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1.
Journal of Clinical Virology Plus ; 3(1):100132, 2023.
Article in English | ScienceDirect | ID: covidwho-2165516

ABSTRACT

The emergence of SARS-CoV-2 has caused worldwide pandemic of COVID-19. Infection is difficult to diagnose early as some patients remain asymptomatic and may carry this virus to other people. Currently, qRT-PCR is the widely accepted mode for detection. However, the need for sophisticated instrument and trained personnel may hinder its application, especially in remote and facility-lacking areas. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) may serve as a potential approach for detection of SARS-CoV-2 as the resources needed in its application is far less complex than those of qRT-PCR. Herein, we evaluated RT-LAMP based analytical method (COVIDNow), relative to qRT-PCR, in detecting SARS-CoV-2 by using 63 clinical respiratory samples. Based on our finding, COVIDNow exhibited sensitivity and specificity values of 87.5% and 80.6%, respectively. Taken together, RT-LAMP based detection of SARS-CoV-2 by utilizing COVIDNow might serves as a valuable diagnostic tool in the management of global COVID-19 pandemic condition.

2.
Current Bioinformatics ; 17(7):586-598, 2022.
Article in English | EMBASE | ID: covidwho-2141263

ABSTRACT

Objectives: Ganoderic acid Me [GA-Me], a major bioactive triterpene extracted from Ganoderma lucidum, is often used to treat immune system diseases caused by viral infections. Although triterpenes have been widely employed in traditional medicine, the comprehensive mechanisms by which GA-Me acts against viral infections have not been reported. Sendai virus [SeV]-infected host cells have been widely employed as an RNA viral model to elucidate the mechanisms of viral infection. Method(s): In this study, SeV-and mock-infected [Control] cells were treated with or without 54.3 muM GA-Me. RNA-Seq was performed to identify differentially expressed mRNAs, followed by qRT-PCR validation for selected genes. GO and KEGG analyses were applied to investigate potential mechanisms and critical pathways associated with these genes. Result(s): GA-Me altered the levels of certain genes' mRNA, these genes revealed are associated pathways related to immune processes, including antigen processing and presentation in SeV-infected cells. Multiple signaling pathways, such as the mTOR pathway, chemokine signaling pathway, and the p53 pathways, significantly correlate with GA-Me activity against the SeV infection process. qRT-PCR results were consistent with the trend of RNA-Seq findings. Moreover, PPI network analysis identified 20 crucial target proteins, including MTOR, CDKN2A, MDM2, RPL4, RPS6, CREBBP, UBC, UBB, and NEDD8. GA-Me significantly changed transcriptome-wide mRNA profiles of RNA polymerase II/III, protein posttranslational and immune signaling pathways. Conclusion(s): These results should be further assessed to determine the innate immune response against SeV infection, which might help in elucidating the functions of these genes affected by GA-Me treatment in virus-infected cells, including cells infected with SARS-CoV-2. Copyright © 2022 Bentham Science Publishers.

