Specimen Collection Procedures Research Articles

Molecular Analysis of Volatile Metabolites Synthesized by Candida albicans and Staphylococcus aureus in In Vitro Cultures and Bronchoalveolar Lavage Specimens Reflecting Single- or Duo-Factor Pneumonia.

Current microbiological methods for pneumonia diagnosis require invasive specimen collection and time-consuming analytical procedures. There is a need for less invasive and faster methods to detect lower respiratory tract infections. The analysis of volatile metabolites excreted by pathogenic microorganisms provides the basis for developing such a method. Given the synergistic role of Candida albicans in increasing the virulence of pathogenic bacteria causing pneumonia and the cross-kingdom metabolic interactions between microorganisms, we compare the emission of volatiles from Candida albicans yeasts and the bacteria Staphylococcus aureus using single and mixed co-cultures and apply that knowledge to human in vivo investigations. Gas chromatography-mass spectrometry (GC-MS) analysis resulted in the identification of sixty-eight volatiles that were found to have significantly different levels in cultures compared to reference medium samples. Certain volatiles were found in co-cultures that mainly originated from C. albicans metabolism (e.g., isobutyl acetate), whereas other volatiles primarily came from S. aureus (e.g., ethyl 2-methylbutyrate). Isopentyl valerate reflects synergic interactions of both microbes, as its level in co-cultures was found to be approximately three times higher than the sum of its amounts in monocultures. Hydrophilic-lipophilic-balanced (HLB) coated meshes for thin-film microextraction (TFME) were used to preconcentrate volatiles directly from bronchoalveolar lavage (BAL) specimens collected from patients suffering from ventilation-associated pneumonia (VAP), which was caused explicitly by C. albicans and S. aureus. GC-MS analyses confirmed the existence of in vitro-elucidated microbial VOCs in human specimens. Significant differences in BAL-extracted amounts respective to the pathogen-causing pneumonia were found. The model in vitro experiments provided evidence that cross-kingdom interactions between pathogenic microorganisms affect the synthesis of volatile compounds. The TFME meshes coated with HLB particles proved to be suitable for extracting VOCs from human material, enabling the translation of in vitro experiments on the microbial volatilome to the in vivo situation involving infected patients. This indicates the direction that should be taken for further clinical studies on VAP diagnosis based on volatile analysis.

A Comprehensive Bone Marrow Aspirate and Biopsy Educational Program Utilizing Task Trainers.

Advanced practice providers (APPs) who care for patients with hematologic malignancies perform bone marrow aspiration and biopsies (BMBXs). Invasive bedside procedures are often taught through the observational training method, which can lead to inconsistencies. The purpose of this project was to create and evaluate a standardized educational curriculum incorporating simulation with a task trainer for bone marrow transplant (BMT) APPs. The project aimed to reduce BMBX incident reporting events, improve BMBX knowledge, and increase APP self-reported confidence. Pre- and post-test surveys were utilized for knowledge assessment of BMBX procedures and specimen allocation. Program delivery occurred on five occasions to accommodate the needs of the team. Each program was delivered over 3 hours and included an educational Microsoft PowerPoint and three breakout sessions: BMBX kit review; simulation on task trainer; and review of BMBX specimen collection procedures. Knowledge assessment surveys were compared through descriptive and statistical analysis. BMBX incident reporting events decreased from 1.92 events per month pre-implementation to 1.2 events per month post-implementation. Overall, BMBX knowledge increased from 41.02% on pre-test surveys to 65.72% on post-test surveys. Participant self-reported confidence improved by a mean difference of -1.85 based on a 5-point Likert scale, t(12) = -1.85 (p ≤ .0001, 95% confidence interval = -2.49 to -1.2). This project suggests that the use of simulation with task trainers is beneficial when paired with a standardized educational curriculum. Simulation training for APPs who perform BMBX improves procedural knowledge, increases self-reported confidence, and can reduce incident reporting events.

