS-CoV, and SARS-CoV-2; this can help in the design of more effective vaccines and therapeutics for countering these pathogenic CoVs.


Subject(s)
COVID-19/virology , Middle East Respiratory Syndrome Coronavirus/genetics , Pathology, Molecular/methods , SARS Virus/genetics , SARS-CoV-2/genetics , Animals , COVID-19/diagnosis , COVID-19/epidemiology , COVID-19/transmission , Female , Global Health/economics , Humans , Male , Mammals , Middle East Respiratory Syndrome Coronavirus/immunology , Middle East Respiratory Syndrome Coronavirus/pathogenicity , SARS Virus/immunology , SARS Virus/pathogenicity , SARS-CoV-2/immunology , SARS-CoV-2/pathogenicity , Virulence
5.
Arch Pathol Lab Med ; 146(3): 272-277, 2022 03 01.
Article in English | MEDLINE | ID: covidwho-1526969

ABSTRACT

CONTEXT.­: The gold standard test to identify the presence of SARS-CoV-2 in COVID-19 patients is the real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR), but inconclusive data and false-positive diagnosis remain the major problem of this approach. OBJECTIVE.­: To compare the fitness of 2 primer sets to the SARS-CoV-2 nucleocapsid phosphoprotein gene (NP) in the molecular diagnosis of COVID-19, we verified the inconclusive data and confidence of high cycle threshold (Ct) values in SARS-CoV-2 detection. DESIGN.­: The 970 patient samples were tested by using United States Centers for Disease Control and Prevention protocol. We compared the fitness of 2 primer sets to 2 different regions of the NP gene. In addition, we checked the consistency of positive samples with high Ct values by retesting extracted SARS-CoV-2 RNA or by second testing of patients. RESULTS.­: N1 and N2 displayed similar fitness during testing, with no differences between Ct values. Then, we verified security range Ct values related to positive diagnostics, with Ct values above 34 failing in 21 of 32 cases (65.6%) after retesting of samples. The patient samples with Ct values above 34.89 that were doubly positive revealed a low sensitivity (52.4%) and specificity (63.6%) of the test in samples with Ct values above 34. CONCLUSIONS.­: It is safe to use 1 primer set for the NP gene to identify SARS-CoV-2 in samples. However, samples with high Ct values may be considered inconclusive and retested to avoid false-positive diagnosis.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Nucleocapsid , Pathology, Molecular , Phosphoproteins/genetics , RNA, Viral/genetics , Sensitivity and Specificity
6.
Analyst ; 146(22): 6917-6923, 2021 Nov 08.
Article in English | MEDLINE | ID: covidwho-1526109

ABSTRACT

Nucleic acid amplification is a widely used diagnostic tool, although it requires a relatively time-consuming and complicated extraction step. To address this issue outside the laboratory, we investigated a sample preparation system and determined that a silica membrane and silica-coated beads are powerful tools for the extraction from raw samples: nucleic acids are kept in the silica membrane, retained during a single wash step, and released at the elution step. The eluent is appropriate for the quantitative real-time polymerase chain reaction (qPCR) and loop-mediated amplification (LAMP) assay in terms of purity and quantity. We also built an innovative equipment-free nucleic acid extraction squeeze system which requires less than 20 min. The sample with improved purity augments the specificity and sensitivity. This system is simple, user-friendly, low-cost, and equipment-free, thus making nucleic acid extraction more accessible and affordable for researchers and untrained users. Furthermore, when combined with the reverse-transcription quantitative real-time polymerase chain reaction method, the method will accelerate the detection of diseases. The same goes when combined with the LAMP assay, especially in developing countries.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Molecular Diagnostic Techniques , Nucleic Acid Amplification Techniques , Pathology, Molecular , RNA, Viral/genetics , Sensitivity and Specificity
8.
Sensors (Basel) ; 21(21)2021 Oct 21.
Article in English | MEDLINE | ID: covidwho-1512556