3.
Microbiol Spectr ; : e0165622, 2022 Nov 15.
Article in English | MEDLINE | ID: covidwho-2117157

ABSTRACT

Selection of reference genes during real-time quantitative PCR (qRT-PCR) is critical to determine accurate and reliable mRNA expression. Nonetheless, not a single study has investigated the expression stability of candidate reference genes to determine their suitability as internal controls in SARS-CoV-2 infection or COVID-19-associated mucormycosis (CAM). Using qRT-PCR, we determined expression stability of the nine most commonly used housekeeping genes, namely, TATA-box binding protein (TBP), cyclophilin (CypA), ß-2-microglobulin (B2M), 18S rRNA (18S), peroxisome proliferator-activated receptor gamma (PPARG) coactivator 1 alpha (PGC-1α), glucuronidase beta (GUSB), hypoxanthine phosphoribosyltransferase 1 (HPRT-1), ß-ACTIN, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in patients with COVID-19 of various severities (asymptomatic, mild, moderate, and severe) and those with CAM. We used statistical algorithms (delta-CT [threshold cycle], NormFinder, BestKeeper, GeNorm, and RefFinder) to select the most appropriate reference gene and observed that clinical severity profoundly influences expression stability of reference genes. CypA demonstrated the most consistent expression irrespective of disease severity and emerged as the most suitable reference gene in COVID-19 and CAM. Incidentally, GAPDH, the most commonly used reference gene, showed the maximum variations in expression and emerged as the least suitable. Next, we determined expression of nuclear factor erythroid 2-related factor 2 (NRF2), interleukin-6 (IL-6), and IL-15 using CypA and GAPDH as internal controls and show that CypA-normalized expression matches well with the RNA sequencing-based expression of these genes. Further, IL-6 expression correlated well with the plasma levels of IL-6 and C-reactive protein, a marker of inflammation. In conclusion, GAPDH emerged as the least suitable and CypA as the most suitable reference gene in COVID-19 and CAM. The results highlight the expression variability of housekeeping genes due to disease severity and provide a strong rationale for identification of appropriate reference genes in other chronic conditions as well. IMPORTANCE Gene expression studies are critical to develop new diagnostics, therapeutics, and prognostic modalities. However, accurate determination of expression requires data normalization with a reference gene, whose expression does not vary across different disease stages. Misidentification of a reference gene can produce inaccurate results. Unfortunately, despite the global impact of COVID-19 and an urgent unmet need for better treatment, not a single study has investigated the expression stability of housekeeping genes across the disease spectrum to determine their suitability as internal controls. Our study identifies CypA and then TBP as the two most suitable reference genes for COVID-19 and CAM. Further, GAPDH, the most commonly used reference gene in COVID-19 studies, turned out to be the least suitable. This work fills an important gap in the field and promises to facilitate determination of an accurate expression of genes to catalyze development of novel molecular diagnostics and therapeutics for improved patient care.

4.
BMC Genomics ; 23(1): 755, 2022 Nov 17.
Article in English | MEDLINE | ID: covidwho-2116898

ABSTRACT

BACKGROUND: Since inception of the COVID-19 pandemic, early detection and isolation of positive cases is one of the key strategies to restrict disease transmission. Real time reverse transcription polymerase chain reaction (qRTPCR) has been the mainstay of diagnosis. Most of the qRTPCR kits were designed against the target genes of original strain of SARS-CoV-2. However, with the emergence of variant strains of SARS-CoV-2, sensitivity of the qRTPCR assays has reportedly reduced. In view of this, it is critical to continuously monitor the performance of the qRTPCR kits in the backdrop of variant strains of SARS-CoV-2. Real world monitoring of assay performance is challenging. Therefore, we developed a two-step in-silico screening process for evaluating the performance of various qRTPCR kits used in India. RESULTS: We analysed 73 qRT-PCR kits marketed in India, against the two SARS-CoV-2 VoCs. Sequences of both Delta (B.1.617.2) and Omicron (B.1.1.529) VoCs submitted to GISAID within a specific timeframe were downloaded, clustered to identify unique sequences and aligned with primer and probe sequences. Results were analysed following a two-step screening process. Out of 73 kits analysed, seven were unsatisfactory for detection of both Delta and Omicron VoCs, 10 were unsatisfactory for Delta VoC whereas 2 were unsatisfactory for only Omicron VoC. CONCLUSION: Overall, we have developed a useful screening process for evaluating the performance of qRTPCR assays against Delta and Omicron VoCs of SARS-CoV-2 which can be used for detecting SARS-CoV-2 VoCs that may emerge in future and can also be redeployed for other evolving pathogens of public health importance.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/genetics , Pandemics , RNA, Viral/genetics , RNA, Viral/analysis , Sensitivity and Specificity , COVID-19/diagnosis , COVID-19/epidemiology
5.
Vet Sci ; 9(11)2022 Nov 16.
Article in English | MEDLINE | ID: covidwho-2116277

ABSTRACT

Porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome coronavirus (SADS-CoV) are four identified porcine enteric coronaviruses. Pigs infected with these viruses show similar manifestations of diarrhea, vomiting, and dehydration. Here, a quadruplex real-time quantitative PCR (qRT-PCR) assay was established for the differential detection of PEDV, TGEV, PDCoV, and SADS-CoV from swine fecal samples. The assay showed extreme specificity, high sensitivity, and excellent reproducibility, with the limit of detection (LOD) of 121 copies/µL (final reaction concentration of 12.1 copies/µL) for each virus. The 3236 clinical fecal samples from Guangxi province in China collected between October 2020 and October 2022 were evaluated by the quadruplex qRT-PCR, and the positive rates of PEDV, TGEV, PDCoV, and SADS-CoV were 18.26% (591/3236), 0.46% (15/3236), 13.16% (426/3236), and 0.15% (5/3236), respectively. The samples were also evaluated by the multiplex qRT-PCR reported previously by other scientists, and the compliance rate between the two methods was more than 99%. This illustrated that the developed quadruplex qRT-PCR assay can provide an accurate method for the differential detection of four porcine enteric coronaviruses.

