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1.
Mayo Clin Proc ; 95(7): 1354-1368, 2020 07.
Article in English | MEDLINE | ID: covidwho-1500136

ABSTRACT

OBJECTIVE: To explore the transcriptomic differences between patients with hypertrophic cardiomyopathy (HCM) and controls. PATIENTS AND METHODS: RNA was extracted from cardiac tissue flash frozen at therapeutic surgical septal myectomy for 106 patients with HCM and 39 healthy donor hearts. Expression profiling of 37,846 genes was performed using the Illumina Human HT-12v3 Expression BeadChip. All patients with HCM were genotyped for pathogenic variants causing HCM. Technical validation was performed using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. This study was started on January 1, 1999, and final analysis was completed on April 20, 2020. RESULTS: Overall, 22% of the transcriptome (8443 of 37,846 genes) was expressed differentially between HCM and control tissues. Analysis by genotype revealed that gene expression changes were similar among genotypic subgroups of HCM, with only 4% (1502 of 37,846) to 6% (2336 of 37,846) of the transcriptome exhibiting differential expression between genotypic subgroups. The qRT-PCR confirmed differential expression in 92% (11 of 12 genes) of tested transcripts. Notably, in the context of coronavirus disease 2019 (COVID-19), the transcript for angiotensin I converting enzyme 2 (ACE2), a negative regulator of the angiotensin system, was the single most up-regulated gene in HCM (fold-change, 3.53; q-value =1.30×10-23), which was confirmed by qRT-PCR in triplicate (fold change, 3.78; P=5.22×10-4), and Western blot confirmed greater than 5-fold overexpression of ACE2 protein (fold change, 5.34; P=1.66×10-6). CONCLUSION: More than 20% of the transcriptome is expressed differentially between HCM and control tissues. Importantly, ACE2 was the most up-regulated gene in HCM, indicating perhaps the heart's compensatory effort to mount an antihypertrophic, antifibrotic response. However, given that the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses ACE2 for viral entry, this 5-fold increase in ACE2 protein may confer increased risk for COVID-19 manifestations and outcomes in patients with increased ACE2 transcript expression and protein levels in the heart.


Subject(s)
Betacoronavirus , Cardiomyopathy, Hypertrophic/genetics , Cardiomyopathy, Hypertrophic/virology , Coronavirus Infections/complications , Peptidyl-Dipeptidase A/genetics , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/complications , Adolescent , Adult , Aged , Angiotensin-Converting Enzyme 2 , COVID-19 , Cardiomyopathy, Hypertrophic/metabolism , Case-Control Studies , Child , Genotype , Humans , Middle Aged , Myocardium/metabolism , Pandemics , RNA, Messenger/metabolism , Real-Time Polymerase Chain Reaction , SARS-CoV-2 , Young Adult
2.
APMIS ; 129(7): 393-400, 2021 Jul.
Article in English | MEDLINE | ID: covidwho-1388189

ABSTRACT

The SARS-CoV-2 pandemic has created an urgent need for diagnostic tests to detect viral RNA. Commercial RNA extraction kits are often expensive, in limited supply, and do not always fully inactivate the virus. Together, this calls for the development of safer methods for SARS-CoV-2 extraction that utilize readily available reagents and equipment present in most standard laboratories. We optimized and simplified a RNA extraction method combining a high molar acidic guanidinium isothiocyanate (GITC) solution, phenol and chloroform. First, we determined the GITC/RNA dilution thresholds compatible with an efficient two-step RT-qPCR for B2M mRNA in nasopharyngeal (NP) or oropharyngeal (OP) swab samples. Second, we optimized a one-step RT-qPCR against SARS-CoV-2 using NP and OP samples. We furthermore tested a SARS-CoV-2 dilution series to determine the detection threshold. The method enables downstream detection of SARS-CoV-2 by RT-qPCR with high sensitivity (~4 viral RNA copies per RT-qPCR). The protocol is simple, safe, and expands analysis capacity as the inactivated samples can be used in RT-qPCR detection tests at laboratories not otherwise classified for viral work. The method takes about 30 min from swab to PCR-ready viral RNA and circumvents the need for commercial RNA purification kits.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , RNA, Viral/isolation & purification , SARS-CoV-2/isolation & purification , Specimen Handling/methods , Humans , Reagent Kits, Diagnostic
3.
Front Pharmacol ; 11: 579330, 2020.
Article in English | MEDLINE | ID: covidwho-1389228