ABSTRACT

This paper proposes a cloud-based software architecture for fully automated point-of-care molecular diagnostic devices. The target system operates a cartridge consisting of an extraction body for DNA extraction and a PCR chip for amplification and fluorescence detection. To facilitate control and monitoring via the cloud, a socket server was employed for fundamental molecular diagnostic functions such as DNA extraction, amplification, and fluorescence detection. The user interface for experimental control and monitoring was constructed with the RESTful application programming interface, allowing access from the terminal device, edge, and cloud. Furthermore, it can also be accessed through any web-based user interface on smart computing devices such as smart phones or tablets. An emulator with the proposed software architecture was fabricated to validate successful operation.


Subject(s)
Cloud Computing , Point-of-Care Systems , Computers , Pathology, Molecular , Software
9.
J Endocrinol Invest ; 44(12): 2675-2684, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1504521

ABSTRACT

PURPOSE: Due to relevant repercussions on reproductive medicine, we aimed to evaluate feasibility of RT-PCR as a detection method of SARS-CoV-2 RNA in seminal fluid. METHODS: A qualitative determination of the RT-PCR assays in semen was performed through different approaches: (1) efficiency of RNA extraction from sperm and seminal plasma was determined using PRM1 and PRM2 mRNA and a heterologous system as control; (2) samples obtained by diluting viral preparation from a SARS-CoV-2 panel (virus cultured in Vero E6 cell lines) were tested; (3) viral presence in different fractions of seminal fluid (whole sample, seminal plasma and post-centrifugation pellet) was evaluated. Semen samples from mild and recovered COVID-19 subjects were collected by patients referring to the Infectious Disease Department of the Policlinico Umberto I Hospital - "Sapienza" University of Rome. Control subjects were recruited at the Laboratory of Seminology-Sperm Bank "Loredana Gandini'' of the same hospital. RESULTS: The control panel using viral preparations diluted in saline and seminal fluid showed the capability to detect viral RNA presence with Ct values depending on the initial viral concentration. All tested semen samples were negative for SARS-CoV-2, regardless of the nasopharyngeal swab result or seminal fluid fraction. CONCLUSION: These preliminary data show that RT-PCR for SARS-CoV-2 RNA testing appears to be a feasible method for the molecular diagnosis of SARS-CoV-2 in seminal fluid, supported by results of the control panel. The ability to detect SARS-CoV-2 in semen is extremely important for reproductive medicine, especially in assisted reproductive technology and sperm cryopreservation.


Subject(s)
COVID-19/diagnosis , Pathology, Molecular/methods , Semen/virology , Adult , Animals , Chlorocebus aethiops , Feasibility Studies , Humans , Male , RNA, Messenger/chemistry , RNA, Viral/chemistry , Real-Time Polymerase Chain Reaction , Reproductive Techniques , Vero Cells
10.
BMJ Glob Health ; 6(8)2021 08.
Article in English | MEDLINE | ID: covidwho-1504484