Effective strategies for Fecal Immunochemical Tests (FIT) programs to improve colorectal cancer screening uptake among populations with limited access to the healthcare system: a rapid review

BackgroundColorectal cancer (CRC) is one of the leading causes of cancer death globally. CRC screening can reduce the incidence and mortality of CRC. However, socially disadvantaged groups may disproportionately benefit less from screening programs due to their limited access to healthcare. This poor access to healthcare services is further aggravated by intersecting, cumulative social factors associated with their sociocultural background and living conditions. This rapid review systematically reviewed and synthesized evidence on the effectiveness of Fecal Immunochemical Test (FIT) programs in increasing CRC screening in populations who do not have a regular healthcare provider or who have limited healthcare system access.MethodsWe used three databases: Ovid MEDLINE, Embase, and EBSCOhost CINAHL. We searched for systematic reviews, meta-analysis, and quantitative and mixed-methods studies focusing on effectiveness of FIT programs (request or receipt of FIT kit, completion rates of FIT screening, and participation rates in follow-up colonoscopy after FIT positive results). For evidence synthesis, deductive and inductive thematic analysis was conducted. The findings were also classified using the Cochrane Methods Equity PROGRESS-PLUS framework. The quality of the included studies was assessed.ResultsFindings from the 25 included primary studies were organized into three intervention design-focused themes. Delivery of culturally-tailored programs (e.g., use of language and interpretive services) were effective in increasing CRC screening. Regarding the method of delivery for FIT, specific strategies combined with mail-out programs (e.g., motivational screening letter) or in-person delivery (e.g., demonstration of FIT specimen collection procedure) enhanced the success of FIT programs. The follow-up reminder theme (e.g., spaced out and live reminders) were generally effective. Additionally, we found evidence of the social determinants of health affecting FIT uptake (e.g., place of residence, race/ethnicity/culture/language, gender and/or sex).ConclusionsFindings from this rapid review suggest multicomponent interventions combined with tailored strategies addressing the diverse, unique needs and priorities of the population with no regular healthcare provider or limited access to the healthcare system may be more effective in increasing FIT screening. Decision-makers and practitioners should consider equity and social factors when developing resources and coordinating efforts in the delivery and implementation of FIT screening strategies.

Target product profile: Trypanosoma brucei gambiense test to verify elimination.

Human African trypanosomiasis is a life-threatening parasitic infection transmitted by the tsetse fly in sub-Saharan Africa. The most common form is caused by Trypanosoma brucei gambiense, with humans as the main reservoir. Diagnosis in the field requires microscopic examination performed by specifically trained personnel. After over two decades of sustained efforts, the incidence of the disease is strongly declining, and some historically endemic countries are no longer detecting cases. The World Health Organization (WHO) has targeted the elimination of transmission of gambiense human African trypanosomiasis by 2030, defined as zero autochthonous cases for at least five consecutive years. Endemic countries reaching this goal must maintain dedicated surveillance to detect re-emergence or re-introduction. With this new agenda, new tools are needed for verification of the absence of transmission. WHO has therefore developed a target product profile calling for development of a method for population-level cross-cutting surveillance of T. b. gambiense transmission. The method needs to be performed in national or sub-national reference laboratories, and to test in parallel numerous samples shipped from remote rural areas. Among other characteristics the product profile specifies: (i)a simple specimen collection procedure; (ii)no cold-chain requirement to transfer specimens to reference laboratories; (iii)high sensitivity and specificity; (iv)high-throughput, substantially automatized; (v)low cost per specimen, when analysed in large batches; and (vi)applicable also in animals.

