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1.
Open Forum Infectious Diseases ; 9(Supplement 2):S734, 2022.
Article in English | EMBASE | ID: covidwho-2189884

ABSTRACT

Background. We sought to compareWWSARS-CoV-2 RNA detection across a range of sites and scales using RTqPCR and RTddPCR. Figure. Methods. Composite-24hWW was collected from aWWtreatment plant (WTP;n=18), a neighborhood (Nb1;n=12) and three hospitals;H-1, H-2, and H-3 (3-sites;A-C)(n=84). RNA was extracted using the 4S-silica column method. RTqPCR (QuantStudio5, Thermo Fisher) and RTddPCR (C1000 Thermal Cycler and QX200 Droplet Reader, BioRad) quantified SARS-CoV-2 RNA nucleocapsid (N2, US CDC) and envelope (E Sarbeco, Corman et al 2020) in triplicate. Fisher's exact test was used to compare assay sensitivity. Correlations between modalities and RNA - clinically-confirmed COVID-19 cases (defined by postal code of primary residence using 5-day rolling average) was assessed using Persons correlation. Results. 114 samples were tested (02/23/2021-04/22/2021). SARS-CoV-2-N2 was identified in 90/114 (79%) by RTqPCR and 89/114 (78%) by ddPCR (p=1). SARS-CoV-2 E was found in 72/114 (63%) by RTqPCR and 90/114 (79%) by ddPCR, p=0.01. Correlations between modalities were strongest for N2 relative to E across all sites (see Table). N2 correlated with clinically diagnosed cases for both modalities greater at the level of the WTP (RTqPCR;r=0.8972, p< 0.0001and ddPCR;0.933, p< 0.0001) relative to neighborhood (RTqPCR;r=0.6, p=0.04 and ddPCR;0.60, p=0.04). E correlated to a lesser degree with cases at WTP (RTqPCR;r=0.65, p=0.0035 and ddPCR;0.88, p=< 0.001) and neighborhoods (RTqPCR;r=0.40, p=0.20 and ddPCR;r=0.43, p=0.16). Conclusion. SARS-CoV-2 detection of N2 was similar between RTqPCR and RTddPCR across a range of sites and scales in the sewershed, and this correlated best with clinical cases whereas E detection was superior with ddPCR.

2.
Journal of Clinical and Diagnostic Research ; 16(8):DC53-DC57, 2022.
Article in English | EMBASE | ID: covidwho-2067196

ABSTRACT

Introduction: In the search of effective medicines against Coronavirus Disease-2019 (COVID-19) besides the conventional mode of treatment many medicines belonging to alternative therapeutics claimed to be effective in this disease. In homeopathy-a branch of alternative medicine some medicines are claimed to be effective in COVID-19 after human trials. Aim: To study whether ultradiluted preparation of Phosphorus 6CH (centesimal (C) dilutions, using Hanhemann's (H) dilution method) can protect damaging action of Delta Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) spike protein Receptor Binding Domain (RBD) in Gallus gallus embryo in relation to their gross appearances, histopathological changes and cytokine changes. Materials and Methods: An in-vivo fertilized chick embryo model experimental analysis was carried out at the Genetic Research Laboratory of Heritage Institute of Technology, Kolkata, West Bengal, India. The whole experimental study was done in a time period of November 2021 to January 2022 and the data collected were analysed using statistical software Minitab. About 14 days old Gallus gallus embryonated eggs were inoculated with the antigen along with the vehicle alcohol controls. The Phosphorus 6CH was used to see whether it can prevent or cure the damaging action of the spike protein in the embryo in different experimental sets. results: The notable finding in this experiment is the remarkable elevated expression of Interleukin (IL)-10 gene in the curative, preventive sets as well as in the medicine control sets in comparison to antigen and alcohol control sets. In case of Transforming Growth Factor, (TGF) β1 there was enhanced expression of TGF β1 gene in the alcohol 6C set and antigen set which gets ameliorated with Phosphorus 6CH. The morbid anatomy of the embryo and the histopathological picture of the liver of the embryo also reflected similar findings in these two experimental sets. After statistical analysis it was found that there was significant correlation in between Interferon (IF) γ and IL-10 in these experimental results which appears very important. conclusion: The homeopathic medicine phosphorus 6CH is capable of maintaining cytokine balance in Delta SARS-CoV-2 spike protein RBD induced pathogenecity in Gallus gallus embryo.

3.
Future Virology ; 17(7):429-439, 2022.
Article in English | EMBASE | ID: covidwho-2032730

ABSTRACT

Aim: This study aimed to evaluate chemokine receptor 5 delta 32 (CCR5-δ32) mutation and HIV-1 surveillance drug-resistance mutations (SDRMs) in peripheral blood mononuclear cells of long-term non progressors (LTNPs) of HIV-1-infected individuals. Materials & methods: This research was performed on 197 treatment-naive HIV-1-infected patients. After follow-up, it was determined that 15 (7.6%) of these people were LTNPs. The PCR assay was performed to identify the CCR5 genotype and HIV-1 SDRMs. Results: One (6.7%) of the LTNPs was heterozygous (wt/δ32) for the CCR5 delta 32 (CCR5δ32). However, none of the individuals was homozygous for this mutation (δ32/δ32). Moreover, none of the LTNPs showed HIV-1 SDRMs. The CRF35-AD subtype was the most dominant subtype, with a percentage of 93.3%. Conclusion: Iranian elite controllers are negative for CCR5-delta 32 homozygous genotype and drug resistance against antiretroviral drugs.