6.
Viruses ; 14(11)2022 Nov 21.
Article in English | MEDLINE | ID: covidwho-2116086

ABSTRACT

Background: The transmissible capacity and toxicity of SARS-CoV-2 variants are continually changing. We report here the follow-up study of hospitalized COVID-19 patients from 2020 to 2022. It is known that the PCR diagnosis for hospitalized patients sometimes causes confusion because of the incompatibility between their diagnosis and symptoms. We applied our sugar chain-immobilized gold-nanoparticles for the extraction and partial purification of RNA from specimens for quantitative RT-PCR assay and evaluated whether the results correlate with patients' symptoms. Methods and Results: Saliva specimens were taken from hospitalized patients with mild or moderate symptoms every early morning. At the time of RT-PCR diagnosis, two methods for the extraction and partial purification of RNA from the specimen were performed: a commonly used Boom (Qiagen) method and our original sugar chain-immobilized gold nanoparticle (SGNP) method. For symptoms, body temperature and oxygen saturation (SpO2) of patients were monitored every 4 h. Conclusions: It was clear that patients infected with the Delta variant needed more time to recover than those with the Omicron variant, and that the SGNP method showed more realistic correlation with the symptoms of patients compared with the common Qiagen method.


Subject(s)
COVID-19 , Metal Nanoparticles , Humans , Reverse Transcriptase Polymerase Chain Reaction , Gold , SARS-CoV-2/genetics , Sugars , Follow-Up Studies , COVID-19/diagnosis , RNA, Viral/genetics , RNA, Viral/analysis , Sensitivity and Specificity , Carbohydrates
7.
Viruses ; 14(11)2022 Oct 25.
Article in English | MEDLINE | ID: covidwho-2082117

ABSTRACT

Wastewater-based epidemiology (WBE) is a tool involving the analysis of wastewater for chemicals and pathogens at the community level. WBE has been shown to be an effective surveillance system for SARS-CoV-2, providing an early-warning-detection system for disease prevalence in the community via the detection of genetic materials in the wastewater. In numerous nation-states, studies have indicated the presence of SARS-CoV-2 in wastewater. Herein, we report the primary time-course monitoring of SARS-CoV-2 RNA in wastewater samples in São José do Rio Preto-SP/Brazil in order to explain the dynamics of the presence of SARS-CoV-2 RNA during one year of the SARS-CoV-2 pandemic and analyze possible relationships with other environmental parameters. We performed RNA quantification of SARS-CoV-2 by RT-qPCR using N1 and N2 targets. The proportion of positive samples for every target resulted in 100% and 96.6% for N1 and N2, respectively. A mean lag of -5 days is observed between the wastewater signal and the new SARS-CoV-2-positive cases reported. A correlation was found between the air and wastewater temperatures and therefore between the SARS-CoV-2 viral titers for N1 and N2 targets. We also observed a correlation between SARS-CoV-2 viral titers and media wastewater flow for the N1 target. In addition, we observed higher viral genome copies within the wastewater samples collected on non-rainy days for the N1 target. Thus, we propose that, based on our results, monitoring raw wastewater may be a broadly applicable strategy that might contribute to resolving the pressing problem of insufficient diagnostic testing; it may represent an inexpensive and early-warning method for future COVID-19 outbreaks, mainly in lower- and middle-income countries.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/genetics , Waste Water , Wastewater-Based Epidemiological Monitoring , COVID-19/epidemiology , RNA, Viral/genetics , Brazil/epidemiology
8.
J Infect Dev Ctries ; 16(8): 1278-1284, 2022 08 30.
Article in English | MEDLINE | ID: covidwho-2030101