Influence of storage conditions and multiple freeze-thaw cycles on N1 SARS-CoV-2, PMMoV, and BCoV signal

Wastewater-based epidemiology/wastewater-based surveillance (WBE/WBS) continues to serve as an effective means of monitoring various diseases, including COVID-19 and the emergence of SARS-CoV-2 variants, at the population level. As the use of WBE expands, storage conditions of wastewater samples will play a critical role in ensuring the accuracy and reproducibility of results. In this study, the impacts of water concentration buffer (WCB), storage temperature, and freeze-thaw cycles on the detection of SARS-CoV-2 and other WBE-related gene targets were examined. Freeze-thawing of concentrated samples did not significantly affect (p > 0.05) crossing/cycle threshold (Ct) value for any of the gene targets studied (SARS-CoV-2 N1, PMMoV, and BCoV). However, use of WCB during concentration resulted in a significant (p < 0.05) decrease in Ct for all targets, and storage at −80 °C (in contrast to −20 °C) appeared preferable for wastewater storage signal stability based on decreased Ct values, although this was only significantly different (p < 0.05) for the BCoV target. Interestingly, when Ct values were converted to gene copies per influent sample, no significant differences (p > 0.05) were observed in any of the targets examined. Stability of RNA targets in concentrated wastewater against freeze-thaw degradation supports archiving of concentrated samples for use in retrospective examination of COVID-19 trends and tracing SARS-CoV-2 variants and potentially other viruses, and provides a starting point for establishing a consistent procedure for specimen collection and storage for the WBE/WBS community.

Occupational exposure to HIV and utilization of post-exposure prophylaxis among healthcare workers at St. Peter’s specialized hospital in Addis Ababa, Ethiopia

Healthcare workers are susceptible to blood borne pathogens, such as human immunodeficiency virus (HIV). Occupational exposure to HIV infection among healthcare workers is becoming a global public health concern. However, there is limited evidence about occupational exposure of healthcare workers to HIV and utilization of post-exposure prophylaxis in Addis Ababa, Ethiopia. Accordingly, this study was conducted to assess the prevalence of occupational exposure to HIV and utilization of post exposure prophylaxis among healthcare workers at St. Peter’s specialized hospital, Addis Ababa, Ethiopia. A health facility-based cross-sectional study was conducted among 308 randomly selected healthcare workers in April 2022. Structured and pretested self-administered questioner was used to collect data. Occupational exposure to HIV was taken as any percutaneous injury or blood or other body fluids exposure while administering medications, specimen collection, and other procedures with HIV confirmed patients. Multivariable binary logistic regression analysis was used to identify factors associated with occupational exposure to HIV and utilization of post-exposure prophylaxis. Statistically significant association was declared on the basis of adjusted odds ratio with 95% confidence interval and p-value less than 0.05. The study found that 42.3% (95% CI 36.6, 47.9%) of the healthcare workers had occupational exposure to HIV during their career time, out of whom 16.1% (95% CI 11.9, 20.3%) used post-exposure prophylaxis. Healthcare workers with lower-level education such as diploma (AOR: 0.41, 95% CI 0.17, 0.96) and BSc (AOR: 0.51, 95% CI 0.26, 0.92), and healthcare workers who received infection prevention training (AOR: 0.55, 95% CI 0.33, 0.90) had less risk of exposure to HIV. On the other hand, nurses (AOR: 1.98, 95% CI 1.07, 3.67), midwifes (AOR: 3.79, 95% CI 1.21, 11.9), and physicians (AOR: 2.11, 95% CI 1.05, 4.22) had high risk of exposure to HIV compared with other professionals. Moreover, healthcare workers with BSc degree compared with healthcare workers with masters degree (AOR: 3.69, 95% CI 1.08, 12.6), healthcare workers with long service year (AOR: 3.75, 95% CI 1.64, 8.57), and healthcare workers who are working in facilities where prophylaxis is available (AOR: 3.41, 95% CI 1.47, 7.91) had higher odds to utilize post-exposure prophylaxis. Significant proportion of healthcare workers included in the current study had occupational exposure to HIV and very few of them used post-exposure prophylaxis. Healthcare workers need to use appropriate personal protective equipment, safely manage contaminated equipment, and safely administered medications and collect specimen to protect themselves from exposure to HIV. Moreover, use of post-exposure prophylaxis should be promoted when exposure exists.