ABSTRACT

BACKGROUND: Early access to diagnosis is crucial for effective management of any disease including tuberculosis (TB). We investigated the barriers and opportunities to maximise uptake and utilisation of molecular diagnostics in routine healthcare settings. METHODS: Using the implementation of WHO approved TB diagnostics, Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) and Line Probe Assay (LPA) as a benchmark, we evaluated the barriers and how they could be unlocked to maximise uptake and utilisation of molecular diagnostics. RESULTS: Health officers representing 190 districts/counties participated in the survey across Kenya, Tanzania and Uganda. The survey findings were corroborated by 145 healthcare facility (HCF) audits and 11 policy-maker engagement workshops. Xpert MTB/RIF coverage was 66%, falling behind microscopy and clinical diagnosis by 33% and 1%, respectively. Stratified by HCF type, Xpert MTB/RIF implementation was 56%, 96% and 95% at district, regional and national referral hospital levels. LPA coverage was 4%, 3% below culture across the three countries. Out of 111 HCFs with Xpert MTB/RIF, 37 (33%) used it to full capacity, performing ≥8 tests per day of which 51% of these were level five (zonal consultant and national referral) HCFs. Likewise, 75% of LPA was available at level five HCFs. Underutilisation of Xpert MTB/RIF and LPA was mainly attributed to inadequate-utilities, 26% and human resource, 22%. Underfinancing was the main reason underlying failure to acquire molecular diagnostics. Second to underfinancing was lack of awareness with 33% healthcare administrators and 49% practitioners were unaware of LPA as TB diagnostic. Creation of a national health tax and decentralising its management was proposed by policy-makers as a booster of domestic financing needed to increase access to diagnostics. CONCLUSION: Our findings suggest higher uptake and utilisation of molecular diagnostics at tertiary level HCFs contrary to the WHO recommendation. Country-led solutions are crucial for unlocking barriers to increase access to diagnostics.


Subject(s)
Mycobacterium tuberculosis , Tuberculosis, Pulmonary , Humans , Mycobacterium tuberculosis/genetics , Pathology, Molecular , Rifampin , Sensitivity and Specificity
11.
IEEE Rev Biomed Eng ; 14: 30-47, 2021.
Article in English | MEDLINE | ID: covidwho-1501335

ABSTRACT

Coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To counter COVID-19 spreading, an infrastructure to provide rapid and thorough molecular diagnostics and serology testing is the cornerstone of outbreak and pandemic management. We hereby review the clinical insights with regard to using molecular tests and immunoassays in the context of COVID-19 management life cycle: the preventive phase, the preparedness phase, the response phase and the recovery phase. The spatial and temporal distribution of viral RNA, antigens and antibodies during human infection is summarized to provide a biological foundation for accurate detection of the disease. We shared the lessons learned and the obstacles encountered during real world high-volume screening programs. Clinical needs are discussed to identify existing technology gaps in these tests. Leverage technologies, such as engineered polymerases, isothermal amplification, and direct amplification from complex matrices may improve the productivity of current infrastructure, while emerging technologies like CRISPR diagnostics, visual end point detection, and PCR free methods for nucleic acid sensing may lead to at-home tests. The lessons learned, and innovations spurred from the COVID-19 pandemic could upgrade our global public health infrastructure to better combat potential outbreaks in the future.


Subject(s)
COVID-19/diagnosis , COVID-19/immunology , Immunoassay/methods , Pathology, Molecular/methods , Animals , Humans , Life Cycle Stages , Pandemics/prevention & control , SARS-CoV-2/immunology , Serologic Tests/methods
13.
Anal Methods ; 13(34): 3744-3763, 2021 09 02.
Article in English | MEDLINE | ID: covidwho-1454826

ABSTRACT

As the COVID-19 pandemic continues to escalate globally and acquires new mutations, accurate diagnostic technologies continue to play a vital role in controlling and understanding the epidemiology of this disease. A plethora of technologies have enabled the diagnosis of individuals, informed clinical management, aided population-wide screening to determine transmission rates and identified cases within the wider community and high-risk settings. This review explores the application of molecular diagnostics technologies in controlling the spread of COVID-19, and the key factors that affect the sensitivity and specificity of the tests used.