ABSTRACT

The Syrian golden hamster (Mesocricetus auratus) has recently been demonstrated as a clinically relevant animal model for SARS-CoV-2 infection. However, lack of knowledge about the tissue-specific expression pattern of various proteins in these animals and the unavailability of reagents like antibodies against this species hampers these models' optimal use. The major objective of our current study was to analyze the tissue-specific expression pattern of angiotensin-converting enzyme 2, a proven functional receptor for SARS-CoV-2 in different organs of the hamster. Using two different antibodies (MA5-32307 and AF933), we have conducted immunoblotting, immunohistochemistry, and immunofluorescence analysis to evaluate the ACE2 expression in different tissues of the hamster. Further, at the mRNA level, the expression of Ace2 in tissues was evaluated through RT-qPCR analysis. Both the antibodies detected expression of ACE2 in kidney, small intestine, tongue, and liver. Epithelium of proximal tubules of kidney and surface epithelium of ileum expresses a very high amount of this protein. Surprisingly, analysis of stained tissue sections showed no detectable expression of ACE2 in the lung or tracheal epithelial cells. Similarly, all parts of the large intestine were negative for ACE2 expression. Analysis of tissues from different age groups and sex didn't show any obvious difference in ACE2 expression pattern or level. Together, our findings corroborate some of the earlier reports related to ACE2 expression patterns in human tissues and contradict others. We believe that this study's findings have provided evidence that demands further investigation to understand the predominant respiratory pathology of SARS-CoV-2 infection and disease.

4.
PLoS One ; 16(6): e0252886, 2021.
Article in English | MEDLINE | ID: covidwho-1269920

ABSTRACT

BACKGROUND: Subgroups of precarious populations such as homeless people are more exposed to infection and at higher risk of developing severe forms of COVID-19 compared to the general population. Many of the recommended prevention measures, such as social distancing and self-isolation, are not feasible for a population living in shelters characterised by physical proximity and a high population density. The objective of the study was to describe SARS-CoV-2 infection prevalence in homeless shelters in Brussels (Belgium), and to identify risk factors and infection control practices associated with SARS-CoV-2 positivity rates. METHODS: A total of 1994 adults were tested by quantitative PCR tests in 52 shelters in Brussels (Belgium) between April and June, 2020, in collaboration with Doctors of the World. SARS-CoV-2 prevalence is here described site by site, and we identify risk factors associated with SARS-CoV-2 positivity rates. We also investigate associations between seropositivity and reported symptoms. RESULTS: We found an overall prevalence of 4.6% for the period, and a cluster of high rates of SARS-CoV-2 positivity (20-30% in two shelters). Among homeless people, being under 40 years of age (OR (CI95%) 2.3 (1.2-4.4), p = 0.02), having access to urgent medical care (AMU) (OR(CI95%): 2.4 (1.4-4.4)], p = 0.02), and sharing a room with someone who tested positive (OR(CI95%): 5.3 (2.9-9.9), p<0.0001) were factors associated with SARS-CoV-2 positivity rates. 93% of those who tested positive were asymptomatic. CONCLUSION: This study shows high rates of SARS-COV-2 infection positive tests in some shelters, with a high proportion of asymptomatic cases. The survey reveals how important testing and isolation measures are, together with actions taken by medical and social workers during the outbreak.


Subject(s)
COVID-19 Nucleic Acid Testing/statistics & numerical data , COVID-19/epidemiology , Homeless Persons/statistics & numerical data , Point-of-Care Testing/statistics & numerical data , SARS-CoV-2/isolation & purification , Adult , Age Factors , Asymptomatic Infections/epidemiology , Belgium/epidemiology , COVID-19/diagnosis , COVID-19/virology , Female , Health Services Accessibility/statistics & numerical data , Humans , Infection Control/organization & administration , Infection Control/standards , Infection Control/statistics & numerical data , Male , Mass Screening/statistics & numerical data , Middle Aged , Pandemics/prevention & control , Pandemics/statistics & numerical data , Prevalence , Risk Factors , SARS-CoV-2/genetics , Young Adult
5.
Neurotoxicol Teratol ; 86: 106982, 2021.
Article in English | MEDLINE | ID: covidwho-1187825