SG-APSIC1011: Factors associated with improved knowledge of COVID-19 prevention and control following a training of healthcare workers in Vietnam

Objectives: SARS-CoV-2 is a novel and highly infectious virus. An effective response requires rapid training of healthcare workers (HCWs). We measured the change in knowledge related to COVID-19 and associated factors before and after training of HCWs in Vietnam. Methods: A quasi-experimental design was used to evaluate HCW knowledge related to prevention and control of SARS-CoV-2 before and after attending a 2-day training-of-trainers course. Between June and September 2020, 963 HCWs from 194 hospitals in 21 provinces received the training. HCW knowledge was assessed using a 20-item questionnaire consisting of multiple-choice questions at the beginning and closing of the training course. A participant received 1 point for each correct answer. He or she was considered to have improved knowledge the posttest score was higher than the pretest score with a score ≥15 on the posttest. We applied the McNemar test and logistic regression model to test the level of association between demographic factors and change in knowledge of COVID-19. Results: Overall, 100% of HCWs completed both the pretest and posttest. At baseline, only 14.7% scored ≥15. Following the training, 78.4% scored ≥15 and 64.3% had improved knowledge according to the predetermined definition. Questions related to the order of PPE donning and doffing and respiratory specimen collection procedures were identified as having the greatest improvement (44.6% and 60.7%, respectively). Being female (OR, 1.5; 95% CI, 1.1–2.0), having a postgraduate degree (OR, 2.5; 95% CI, 1.4–4.4), working in a nonmanager position (OR, 1.5; 95% CI, 1.1–2.1), previous contact with a COVID-19 patient (OR, 1.5; 95% CI, 1.1–2.0), and working in northern Vietnam (OR, 2.0; 95% CI, 1.4–2.6), were associated with greater knowledge improvement. Conclusions: Most HCWs demonstrated improved knowledge of COVID-19 prevention and control after attending the training. Particular groups may benefit from additional training: those who are male, leaders and managers, those who hold an undergraduate degree, and those who work in the southern provinces.

Study on Intestinal Helminthoses and the Efficacy of Anthelminthoses Drugs among School Children in Girei Local Government Area, Adamawa State

Intestinal helminthoses have adverse effects on the physical and mental development in poorly nourished community populations. This study was conducted to determine the prevalence of helminthoses and to evaluate the efficacy of two antihelminthoses drugs (albendazole and levamisole) among school aged pupils in Girei local government area of Adamawa State, Nigeria. A total of 384 primary school pupils were randomly selected from six public primary schools in Girei local government area of Adamawa state. Statistical tools used for data collection was a structured questionnaire, while simple percentages and chi-square test of association were used for data analysis with the help of statistical package for social sciences (SPSS) version 26. Clean plastic containers were distributed to the participants at enrollment with detailed instructions about the procedure for stool specimen collection. All stool samples were collected early in the morning and conserved in ice boxes before transportation to the laboratory. Each specimen was examined for the presence of intestinal parasitic organisms by the concentration techniques and kato-katz was used for egg count. The age specific prevalence were 21.1% among pupils with age 6-9 years, 17.1% among pupils with age ≥10 years and 10.4% among the youngest children ≤ 5years of age.&#x0D; Keywords: Intestinal Helmnthosis, Efficacy; Anthelminthic; Drugs

Reliable measurement of plasma kinin peptides: Importance of preanalytical variables

BackgroundThe kallikrein‐kinin system is involved in many (patho)physiological processes and kinin peptides are considered potential clinical biomarkers. Variance in blood specimen collection and processing, artificial ex vivo bradykinin formation, and rapid degradation of kinins have contributed to divergence in published plasma levels, therefore limiting their significance. Thus, reliable preanalytical settings are highly required. ObjectivesThis study aimed to develop and evaluate a standardized preanalytical procedure for reliable kinin quantification. The procedure was based on identification of the most impactful variables on ex vivo plasma level alterations. MethodsSuitable protease inhibitors and blood specimen collection and handling conditions were systematically investigated. Their influence on plasma levels of seven kinins was monitored using an established in‐house liquid chromatography–tandem mass spectrometry platform. ResultsIn nonstandardized settings, ex vivo rise of bradykinin was found to already occur 30 seconds after blood sampling with high interindividual variation. The screening of 17 protease inhibitors resulted in a customized seven‐component protease inhibitor, which efficiently stabilized ex vivo kinin levels. The reliability of kinin levels was substantially jeopardized by prolonged rest time until centrifugation, phlebotomy methodology (eg, straight needles, catheters), vacuum sampling technique, or any time delays during venipuncture. The subsequently developed standardized procedure was applied to healthy volunteers and proved it significantly limited interday and interindividual kinin level variability. ConclusionThe developed procedure for blood specimen collection and handling is feasible in clinical settings and allows for determination of reliable kinin levels. It may contribute to further elucidating the role of the kallikrein‐kinin system in diseases like angioedema, sepsis, or coronavirus disease 2019.

