scholarly journals Self-collected gargle specimen as a patient-friendly sample collection method for COVID-19 diagnosis in a population context

2021 ◽  
Author(s):  
Revata Utama ◽  
Rebriarina Hapsari ◽  
Iva Puspitasari ◽  
Desvita Sari ◽  
Meita Hendrianingtyas ◽  
...  
Keyword(s):  
Healthcare Workers ◽  
Room Temperature ◽  
Rna Extraction ◽  
User Preference ◽  
Sample Collection ◽  
Rt Pcr ◽  
Collection Method ◽  
Qrt Pcr ◽  
Alternative Specimen ◽  
Temperature Storage

Abstract Scaling up SARS-CoV-2 testing and tracing continues to be plagued with the limitation of the sample collection method, which requires trained healthcare workers to perform and causes discomfort to the patients. In response, we assessed the performance and user preference of gargle specimens for qRT-PCR-based detection of SARS-CoV-2 in Indonesia. Inpatients who had recently been diagnosed with COVID-19 and outpatients who were about to perform qRT-PCR testing were asked to provide nasopharyngeal and oropharyngeal (NPOP) swabs and self-collected gargle specimens. We demonstrated that self-collected gargle specimens can be an alternative specimen to detect SARS-CoV-2 and the viral RNA remained stable for 31 days at room temperature storage. The developed method was validated for use on multiple RNA extraction kits and commercially available COVID-19 RT-PCR kits. Our developed method achieved a sensitivity of 91.38% when compared to paired NPOP swab specimens (Ct < 35), with 97.10% of patients preferring the self-collected gargle method.

scholarly journals Self-collected gargle specimen as a patient-friendly sample collection method for COVID-19 diagnosis in a population context

2021 ◽  
Author(s):  
Revata Utama ◽  
Rebriarina Hapsari ◽  
Iva Puspitasari ◽  
Desvita Sari ◽  
Meita Hendrianingtyas ◽  
...  
Keyword(s):  
Healthcare Workers ◽  
Room Temperature ◽  
Rna Extraction ◽  
User Preference ◽  
Sample Collection ◽  
Rt Pcr ◽  
Collection Method ◽  
Qrt Pcr ◽  
Alternative Specimen ◽  
Temperature Storage

Abstract Scaling up SARS-CoV-2 testing and tracing continues to be plagued with the limitation of the sample collection method, which requires trained healthcare workers to perform and causes discomfort to the patients. In response, we assessed the performance and user preference of gargle specimens for qRT-PCR-based detection of SARS-CoV-2 in Indonesia. Inpatients who had recently been diagnosed with COVID-19 and outpatients who were about to perform qRT-PCR testing were asked to provide nasopharyngeal and oropharyngeal (NPOP) swabs and self-collected gargle specimens. We demonstrated that self-collected gargle specimens can be an alternative specimen to detect SARS-CoV-2 and the viral RNA remained stable for 31 days at room temperature storage. The developed method was validated for use on multiple RNA extraction kits and commercially available COVID-19 RT-PCR kits. Our developed method achieved a sensitivity of 91.38% when compared to paired NPOP swab specimens (Ct < 35), with 97.10% of patients preferring the self-collected gargle method.

Self-collected gargle as a patient-friendly sample collection method for COVID-19 diagnosis in population context

2021 ◽  
Author(s):  
Revata Utama ◽  
Rebriarina Hapsari ◽  
Iva Puspitasari ◽  
Desvita Sari ◽  
Meita Hendrianingtyas ◽  
...  
Keyword(s):  
Healthcare Workers ◽  
Room Temperature ◽  
Rna Extraction ◽  
User Preference ◽  
Sample Collection ◽  
Rt Pcr ◽  
Collection Method ◽  
Qrt Pcr ◽  
Alternative Specimen ◽  
Temperature Storage

Abstract Scaling up SARS-CoV-2 testing and tracing continues to be plagued with limitation of sample collection method that requires trained healthcare workers to perform and cause discomfort to the patients. In response, we assessed the performance and user preference of gargle specimens for qRT-PCR based detection of SARS-CoV-2 in Indonesia. Inpatients who had recently been diagnosed with COVID-19 and outpatients who were about to perform qRT-PCR testing were asked to provide nasopharyngeal and oropharyngeal (NPOP) swabs and self-collected gargle specimens. We demonstrated that self-collected gargle specimens can be an alternative specimen to detect SARS-CoV-2 and the viral RNA remained stable for 31 days on room temperature storage. The developed method was validated for use on multiple RNA extraction kit and commercially available COVID-19 RT-PCR kits. Our developed method achieved sensitivity of 91.38% when compared to paired NPOP swab specimens (Ct < 35) with 95.16% of patients prefer the self-collected gargle method.