4.
HemaSphere ; 6:3304-3305, 2022.
Article in English | EMBASE | ID: covidwho-2032139

ABSTRACT

Background: Treatment-free remission (TFR) in CML is defined as achieving a deep molecular remission (DMR) corresponding to molecular remissions of grade 4 or >4. Classically, Imatinib allows it to be achieved in nearly 20% of cases. Aims: We present our experience of stopping Imatib (IMc) (a copy of Imatinib) Methods: Methods and patients This was a study of 25 patients (pts): median age at diagnosis 40 years (9-62) including 2 children and 23 adults, at the time of discontinuation 50 years (18- 73), sex ratio M/F=0.92 (12H/13F);the sokal: 11 pts are intermediate risk, 8 low risk and 6 high risk. According to ELTS, 10 pts low-risk, 10 intermediate-risk, and 5 high-risk, all in chronic phase, all Mbcr/abl and received IMc at 400 mg/day, the first since June 2007 and the last in October 2013. The pts were followed by GeneXpert automated PCR, until deep MR [MMR4 and MMR4.5 ](Xpert BCR-ABL Ultra test) was achieved and maintained for more than 24 months with a hindsight of more than 5 years. Follow-up of discontinuation was done by PCR every month (2 to 3 months during covid-19);resumption of IMc if BCR/ABL transcript > 0.1% and maintenance of IMc discontinuation, if transcript ≤ 0.1% with monitoring. Deep MR (DMR) was completed at a mean of 70 months;2 pts (8.3%) were in MMR4 and 23 pts (91.7%) in MMR4.5. with a median follow-up of 8.5 years (6 - 12 years) . Imatib discontinuation was started in January 2019 Results: Results of discontinuation CHR was maintained in 24 pts, relapse at 6 months in one pt. TFR was lost in 9 pts (36%): [2 pts (20%) at 1 month, 3 pts (30%) at 2 months, 5 pts (50%) at 6 months]. The median duration of TFR was 23.5 months, 16 pts (64%) remained in DMR (2 children and 14 adults), no progression, withdrawal syndrome noted in 36% (9pts) at one month of stopping, mainly musculoskeletal pain in 28% (7pts), skin rash with pruritus in 12% (3 pts), asthenia in12%, palpebral myositis;among patients who presented these events 66.6% (6 pts) are still in TFR. For relapsed patients, the resumption of IMc was effective in 9 patients, with MMR4 and MMR4.5 achieved on average at 6 months, Summary/Conclusion: Comments: the FIT rate is 64% in our cohort, the relapses (9/25) occurred very quickly, during the first 6 months, the duration of impregnation in our patients who lost their TFR was on average 9 years (6-12 years), without difference with the patients who maintained the FIT which is 8.7 years (6- 13). Sokal and ELTS scores had no influence on FIT since 44.4% (4pts) of intermediate, 33.3% (3pts) of low and 2 of high risk relapsed vs 37.5% (6pts) of intermediate, 25% (4pts) of low and 4 of high risk in FIT, an influence of gender since 77.7% of relapses were men, age and even depth of MMR are all potential factors for cessation but the results in our cohort. The results in our cohort do not confirm their influence since 88.8% of our relapses were in MMR4.5 with a median age of 45 years (42-69) Conclusion: The A-STIM and EURO-SKI studies demonstrate that the depth of response before stopping does not play a prognostic factor for a good FIT, which is confirmed by our study;therapeutic impregnation is the only factor ensuring the maintenance of the FIT found in the studies less in our cohort, hence the factors ensuring the maintenance of the FIT in CML are still to be determined, in order to select the eligible patients.

5.
Annals of the Rheumatic Diseases ; 81:1689-1690, 2022.
Article in English | EMBASE | ID: covidwho-2009071

ABSTRACT

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral load and its impact on disease outcome in patients with autoimmune rheumatic disease (ARD) are lacking. Also, whether patients with ARD receiving immunomodulators have different viral loads compared to the general population is unknown. Objectives: To compare the viral load of SARS-CoV-2 and its trending between patients without and with ARD. Methods: Retrospectively, patients with ARD infected with SARS-CoV-2 were matched by age and sex at a ratio of 1:2 to patients without ARD and not receiving immunosuppression or immunomodulator drugs. Viral load was determined by the cycle threshold (CT) value measured by a number of platforms: (a) Automated Platforms-the Roche Cobas 6800 system using the Cobas SARS-CoV-2 Test targeting the E and orf1a/b genes (Roche, Switzerland) and the Xpert Xpress SARS-CoV-2 targeting the E and N genes (Cepheid, USA);(b) Manual platforms-EZ1 (QIAGEN, USA), QIAsymphony (QIAGEN, USA), and Bioneer ExiPrepTM 96 Virus DNA/RNA kits Catalogue No K4614 (Bioneer, South Korea) extraction with thermal cycling using TaqPath™ PCR COVID-19 Combo Kit targeting the N, S and orf1a/b genes (Thermo Fisher Scientific, USA) on ABI 7500 thermal cyclers. Independent samples t-test was used to compare the mean CT values of the study groups at baseline and at 5 subsequent intervals (1-5.9, 6-11.9, 12-17.9, 18-23.9 and 24-30 days). Results: Mean age (SD) of 197 cases and 420 controls were 45.2 (11.8) and 44.1 (12.3) years, respectively. Females were predominant in both groups 60% vs. 52%, P=0.053. The most common ARD was rheumatoid arthritis in 82 cases (41.6%), followed by spondyloarthropathy in 33 (16.8%) and systemic lupus ery-thematosus in 31 (15.7%). Of the cases, 67% were on conventional synthetic disease modifying anti-rheumatic drugs (DMARDs), 15.2% on biological DMARDs and 4.6% patients were on rituximab. The mean CT values was signifcantly lower in the ARD group at baseline and persisted till day 24. Conclusion: Compared to patients without ARD, the viral load of SARS-CoV-2 in patients with ARD is signifcantly higher at baseline testing and persists till day 24. This fnding may indicate that patients with ARD are at higher risk of severe SARS-CoV-2 infection and prolonged potential transmission. Clinical outcome correlation is needed.