ABSTRACT

INTRODUCTION: Mass testing is essential in the surveillance strategy for fighting the COVID-19 pandemic. It allows early detection of suspected cases and subsequently early isolation to mitigate spread. However, the high cost and limited consumables and reagents hinder the mass testing strategy in developing countries such as Indonesia. The specimen pooling strategy is an option to perform mass screening with limited resources. This study aims to determine the positivity rate cut-off and to evaluate the efficiency of pooling strategy for the laboratory diagnosis of COVID-19. METHODOLOGY: Between August 4th, 2020, and November 11th, 2020, a four-sample pooling strategy testing to detect SARS-CoV-2 was carried out at the Microbiology Diagnostic Laboratory of Diponegoro National Hospital, Semarang, Indonesia. Pools with positive results were subjected to individual specimen retesting. Spearman's correlation and linear regression analysis were used to determine the best positivity rate cut-off to apply pooling strategy. RESULTS: A total of 15,216 individual specimens were pooled into 3,804 four-sample pools. Among these pools, 1,007 (26.47%) were positive. Five hundred and ten (50.64%) were 1/4 positive. A maximum positivity rate of 22% is needed to save at least 50% extraction and qRT-PCR reactions in a four-sample pooling strategy. CT values between individual specimens and pools showed a good interval agreement. CONCLUSIONS: Pooling strategy could reduce personnel workload and reagent cost, and increase laboratory capacity by up to 50% when the positivity rate is less than 22%.


Subject(s)
COVID-19 , COVID-19/diagnosis , COVID-19 Testing , Humans , Pandemics , SARS-CoV-2 , Sensitivity and Specificity , Specimen Handling/methods
9.
Medical Journal of Dr. D.Y. Patil Vidyapeeth ; 15(7):S46-S48, 2022.
Article in English | Scopus | ID: covidwho-2024851

ABSTRACT

Background: COVID-19 has caused a pandemic since the end of the year 2019. Controversy regarding the presence of the SARS-CoV-2 in tears and conjunctival sac has created suspense throughout. Moderate to severe dry eye has been observed in the patients infected with COVID-19. Material and Method: We conducted a descriptive, prospective observational study. We enrolled 16 patients fulfilling the inclusion criteria for our study. Patients affected with acute COVID-19 from 28-9-2020 to 9-10-2020 registered at our hospital and willing to participate were included. Tears and conjunctival swabs were taken at the interval of 24 h;consecutively three samples were taken from one COVID-19 infected patient. In total, 48 samples were analyzed by qRT-PCR. Result: We had 16 participants, 47% were females and 53% were males;2/16 [12.5%] patients showed the presence of SARS-CoV-2 in tears and conjunctival swabs. Both the infected patients were females, and they did not have any obvious abnormal ocular conditions. Schirmer strip reading, however, showed a severe dry eye in both the patients. Conclusion: We identified dry eye ranging from mild to moderate to severe [Schirmer 1 reading ranged from 2 to 8 mm] in all our patients admitted to the COVID-19 ward. The SARS-CoV-2 gene seems to be mutating. More work needs to be done to find the viral load in the tears and any abnormalities in the eyes accordingly. © Medical Journal of Dr. D.Y. Patil Vidyapeeth 2022.

10.
Front Cell Infect Microbiol ; 12: 953027, 2022.
Article in English | MEDLINE | ID: covidwho-2022658

ABSTRACT

Quick differentiation of the circulating variants and the emerging recombinant variants of SARS-CoV-2 is essential to monitor their transmission. However, the widely used gene sequencing method is time-consuming and costly when facing the viral recombinant variants, because partial or whole genome sequencing is required. Allele-specific real time RT-PCR (qRT-PCR) represents a quick and cost-effective method in SNP genotyping and has been successfully applied for SARS-CoV-2 variant screening. In the present study, we developed a panel of 3 multiplex allele-specific qRT-PCR assays targeting 12 key differential mutations for quick differentiation of SARS-CoV-2 recombinant variants (XD and XE) and Omicron subvariants (BA.1 and BA.2). Two parallel multiplex qRT-PCR reactions were designed to separately target the protype allele and the mutated allele of the four mutations in each allele-specific qRT-PCR assay. The variation of Cp values (ΔCp) between the two multiplex qRT-PCR reactions was applied for mutation determination. The developed multiplex allele-specific qRT-PCR assays exhibited outstanding analytical sensitivities (with limits of detection [LoDs] of 2.97-27.43 copies per reaction), wide linear detection ranges (107-100 copies per reaction), good amplification efficiencies (82% to 95%), good reproducibility (Coefficient of Variations (CVs) < 5% in both intra-assay and inter-assay tests) and clinical performances (99.5%-100% consistency with Sanger sequencing). The developed multiplex allele-specific qRT-PCR assays in this study provide an alternative tool for quick differentiation of SARS-CoV-2 recombinant variants (XD and XE) and Omicron subvariants (BA.1 and BA.2).