Paper or Plastic: RT-PCR of BCR-ABL from Blood Spots Stored and Shipped on Paper

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4566-4566
Author(s):  
Olga Sala Torra ◽  
Lan Beppu ◽  
Susan Branford ◽  
Linda Fletcher ◽  
Gooley Ted ◽  
...  
Keyword(s):  
Room Temperature ◽  
Time Course ◽  
Rna Extraction ◽  
Dried Blood Spots ◽  
Rt Pcr ◽  
Fresh Blood ◽  
Blood Samples ◽  
Blood Spots ◽  
Qrt Pcr ◽  
Sample Age

Abstract In many parts of the world, diagnosis and monitoring of CML patients is limited by the availability and cost of molecular testing. In countries without molecular diagnostic capabilities, blood samples can be shipped to central labs, but this is both hampered by sample degradation, and the high costs of shipping. This study explores the method of directly spotting peripheral blood onto a paper template (dried blood spots), with subsequent shipping, RNA extraction, and BCR-ABL testing. Methods: Blood Spots and Shipment. We received dried blood spots from Australia and African countries by mail or courier, and blood from CML patients from our institution were also used for these experiments. 200μL of blood (PB) was pipetted onto Whatman 503 Protein Saver Cards (PSC; Sigma-Aldrich), where each card contains four 50μL spots. Cards were allowed to dry for at least 24 hours at room temperature. For mailing, PSCs were sealed into glassine envelopes with a packet of desiccant, and then placed inside a mailing envelope following DOT and IATA regulation for shipping non-regulated, exempt human specimens. RNA Extraction from Cards and %BCR-ABL determination. Blood spots were incubated with proteinase K followed by RNA isolation using RNeasy Mini Kits (Qiagen). Extracted RNA was quantified using a NanoDrop spectrometer (Thermo Scientific). %BCR-ABL was determined using the automated Cepheid GeneXpert platform or manual two-step quantitative RT-PCR on the 7900HT Fast Real-Time PCR System (Applied Biosystems). Results: Bench top time course: To test for effects of long transit times on RNA quality, we performed a time course study of cards at room temperature (RT) with 5 samples. For each sample, multiple cards were spotted with PB. The cards were then allowed to sit at RT for predetermined amounts of time, up to 42 days, before extracting RNA. We measured RNA integrity for one of the specimens (CML # 5) and found rapid degradation with the RIN number going from 8.7 for the fresh blood to 2.8 after 28 days on the card. However the amplification for both BCR-ABL and ABL differed less than one cycle between the fresh blood and the last time point by manual qRT-PCR (BCR-ABL Ct = 23.63 for fresh blood and 24.06 for day 28 PSC; ABL Ct = 26.69 for fresh blood and 27.64 for day 28 PSC). Figure 1 shows the results of the time course experiment for the 5 samples as a plot of ΔCt versus time in days. BCR-ABL qRT-PCR concordance studies: We compared the %BCR-ABL results obtained in fresh specimen at the institution sending the sample with the %BCR-ABL results we obtained from RNA extracted from PSC using the Cepheid GeneXpert. Paired evaluable results were available for 9 samples with a median WBC = 9.8 x 109/L (range: 3.37x109/L – 85.5x109/L). Samples were 8 to 49 days old at the time of extraction. The amount of RNA input into the GeneXpert reaction ranged from 38.75ng to 1μg. The %BCR-ABL detected ranged from 0.37% to 27% (see Table). The mean absolute difference between fresh blood and PSC BCR-ABL% is 2%; the relative mean percent change for BCR-ABL, using fresh blood as the reference is 13.1% (S.D., 31.2), P = 0.24. Conclusions and future directions: Dried blood spots are relatively inexpensive method to transport blood that preserves enough RNA stability to allow highly accurate BCR-ABL detection, when compared to results performed on an identical platform using fresh peripheral blood samples. Further studies are undergoing to accurately determine the sensitivity of this method and the feasibility of using regular mail for inexpensive transport of specimens. Table 1IDWBC (1000/μL)Sample Age at Spotting (Days)Sample Age at RNA extraction (Days)RNA ng/μlVolume GeneXPert (μL)Paper %BCR-ABL (IS)GeneXpertFresh Blood % BCR-ABL (IS) GeneXpertI1na010426349naI224.101311092745I38009181544naI47.4285102.4*3.1I55.50495241.92I63.61307.4225912I785.5130102102439I812.212912.415128.8I9na1281.5250.37*0.71I103.370273257.85.7I1115.912731102325I126.612714.415na2.3 *%BCR-ABL was manually calculated due to late ABL Cts because of low starting material. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Effective optimization of SARS-CoV-2 laboratory testing variables in an era of supply chain constraints