6.
Annals of the Rheumatic Diseases ; 81:961, 2022.
Article in English | EMBASE | ID: covidwho-2009057

ABSTRACT

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral load and its impact on disease outcome in patients with autoimmune rheumatic disease (ARD) are lacking. Also, whether patients with ARD receiving immunomodulators have different viral loads compared to the general population is unknown. Objectives: To compare the viral load of SARS-CoV-2 and its trending between patients without and with ARD. Methods: Retrospectively, patients with ARD infected with SARS-CoV-2 were matched by age and sex at a ratio of 1:2 to patients without ARD and not receiving immunosuppression or immunomodulator drugs. Viral load was determined by the cycle threshold (CT) value measured by a number of platforms: (a) Automated Platforms-the Roche Cobas 6800 system using the Cobas SARS-CoV-2 Test targeting the E and orf1a/b genes (Roche, Switzerland) and the Xpert Xpress SARS-CoV-2 targeting the E and N genes (Cepheid, USA);(b) Manual platforms-EZ1 (QIAGEN, USA), QIAsymphony (QIAGEN, USA), and Bioneer ExiPrepTM 96 Virus DNA/RNA kits Catalogue No K4614 (Bioneer, South Korea) extraction with thermal cycling using TaqPath™ PCR COVID-19 Combo Kit targeting the N, S and orf1a/b genes (Thermo Fisher Scientific, USA) on ABI 7500 thermal cyclers. Independent samples t-test was used to compare the mean CT values of the study groups at baseline and at 5 subsequent intervals (1-5.9, 6-11.9, 12-17.9, 18-23.9 and 24-30 days). Results: Mean age (SD) of 197 cases and 420 controls were 45.2 (11.8) and 44.1 (12.3) years, respectively. Females were predominant in both groups 60% vs. 52%, P=0.053. The most common ARD was rheumatoid arthritis in 82 cases (41.6%), followed by spondyloarthropathy in 33 (16.8%) and systemic lupus ery-thematosus in 31 (15.7%). Of the cases, 67% were on conventional synthetic disease modifying anti-rheumatic drugs (DMARDs), 15.2% on biological DMARDs and 4.6% patients were on rituximab. The mean CT values was signifcantly lower in the ARD group at baseline and persisted till day 24. Conclusion: Compared to patients without ARD, the viral load of SARS-CoV-2 in patients with ARD is signifcantly higher at baseline testing and persists till day 24. This fnding may indicate that patients with ARD are at higher risk of severe SARS-CoV-2 infection and prolonged potential transmission. Clinical outcome correlation is needed.

7.
Journal of Public Health in Africa ; 13:26-27, 2022.
Article in English | EMBASE | ID: covidwho-2006896

ABSTRACT

Introduction/ Background: Tuberculosis(TB) and COVID-19 pandemics are airbone diseases of public health threat globally. They also share some clinical signs and symptoms. We, therefore, took advantage of collected sputum samples at the early stage of COVID-19 outbreak in Ghana to conduct differential diagnoses of long standing endemic respiratory illness, particularly tuberculosis. Methods: Sputum samples collected through the enhanced national surveys from suspected COVID-19 patients and contact tracing cases were analyzed for TB. The sputum samples were processed using Cepheid's GeneXpert-MTB/RIF assay in pools of 4 samples to determine the presence of Mycobacterium tuberculosis complex. Positive pools were then decoupled and analyzed individually. Details of positive TB samples were forwarded to the NTP for appropriate case management. Chi-square-test, Fischer's exact test, or logistic regression where appropriate was used to test for statistical significance. P=values <0.05 were regarded as statistically significant at a 95% confidence level. All statistical analyses were carried out in Stata. Results: Seven-hundred and seventy-four sputum samples were analyzed for Mycobacterium tuberculosis in both suspected COVID-19 cases (679/774, 87.7%) and their contacts (95/774,12.3%). A total of 111(14.3%) were diagnosed with SARS CoV2 infection and six (0.8%) out of the 774 individuals tested positive for pulmonary tuberculosis: five(83.3%) males and one(16.7%) female. Drug susceptibility analysis identified 1(16.7%) rifampicin-resistant tuberculosis case. Out of the six TB positive cases, 2 (33.3%) tested positive for COVID- 19 indicating a coinfection. Stratifying by demography, three out of the six (50%) were from the Ayawaso-West-District. All positive cases received appropriate treatment at the respective sub district according to the national guidelines. Impact: Misdiagnosis of respiratory infections potentially leads to mismanagement with its concomitant clinical and public health implications. There is need for differential diagnosis of respiratory infections to advice proper clinical management. Conclusion: The discovery of TB positive patients among suspected COVID-19 patients harbingers the untold stories of undiagnosed TB in our communities. Our findings therefore highlight the need for differential diagnosis among COVID-19 suspected cases and regular active TB surveillance in TB endemic settings.

8.
Journal of Public Health in Africa ; 13:60, 2022.
Article in English | EMBASE | ID: covidwho-2006859

ABSTRACT

Introduction/ Background: The global spread of SARS-CoV-2 and high demand for reagents may cause a challenge in procurement and the testing process, impacting turnaround times and the epidemiological data for optimal response mainly in low-income countries. To overcome this bottleneck, evaluating pooling system in the testing could be a solution Methods: For pooling strategy, 100 ul of each sample were pooled for up to 4 samples and extract using the same technology. The swabs were Oropharyngeal and nasopharyngeal swabs collected from individual attending clinic at Thies région and tested by Real-Time PCR after inactivation and RNA extraction using KingFisher Flex machine from ThermoFisher according to the manufacturer. SARS-CoV-2 detection were done using AllplexTM 2019-nCoV assay from Seegene targetting N, E, RdRp Gene in Biorad CFX96. All Samples are test individually and pooled. Ct values were compared between positives samples and result obtained with pooling. Results: We include in this analysis 43 pool of 4 samples each including 54 positive SARS-CoV-2 pooled with 114 negative samples and 40 pool of 4 negatives samples. Among positives sample included in the pool, 19 had Ct between 17 and 25, 23 between 25 to 30 and 12 had more than 30. Our result confirms that all 54 positives samples pooled in negatives samples were detected by pooling. The pooling was associated with a loss of 0.8 average of Ct ranging between -1.2 to 2.8. All samples individually negatives were also negatives in the pooling. The complete analysis is ongoing. Impact: This study indicates that pooling sample is practical and can be used for community surveillance, testing of low-risk populations, and in resourcelimited settings to mitigate reagent stock out. This can allow to reduce testing turnaround times and faster public health authorities' response to the global pandemic, especially in low-income countries. Conclusion: Our preliminary data confirms that pooling sample correctly identifies SARS-CoV-2 infected individual in 100% of our sample with an expected average of loss of ct of 0.8. This strategy can increase testing throughput in RLS and reduce turnaround time.