Subject(s)
COVID-19 , SARS-CoV-2 , Alleles , COVID-19/diagnosis , COVID-19 Testing , Humans , RNA, Viral/genetics , Reproducibility of Results , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2/genetics
11.
Diagnostics (Basel) ; 12(9)2022 Sep 02.
Article in English | MEDLINE | ID: covidwho-2009974

ABSTRACT

The rapid identification of patients infected with COVID-19 during the SARS-CoV-2 pandemic is critical to operating emergency rooms effectively. Xpert Xpress SARS-CoV-2 (Xpert) assays are increasingly being used in the rapid screening of COVID-19. We evaluated the clinical performance of Xpert by comparing findings with those of qRT-PCR evaluations and included the clinical features of patients visiting the emergency department. Positive results with Xpert testing (n = 370) were compared with qRT-PCR findings, demonstrating a 91.9% intertest agreement. We reviewed the subsequent COVID-19 test results and SARS-CoV-2 infection histories for individuals showing discrepancies in Xpert and qRT-PCR testing and determined whether the findings were true-positive or false-positive. The true-positive rate for Xpert testing was 95.4% (353/370); the remaining 17 samples (4.6%) were false-positive. All false-positive data for Xpert testing showed N2 signals amplified to Ct values of ≥40 with no E gene signals. Rapid Xpert testing is highly sensitive and shows a good performance overall in challenging situations, such as an emergency room. However, we considered the possibility of false-positive Xpert results given an N2 gene signal only, especially given high Ct values. We recommend interpreting test data with caution and considering retesting over time.

12.
Front Med (Lausanne) ; 9: 869818, 2022.
Article in English | MEDLINE | ID: covidwho-2009875

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is believed to have originated in Wuhan City, Hubei Province, China, in December 2019. Infection with this highly dangerous human-infecting coronavirus via inhalation of respiratory droplets from SARS-CoV-2 carriers results in coronavirus disease 2019 (COVID-19), which features clinical symptoms such as fever, dry cough, shortness of breath, and life-threatening pneumonia. Several COVID-19 waves arose in Taiwan from January 2020 to March 2021, with the largest outbreak ever having a high case fatality rate (CFR) (5.95%) between May and June 2021. In this study, we identified five 20I (alpha, V1)/B.1.1.7/GR SARS-CoV-2 (KMUH-3 to 7) lineage viruses from COVID-19 patients in this largest COVID-19 outbreak. Sequence placement analysis using the existing SARS-CoV-2 phylogenetic tree revealed that KMUH-3 originated from Japan and that KMUH-4 to KMUH-7 possibly originated via local transmission. Spike mutations M1237I and D614G were identified in KMUH-4 to KMUH-7 as well as in 43 other alpha/B.1.1.7 sequences of 48 alpha/B.1.1.7 sequences deposited in GISAID derived from clinical samples collected in Taiwan between 20 April and July. However, M1237I mutation was not observed in the other 12 alpha/B.1.1.7 sequences collected between 26 December 2020, and 12 April 2021. We conclude that the largest COVID-19 outbreak in Taiwan between May and June 2021 was initially caused by the alpha/B.1.1.7 variant harboring spike D614G + M1237I mutations, which was introduced to Taiwan by China Airlines cargo crew members. To our knowledge, this is the first documented COVID-19 outbreak caused by alpha/B.1.1.7 variant harboring spike M1237I mutation thus far. The largest COVID-19 outbreak in Taiwan resulted in 13,795 cases and 820 deaths, with a high CFR, at 5.95%, accounting for 80.90% of all cases and 96.47% of all deaths during the first 2 years. The high CFR caused by SARS-CoV-2 alpha variants in Taiwan can be attributable to comorbidities and low herd immunity. We also suggest that timely SARS-CoV-2 isolation and/or sequencing are of importance in real-time epidemiological investigations and in epidemic prevention. The impact of G614G + M1237I mutations in the spike gene on the SARS-CoV-2 virus spreading as well as on high CFR remains to be elucidated.