ABSTRACT

Despite reports that quinoline antimalarials including chloroquine (Chq) exhibit idiosyncratic neuropsychiatric effects even at low doses, the drug continues to be in widespread use during pregnancy. Surprisingly, very few studies have examined the potential neurotoxic action of Chq exposure at different points of gestation or how this phenomenon may affect neurophysiological well-being in later life. We therefore studied behavior, and the expression of specific genes and neurochemicals modulating crucial neural processes in offspring of rats exposed to prophylactic dose of Chq during different stages of gestation. Pregnant rats were injected 5 mg/kg/day (3 times) of Chq either during early- (first week), mid- (second week), late- (third week), or throughout- (all weeks) gestation, while controls received PBS injection. Behavioral characterization of offspring between postnatal days 15-20 in the open field, Y-maze, elevated plus and elevated zero mazes revealed that Chq evoked anxiogenic responses and perturbed spatial memory in rats, although locomotor activity was generally unaltered. In the prefrontal cortex (PFC), hippocampus and cerebellum of rats prenatally exposed to Chq, RT-qPCR analysis revealed decreased mRNA expression of presynaptic marker synaptophysin, which was accompanied by downregulation of postsynaptic marker PSD95. Synaptic marker PICK1 expression was also downregulated in the hippocampus but was unperturbed in the PFC and cerebellum. In addition to recorded SOD downregulation in cortical and hippocampal lysates, induction of oxidative stress in rats prenatally exposed to Chq was corroborated by lipid peroxidation as evinced by increased MDA levels. Offspring of rats infused with Chq at mid-gestation and weekly treatment throughout gestation were particularly susceptible to neurotoxic changes, especially in the hippocampus. Interestingly, Chq did not cause histopathological changes in any of the brain areas. Taken together, our findings causally link intrauterine exposure to Chq with postnatal behavioral impairment and neurotoxic changes in rats.


Subject(s)
Behavior, Animal/drug effects , Brain Chemistry/drug effects , Chloroquine/toxicity , Neuronal Plasticity/drug effects , Prenatal Exposure Delayed Effects/metabolism , Prenatal Exposure Delayed Effects/psychology , Animals , Anxiety/chemically induced , Anxiety/psychology , Female , Gene Expression/drug effects , Gestational Age , Maze Learning/drug effects , Motor Activity/drug effects , Pregnancy , Rats , Spatial Memory/drug effects
6.
Int J Gen Med ; 14: 2069-2078, 2021.
Article in English | MEDLINE | ID: covidwho-1256167

ABSTRACT

BACKGROUND: Effective management of foreign-imported COVID-19 cases is a new and great challenge for China. Our study focused on the foreign-imported COVID-19 cases to provide detailed data for insights into the prevention, early diagnosis, treatment and control of imported COVID-19. METHODS: For this observational and retrospective study, we investigated the clinical characteristics of imported COVID-19 cases that were confirmed by real-time RT-PCR in the Xi'an Public Health Center from 29 March 2020 to 31 August 2020. RESULTS: Of the 79 patients with COVID-19, 19 (24.1%) had exposure to confirmed COVID-19 patients, 15 (19.0%) had exposure to suspicious COVID-19 patients, and 45 (56.9%) had an unclear history of exposure to confirmed patients. The mean age of the patients was 38 years, and 70 (88.7%) patients were male. Except for 2 severe cases, the remaining 58 (73.4%) cases displayed mild or moderate symptoms, and 19 (24.2%) infected patients were asymptomatic. Twenty-one (26.6%) patients were not diagnosed until a third or later nucleic acid test. Ten (12.7%) patients had chronic diseases. The most common manifestations of the patients were cough [18 (22.8%) cases], fever [9 (11.4%) cases] and sore throat [9 (11.4%) cases]. Forty-one (51.9%) cases showed abnormal chest CT images, To date, all patients have been discharged, and no patient has died. CONCLUSION: The imported COVID-19 cases in Xi'an were mainly young and middle-aged adults with mild or moderate symptoms who had a low rate of comorbidity, showed favourable laboratory and chest CT images, and had a better prognosis. Notably, for suspected COVID-19 cases, at least three consecutive nucleic acid tests should be carried out to avoid missed detection of infected patients. Except for severe cases, high-level medical resources are not necessary in most cases.

7.
Can Commun Dis Rep ; 47(4): 216-223, 2021 May 07.
Article in English | MEDLINE | ID: covidwho-1244374

ABSTRACT

BACKGROUND: During the coronavirus disease 2019 (COVID-19) pandemic, Ontario created a three-phase reopening framework for the economy. Outbreaks were expected at each phase. One week after Phase Two of reopening in the provincial public health administration region of Kingston, Frontenac, Lennox and Addington (KFL&A), a positive case was reported after three weeks of zero new COVID-19 cases. The objective of this report is to describe this COVID-19 outbreak, linked to a personal service setting (PSS), and the public health response to contain the outbreak. METHODS: The outbreak investigation included all COVID-19 cases in KFL&A between June 20, 2020 and July 3, 2020. Public health inspectors and nurses were rapidly deployed to inspect the PSS. A multimodal approach to high-volume testing involved fixed assessment centres, drive-through testing capacity and targeted testing at the outbreak site. Testing was conducted through a real-time polymerase chain reaction assay at the local Public Health Ontario laboratory. RESULTS: Thirty-seven cases were associated with the outbreak: 38% through direct PSS exposure; 32% through household contact; and 30% through social and workplace contact. A superspreading event contributed to 38% of total cases. The majority of cases were in the low to mid-quintiles when analyzed for material deprivation. Testing rates increased four-fold compared to the prior baseline weeks in response to media attention and public health messaging, resulting in a low percent positivity. CONCLUSION: The interplay of aggressive accessible testing, quick lab turnaround time, contact tracing within 24 hours of positive laboratory results as per provincial standards, frequent public communication, rapid inspections, mandatory self-isolation and face coverings were measures successful in halting the outbreak. Inspections or self-audits should be required at all PSSs prior to reopening and outbreak management must work with PSSs to reduce the possibility of superspreading events.