9.
Journal of Public Health in Africa ; 13:75-76, 2022.
Article in English | EMBASE | ID: covidwho-2006816

ABSTRACT

Introduction/ Background: The Africa CDC organized continental training to establish testing capacity in MS. Africa CDC developed and implemented a training dashboard to track all training programs and use for planning and decision making. This study describes the implementation and utility of the laboratory training dashboard in the context of COVID-19 response. Methods: Google Spreadsheets was used to keep track of the training data. A comma-separated values training data file was used to create a training dashboard in Google Data Studio, where it was broken down into metrics and dimensions that could be used in dashboard reports connected between training data and dashboard reports. Data was imported and explored in an excel file, and a dashboard data modelling was created. Using training data KPIs, graphical displays were created, and data was examined.. The reports and visualizations on the developed dashboard are automatically updated in real-time with the most recent data from the datasets. Results: The collection of training data and visualization on a dashboard proved useful for immediate generation of report. As of October 2021, a total of 15,548 laboratory personnel had been trained. Of these, 1,117 (7.7%) received hands-on and virtual training on RT-PCR, 430 (2.9%) on Biosafety and Biosecurity, 137 (0.9%) on LQMS, 78 (0.7%) on COVID- 19 Genome Sequencing, 99 (0.8%) on GeneXpert and 13,627 (87%) on Ag-RDT testing in 55 Member States. Over 50 webinar sessions with total 23,063 participants were held to build the capacity of national and subnational level staff in all countries in the Africa Region. Impact: The dashboard primarily reported training data trends and disaggregated data by country, laboratory disciplines, and maps and graphs to make the data more appealing, shareable, and convertible into a report. By enhancing reporting and promoting timely decision-making, the training dashboard improved laboratory trainings in the context of COVID-19 response efforts. Conclusion: Tracking of all training programs in real time using interactive training dashboard will improve the use of data in subsequent decision making and measuring the progress during outbreak response. The dashboard reports and visualizations are shared with key stakeholders and used for workforce planning and monitoring.

10.
Open Access Macedonian Journal of Medical Sciences ; 10:187-190, 2022.
Article in English | EMBASE | ID: covidwho-1939089

ABSTRACT

BACKGROUND: Coronavirus disease 2019 (COVID-19) has distracted the global health system due to significant morbidity and mortality. There are increasing mortality rates related to the existence of comorbidities. Due to immunologic conditions, other infectious diseases, such as multidrug-resistant tuberculosis (MDR-TB), might coinfect with COVID-19. We describe a case of MDR-TB with diabetes mellitus and critical COVID-19 patient with fatal outcome. CASE REPORT: A 60-year-old man was admitted to our hospital with shortness of breath for 2 days. A history of recurrent shortness of breath has had developed for about 7 months. Room air oxygen saturation was at 66%. RT-PCR SARS-CoV-2 nasopharynx swab result was positive. The chest X-ray series result showed destroyed left lung with increasing infiltrate in the lower right lung. The patient was diagnosed with pulmonary MDR-TB based on GeneXpert and LPA (Line Probe Assay) test 6 months prior and also has had history of diabetes mellitus for 7 years. Then, the patient was diagnosed with COVID-19, pulmonary MDR-TB, and diabetes mellitus. MDR-TB regimen, anti-diabetic medication, and management of COVID-19 were carried out. On the 6th day, the patient’s condition worsened to the point, where he needed intubation. The patient eventually passed away. CONCLUSION: The treatment outcome was highly related to the severity of COVID-19 symptoms and complications of comorbidities when patients are admitted to the hospital. The early screening and treatment of COVID-19 are important to prevent deteriorating clinical conditions caused by comorbidities.

11.
American Journal of Respiratory and Critical Care Medicine ; 205(1), 2022.
Article in English | EMBASE | ID: covidwho-1927918

ABSTRACT

Background: COVID-19 is an emerging infection that has reached pandemic levels with a reported case fatality rate of 3-4%. This study aimed to determine risk factors at baseline that predict inhospital death from COVID-19 patients admitted at Lung Center of the Philippines (LCP). The goal was not only to identify preventable causes ofdeath of COVID-19 cases, but also prognosticate those with severe disease. Method: This was a retrospective, cohort, observational, and analytical study using chart review. The study subjects included COVID-19 cases confirmed by a positive result via RT-PCR testing or SARS-CoV-2 GeneXpert admitted and or discharged/expired at LCP from March 7 to August 31, 2020. Results: Out of 531 admitted patients, 258 were included in this study. There were 84 non-survivors, and 174 survivors. Non-survivors were older and have more than one co-morbid illness. Fever, cough, and dyspnea were the most common symptoms on disease onset. Among the inflammatory markers: AST, C-reactive protein (CRP), lactate dehydrogenase (LDH), procalcitonin and troponin I were significantly elevated on non-survivors. After multi variate analysis, oxygen saturation (OR 0.952 C 0.92-0.99 p 0.015), Glasgow Coma Scale (GCS) score (OR 0.4722 CI0.27 - 0.83), estimated glomerular filtration rate (eGFR) (OR 0.9681 CI0.95-0.98 p<0.001), neutrophilia (OR 1.0485 p 0.036), and increased LDH (OR 1.0038 CI 1.002 - 1.006 p<0.001) were found to correlate with mortality. Conclusions: Decreased oxygen saturation, low GCS score, and baseline findings of increased neutrophils, increased LDH, and decreased eGFR may warrant more aggressive management as they confer increased mortality risk.