Detecting Preanalytical Errors Using Quality Indicators in a Hematology Laboratory.

Background and Objectives:Monitoring laboratory performance continuously is crucial for recognizing errors and fostering further improvements in laboratory medicine. This study aimed to review the quality indicators (QIs) and describe the laboratory errors in the preanalytical phase of hematology testing in a clinical laboratory.Methods:All samples received in the Hematology Laboratory of the Maternity and Pediatric Hospital in Hail for 3 years were retrospectively reviewed and evaluated for preanalytical issues using a set of QIs. The rate of each QI was compared to the quality specifications cited in the literature.Results:A total of 95002 blood samples were collected for analysis in the hematology laboratory from January 2017 through December 2019. Overall, 8852 (9.3%) were considered to show preanalytical errors. The most common were “clotted specimen” (3.6%) and “samples not received” (3.5%). Based on the quality specifications, the preanalytical QIs were classified generally as low and medium level of performance. In contrast, the sigma-based performance level indicates acceptable performance on all the key processes. Further analysis of the study showed a decreasing rate of preanalytical errors from 11.6% to 6.5%.Conclusions:Preanalytical errors remain a challenge to hematology laboratories. The errors in this case were predominantly related to specimen collection procedures that compromised the specimen quality. Quality indicators are a valuable instrument in the preanalytical phase that allows an opportunity to improve and explore clinical laboratory process performance and progress. Continual monitoring and management of QI data are critical to ensure ongoing satisfactory performance and to enhance the quality in the preanalytical phase.

Accuracy and Acceptance of a Self-Collection Model for Respiratory Tract Infection Diagnostics: A Concise Clinical Literature Review

BackgroundNurses are the primary clinicians who collect specimens for respiratory tract infection testing. The specimen collection procedure is time and resource-consuming, but more importantly, it places nurses at risk for potential infection. The practice of allowing patients to self-collect their diagnostic specimens may provide an alternative testing model for the current COVID-19 outbreaks. The objective of this paper was to evaluate the accuracy and patient perception of self-collected specimens for respiratory tract infection diagnostics. MethodsA concise clinical review of the recently published literature was conducted. ResultsA total of 11 articles were included the review synthesis. The concept of self-collected specimens has a high patient acceptance rate of 83-99%. Self-collected nasal-swab specimens demonstrated strong diagnostic fidelity for respiratory tract infections with a sensitivity between 80-100%, this is higher than the 76% sensitivity observed with self-collected throat specimens. In a comparative study evaluating a professionally collected to a self-collected specimen for COVID-19 testing, a high degree of agreement (k = 0.89) was observed between the two methods. ConclusionAs we continue to explore for testing models to combat the COVID-19 pandemic, self-collected specimens is a practical alternative to nurse specimen collection.