2020 ◽  
Vol 15 (15) ◽  
pp. 1483-1487
Author(s):  
Nikhil S Sahajpal ◽  
Ashis K Mondal ◽  
Allan Njau ◽  
Sudha Ananth ◽  
Kimya Jones ◽  
...  
Keyword(s):  
Supply Chain ◽  
Rna Extraction ◽  
Nasopharyngeal Swab ◽  
Sample Collection ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Short Supply ◽  
Viral Transport ◽  
3D Printed ◽  
Bridging Studies

RT-PCR-based assays for the detection of SARS-CoV-2 have played an essential role in the current COVID-19 pandemic. However, the sample collection and test reagents are in short supply, primarily due to supply chain issues. Thus, to eliminate testing constraints, we have optimized three key process variables: RNA extraction and RT-PCR reactions, different sample types and media to facilitate SARS-CoV-2 testing. By performing various validation and bridging studies, we have shown that various sample types such as nasopharyngeal swab, bronchioalveolar lavage and saliva, collected using conventional nasopharyngeal swabs, ESwab or 3D-printed swabs and, preserved in viral transport media, universal transport media, 0.9% sodium chloride or Amies media are compatible with RT-PCR assay for COVID-19. Besides, the reduction of PCR reagents by up to fourfold also produces reliable results.

scholarly journals Development of an RNA Extraction Protocol for a Norovirus from Raw Oysters and Detection by qRT-PCR and Droplet-Digital RT-PCR

Foods ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1804
Author(s):  
Daniel Plante ◽  
Julio Alexander Bran Barrera ◽  
Maude Lord ◽  
Irène Iugovaz ◽  
Neda Nasheri
Keyword(s):  
Hepatitis A Virus ◽  
Rna Extraction ◽  
Complex Nature ◽  
Murine Norovirus ◽  
Rt Pcr ◽  
Pcr Inhibitors ◽  
Qrt Pcr ◽  
Extraction Protocol ◽  
Viral Particles ◽  
Hands On

Foodborne viruses such as norovirus and hepatitis A virus cause frequent outbreaks associated with the consumption of raw or undercooked oysters. Viral particles are bioaccumulated in the oyster’s digestive glands, making RNA extraction and RT-PCR detection difficult due to the complex nature of the food matrix and the presence of RT-PCR inhibitors. Herein, we have developed a viral RNA extraction protocol from raw oysters using murine norovirus (MNV) as a surrogate for human noroviruses. The method combines lysis in Tri-Reagent reagent, followed by RNA extraction using Direct-Zol purification columns and lithium chloride precipitation. Viral load quantification was performed by both qRT-PCR and droplet-digital RT-PCR. We have demonstrated that this method can efficiently remove RT-PCR inhibitors, and is sensitive enough to reliably detect viral contamination at 25 PFU/0.2 g. We have also compared the efficiency of this method with the ISO 15216-1:2017 method and Method E developed by Quang and colleagues, and observed significantly higher efficiency compared with the ISO 15216-1 method and comparable efficiency with Method E, with less steps, and shorter hands-on time.

Glycosylated hemoglobin measured by affinity chromatography: micro-sample collection and room-temperature storage.

1983 ◽  
Vol 29 (6) ◽  
pp. 1080-1082 ◽  
Author(s):  
R R Little ◽  
J D England ◽  
H M Wiedmeyer ◽  
D E Goldstein
Keyword(s):  
Affinity Chromatography ◽  
Whole Blood ◽  
High Performance ◽  
Room Temperature ◽  
Hemoglobin A1c ◽  
Glycosylated Hemoglobin ◽  
Exchange Chromatography ◽  
Sample Collection ◽  
Room Temperature Storage ◽  
Temperature Storage

Abstract Under proper conditions, whole blood can be stored at room temperature for as long as 21 days before measurement of glycosylated hemoglobin by affinity chromatography. Whole blood (anticoagulated with EDTA or heparin) was placed in capillary tubes, which were then sealed at both ends and stored at room temperature. Just before assay, whole blood was rinsed from the tubes and diluted 10-fold with water. Samples of each patient's blood were assayed as whole-blood hemolysates by affinity chromatography after zero, seven, 14, and 21 days of storage. Values for glycosylated hemoglobin did not change over 21 days of storage and values for each storage day correlated well (r = 0.97, p less than .0001) with hemoglobin A1C measured in fresh erythrocyte hemolysates by "high-performance" liquid ion-exchange chromatography.