8.
J Thorac Dis ; 13(4): 2288-2299, 2021 Apr.
Article in English | MEDLINE | ID: covidwho-1222569

ABSTRACT

BACKGROUND: We would evaluate the epidemiology, clinical aspects, and prognostic factors of patients of all ages admitted with human corona virus (HCoV). METHODS: This study was retrospectively performed at five university teaching hospitals between 1st January 2018 and 31th March 2020. Routine molecular testing using for multiplex real-time reverse transcription-polymerase chain reaction (RT-PCR) methods was conducted on the respiratory viruses. We assessed the demographics, laboratory findings, and treatment of patients infected with coronavirus. RESULTS: There were 807 coronavirus-infected patients from 24,311 patients with respiratory virus PCR test admitted to five hospitals over 27 months. All-cause mortality rates of patients admitted for seasonal HCoV disease were 3.1% in all patients and 10.8% in patients aged ≥18 years. The Cox proportional hazard regression analysis was performed in patients aged ≥18 years. After adjusting for other clinical variables, general weakness symptoms [hazard ratio (HR), 2.651; 95% confidence interval (CI), 1.147-6.125, P=0.023], National Early Warning Score (NEWS) ≥2 (HR, 5.485; 95% CI, 1.261-23.858, P=0.023), and coronavirus subtype OC43 (HR, 2.500; 95% CI, 1.060-5.897, P=0.036) were significantly associated with death from coronavirus. CONCLUSIONS: Coronavirus infection can reveal a higher mortality rate in patients of ≥18 than those of <18 years, thus, adult patients require more careful treatment. Furthermore, in adult patients, the factors associated with death from coronavirus include general weakness symptoms, NEWS higher than 2, and OC43 subtype.

9.
Indian J Clin Biochem ; 36(4): 473-484, 2021 Oct.
Article in English | MEDLINE | ID: covidwho-1202851

ABSTRACT

To evaluate the role of routine laboratory biomarkers like C Reactive Protein (CRP), Lactate Dehydrogenase (LDH), Interleukin 6 (IL6), Ferritin, Creatinine, Procalcitonin (PCT), Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Serum Albumin, Total Bilirubin (T Bil), High Sensitive Troponin I (hs troponin I), N Terminal-pro B-type Natriuretic Peptide (NT proBNP), Blood Urea Nitrogen (BUN) and Blood Gases in COVID 19 patients who are admitted with SARS CoV-2 positive test results by real-time reverse transcriptase polymerase chain reaction (rRT PCR) in Kokilaben Dhirubhai Ambani Hospital & Medical Research Institute, Mumbai, India. 100 individuals detected with COVID-19 belonging to the age group 12-83 years (median age 62 years) within the period of 1st March 2020 to 10th July 2020 were studied. The case group consisted of 72 males and 28 females. 40 healthy adults without any history or clinical evidence suggestive of COVID-19 and without any comorbidities, like diabetes, hypertension chronic lung disease, cardiac disease, cancer, and immune-compromised individuals were considered as a control group for the study. Routine laboratory findings of these 100 patients were used to evaluate the abnormalities found in COVID-19 patients. Statistical analysis was carried out on the data after determining whether the data had a normal/log-normal distribution and their significance was determined by calculating the p-value. The percentage of patients showing a decrease or increase from the normal value was calculated. Trend analysis was carried out for the 100 patients considered in the case group. Among them, 6 patients were used as representatives to show the trend in these biomarkers during the course of hospital stay. These 5 severe cases consisted of 2 adult males, 2 adult females, and 1 adolescent girl. This selection is to demonstrate the representation of COVID-19 infection in adult males and females and pediatric multisystem inflammatory syndrome associated with COVID-19 in the younger age group. One mild case (adult male) was also selected in the case study. We found a significant increase in mean values of AST, ALT, Total Billirubin, Creatinine, CRP, PCT, LDH, IL6, Ferritin, Lactate, hsTroponin I, NT Pro BNP and decrease in mean values of Albumin, SO2, and PO2 in COVID 19 cases than control. We applied Receiver Operating Curve (ROC) curve to discriminate case population more precisely than the control population. Therefore, Routine laboratory biomarkers appear to play a significant role in COVID-19 patients.