13.
Am J Infect Control ; 50(8): 890-897, 2022 08.
Article in English | MEDLINE | ID: covidwho-2000216

ABSTRACT

BACKGROUND: SARS-CoV-2 emerged in 2019 and resulted in a pandemic causing millions of infections worldwide. Gold-standard for SARS-CoV-2 detection uses quantitative RT-qPCR on respiratory secretions to detect viral RNA (vRNA). Acquiring these samples is invasive, can be painful for those with xerostomia and other health conditions, and sample quality can vary greatly. Frequently only symptomatic individuals are tested even though asymptomatic individuals can have comparable viral loads and efficiently transmit virus. METHODS: We utilized a non-invasive approach to detect SARS-CoV-2 in individuals, using polyvinyl alcohol (PVA) strips embedded in KN95 masks. PVA strips were tested for SARS-CoV-2 vRNA via qRT-PCR and infectious virus. RESULTS: We show efficient recovery of vRNA and infectious virus from virus-spiked PVA with detection limits comparable to nasal swab samples. In infected individuals, we detect both human and SARS-CoV-2 RNA on PVA strips, however, these levels are not correlated with length of time mask was worn, number of times coughed or sneezed, or level of virus in nasal swab samples. We successfully cultured and deep-sequenced PVA-associated virus. CONCLUSIONS: These results demonstrate the feasibility of using PVA-embedded masks as a non-invasive platform for detecting SARS-CoV-2 in exhaled air in COVID-positive individuals regardless of symptom status.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnosis , COVID-19 Testing , Humans , Pandemics , RNA, Viral/analysis , RNA, Viral/genetics
14.
Gastro Hep Adv ; 1(5): 844-852, 2022.
Article in English | MEDLINE | ID: covidwho-1959542

ABSTRACT

Background and Aims: Recent evidence suggests that the gut is an additional target for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. However, whether SARS-CoV-2 spreads via gastrointestinal secretions remains unclear. To determine the prevalence of gastrointestinal SARS-CoV-2 infection in asymptomatic subjects, we analyzed gastrointestinal biopsy and liquid samples from endoscopy patients for the presence of SARS-CoV-2. Methods: We enrolled 100 endoscopic patients without known SARS-CoV-2 infection (cohort A) and 12 patients with a previous COVID-19 diagnosis (cohort B) in a cohort study performed at a regional hospital. Gastrointestinal biopsies and fluids were screened for SARS-CoV-2 by polymerase chain reaction (PCR), immunohistochemistry, and virus isolation assay, and the stability of SARS-CoV-2 in gastrointestinal liquids in vitro was analyzed. Results: SARS-CoV-2 ribonucleic acid was detected by PCR in the colonic tissue of 1/100 patients in cohort A. In cohort B, 3 colonic liquid samples tested positive for SARS-CoV-2 by PCR and viral nucleocapsid protein was detected in the epithelium of the respective biopsy samples. However, no infectious virions were recovered from any samples. In vitro exposure of SARS-CoV-2 to colonic liquid led to a 4-log-fold reduction of infectious SARS-CoV-2 within 1 hour (P ≤ .05). Conclusion: Overall, the persistent detection of SARS-CoV-2 in endoscopy samples after resolution of COVID-19 points to the gut as a long-term reservoir for SARS-CoV-2. Since no infectious virions were recovered and SARS-CoV-2 was rapidly inactivated in the presence of colon liquids, it is unlikely that performing endoscopic procedures is associated with a significant infection risk due to undiagnosed asymptomatic or persistent gastrointestinal SARS-CoV-2 infections.

15.
Int J Environ Res Public Health ; 19(9)2022 05 08.
Article in English | MEDLINE | ID: covidwho-1953351

ABSTRACT

Governments have implemented measures to minimize SARS-CoV-2 spread. However, these measures were relaxed, and the appearance of new variants has prompted periods of high contagion known as waves. In Mexico, four waves distributed between July and August 2020, January and February 2021, August and September 2021, and January and February 2022 have appeared. Current health policies discourage mass sampling, preferring to focus on the corrective treatment of severe cases. Outpatients are only advised to undergo brief voluntary confinement and symptomatic treatment, with no follow-up. Therefore, the present study aimed to analyze sex, age, and viral load in outpatients during the four waves in a medium-sized city in Mexico. For each wave, the date of peak contagion was identified, and data were collected within ±15 days. In this regard, data from 916 patients (434 men and 482 women) were analyzed. The age range of positive patients (37-45 years) presented a higher frequency during the first and third waves, while 28-36 years was the most frequent age range during the second and fourth waves, while the viral load values were significantly higher, for both sexes, during the fourth wave. Obtained data of COVID-19 prevalence in population segments can be used for decision-making in the design of effective public health policies.