12.
Pakistan Journal of Medical and Health Sciences ; 16(6):37-39, 2022.
Article in English | EMBASE | ID: covidwho-1918387

ABSTRACT

Aim: The assessment of serum electrolytes at the time of initial presentation of the patient with respiratory tract infection possibly causing lung parenchyma and pulmonary vasculature damage and serial monitoring during the stay could be beneficial in order to determine when and how to take remedial action when necessary. Methodology: A non-probability sampling was done on 139 subjects with suspected respiratory tract infection. For confirmation, culture, MTB PCR, COVID-19 testing was done to diagnose the nature of infection. Serum electrolytes were tested on chemical analyses Alinity instrument. Results: Most common infections found were COVID-19 and bacterial (n=59) collectively in a co-morbid state. Mycobacterium tuberculosis and fungal infections were also found in (n=8) each. Electrolytes imbalance was markedly observed in high prevalence amongst Tuberculosis and COVID-19 patients but also showed significant association with other respiratory investigated infections. Conclusion: A robust association of electrolyte imbalance was found in all cases presented with upper or lower respiratory tract infections.

13.
Clinica Chimica Acta ; 530:S344, 2022.
Article in English | EMBASE | ID: covidwho-1885661

ABSTRACT

Background-aim: World Health Organization (WHO) announced that diagnostic testing for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-COV2) should be performed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Most of these methods use different gene props and therefore the sensitivity and specificity of each method may different. In this study, we have compared two RT-PCR methods using two different genes for detection SARS-COV2. Methods: A total of random 40 nasopharyngeal swab samples were collected, transported and received in iced box shipment. All samples were performed on two separate semi-automated PCR systems (Qiagen and Abbott m2000). For Qiagen method, 200uL from each sample were added in 96-well QiAcube plate which loaded in QiAcube HT (SN:019658;Qiagen, Germany) to extract RNA. Following extraction, master mix prepared using SARS-COV2-RT-PCR kit 1.0 (REF: 821005;Altona, Germany) for 44 samples including 40 patient samples, two negative controls using nuclease-free water (with and without internal control), and two positive controls (with and without internal control). Extraction elute of each sample (20uL) added to master mix (10uL) to have a total volume of 30uL which uploaded into Rotor-Gene Q (SN:R0219307;Qiagen, Germany). The primer pair used to amplify S gene and E gene in SARS-COV2. Amplifications were done as follow: reverse transcriptase (20 minutes at 55oC);initial denaturation (2 minutes at 95oC);45 cycles of denaturation (15 seconds at 95oC), annealing for (45 seconds at 55oC), and extension (15 seconds at 72oC). Results reported as valid for internal control less than 35 cycle threshold (CT). The Abbott m2000 System uses SARS-CoV-2 assay was a dual target assay for the RdRp and N genes. All 40 samples were extracted using m2000sp (Abbott, United States) as recommend by manufacture using 100uL. An RNA sequence that was unrelated to the SARS-CoV-2 target sequence was introduced into each specimen at the beginning of sample preparation. This unrelated RNA sequence was simultaneously amplified by RT-PCR and serves as an internal control (IC) to demonstrate that the process has proceeded correctly for each sample. Following extraction, master mix (20uL) added to extraction elute of each sample (30uL) to have a total volume of 50uL which uploaded into m2000rp (Abbott, United States). Amplification were done as follow: reverse transcriptase (25 minutes at 55oC);initial denaturation (5 minutes at 94oC);40 cycles of denaturation (20 seconds at 94oC), annealing for (55 seconds at 55oC), and extension (15 seconds at 72oC). Result: All samples had valid extraction process with a CT value of internal control between 26.97 to 28.89. A total of 30 samples displayed positive results and 10 samples exhibited negative results with 100% agreement for both methods. This has resulted with a 100% accuracy between both methods. Conclusions: Both semi-automated methods from Qiagen and Abbott are comparable and accurate despite different technology and different primer genes.

14.
Topics in Antiviral Medicine ; 30(1 SUPPL):331-332, 2022.
Article in English | EMBASE | ID: covidwho-1880280

ABSTRACT

Background: SARS-CoV2 antibody testing is an important auxillary test especially for retrospective diagnosis or in patients with long COVID-19 or multisystem inflammatory syndrome of childhood. Epidemiological serology studies may also assist public health planning. Access to formal laboratory testing is not universal in many low-and middle-income (LMIC) countries and rapid lateral flow antibody tests are an attractive alternative. Performance of these tests has been inconsistent. A large-scale study was undertaken in South Africa, during the beta and delta waves, to assess the field-based performance of rapid point of care (POC) COVID-19 antibody tests. Methods: Symptomatic, ambulatory persons under investigation (PUIs) aged 18 years and older, presenting for SARS-CoV-2 diagnosis at public health facilities in three provinces, South Africa were enrolled at baseline. All patients completed a questionnaire regarding symptoms. Nasopharyngeal swabs were taken and processed for SARS-CoV-2 PCR testing using a GeneXpert (Cepheid, USA), or manual assay (ThermoFisher TaqPath assay or Seegene Allplex assay) on a real-time platform at routine accredited National Health Laboratory Service laboratories as per routine national protocols. Concomitantly, trained study staff performed three facility-based POC lateral flow antibody tests on a on a fingerstick sample and blood was collected for formal serology. POC tests were selected following a rapid in-laboratory evaluation. Asymptomatic contacts of people with confirmed COVID-19 were recruited into the asymptomatic study arm and rapid tests and PCR were performed. PCR and rapid positive patients and 500 negative controls were followed up at 5-14 days. Antibody tests were compared with formal serology performed on 2 platforms-Euroimmun (Euroimmun, Lubeck) IgA and IgG anti-S antibodies and Abbott Architect IgG test. Results: The sensitivity (S), specificity (Sp), positive (PPV) and negative predictive (NPV) values of tests for PUIs and contacts were calculated (Table 1)∗. Analyses using serology as a reference are forthcoming. Conclusion: Compared with PCR, performance of rapid POC COVID-19 antibody tests was poor with low sensitivity. This may reflect the patient cohort tested as humoral responses typically develop from day 7-14. The tests are unlikely to be useful for acute diagnosis but sensitivity may improve at later timepoints and further follow up data will be analysed by duration of symptom onset, severity of symptoms and wave (beta versus delta).

