scholarly journals Pooled testing conserves SARS-CoV-2 laboratory resources and improves test turn-around time: experience on the Kenyan Coast

2021 ◽  
Vol 5 ◽  
pp. 186 ◽  
Author(s):  
Charles N. Agoti ◽  
Martin Mutunga ◽  
Arnold W. Lambisia ◽  
Domtila Kimani ◽  
Robinson Cheruiyot ◽  
...  
Keyword(s):  
Low Income ◽  
Laboratory Testing ◽  
Rna Extraction ◽  
Threshold Value ◽  
Rt Pcr ◽  
Laboratory Equipment ◽  
Pooled Testing ◽  
Kenyan Coast ◽  
Multiple Samples

Background. International recommendations for the control of the coronavirus disease 2019 (COVID-19) pandemic emphasize the central role of laboratory testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent, at scale. The availability of testing reagents, laboratory equipment and qualified staff are important bottlenecks to achieving this. Elsewhere, pooled testing (i.e. combining multiple samples in the same reaction) has been suggested to increase testing capacities in the pandemic period. Methods. We discuss our experience with SARS-CoV-2 pooled testing using real-time reverse transcription polymerase chain reaction (RT-PCR) on the Kenyan Coast. Results. In mid-May, 2020, our RT-PCR testing capacity for SARS-CoV-2 was improved by ~100% as a result of adoption of a six-sample pooled testing strategy. This was accompanied with a concomitant saving of ~50% of SARS-CoV-2 laboratory test kits at both the RNA extraction and RT-PCR stages. However, pooled testing came with a slight decline of test sensitivity. The RT-PCR cycle threshold value (ΔCt) was ~1.59 higher for samples tested in pools compared to samples tested singly. Conclusions. Pooled testing is a useful strategy to increase SARS-CoV-2 laboratory testing capacity especially in low-income settings.

scholarly journals Pooled testing conserves SARS-CoV-2 laboratory resources and improves test turn-around time: experience on the Kenyan Coast

2020 ◽  
Vol 5 ◽  
pp. 186 ◽  
Author(s):  
Charles N. Agoti ◽  
Martin Mutunga ◽  
Arnold W. Lambisia ◽  
Domtila Kimani ◽  
Robinson Cheruiyot ◽  
...  
Keyword(s):  
Low Income ◽  
Laboratory Testing ◽  
Rna Extraction ◽  
Threshold Value ◽  
Rt Pcr ◽  
Laboratory Equipment ◽  
Pooled Testing ◽  
Kenyan Coast ◽  
Multiple Samples

Background. International recommendations for the control of the coronavirus disease 2019 (COVID-19) pandemic emphasize the central role of laboratory testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent, at scale. The availability of testing reagents, laboratory equipment and qualified staff are important bottlenecks to achieving this. Elsewhere, pooled testing (i.e. combining multiple samples in the same reaction) has been suggested to increase testing capacities in the pandemic period. Methods. We discuss our experience with SARS-CoV-2 pooled testing using real-time reverse transcription polymerase chain reaction (RT-PCR) on the Kenyan Coast. Results. In mid-May, 2020, our RT-PCR testing capacity for SARS-CoV-2 was improved by ~100% as a result of adoption of a six-sample pooled testing strategy. This was accompanied with a concomitant saving of ~50% of SARS-CoV-2 laboratory test kits at both the RNA extraction and RT-PCR stages. However, pooled testing came with a slight decline of test sensitivity. The RT-PCR cycle threshold value (ΔCt) was ~1.59 higher for samples tested in pools compared to samples tested singly. Conclusions. Pooled testing is a useful strategy to increase SARS-CoV-2 laboratory testing capacity especially in low-income settings.