10.
PLoS One ; 16(3): e0248371, 2021.
Article in English | MEDLINE | ID: covidwho-1147457

ABSTRACT

Since its emergence in China, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide including Pakistan. During the pandemic, whole genome sequencing has played an important role in understanding the evolution and genomic diversity of SARS-CoV-2. Although an unprecedented number of SARS-CoV-2 full genomes have been submitted in GISAID and NCBI, data from Pakistan is scarce. We report the sequencing, genomic characterization, and phylogenetic analysis of five SARS-CoV-2 strains isolated from patients in Pakistan. The oropharyngeal swabs of patients that were confirmed positive for SARS-CoV-2 through real-time RT-PCR at National Institute of Health, Pakistan, were selected for whole-genome sequencing. Sequencing was performed using NEBNext Ultra II Directional RNA Library Prep kit for Illumina (NEW ENGLAND BioLabs Inc., MA, US) and Illumina iSeq 100 instrument (Illumina, San Diego, US). Based on whole-genome analysis, three Pakistani SARS-CoV-2 strains clustered into the 20A (GH) clade along with the strains from Oman, Slovakia, United States, and Pakistani strain EPI_ISL_513925. The two 19B (S)-clade strains were closely related to viruses from India and Oman. Overall, twenty-nine amino acid mutations were detected in the current study genome sequences, including fifteen missense and four novel mutations. Notably, we have found a D614G (aspartic acid to glycine) mutation in spike protein of the sequences from the GH clade. The G614 variant carrying the characteristic D614G mutation has been shown to be more infectious that lead to its rapid spread worldwide. This report highlights the detection of GH and S clade strains and G614 variant from Pakistan warranting large-scale whole-genome sequencing of strains prevalent in different regions to understand virus evolution and to explore their genetic diversity.


Subject(s)
Genetic Variation , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Aged, 80 and over , COVID-19/pathology , COVID-19/virology , Female , Gene Deletion , Humans , Male , Middle Aged , Mutation, Missense , Oropharynx/virology , Pakistan , Phylogeny , RNA, Viral/chemistry , RNA, Viral/isolation & purification , RNA, Viral/metabolism , Real-Time Polymerase Chain Reaction , SARS-CoV-2/classification , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/metabolism , Whole Genome Sequencing , Young Adult
11.
Expert Syst Appl ; 176: 114883, 2021 Aug 15.
Article in English | MEDLINE | ID: covidwho-1135324

ABSTRACT

In recent months, a novel virus named Coronavirus has emerged to become a pandemic. The virus is spreading not only humans, but it is also affecting animals. First ever case of Coronavirus was registered in city of Wuhan, Hubei province of China on 31st of December in 2019. Coronavirus infected patients display very similar symptoms like pneumonia, and it attacks the respiratory organs of the body, causing difficulty in breathing. The disease is diagnosed using a Real-Time Reverse Transcriptase Polymerase Chain reaction (RT-PCR) kit and requires time in the laboratory to confirm the presence of the virus. Due to insufficient availability of the kits, the suspected patients cannot be treated in time, which in turn increases the chance of spreading the disease. To overcome this solution, radiologists observed the changes appearing in the radiological images such as X-ray and CT scans. Using deep learning algorithms, the suspected patients' X-ray or Computed Tomography (CT) scan can differentiate between the healthy person and the patient affected by Coronavirus. In this paper, popular deep learning architectures are used to develop a Coronavirus diagnostic systems. The architectures used in this paper are VGG16, DenseNet121, Xception, NASNet, and EfficientNet. Multiclass classification is performed in this paper. The classes considered are COVID-19 positive patients, normal patients, and other class. In other class, chest X-ray images of pneumonia, influenza, and other illnesses related to the chest region are included. The accuracies obtained for VGG16, DenseNet121, Xception, NASNet, and EfficientNet are 79.01%, 89.96%, 88.03%, 85.03% and 93.48% respectively. The need for deep learning with radiologic images is necessary for this critical condition as this will provide a second opinion to the radiologists fast and accurately. These deep learning Coronavirus detection systems can also be useful in the regions where expert physicians and well-equipped clinics are not easily accessible.