15.
Topics in Antiviral Medicine ; 30(1 SUPPL):331, 2022.
Article in English | EMBASE | ID: covidwho-1880279

ABSTRACT

Background: Access to SARS-CoV-2 polymerase chain reaction (PCR) testing is a bottleneck globally, especially in low-and middle-income countries (LMICs). Reliable point-of-care (POC) diagnostics for coronavirus disease 2019 (COVID-19) are cheaper and easier to scale-up than PCR especially in LMICs, and will facilitate interruption of transmission. We report the field-based effectiveness of rapid point-of-care (POC) antigen COVID-19 tests during the beta and delta waves, in South Africa. Methods: We enrolled symptomatic, ambulatory persons under investigation (PUIs) aged 18 years and older, presenting for SARS-CoV-2 diagnosis at public health facilities in three provinces, South Africa. All patients completed a questionnaire regarding symptoms. Nasopharyngeal swabs were taken and processed for SARS-CoV-2 PCR testing using either GeneXpert (Cepheid, USA), or with a manual assay (ThermoFisher TaqPath assay or Seegene Allplex assay) on a real-time PCR platform at routine, accredited National Health Laboratory Service laboratories, as per routine national protocols. Concomitantly, trained study staff performed three facility-based POC antigen tests on a nasal/nasopharyngeal swab, as recommended by the manufacturer. Asymptomatic contacts of people with confirmed COVID-19 were recruited into the asymptomatic study arm and rapid tests and PCR were performed. The sensitivity (S), specificity (Sp), positive (PPV) and negative predictive (NPV) values of tests for PUIs and contacts were calculated using PCR as the reference standard. Results: Between Oct 2020-2021 1816 participants were enrolled;472 (26%) tested PCR or rapid test positive;235 positives (49.8%) and 532 negatives were followed up at 5-14 days;574 asymptomatic contacts were enrolled, of which 21 (3.7%) were PCR positive. Performance of the three antigen tests are shown in Table 1∗. Conclusion: In a real world setting, during the beta and delta waves, compared with PCR the sensitivity of rapid antigen tests ranged from 35-68%. This may reflect low viral loads at diagnosis. Further work will compare antigen test performance in patients with high versus lower cycle threshold (Ct) values. Meanwhile, PCR testing capacity needs urgent scale-up in LMICs and improved POC diagnostics are needed to facilitate COVID-19 diagnosis in LMICs.

16.
Egyptian Journal of Medical Human Genetics ; 23(1), 2022.
Article in English | EMBASE | ID: covidwho-1822227

ABSTRACT

Background: Blood group has been stated to be one of the risk factors associated with viral diseases like dengue, hepatitis virus, Norwalk virus and even the coronavirus associated with 2003 severe acute respiratory syndrome (SARS) outbreak. In addition, anti-A antibodies in experimental models have been shown to inhibit the interaction between coronavirus and angiotensin converting enzyme (ACE) receptor of the host target cell, the major receptor involved in viral pathogenesis. Thus, several workers propose an association between ABO blood type and coronavirus disease- 2019 (COVID-19) disease in many previous studies. The present study was undertaken in the Eastern part of India in line with these authors to study the association of ABO blood group of patients with COVID susceptibility and severity. Methods: This is a retrospective study over a period of 6 months from June 2020 to November 2020 where patients who underwent quantitative real-time polymerase chain reaction (qRT-PCR) test for SARS-COV2 and having a recorded patient blood group type were considered. The qRT-PCR positive admitted cases were considered as cases, and qRT-PCR negative cases were considered as controls. Data were entered in Microsoft Excel format and analyzed by statistical method to obtain association. Results: Consecutively obtained 5000 qRT-PCR positive patients (cases) and 11,700 (controls) were included in the present study. The mean age of cases was higher (54.24 vs. 34. 67) than the controls. Among the cases, the highest number (2379;47.6%) of samples belonged to A blood group followed by B (1278;25.6%) while among the control group O blood group had the highest prevalence (4215;36%). Blood group A had a higher odd of testing positive (Odds ratio-2.552;CI 2.381–2.734;p < 0.0001) than all other blood groups. A blood group is also associated with higher risk of ICU admission (Odds ratio- 1.699;95% CI 1.515–1.905) and 65.3% of this group is also associated with high viral load which gives an indication of higher disease severity. Conclusion: Blood group A is associated with an increased susceptibility to COVID 19 infection than other blood groups. Cases of this blood group are also associated with more critical care needs and a higher viral load on testing.