Subject(s)
COVID-19 , SARS-CoV-2 , Adult , COVID-19/epidemiology , Female , Humans , Male , Middle Aged , Outpatients , Serologic Tests , Viral Load
16.
Microorganisms ; 10(7)2022 Jul 01.
Article in English | MEDLINE | ID: covidwho-1917628

ABSTRACT

Precise reasons for severe manifestation of SARS-CoV-2 remain unanswered, and efforts have been focused on respiratory system management. Demonstration of unequivocal presence of SARS-CoV-2 in vital body organs by cadaver autopsy was the only way to prove multi-organ involvement. Hence, the primary objective of the study was to determine presence of the SARS-CoV-2 in various organs of patients succumbing to SARS-CoV-2 infection. A total of 246 samples from different organs of 21 patients who died due to severe COVID-19 illness were investigated by qRT-PCR, and SARS-CoV-2 was detected in 181 (73.57%) samples and highest positivity of SARS-CoV-2 being (expectedly) found in nasopharynx (90.4%) followed by bilateral lungs (87.30%), peritoneal fluid (80%), pancreas (72.72%), bilateral kidneys (68.42%), liver (65%) and even in brain (47.2%). The deceased patients were categorized to three subgroups based upon the extent of organs in which SARS-CoV-2 was detected by qRT-PCR (high intensity ≥80%, intermediate intensity = 65-80% and low intensity ≤65% organs involvement). It was conclusively established that SARS-CoV-2 has the property of invasion beyond lungs and even crosses the blood-brain barrier, resulting in multi-system disease; this is probably the reason behind cytokine storm, though it is not clear whether organ damage is due to direct injury caused by the virus or result of inflammatory assault. Significant inverse correlation was found between the Ct value of lung samples and number of organs involved, implying that higher viral load in lungs is directly proportionate to involvement of extrapulmonary organs and patients with higher viral load in respiratory secretions should be monitored more closely for any warning signs and the treatment strategies should also address involvement of other organs for better outcome, because lungs, though the primary site of infection, are not the only organ system responsible for pathogenesis of systemic illness.

17.
Viruses ; 14(6)2022 06 16.
Article in English | MEDLINE | ID: covidwho-1911640

ABSTRACT

As the SARS-CoV-2 virus evolves, mutations may result in diminished sensitivity to qRT-PCR diagnostic assays. We investigated four polymorphisms circulating in the SARS-CoV-2 Delta lineage that result in N gene target failure (NGTF) on the TaqPath COVID-19 Combo Kit. These mutations were detected from the SARS-CoV-2 genome sequences that matched with the diagnostic assay results of saliva specimens. Full length N genes from the samples displaying NGTF were cloned into plasmids and assayed using three SARS-CoV-2 qRT-PCR assays. These constructs resulted in reduced sensitivity to the TaqPath COVID-19 Combo Kit compared to the controls (mean Ct differences of 3.06, 7.70, 12.46, and 14.12), but were detected equivalently on the TaqPath COVID-19 Fast PCR Combo 2.0 or CDC 2019_nCoV_N2 assays. This work highlights the importance of genomic sequencing to monitor circulating mutations and provide guidance in improving diagnostic assays.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnosis , Humans , Mutation , Pathology, Molecular , SARS-CoV-2/genetics , Sensitivity and Specificity
18.
Diagnostics (Basel) ; 12(7)2022 Jun 27.
Article in English | MEDLINE | ID: covidwho-1911244

ABSTRACT

BACKGROUND: Rapid diagnosis of COVID-19 is essential in order to restrict the spread of the pandemic, and different approaches for SARS-CoV-2 testing have been proposed as cost-effective and less time-consuming alternatives. For virus detection, the real-time reverse transcriptase-polymerase chain reaction (RT-PCR) technique is still the "gold standard" for accuracy and reliability, but its performance is affected by the efficiency of nucleic acid extraction methods. OBJECTIVE: In order to improve the SARS-CoV-2 diagnostic workflow, we compared a "standard" commercially available kit, based on viral RNA extraction from human swab samples by magnetic beads, with its technological evolution. The two methods differ mainly in their time consumption (9 vs. 35 min). METHODS: We adopted the MAGABIO PLUS VIRUS DNA/RNA PURIFICATION KIT II (BIOER), defined as "standard", with the automatic extractor BIOER (GenePure Pro fully automatic nucleic acid purification system) to isolate RNA from nasopharyngeal swabs for the detection of SARS-CoV-2 by RT-PCR. We tested this kit with a new faster version of the first one, defined as "rapid" (MAGABIO PLUS VIRUS RNA PURIFICATION KIT II). RESULTS AND CONCLUSION: The two evaluated procedures provided similar analytical results, but the faster method proved to be a more suitable tool for the detection of SARS-CoV-2 from nasopharyngeal swabs, due to a more rapid availability of results, which may contribute to improving both clinical decision making and patient safety.