12.
Glob Health Med ; 3(2): 107-111, 2021 Apr 30.
Article in English | MEDLINE | ID: covidwho-1128386

ABSTRACT

The quantitative reverse transcription polymerase chain reaction method using nasopharyngeal swabs (NPS RT-qPCR) is regarded as the reference standard for diagnosing coronavirus disease 2019 (COVID-19). However, when using NPS RT-qPCR at busy airport quarantine stations, there are constraints on testing capacity, time, travelerstolerance, and availability of personal protective equipment for quarantine officers. A feasible alternative is therefore needed to test incoming travelers, especially when passenger numbers increase with the resumption of business, tourism, and economic activities. To explore alternatives to NPS RT-qPCR, we collected nasopharyngeal, anterior nasal, and saliva samples chronologically over days 1-7 from asymptomatic COVID-19 air travelers who were under quarantine at a designated facility, and we then compared test results for 9 different methods, comprising RT-qPCR (including the reference method), loop-mediated isothermal amplification (LAMP), and qualitative and quantitative antigen testing. We evaluated sensitivity for 97 person-day samples independently to evaluate asymptomatic travelers regardless of their testing date and period of asymptomatic status upon entry. Sensitivity of the different tests varied from 46.6% to 81.0%, but this was improved from 72.7% to 100.0% when the viral load was > 10 4 copies/sample on NPS RT-qPCR. Thus, most high-risk asymptomatic travelers with higher viral load would be detected by the tests evaluated. Quantitative antigen testing using saliva samples showed 90.9% sensitivity and provided quicker results, and should be an acceptable alternative to NPS RT-qPCR at busy airport quarantine stations. We discuss the implications of our exploratory findings for establishing a comprehensive and feasible testing strategy for COVID-19 among air passengers.

13.
Sci Total Environ ; 778: 146198, 2021 Jul 15.
Article in English | MEDLINE | ID: covidwho-1121857

ABSTRACT

Human sewage from Florianopolis (Santa Catarina, Brazil) was analyzed for severe acute respiratory syndrome coronavirus-2 (SARS-CoV2) from October 2019 until March 2020. Twenty five ml of sewage samples were clarified and viruses concentrated using a glycine buffer method coupled with polyethylene glycol precipitation, and viral RNA extracted using a commercial kit. SARS-CoV-2 RNA was detected by RT-qPCR using oligonucleotides targeting N1, S and two RdRp regions. The results of all positive samples were further confirmed by a different RT-qPCR system in an independent laboratory. S and RdRp amplicons were sequenced to confirm identity with SARS-CoV-2. Genome sequencing was performed using two strategies; a sequence-independent single-primer amplification (SISPA) approach, and by direct metagenomics using Illumina's NGS. SARS-CoV-2 RNA was detected on 27th November 2019 (5.49 ± 0.02 log10 SARS-CoV-2 genome copies (GC) L-1), detection being confirmed by an independent laboratory and genome sequencing analysis. The samples in the subsequent three events were positive by all RT-qPCR assays; these positive results were also confirmed by an independent laboratory. The average load was 5.83 ± 0.12 log10 SARS-CoV-2 GC L-1, ranging from 5.49 ± 0.02 log10 GC L-1 (27th November 2019) to 6.68 ± 0.02 log10 GC L-1 (4th March 2020). Our findings demonstrate that SARS-CoV-2 was likely circulating undetected in the community in Brazil since November 2019, earlier than the first reported case in the Americas (21st January 2020).


Subject(s)
COVID-19 , RNA, Viral , Brazil , Humans , SARS-CoV-2 , Sewage
14.
Eur J Clin Invest ; 51(6): e13501, 2021 Jun.
Article in English | MEDLINE | ID: covidwho-1054522

ABSTRACT

BACKGROUND: The presence of SARS-CoV-2 RNA in plasma has been linked to disease severity and mortality. We compared RT-qPCR to droplet digital PCR (ddPCR) to detect SARS-CoV-2 RNA in plasma from COVID-19 patients (mild, moderate, and critical disease). METHODS: The presence/concentration of SARS-CoV-2 RNA in plasma was compared in three groups of COVID-19 patients (30 outpatients, 30 ward patients and 30 ICU patients) using both RT-qPCR and ddPCR. Plasma was obtained in the first 24h following admission, and RNA was extracted using eMAG. ddPCR was performed using Bio-Rad SARS-CoV-2 detection kit, and RT-qPCR was performed using GeneFinder™ COVID-19 Plus RealAmp Kit. Statistical analysis was performed using Statistical Package for the Social Science. RESULTS: SARS-CoV-2 RNA was detected, using ddPCR and RT-qPCR, in 91% and 87% of ICU patients, 27% and 23% of ward patients and 3% and 3% of outpatients. The concordance of the results obtained by both methods was excellent (Cohen's kappa index = 0.953). RT-qPCR was able to detect 34/36 (94.4%) patients positive for viral RNA in plasma by ddPCR. Viral RNA load was higher in ICU patients compared with the other groups (P < .001), by both ddPCR and RT-qPCR. AUC analysis revealed Ct values (RT-qPCR) and viral RNA load values (ddPCR) can similarly differentiate between patients admitted to wards and to the ICU (AUC of 0.90 and 0.89, respectively). CONCLUSION: Both methods yielded similar prevalence of RNAemia between groups, with ICU patients showing the highest (>85%). RT-qPCR was as useful as ddPCR to detect and quantify SARS-CoV-2 RNAemia in plasma.