Role of endothelin receptors in the hypertensive state of kinin B2 knockout mice subjected to a high-salt diet

2002 ◽  
Vol 103 (s2002) ◽  
pp. 380S-384S ◽  
Author(s):  
Isabelle BROCHU ◽  
Julie LABONTÉ ◽  
Ghassan BKAILY ◽  
Pedro D'ORLÉANS-JUSTE
Keyword(s):  
Blood Pressure ◽  
Angiotensin Ii ◽  
Receptor Antagonist ◽  
Tap Water ◽  
Blood Pressure Measurement ◽  
Rna Extraction ◽  
Angiotensin Receptors ◽  
Rt Pcr ◽  
Arterial Blood

Mice with disruption of the kinin B2 receptor (B2KO mice) are sensitive to salt-rich diets, which causes hypertension. The aim of the study was to assess the role of endothelin-1 (ET-1) and angiotensin-II in hypertensive B2KO mice on a salt-rich diet. We also wanted to verify if there is an upregulation of the mRNA expression of the precursors or receptors for these hormones. Two groups of B2KO mice (20–25g) were investigated. The first group received an 8% NaCl diet with 1% NaCl in drinking water (HS) and the second was fed with normal food with tap water (NS). The antagonists tested were the ETA receptor antagonist BQ-123 (1 and 5mg/kg), the ETB receptor antagonist BQ-788 (0.25 and 1mg/kg), the angiotensin receptor type 1 antagonist losartan (10mg/kg) and the angiotensin-converting enzyme inhibitor captopril (3mg/kg). These were injected intraperitoneally 30min prior to blood pressure measurement by the tail-cuff method. We also studied the level of expression of preproET-1, ET-1 receptors, angiotensinogen and angiotensin receptors by RNA extraction from the heart and kidneys of these mice followed by reverse transcriptase (RT)-PCR. B2KO mice (HS) were hypertensive after 8 weeks compared with B2KO mice on normal diet (HS, 93.4±1.5mmHg, n = 7; NS, 61.4±2.7mmHg, n = 7). In the HS group, the mean arterial blood pressure was significantly reduced by BQ-123 (5mg/kg) to 61.9±1.8mmHg (n = 7), by BQ-788 (1mg/kg) to 58.8±2.6mmHg (n = 6), by losartan (10mg/kg) to 73.2±1.7mmHg (n = 8) and by captopril (3mg/kg) to 86.0±2.3mmHg (n = 8). The expression studied by RT-PCR did not show any difference (either in precursors or receptors expression) between hypertensive and normal mice. The four antagonists used seemed to reverse the hypertension. These results suggest that ET-1 and angiotensin-II are probably involved in the mechanism that leads to hypertension since the effect of these hormones is probably not compensated by kinins in B2KO mice. Further studies are necessary to understand the implication of the cross-talk between these hormones in the hypertensive state.

scholarly journals SalivaAll: Clinical validation of a sensitive test for saliva collected in healthcare and community settings with pooling utility for SARS-CoV-2 mass surveillance.

2020 ◽  
Author(s):  
Nikhil Shri Sahajpal ◽  
Ashis K Mondal ◽  
Sudha Ananth ◽  
Allan Njau ◽  
Pankaj Ahluwalia ◽  
...  
Keyword(s):  
Phase I ◽  
Phase Ii ◽  
Detection Rate ◽  
Limit Of Detection ◽  
Rna Extraction ◽  
Community Settings ◽  
Rt Pcr ◽  
Effective Measure ◽  
Pooled Testing ◽  
Mass Surveillance

Background: The adoption of saliva as a specimen type for SARS-CoV-2 mass surveillance can significantly increase population compliance with decreased exposure risk for healthcare workers. However, studies evaluating the clinical performance of saliva compared to nasopharyngeal swab (NPS) samples have demonstrated conflicting results regardless of the collection being in healthcare or community settings. Further, pooled testing with saliva remains a challenge owing to the ambiguous sensitivity, limit of detection (LoD), and processing challenges. To overcome these limitations, SalivaAll protocol was developed and validated as a cost-effective measure that must be used on saliva collected in health care or community settings with pooling utility for SARS-CoV-2 mass surveillance. Methods: The study evaluated 429 matched NPS and saliva samples collected from 344 individuals in either healthcare or community setting. In phase I (protocol U), 240 matched NPS, and saliva samples were tested for SARS-CoV-2 detection by RT-PCR. In phase II (SalivaAll protocol), 189 matched NPS and saliva samples were tested, with an additional sample homogenization step for saliva before RNA extraction, followed by RT-PCR. Eighty-five saliva samples were evaluated with both protocols (U and SalivaAll). Subsequently, adopting SalivaAll protocol, a five-sample pooling strategy was evaluated for saliva samples based on FDA recommendations. Results: In phase I, 28.3% (68/240) samples tested positive for SARS-CoV-2 from either saliva, NPS, or both. The detection rate was lower in saliva compared to NPS samples (50.0% vs. 89.7%). In phase II, 50.2% (95/189) samples tested positive for SARS-CoV-2 from either saliva, NPS, or both. The detection rate for SARS-CoV-2 was higher in saliva compared to NPS testing (97.8% vs. 78.9%). Of the 85 saliva samples evaluated by both protocols, 57.6% (49) tested positive for SARS-CoV-2 with either protocol U, SalivaAll, or both. The detection rate was 100% for samples tested with SalivaAll, whereas it was 36.7% with protocol U. Also, the LoD with SalivaAll protocol was 20 copies/ml. The pooled testing approach demonstrated a 95% positive and 100% negative percent agreement. Conclusion: This single-site study demonstrated the variability of results reported in the literature for saliva samples, and found that the discrepancies are explained by processing challenges associated with saliva samples. We have optimized a protocol for saliva samples that results in higher sensitivity compared to NPS samples and also breaks the barrier to using pooled saliva testing for SARS-CoV-2.