17.
Cancer Research ; 82(4 SUPPL), 2022.
Article in English | EMBASE | ID: covidwho-1779441

ABSTRACT

Background Detection of circulating tumour DNA (ctDNA) in patients (pts) who have completed treatment for early-stage triple negative breast cancer (TNBC) is associated with a very high risk of future relapse. Identifiying those at high risk of subsequent relapse may allow tailoring of further therapy to delay or prevent recurrence. The c-TRAK TN trial assessed the utility of prospective ctDNA surveillance in pts treated for TNBC and the activity of pembrolizumab (P) in pts with ctDNA detected. Methods c-TRAK TN, a multi-centre phase II trial with integrated prospective screening component, enrolled pts with early-stage TNBC and either residual disease following neoadjuvant chemotherapy, or tumour size >20mm and/or axillary lymph node involvement if adjuvant chemotherapy was given. Tumour tissue was sequenced to identify somatic mutations suitable for tracking using personalised digital PCR ctDNA assays (BioRad QX200). Pts had "active" ctDNA surveillance via blood sample testing every 3 months to 12 months (potential up to 18 months if S samples missed due to COVID) during which time if ctDNA was detected (ctDNA+) pts could be randomised 2:1 to P (200mg i.v. q 3 weeks for 1 year) or observation (Obs). Pts and clinicians were blinded to ctDNA+ results unless they were allocated P, when staging scans were done and those free of clinical recurrence started treatment. Following advice from the Independent Data Monitoring Committee, the Obs arm closed on 16/06/2020 with all subsequent ctDNA+ pts allocated P. Following the completion of active ctDNA surveillance, 3-monthly visits continued to 24 months to be analysed retrospectively. The aim was to recruit 150 pts to ctDNA surveillance, assuming 30% would be ctDNA+ within 12 months, allowing ctDNA+ rate to be estimated with a 2-sided 95%CI of +/-7.3%. Co-primary endpoints are i) rates of ctDNA detection by 12 and 24 months from start of ctDNA surveillance;ii) rates of sustained ctDNA clearance on P defined as absence of detectable ctDNA, or disease recurrence 6 months after starting P. Results 208 pts were registered between 30/01/18 and 06/12/19, 185 had tumour sequenced, 171 (92.4%) had trackable mutations, and 161 entered ctDNA surveillance. The rate of ctDNA detection by 12 months after start of surveillance was 27.3% (44/161, 95% CI 20.6-34.9). ctDNA+ rates from baseline, 3, 6, 9 and 12 month ctDNA samples were 23/161 (14.3%), 6/115 (5.2%), 6/99 (5.1%), 7/84 (8.3%), and 2/84 (2.4%) respectively. An additional 2 pts were ctDNA+ on COVID extended active surveillance at 15 (1/51, 2%) or 18 months (1/11, 9%). 7 pts relapsed without prior ctDNA detection. 45 pts entered the therapeutic component of the trial (initially 31 to P and 14 to Obs). 1 Obs pt was re-allocated to P. Of pts allocated to P, 72% (23/32) had metastatic disease at time of ctDNA detection on staging scans (75% (12/16) who were ctDNA+ at baseline and 69% (11/16) at other timepoints). 4 pts declined to start P, largely due to COVID concerns. Of the 5 pts who commenced P, at the time of analysis none achieved sustained ctDNA clearance and 4 had recurred. In pts allocated to Obs, median time to recurrence was 4.1 months (95% CI: 3.2-not-defined). Conclusion The c-TRAK TN trial is to our knowledge the first study to assess the proof-of-principle of whether ctDNA assays have clinical utility in guiding further therapy in TNBC. Relatively few pts commenced P treatment precluding assessment of potential activity. At enrollment, patients had a relatively high of rate of undiagnosed metastatic disease when imaged. Our findings have implications for future trial design, emphasizing the importance of early start of ctDNA testing, and more sensitive and/or more frequent ctDNA testing regimes.

18.
Molecular Genetics and Metabolism ; 132:S270-S271, 2021.
Article in English | EMBASE | ID: covidwho-1768661

ABSTRACT

Introduction: Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease that results from mutation of the survival motor neuron 1 gene (SMN1) and the most common genetic cause of infant death. Approximately 95% of SMA cases are caused by a deletion in both alleles of exon 7 in the SMN1 gene. The copy number of the highly homologous SMN2 gene is an important predictor of the severity of SMA as it has been shown to decrease disease severity in a dose-dependent manner. SMN1 and SMN2 only differ by a few nucleotides, presenting a challenge in determining copy numbers. While carriers typically have one copy of SMN1, cis duplication of SMN1 can produce “silent carrier” (2 + 0) genotypes, which are often associated with two SMN1 variants, c.*3 + 80T>G and c.*211_*212del, that can improve the overall carrier detection rate. SMA treatments SPINRAZA®,, Evrysdi™, and ZOLGENSMA® achieve profound benefits on survival and motor milestones by modifying SMN2 splicing or using gene replacement with functional SMN genes. Early detection of SMA (including SMN2 copy number status) and identification of at-risk couples through carrier screening is critical to aid in early intervention and family planning decisions. We developed an accurate and robust single-tube PCR assay and companion software (AmplideX® PCR/CE SMN1/2 Plus Kit*) that uses capillary electrophoresis (CE) to quantify SMN1 and SMN2 copy numbers (0 to ≥4) and determines the presence/absence of the two SMN1 gene duplication “silent carrier” variants, c.*3 + 80T>G and c. *211_*212del, and the SMN2 disease modifier variant c.859G>C. The SMN1/2 Plus Kit has been previously validated for use with DNA isolated from blood. Here, we verify that DNA isolated from buccal swabs can also be used to determine SMN1 and SMN2 copy number and expanded content using this kit. Materials and Methods: A total of 60 DNA samples isolated from buccal swabs, with varying SMN1/2 copies and other positive and negative variants,were tested using the SMN1/2 Plus kit at a single site (Asuragen). Samples were tested in two cohorts: an initial cohort containing 17 samples isolated from buccal swabs with column or magnetic bead-based methods, and a second cohort of 43 samples isolated from matched blood and buccal samples using column-based methods. PCR products were generated using a Veriti thermal cycler and resolved on Applied Biosystems™ 3500xL, 3130xl, 3730xl, and SeqStudio™ Genetic Analyzers. Raw electrophoresis data (.fsa) files were directly imported into an assay-specific analysis module of the AmplideX® Reporter software that automates peak detection and sizebased classification, SMN1 and SMN2 exon 7 copy number quantification, detection of gene duplication and disease modifier variants, and sample- and batch-level quality control checks. Samples were analyzed using the default (kit calibrator) and user-defined calibration (UDC) (buccal DNA) workflows as described in the protocol. Results: For the initial cohort of 17 Buccal swab samples, SMN1 copy number calls were concordant with MLPA reference results (reported as 0, 1, 2, or ≥3) for 16/17 (94.1%) of samples with default calibration and 17/17 (100%) of samples with UDC. Further, concordance for carrier samples (1 SMN1 copy) were 7/7 (100%) using both methods. SMN2 copy numbercallswere concordant with MLPA reference results for 17/17 (100%) of samples with either default calibration or UDC. For the second cohort of 43 buccal swab samples with matched blood samples, SMN1 and SMN2 copy number calls were concordant with the results from the paired whole blood for at least 95% of samples assessed across the four different CE platforms. All variant status calls were concordant between the buccal swab and whole blood results. Conclusions: Here, we demonstrate that buccal swabs are a compatible DNA source for the quantification of 0, 1, 2, 3, and ≥4 gene copies of both SMN1 and SMN2 and the status determination of three clinically significant variants using the single-tube PCR/CE SMN1/2 Plus kit. Although d fault calibration yielded high rates of agreement between copy number results from buccal swabs and reference results, analyzing samples with user-defined calibration (i.e. calibrating to a buccal swab sample) modestly improved concordance. These results suggest that DNA samples isolated from buccal swabs are compatible with this assay and has implications for more facile sample collection and handling, particularly given the strain of COVID-19 on healthcare infrastructure.