scholarly journals Cross-sectional quantitative RT-PCR study of feline coronavirus viremia and replication in peripheral blood of healthy shelter cats in Southern California

2017 ◽  
Vol 20 (4) ◽  
pp. 295-301 ◽  
Author(s):  
Eric J Fish ◽  
Pedro Paulo VP Diniz ◽  
Yen-Chen Juan ◽  
Frank Bossong ◽  
Ellen W Collisson ◽  
...  
Keyword(s):  
Peripheral Blood ◽  
Southern California ◽  
Buffy Coat ◽  
Sample Collection ◽  
Rt Pcr ◽  
Case Control Studies ◽  
M Gene ◽  
Cross Sectional ◽  
Qrt Pcr ◽  
Feline Coronavirus

Objectives The objectives of this study were to determine the prevalence of feline coronavirus (FCoV) viremia, and its replication in peripheral blood using quantitative RT-PCR (qRT-PCR) methodology in a population of 205 healthy shelter cats in Southern California, as well as to assess any possible connection to longitudinal development of feline infectious peritonitis (FIP). Methods The study was performed on buffy-coat samples from EDTA-anticoagulated whole blood samples of 205 healthy shelter cats. From 50 of these cats, fecal samples were also examined. FCoV genomic and subgenomic RNA in the buffy coats was amplified by a total FCoV RNA qRT-PCR. Evidence for FCoV replication in peripheral blood and feces was obtained by M gene mRNA qRT-PCR. Results Nine of 205 cats (4.4%) were viremic by the total FCoV RNA qRT-PCR, and one of these cats had evidence of peripheral FCoV blood replication by an FCoV mRNA qRT-PCR. The single cat with peripheral blood replication had a unique partial M gene sequence distinct from positive controls and previously published FCoV sequences. Neither seven of the nine viremic cats with follow-up nor the single cat with replicating FCoV with positive qRT-PCR results developed signs compatible with FIP within 6 months of sample collection. Conclusions and relevance FCoV viremia and peripheral blood replication in healthy shelter cats have a low prevalence and do not correlate with later development of FIP in this study population, but larger case-control studies evaluating the prognostic accuracy of the qRT-PCR assays are needed.

scholarly journals Assessment of Successful qRT-PCR of SARS-CoV-2 Assay in Pool Screening Using Isopropyl Alcohol Purification Step in RNA Extraction

2021 ◽  
Vol 2021 ◽  
pp. 1-6
Author(s):  
Mayank Gangwar ◽  
Alka Shukla ◽  
Virendra Kumar Patel ◽  
Pradyot Prakash ◽  
Gopal Nath
Keyword(s):  
Isopropyl Alcohol ◽  
Rna Extraction ◽  
Group Testing ◽  
Research Work ◽  
Rt Pcr ◽  
Purification Step ◽  
E Gene ◽  
Qrt Pcr ◽  
Pcr Targeting ◽  
Pooled Samples

The study is aimed at establishing the optimal parameters for RNA purification of pooled specimens, in SARS-CoV-2 assay. This research work evaluates the difference of extracted RNA purity of pooled samples with and without treatment with isopropyl alcohol and its effect on real-time RT-PCR. As per the protocol of the Indian Council of Medical Research (ICMR), 5 sample pools were analysed using qRT-PCR. A total of 100 pooled samples were selected for the study by mixing 50 μL of one COVID-19 positive nasopharyngeal/oropharyngeal (NP/OP) specimen and 50 μL each of 4 known negative specimens. Pool RNA was extracted using the column-based method, and 1 set of pooled extracted RNA was tested as such, while RNA of the second set was treated additionally with chilled isopropyl alcohol (modified protocol). Further, the purity of extracted RNA in both the groups was checked using Microvolume Spectrophotometers (Nanodrop) followed by RT-PCR targeting E-gene and RNaseP target. The results showed that the purity index of extracted RNA of untreated pooled specimens was inferior to isopropyl alcohol-treated templates, which was observed to be 85% sensitivity and 100% specificity. The average Cq (E gene) in the unpurified and purified pool RNA group was 34.66 and 31.48, respectively. The nanodrop data suggested that purified RNA concentration was significantly increased with an average value of 24.73 ± 1.49   ng / uL , which might be the reason for high sensitivity and specificity. Thus, this group testing of SARS-CoV-2 cases using pools of 5 individual samples would be the best alternative for saving molecular reagents, personnel time, and can increase the overall testing capacity. However, purity of RNA is one of the important determinants to procure unfailing results, thus, this additional purification step must be included in the protocol after RNA has been extracted using commercially available kit before performing qRT-PCR.