Subject(s)
COVID-19/blood , RNA, Viral/blood , Real-Time Polymerase Chain Reaction/methods , Aged , Ambulatory Care , Female , Humans , Intensive Care Units , Male , Middle Aged , Patients' Rooms , Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , Severity of Illness Index
15.
Graefes Arch Clin Exp Ophthalmol ; 259(6): 1605-1608, 2021 Jun.
Article in English | MEDLINE | ID: covidwho-1052975

ABSTRACT

OBJECTIVES: The aims of this study were to evaluate the isolated prevalence of real-time reverse transcriptase-polymerase chain reaction (RT-PCR)-confirmed SARS-CoV-2 on the ocular surface without systemic infection in hospitalized asymptomatic patients and to determine the risk for ophthalmologists and medical staff to be infected by prescreened asymptomatic patients in a tertiary eye care center. METHODS: In this prospective, observational study, bilateral swaps of the conjunctiva in the lower fornices as well as nasopharyngeal swaps were collected in 1145 hospitalized asymptomatic patients of a tertiary eye care center. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was performed for each swap to evaluate the prevalence of SARS-CoV-2. Demographic data and potential risk factors for an isolated infection of the ocular surface were noted. RESULTS: Two thousand two hundred eighty-eight (99.9%) of all 2290 tested eyes had negative results in the RT-PCR analysis of the conjunctival swabs. One patient had bilateral false-positive results in the conjunctival swabs. None of the 1145 patients had any positive RT-PCR-confirmed result in the nasopharyngeal swabs. CONCLUSIONS: The risk for an isolated conjunctival viral activity in patients with a negative nasopharyngeal swab-based RT-PCR seems to be absent or extremely low, suggesting no need to perform additional conjunctival swabs in patients with negative nasopharyngeal swabs. Furthermore, the risk of a work-related SARS-CoV-2 infection due to direct contact with preselected asymptomatic patients in an eye care center is very low, especially when additional hygiene standards and safe distances are respected carefully. This might reassure medical staff and reduce the fear of SARS-CoV-2 infection.


Subject(s)
COVID-19/diagnosis , COVID-19/virology , Conjunctiva/virology , Eyelids/virology , Nasopharynx/virology , SARS-CoV-2/isolation & purification , Adolescent , Adult , Aged , Aged, 80 and over , Asymptomatic Infections , COVID-19/epidemiology , COVID-19 Nucleic Acid Testing , Child , Child, Preschool , Female , Germany/epidemiology , Hospitalization , Humans , Infant , Infant, Newborn , Male , Middle Aged , Prospective Studies , RNA, Viral/genetics , SARS-CoV-2/genetics , Tertiary Care Centers
16.
J Virol Methods ; 290: 114083, 2021 04.
Article in English | MEDLINE | ID: covidwho-1051816

ABSTRACT

In the current pandemic of SARS-CoV-2, rapid identification of infected individuals is crucial for management and control of the outbreak. However, transport of samples, sample processing and RT-qPCR analysis in laboratories are time-consuming. Here we present a prototype of a novel nucleic acid-based test format - pulse controlled amplification - that allows detection of SARS-CoV-2 directly from up to eight swab samples simultaneously without the need for RNA extraction within 25 min with a sensitivity of 100 % for samples with a viral load of ≥ 1.6 × 10e3 copies/µl This new principle might pave the way to rapid and sensitive point of care testing.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , Nucleic Acid Amplification Techniques/methods , SARS-CoV-2/isolation & purification , COVID-19 Nucleic Acid Testing/standards , Humans , Nucleic Acid Amplification Techniques/standards , Point-of-Care Testing , RNA, Viral/genetics , SARS-CoV-2/genetics , Sensitivity and Specificity
17.
J Indian Soc Periodontol ; 25(1): 86-88, 2021.
Article in English | MEDLINE | ID: covidwho-1040151