19.
Open Forum Infectious Diseases ; 8(SUPPL 1):S287, 2021.
Article in English | EMBASE | ID: covidwho-1746625

ABSTRACT

Background. The SARS-CoV-2 pandemic has demonstrated the need for streamlined workflows in high-throughput testing. In extraction-based testing, limited extraction reagents and required proprietary instrumentation may pose a bottleneck for labs. As a solution, ChromaCode developed a Direct Extraction protocol for the HDPCR™ SARS-CoV-2 Assay, distributed in accordance with the guidance on Policy for Coronavirus Disease-2019 Tests During the Public Health Emergency, Section IV.C., which allows for the processing of specimens without an extraction system. In lieu of an extraction system, the Direct Extraction protocol uses a thermal cycler to lyse and inactivate specimens which are directly added to the Polymerase Chain Reaction (PCR). Methods. The Limit of Detection (LoD), Clinical Performance, and effect of Interfering Substances was determined for the Direct Extraction protocol. The LoD was established on 6 PCR platforms with dilutions of inactivated SARS-CoV-2 virus spiked into residual, negative nasopharyngeal swab (NPS) matrix. Clinical performance was assessed with 48 positive and 50 negative frozen retrospective samples using the Direct Extraction protocol compared to an external Emergency Use Authorized (EUA) comparator assays (cobas® Liat® SARS-CoV-2 & Influenza A/B assay and the Hologic Panther Fusion® SARS-CoV-2 Assay respectively) on three PCR platforms. The Direct Extraction protocol was evaluated for performance in the presence of 13 potentially interfering substances that can be present in a respiratory specimen. Results. The LoD of the Direct Extraction protocol ranges from 1000 - 3000 genomic equivalents (GE)/mL. The clinical performance of the assay was 95.8% positive agreement (95% CI of 84.6% - 99.3%) and 100% negative agreement (95% CI of 90.9% - 100% or 91.1% - 100%) across all three PCR platforms tested. The viral target was detected at 3X LoD for all interferents tested. Conclusion. The Direct Extraction protocol of ChromaCode's SARS-CoV-2 Assay is a sensitive test that eliminates the need for sample extraction and performs very well against traditional extraction-based workflows. The inclusion of this protocol can reduce costs, reliance on extraction systems, and time associated with extraction-based protocols.

20.
Open Forum Infectious Diseases ; 8(SUPPL 1):S297, 2021.
Article in English | EMBASE | ID: covidwho-1746604

ABSTRACT

Background. Federal mandate requires NHs to perform weekly COVID-19 testing of staff. Testing is effective due to barriers to disclosing mild illness, but it is unclear how long the mandate will last. We explored if environmental samples can be used to signal staff COVID-19 cases as an alternative screening tool in NHs. Methods. We conducted a cross sectional study to assess the value of environmental sampling as a trigger for COVID-19 testing of NH staff using data from currently performed weekly staff sweeps. We performed 35 sampling sweeps across 21 NHs from 6/2020-2/2021. For each sweep, we sampled up to 24 high touch objects in NH breakrooms (N=226), entryways (N=216), and nursing stations (N=194) assuming that positive samples were due to contamination from infected staff. Total staff and positive staff counts were tallied for the staff testing sweeps performed the week of and week prior to environmental sampling. Object samples were processed for SARSCoV-2 using PCR (StepOnePlus) with a 1 copy/mL limit of detection. We evaluated concordance between object and staff positivity using Cohen's kappa and calculated the positive and negative predictive value (PPV, NPV) of environmental sweeps for staff positivity, including the attributable capture of positive staff. We tested the association between the proportion of staff positivity and object contamination by room type in a linear regression model when clustering by NH. Results. Among 35 environmental sweeps, 49% had SARS-CoV-2 positive objects and 69% had positive staff in the same or prior week. Mean positivity was 16% (range 0-83%) among objects and 4% (range 0-22%) among staff. Overall, NPV was 61% and Cohen's kappa was 0.60. PPV of object sampling as an indicator of positive staff was 100% for every room type, with an attributable capture of positive staff of 76%, with values varying by room type (Table). Breakroom samples were the strongest indicator of any staff cases. Each percent increase in object positivity was associated with an increase in staff positivity in entryways (7.2% increased staff positivity, P=0.01) and nursing stations (5.7% increased staff positivity, P=0.05). Conclusion. If mandatory weekly staff testing ends in NHs, environmental sampling may serve as an effective tool to trigger targeted COVID-19 testing sweeps of NH staff.

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