Role of Plasma miRNA-501 Expression Profile as a Marker of Hepatocellular Carcinoma (HCC) Progression With Hepatitis C Virus Infection

2019 ◽  
Vol 152 (Supplement_1) ◽  
pp. S134-S134
Author(s):  
Mohamed Shedid ◽  
Mohamed Abdelmonem ◽  
Ayman Boraik ◽  
Alaa Elmetwalli ◽  
Ahmed Esmael
Keyword(s):  
Rna Extraction ◽  
Control Group ◽  
Hepatitis C Virus Infection ◽  
Rt Pcr ◽  
The World ◽  
Noninvasive Biomarker ◽  
Biological Aspects ◽  
Plasma Mirna

Abstract Introduction HCV is a health problem that confronts many countries in the world. Those patients will develop complications like cirrhosis and HCC, which is one of the most common cancers in the world, especially in Egypt, and considered the third leading cause of death worldwide. The prognosis of HCC is still dismal due to the late diagnosis. miRNAs are small, short noncoding RNAs, which have roles in the diagnosis of HCC. In our study, we focus on biological aspects of miRNAs. We report that miR-501 is strongly expressed and observed in the process of HCC development. miR-501 regulation is important as an oncofetal relevant to the diagnosis of HCC. Method This study was conducted on 100 adult patients; 25 patients were positive for anti-HCV and 25 patients were negative for HCV and enrolled as a control group. Patients were categorized into three groups: fibrosis (n = 25), CHC (n = 25), and HCC (n = 25) related to HCV evident by CT abdomen. All patients and controls were subjected to full clinical assessment and laboratory investigation. Blood (8 mL) was withdrawn from subjects, and 3 mL was collected in EDTA tubes for processing total RNA extraction and miRNA. The remaining 5 mL was left for determination of biochemical parameters. miRN-501 expression levels were determined by RT-PCR. Results The data revealed a significant increase in levels of AST, ALT, ALP, and CBC in both HCC and CHC groups compared to controls. The results of miRNA expression showed that miR-501 was higher in the HCC group than non-HCC group at P1.1. Conclusion miR-501 can be used as a noninvasive biomarker for early diagnosis of HCC among patients with HCV on account of its affectability for HCC.

scholarly journals Hydrogel particles improve detection of SARS-CoV-2 RNA from multiple sample types

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
R. A. Barclay ◽  
I. Akhrymuk ◽  
A. Patnaik ◽  
V. Callahan ◽  
C. Lehman ◽  
...  
Keyword(s):  
Rna Extraction ◽  
Threshold Value ◽  
Acid Extraction ◽  
Affinity Capture ◽  
Transport Medium ◽  
Pooled Testing ◽  
Hydrogel Particles ◽  
Magnetic Hydrogel ◽  
Multiple Sample ◽  
Viral Titers