ABSTRACT

CONTEXT: Dentists across the globe are witnessing a completely unforeseen and uncertain professional situation during these times of COVID-19 pandemic. There is conflicting evidence regarding the effectiveness of routinely used mouthwashes and especially Chlorhexidine, to reduce the viral load in oral cavity and the aerosols during oral procedures. AIMS: Comparative evaluation of the effectiveness of the current 'gold standard' chlorhexidine and povidone iodine as a control agent, through an in-vitro analysis. SETTINGS AND DESIGN: In-vitro laboratory analysis. METHODS AND MATERIAL: All the experiments for analysis of antiviral efficacy of chlorhexidine digluconate (2%)and povidone iodine(1%), against SARS-CoV-2 virus were performed in the BSL3 facility at the Council of Scientific and Industrial Research-Institute of Microbial Technology, using the VeroE6 cell lines. The analysis of the virus inactivation was based on quantification of viral RNA (Cycle threshold (Ct) profile) present in the culture supernatant using Real-Time Quantitative Reverse Transcription PCR (qRT-PCR). STATISTICAL ANALYSIS USED: Descriptive analysis (Statistical package for social sciences (SPSS Inc., Chicago, IL, version 15.0 for Windows). RESULTS: Chlorhexidine digluconate in 0.2% concentration inactivated more than 99.9% of SARS CoV 2 virus, in minimal contact time of 30 seconds, which was considered better efficacy than povidone-iodine utilized for 30 and 60 seconds. Subtle differences were observed in the activity of both the compounds in terms of percent inactivation of virus, though a greater relative change in Ct values was observed for chlorhexidine. CONCLUSIONS: Within the limitations of the present study, it can be concluded that Chlorhexidine digluconate in 0.2% concentration inactivated SARS CoV 2 in minimal contact time i.e 30 secs, however both compounds tested i.e Chlorhexidine and povidone-iodine were found to have antiviral activity against SARS CoV2 virus.

18.
Am J Transl Res ; 12(4): 1348-1354, 2020.
Article in English | MEDLINE | ID: covidwho-1024940

ABSTRACT

BACKGROUND: Since December 2019, there had been an outbreak of COVID-19 in Wuhan, China. At present, diagnosis COVID-19 were based on real-time RT-PCR, which have to be performed in biosafe laboratory and is unsatisfactory for suspect case screening. Therefore, there is an urgent need for rapid diagnostic test for COVID-19. OBJECTIVE: To evaluate the diagnostic performance and clinical utility of the colloidal gold immunochromatography assay for SARS-Cov-2 specific IgM/IgG anti-body detection in suspected COVID-19 cases. METHODS: In the prospective cohort, 150 patients with fever or respiratory symptoms were enrolled in Taizhou Public Health Medical Center, Taizhou Hospital, Zhejiang province, China, between January 20 to February 2, 2020. All patients were tested by the colloidal gold immunochromatography assay for COVID-19. At least two samples of each patient were collected for RT-PCR assay analysis, and the PCR results were performed as the reference standard of diagnosis. Meanwhile 26 heathy blood donor were recruited. The sensitivity and specificity of the immunochromatography assay test were evaluated. Subgroup analysis were performed with respect to age, sex, period from symptom onset and clinical severity. RESULTS: The immunochromatography assay test had 69 positive result in the 97 PCR-positive cases, achieving sensitivity 71.1% [95% CI 0.609-0.797], and had 2 positive result in the 53 PCR-negative cases, achieving specificity 96.2% [95% CI 0.859-0.993]. In 26 healthy donor blood samples, the immunochromatography assay had 0 positive result. In subgroup analysis, the sensitivity was significantly higher in patients with symptoms more than 14 days 95.2% [95% CI 0.741-0.998] and patients with severe clinical condition 86.0% [95% CI 0.640-0.970]. CONCLUSIONS: The colloidal gold immunochromatography assay for SARS-Cov-2 specific IgM/IgG anti-body had 71.1% sensitivity and 96.2% specificity in this population, showing the potential for a useful rapid diagnosis test for COVID-19. Further investigations should be done to evaluate this assay in variety of clinical settings and populations.

19.
Nat Biomed Eng ; 4(12): 1159-1167, 2020 12.
Article in English | MEDLINE | ID: covidwho-960319

ABSTRACT

The diagnosis of severe acute respiratory syndrome 2 (SARS-CoV-2) infection by quantitative PCR with reverse transcription (RT-qPCR) typically involves bulky instrumentation in centralized laboratories and an assay time of 1-2 h. Here, we show that SARS-CoV-2 RNA can be detected in 17 min via a portable device integrating reverse transcription, fast thermocycling (via plasmonic heating through magneto-plasmonic nanoparticles) and in situ fluorescence detection following magnetic clearance of the nanoparticles. The device correctly classified all nasopharyngeal, oropharyngeal and sputum samples from 75 patients with COVID-19 and 75 healthy controls, with good concordance in fluorescence intensity with standard RT-qPCR (Pearson coefficients > 0.7 for the N1, N2 and RPP30 genes). Fast, portable and automated nucleic acid detection should facilitate testing at the point of care.

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