Subject(s)
COVID-19 , Humans , Pandemics , Pathology, Molecular , SARS-CoV-2 , Sensitivity and Specificity
14.
Rev Esp Quimioter ; 34 Suppl 1: 49-51, 2021 Sep.
Article in English | MEDLINE | ID: covidwho-1449584

ABSTRACT

The diagnosis of SARS-CoV-2 is based on the use of nucleic acid amplification tests (NAAT), especially rRT-PCR. The latter also allows us to quickly identify variants of concern. However, its use in follow-up of patients and the correlation between Ct value and the viability of the virus is controversial.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19 Testing , Humans , Pathology, Molecular
15.
Chin J Integr Med ; 28(1): 88-95, 2022 Jan.
Article in English | MEDLINE | ID: covidwho-1442168

ABSTRACT

COVID-19 virus is a causative agent of viral pandemic in human beings which specifically targets respiratory system of humans and causes viral pneumonia. This unusual viral pneumonia is rapidly spreading to all parts of the world, currently affecting about 105 million people with 2.3 million deaths. Current review described history, genomic characteristics, replication, and pathogenesis of COVID-19 with special emphasis on Nigella sativum (N. sativum) as a treatment option. N. sativum seeds are historically and religiously used over the centuries, both for prevention and treatment of different diseases. This review summarizes the potential role of N. sativum seeds against COVID-19 infection at levels of in silico, cell lines and animal models.


Subject(s)
COVID-19 , Nigella , Animals , Humans , Pandemics , Pathology, Molecular , SARS-CoV-2
16.
ACS Biomater Sci Eng ; 7(9): 4669-4676, 2021 09 13.
Article in English | MEDLINE | ID: covidwho-1373347

ABSTRACT

The COVID-19 pandemic has exposed the dependence of diagnostic laboratories on a handful of large corporations with market monopolies on the worldwide supply of reagents, consumables, and hardware for molecular diagnostics. Global shortages of key consumables for RT-qPCR detection of SARS-CoV-2 RNA have impaired the ability to run essential, routine diagnostic services. Here, we describe a workflow for rapid detection of SARS-CoV-2 RNA in upper respiratory samples including nasal swabs and saliva, utilizing low-cost equipment and readily accessible reagents. Using repurposed Creality3D Ender-3 three-dimensional (3D) printers, we built a semiautomated paramagnetic bead RNA extraction platform. The hardware for the system was built for $300 USD, and the material cost per reaction was $1 USD. Named the Ender VX500, instrument performance when paired with RT-qPCR for SARS-CoV-2 detection in nasal and saliva specimens was two virus copies per microliter. There was a high-performance agreement (assessed using 458 COVID-19 nasal swab specimens) with the Aptima SARS-CoV-2 assay run on the Hologic Panther, a commercial automated RNA extraction and detection platform. Inter- and intrainstrument precision was excellent (coefficients of variation (CoV) of 1.10 and 0.66-1.32%, respectively) across four instruments. The platform is scalable with throughput ranging from 23 specimens on a single instrument run by one user in 50 min to 364 specimens on four instruments run by four users in 190 min. Step-by-step instructions and protocols for building and running the Ender VX500 have been made available without restriction.


Subject(s)
COVID-19 , Humans , Pandemics , Pathology, Molecular , RNA, Viral/genetics , SARS-CoV-2
17.
Talanta ; 235: 122797, 2021 Dec 01.
Article in English | MEDLINE | ID: covidwho-1347835

ABSTRACT

As the outbreak of coronavirus disease 2019 (COVID-19), on-site molecular diagnosis is becoming increasingly important. In this study, a freeze-drying method was introduced for PCR reagents to meet the requirements of microfluidic molecular diagnosis. Using this method, PCR components were pre-mixed and freeze-dried as a bead, which could be transferred into microfluidic chips easily. As this bead only required reconstitution in water, operational steps of PCR were simplified, pipetting errors and errors associated with improper handling of wet reagents could also be reduced. In addition, 19 PCR mixes for different targets (including both RNA and DNA) detection were stable when stored at room temperature (18-25 °C) for 1-2 years and may be stored longer as activity monitoring remains ongoing. To shorten the stability testing time, accelerated stability testing at higher temperatures was proposed. The evaluation periods of the freeze-dried PCR mixes were shortened to less than one month when stored at 56 °C and 80 °C. When attempts were further tried to predict the shelf lives for freeze-dried PCR mixes, our findings challenged the classic view of the Q10 method as a prediction model for freeze-dried PCR mixes and confirmed for the first time that this prediction was influenced by different factors at varying degrees. These studies and findings are important for the development of molecular diagnosis at both central laboratories and resource-limited areas.