AbstractHere we present a rapid and versatile method for capturing and concentrating SARS-CoV-2 from contrived transport medium and saliva samples using affinity-capture magnetic hydrogel particles. We demonstrate that the method concentrates virus from 1 mL samples prior to RNA extraction, substantially improving detection of virus using real-time RT-PCR across a range of viral titers (100–1,000,000 viral copies/mL) and enabling detection of virus using the 2019 nCoV CDC EUA Kit down to 100 viral copies/mL. This method is compatible with commercially available nucleic acid extraction kits (i.e., from Qiagen) and a simple heat and detergent method that extracts viral RNA directly off the particle, allowing a sample processing time of 10 min. We furthermore tested our method in transport medium diagnostic remnant samples that previously had been tested for SARS-CoV-2, showing that our method not only correctly identified all positive samples but also substantially improved detection of the virus in low viral load samples. The average improvement in cycle threshold value across all viral titers tested was 3.1. Finally, we illustrate that our method could potentially be used to enable pooled testing, as we observed considerable improvement in the detection of SARS-CoV-2 RNA from sample volumes of up to 10 mL.

scholarly journals SARS-CoV-2 direct real-time polymerase chain reaction testing in laboratories with shortage challenges

2021 ◽  
Vol 16 (2) ◽  
pp. 133-139
Author(s):  
Taghreed Alaifan ◽  
Asmaa Altamimi ◽  
Dalia Obeid ◽  
Turki Alshehri ◽  
Shaihana Almatrrouk ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Rna Extraction ◽  
Threshold Value ◽  
Limited Resources ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Viral Transport ◽  
Extraction Step ◽  
Polymerase Chain

Aim: In our study, we propose the use of direct real-time polymerase chain reaction (RT-PCR), this test does not require extraction or a preheating step, which saves a lot of cost, labor, processing time and provides a solution for supply shortage. Materials & methods: We assayed 185 nasopharyngeal samples stored in viral transport media. The indirect method was done with RNA extraction step, and the direct RT-PCR was done without an extraction step, both assays were evaluated on a commercially validated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) kit targeting E gene. Results: Our assay showed a sensitivity of 79%, a specificity of 100% and the agreement between methods was 72%. Conclusion: Overall, this simple direct RT-PCR approach can be utilized as a qualitative diagnostic tool for emergency SARS-CoV-2 surveillance in countries with limited resources and may help laboratories to continue testing and at greater frequency despite supply shortages, although with delay in cycle threshold value in comparison with indirect RT-PCR.

Epidemiology and antimicrobial resistance of Acinetobacter

2018 ◽  
Vol 2 (4) ◽  
pp. 46-59
Author(s):  
A.G. Salmanov ◽  
O.M. Verner ◽  
L.F. Slepova
Keyword(s):  
Antibiotic Resistance ◽  
Laboratory Testing ◽  
Immunocompromised Host ◽  
Clinical Problem ◽  
Healthcare Associated Infections ◽  
Opportunistic Bacteria ◽  
Acinetobacter Spp ◽  
Healthcare Associated ◽  
Major Clinical Problem

Species of the Acinetobacter represent opportunistic bacteria with a growing clinical significance for Healthcare-associated infections (HAIs). In this literature review, we focus on the current role of Acinetobacter in infectious pathology and describe taxonomy, pathogenicity, and antibiotic resistance of these bacteria. Pathogenesis and regulation of virulence factors in Acinetobacter spp. are described in detail. The majority of acinetobacterial infections are associated with A. baumannii and occur predominantly in an immunocompromised host. Usually, acinetobacterial  infections  are characterized by local purulent inflammation; in severe cases, meningitis and sepsis may develop. Antibiotic resistance of Acinetobacter is a major clinical problem; therefore we give special attention to laboratory testing of resistance to antibiotics as well as identification of Acinetobacter.

The Fight to Save Welfare for Low-Income Older Asian Immigrants: The Role of National Asian American Organizations

2003 ◽  
Vol 1 (1) ◽  
pp. 85-101 ◽  
Author(s):  
Grace Yoo
Keyword(s):  
Welfare Reform ◽  
Asian American ◽  
Low Income ◽  
Bill Clinton ◽  
Asian Immigrants ◽  
Central Question ◽  
Political Debate ◽  
Welfare Reform Law ◽  
Selection Of

The welfare reform law of August 1996 signed by President Bill Clinton put an end to immigrants’ eligibility of federal means tested entitlements. The rollbacks on welfare are the most drastic for older, low-income Asian immigrants who are on Supplemental Security Income. The article’s focus is in on national Asian American organizations who are involved in this political debate. The central question discuss is how did national Asian American organizations characterize and affect the 1996 federal welfare reform and immigrant debate. The selection of organizations that was studied and the findings of that investigation, along with the assessment of its effectiveness and the resources barriers they face are discussed.