19.
J Clin Med ; 11(12)2022 Jun 09.
Article in English | MEDLINE | ID: covidwho-1884245

ABSTRACT

In the tissue donation field, to prevent pathogen transmission, all donors are screened by postmortem swabs for SARS-CoV-2 using qRT-PCR. Corneas from donors who tested positive for SARS-CoV-2 were subjected to further investigations. Corneal transplants and culture medium from positive donors were cultured under appropriate safety conditions for further analyses. Cornea tissue samples, including sclera/limbus/cornea, and culture media were taken at different time points for testing for SARS-CoV-2 using qRT-PCR, immunohistochemistry (IHC) and subgenomic RNA (sgRNA) analysis. Between January and May 2021, in four donors with initial negative premortem rapid tests, SARS-CoV-2 was detected post-mortem using qRT-PCR. In these cases, SARS-CoV-2 was observed at the beginning of cultivation in both tissue and culture medium using qRT-PCR and IHC. The virus was mainly localized in the limbus epithelial cells, with a stable detection level. Premortem rapid tests are potentially insufficient to exclude SARS-CoV-2 infection in corneal donors. While, for SARS-CoV-2, the risk of infection via transplants is considered low, a residual risk remains for presymptomatic new infections. However, our investigations provide the first indications that, with organ cultures, the risk of virus transmission is minimized due to the longer minimum culture period.

20.
Virol J ; 19(1): 77, 2022 05 02.
Article in English | MEDLINE | ID: covidwho-1879243

ABSTRACT

BACKGROUND: To retain the spread of SARS-CoV-2, fast, sensitive and cost-effective testing is essential, particularly in resource limited settings (RLS). Current standard nucleic acid-based RT-PCR assays, although highly sensitive and specific, require transportation of samples to specialised laboratories, trained staff and expensive reagents. The latter are often not readily available in low- and middle-income countries and this may significantly impact on the successful disease management in these settings. Various studies have suggested a SARS-CoV-2 loop mediated isothermal amplification (LAMP) assay as an alternative method to RT-PCR. METHODS: Four previously published primer pairs were used for detection of SARS-CoV-2 in the LAMP assay. To determine optimal conditions, different temperatures, sample input and incubation times were tested. Ninety-three extracted RNA samples from St. George's Hospital, London, 10 non-extracted nasopharyngeal swab samples from Great Ormond Street Hospital for Children, London, and 92 non-extracted samples from Queen Elisabeth Central Hospital (QECH), Malawi, which have previously been tested for SARS-Cov-2 by quantitative reverse-transcription RealTime PCR (qRT-PCR), were analysed in the LAMP assay. RESULTS: In this study we report the optimisation of an extraction-free colourimetric SARS-CoV-2 LAMP assay and demonstrated that a lower limit of detection (LOD) between 10 and 100 copies/µL of SARS-CoV-2 could be readily detected by a colour change of the reaction within as little as 30 min. We further show that this assay could be quickly established in Malawi, as no expensive equipment is necessary. We tested 92 clinical samples from QECH and showed the sensitivity and specificity of the assay to be 86.7% and 98.4%, respectively. Some viral transport media, used routinely to stabilise RNA in clinical samples during transportation, caused a non-specific colour-change in the LAMP reaction and therefore we suggest collecting samples in phosphate buffered saline (which did not affect the colour) as the assay allows immediate sample analysis on-site. CONCLUSION: SARS-CoV-2 LAMP is a cheap and reliable assay that can be readily employed in RLS to improve disease monitoring and management.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnosis , Child , Humans , Molecular Diagnostic Techniques , Nucleic Acid Amplification Techniques/methods , RNA , SARS-CoV-2/genetics
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