Subject(s)
COVID-19 , Microfluidics , Humans , Pathology, Molecular , Polymerase Chain Reaction , SARS-CoV-2 , Temperature
19.
Int Rev Immunol ; 40(1-2): 143-156, 2021.
Article in English | MEDLINE | ID: covidwho-1236150

ABSTRACT

The pandemic causing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has globally infected more than 50 million people and ∼1.2 million have succumbed to this deadly pathogen. With the vaccine trials still in clinical phases, mitigation of Coronavirus Disease 2019 (COVID-19) relies primarily on robust virus detection methods and subsequent quarantine measures. Hence, the importance of rapid, affordable and reproducible virus testing will serve the need to identify and treat infected subjects in a timely manner. Based on the type of diagnostic assay, the primary targets are viral genome (RNA) and encoded proteins. Currently, COVID-19 detection is performed using various molecular platforms as well as serodiagnostics that exhibit approximately 71% sensitivity. These methods encounter several limitations including sensitivity, specificity, availability of skilled expertise and instrument access. Saliva-based COVID-19 diagnostics are emerging as a superior alternative to nasal swabs because of the ease of sample collection, no interaction during sampling, and high viral titers during early stages of infection. In addition, SARS-CoV-2 is detected in the environment as aerosols associated with suspended particulate matter. Designing virus detection strategies in diverse samples will allow timely monitoring of virus spread in humans and its persistence in the environment. With the passage of time, advanced technologies are overcoming limitations associated with detection. Enhanced sensitivity and specificity of next-generation diagnostics are key features enabling improved prognostic care. In this comprehensive review, we analyze currently adopted advanced technologies and their concurrent use in the development of diagnostics for SARS-CoV-2 detection.


Subject(s)
Biosensing Techniques/methods , COVID-19 Nucleic Acid Testing/methods , COVID-19 Serological Testing/methods , COVID-19/diagnosis , Molecular Diagnostic Techniques/methods , Humans , Pathology, Molecular/methods , Point-of-Care Testing , RNA, Viral/genetics , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Sensitivity and Specificity , Viral Proteins/analysis
20.
ACS Nano ; 15(6): 10194-10202, 2021 06 22.
Article in English | MEDLINE | ID: covidwho-1233686

ABSTRACT

Advent and fast spread of pandemic diseases draw worldwide attention to rapid, prompt, and accurate molecular diagnostics with technical development of ultrafast polymerase chain reaction (PCR). Microfluidic on-chip PCR platforms provide highly efficient and small-volume bioassay for point-of-care diagnostic applications. Here we report ultrafast, real-time, and on-chip nanoplasmonic PCR for rapid and quantitative molecular diagnostics at point-of-care level. The plasmofluidic PCR chip comprises glass nanopillar arrays with Au nanoislands and gas-permeable microfluidic channels, which contain reaction microchamber arrays, a precharged vacuum cell, and a vapor barrier. The on-chip configuration allows both spontaneous sample loading and microbubble-free PCR reaction during which the plasmonic nanopillar arrays result in ultrafast photothermal cycling. After rapid sample loading less than 3 min, two-step PCR results for 40 cycles show rapid amplification in 264 s for lambda-DNA, and 306 s for plasmids expressing SARS-CoV-2 envelope protein. In addition, the in situ cyclic real-time quantification of amplicons clearly demonstrates the amplification efficiencies of more than 91%. This PCR platform can provide rapid point-of-care molecular diagnostics in helping slow the fast-spreading pandemic.


Subject(s)
COVID-19 , Lab-On-A-Chip Devices , Humans , Pathology, Molecular , Real-Time Polymerase Chain Reaction , SARS-CoV-2
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