Monitoring the marine environment for small round structured viruses (SRSVS): a new approach to combating the transmission of these viruses by molluscan shellfish

1998 ◽  
Vol 38 (12) ◽  
pp. 51-56 ◽  
Author(s):  
K. Henshilwood ◽  
J. Green ◽  
D. N. Lees
Keyword(s):  
Enteric Virus ◽  
Winter Period ◽  
Rt Pcr ◽  
Cloning And Sequencing ◽  
New Approach ◽  
Human Enteric Virus ◽  
Different Strains ◽  
The Uk ◽  
Public Health Protection

This study investigates human enteric virus contamination of a shellfish harvesting area. Samples were analysed over a 14-month period for Small Round Structured Viruses (SRSVs) using a previously developed nested RT-PCR. A clear seasonal difference was observed with the largest numbers of positive samples obtained during the winter period (October to March). This data concurs with the known winter association of gastroenteric illness due to oyster consumption in the UK and also with the majority of the outbreaks associated with shellfish harvested from this area during the study period. RT-PCR positive amplicons were further characterised by cloning and sequencing. Sequence analysis of the positive samples identified eleven SRSV strains, of both Genogroup I and Genogroup II, occurring throughout the study period. Many shellfish samples contained a mixture of strains with a few samples containing up to three different strains with both Genogroups represented. The observed common occurrence of strain mixtures may have implications for the role of shellfish as a vector for dissemination of SRSV strains. These results show that nested RT-PCR can identify SRSV contamination in shellfish harvesting areas. Virus monitoring of shellfish harvesting areas by specialist laboratories using RT-PCR is a possible approach to combating the transmission of SRSVs by molluscan shellfish and could potentially offer significantly enhanced levels of public health protection.

The Immunomodulatory Role of G2013 (α-L-Guluronic Acid) on the Expression of TLR2 and TLR4 in HT29 cell line

2019 ◽  
Vol 16 (1) ◽  
pp. 91-95 ◽  
Author(s):  
Hamid Farhang ◽  
Laleh Sharifi ◽  
Mohammad Mehdi Soltan Dallal ◽  
Mona Moshiri ◽  
Zahra Norouzbabaie ◽  
...  
Keyword(s):  
Inflammatory Responses ◽  
Inflammatory Diseases ◽  
Rna Extraction ◽  
Cell Plate ◽  
Inhibitory Effect ◽  
Control Group ◽  
Ht29 Cells ◽  
Guluronic Acid ◽  
Tlr4 Mrna

Background: The non-steroidal anti-inflammatory drugs (NSAIDs) play crucial role in the controlling of inflammatory diseases. Due to the vast side effects of NSAIDs, its use is limited. G2013 or &amp;#945;-L-Guluronic Acid is a new NSAID with immunomodulatory features. Objectives: Considering the leading role of TLRs in inflammatory responses, in this study, we aimed to evaluate G2013 cytotoxicity and its effect on the expression of TLR2 and TLR4 molecules. Methods: HEK293-TLR2 and HEK293-TLR4 cells were cultured and seeded on 96-well cell plate, and MTT assay was performed for detecting the viability of the cells after treatment with different concentrations of G2013. HT29 cells were grown and treated with low and high doses of G2013. After total RNA extraction and cDNA synthesis, quantitative real-time PCR were performed to assess the TLR2 and TLR4 mRNA synthesis. Results: We found that concentrations of ≤125 &amp;#181;g/ml of G2013 had no apparent cytotoxicity effect on the HEK293-TLR2 and -TLR4 cells. Our results indicated that after G2013 treatment (5 &amp;#181;g/ml) in HT29 cells, TLR2 and TLR4 mRNA expression decreased significantly compared with the untreated control group (p=0.02 and p=0.001 respectively). Conclusion: The results of this study revealed that G2013 can down regulate the TLR2 and TLR4 gene expression and exerts its inhibitory effect. Our findings are parallel to our previous finding which showed G2013 ability to down regulate the signaling pathway of TLRs. However, further studies are needed to identify the molecular mechanism of G2013.<p&gt;

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