scholarly journals Extraction-free rapid cycle RT-qPCR and extreme RT-PCR for SARS-CoV-2 virus detection

2021 ◽  
Vol 156 (Supplement_1) ◽  
pp. S139-S139
Author(s):  
J C Lownik ◽  
J S Farrar ◽  
G Way ◽  
R K Martin
Keyword(s):  
Supply Chain ◽  
Diagnostic Testing ◽  
Rna Extraction ◽  
Virus Detection ◽  
Detection Methods ◽  
Molecular Diagnostic ◽  
Sample Collection ◽  
Rt Pcr ◽  
Hydrolysis Probe ◽  
Time Requirements

Abstract Introduction/Objective Since the start of the coronavirus disease 2019 (COVID-19) pandemic, molecular diagnostic testing for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has faced substantial supply chain shortages and noteworthy delays in result reporting after sample collection. Supply chain shortages have been most evident in reagents for RNA extraction and rapid diagnostic testing. In this study, we explored the kinetic limitations of extraction-free rapid cycle RT-qPCR for SARS-CoV-2 virus detection using the commercially available capillary based LightCycler. Methods/Case Report We optimized reverse transcription and PCR under extraction-free and rapid thermocycling conditions utilizing hydrolysis probe-based detection methods using a Roche LightCycler. Results (if a Case Study enter NA) This protocol improves detection speed while maintaining the sensitivity and specificity of hydrolysis probe-based detection. Percentage agreement between the developed assay and previously tested positive patient samples was 97.6% (n= 40/41) and negative patient samples was 100% (40/40). We further demonstrate that using purified RNA, SARS-CoV-2 testing using extreme RT-PCR and product verification by melting can be completed in less than 3 minutes. Conclusion We developed a protocol for sensitive and specific RT-qPCR of SARS-CoV-2 RNA from nasopharyngeal swabs in less than 20 minutes, with minimal hands-on time requirements. Overall, these studies provide a framework for increasing the speed of SARS-CoV-2 and other infectious disease testing.

scholarly journals A Study on Requirement of Cold Chain Maintenance for Reliable Testing of SARS-CoV-2 Samples

2021 ◽  
Author(s):  
Varunika Vijayvergia ◽  
Aruna Vyas ◽  
Nazneen Pathan ◽  
Rajni Sharma ◽  
Snigdha Purohit ◽  
...  
Keyword(s):  
Room Temperature ◽  
Standard Test ◽  
Cold Chain ◽  
Effect Of Temperature ◽  
Sample Collection ◽  
Rt Pcr ◽  
Significant Deterioration ◽  
Clinical Sensitivity ◽  
Ambient Room Temperature ◽  
The World

Introduction: Coronavirus Disease 2019 (COVID-19) has been haunting the world since December 2019 and has grown to pandemic proportions from March 2020. Even after a full year of research and study, the most effective way to control the spread of this infection is early diagnosis and isolation of the cases. Real-time Reverse Transcription Polymerase Chain Reaction (RT-PCR) is considered the standard test all over the world for the diagnosis of Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) infection. All the sample collection guidelines have recommended stringent maintenance of the cold chain for the sample transport. However, it is not possible for the resource constrained developing countries with inadequate infrastructure to comply with these guidelines all the time. Aim: To determine necessity of stringent transport criteria and the effect of temperature on the clinical sensitivity of a RT-PCR assay for diagnosis of SARS-CoV-2 infection. Materials and Methods: In this prospective experimental study conducted in November 2020, 49 positive samples were kept at ambient room temperature and were tested everyday with RT- PCR for the detection of SARS-CoV-2 Ribonucleic Acid (RNA). The samples were also kept under refrigeration at 4°C and were also tested by RT-PCR and the results were compared with their respective counterparts kept at room temperature till nine days. Python Jupiter notebook SciPy and Anaconda software was used for statistical analysis. Results: It was observed that the positivity of the RT-PCR results were not deteriorated till five days and there was no significant deterioration even after nine days of samples being stored at room temperature suggesting that even if the viral RNA itself is not stable outside strict temperature control but small fragment or target genetic sequences are enough for detection of virus by RT-PCR. Conclusion: It is possible to keep samples at this ambient temperature for five days without any loss of positivity in RT-PCR.

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