Survey of renin and aldosterone testing practices by Ontario laboratories – Providing insight into best practices

ObjectivesTesting for renin and aldosterone in clinical laboratories is complicated by pre-analytical considerations such as the posture for blood collection and susceptibility to cryoactivation of renin. From an analytical perspective, there are both renin activity and renin mass or concentration assays available. There can also be variability in result reporting practices and the aldosterone-renin ratio (ARR) cut-off applied to screen for primary aldosteronism (PA). The Institute for Quality Management in Healthcare (IQMH) Centre for Proficiency Testing surveyed laboratories on their handling of renin and aldosterone testing to better understand current practices. Design and methodsAn online survey was prepared and sent to 134 Canadian laboratories enrolled in endocrinology proficiency testing with IQMH. ResultsOne hundred twenty Ontario laboratories submitted responses. While only six (5%) laboratories perform testing for both renin and aldosterone, 108 (90%) collect and process specimens to be tested by reference laboratories. The survey revealed considerable variation in practices including the recommended state of patients prior to sample collection (for example, regarding medications or salt intake), the patient posture specifications for sample collection, the precautions taken against cryoactivation of renin, the choice of renin activity or mass assay, and the ARR cut-off used. The available literature on these factors was then reviewed. ConclusionsAlthough there is no standardized procedure for specimen collection, analysis, or result reporting for renin or aldosterone testing, we have attempted to summarize the available literature to develop evidence-based recommendations. Where laboratory practice differs from peers and/or recommended protocols, laboratories should review their practices.

The Effect of High Intensities and Moderate Intensities Football Specific Circuit Training on Selected Hematological Variables among Under-17 Football Players

Objective: The purpose of the study was to investigate the comparative effect of high intensities andmoderate intensities football specific circuit training on selected Hematological Variables among Under-17football players in Ethiopia.Method: Randomized control group pretest-posttest design was implemented for this experimental study.For the purpose of this study from a total of ninety (90) football players of Adigrat city sixty (60) footballerswere taken as a sample size. The subjects were at the age of 16.15+.71 years, height of 1.62+.54 meter andweight of 50.67+3.71. These subjects were distributed into the experimental groups of high intensity circuittraining group (HICT group) N1=20, moderate intensity circuit training group(MICT group)N2=20andcontrol group (CG) N=20. Only HICT group and MICT group were engaged in high intensity circuit trainingand moderate intensity circuit training for sixteen weeks respectively. The selected hematological parameterswere Red blood cells (RBC), White blood cells (WBC), Hemoglobin (Hgb), Hematocrit (HCT), Mean cellvolume (MCV) and Mean cell hemoglobin (MCH). The data was collected from cubital vein as per the guidefor blood specimen collection procedure at both pre and post-tests. The data was analyzed using ANCOVAand post hoc test for paired mean comparison when significant difference exists.Result: Our finding indicated that the High intensity circuit training group increased significantly in RBC,WBC, Hgb and MCV than control group with (519, P=.000), (91.595, P=.000), (.903, P=.019) and (5.668,P=.000) respectively. Moderate intensity circuit training Group increased significantly from the controlgroup on RBC, WBC, Hgb and MCV with a mean difference of (.746, P=.000), (1.894, P=.000), (1.238,P=.000) and (2.777, P=.000) respectively. The High intensity circuit training group decreased significantly inHCT and MCH with (-3.175, P=.000) and (-1.344, P=.000) respectively. Moderate intensity circuit trainingGroup decreased significantly from the control group on HCT and MCH with (-4.310, P=.000) and (-2.076,P=.000) respectively. In addition to this the Moderate intensity circuit training Group showed significantincrease in RBC and decrease in MCH from High intensity circuit training group with (.227, P=0.014) and(.732, P=.032) respectively. The High intensity circuit training group increased significantly in MCV thanthe Moderate intensity circuit training Group with (2.892, P=.001).Conclusion: From our finding we conclude that different intensity of training brings different changes indifferent Hematological parameters and similar changes in some variables as well. Thus the circuit training intensity should be considered in the coachingplan for junior players.

Cost Estimation of the New HPV/DNA Test Screening Procedure per Person among 35 Years Old Ever-Married Women in a District of Sri Lanka

Background: HPV/DNA screening test has an optimum sensitivity (92.9%) to detect cervical lesions. The objective of the study was to estimate the cost of cobas 4800 HPV/DNA test in thin prep cell collection media. Methods: The cost estimation of the HPV/DNA specimen collection was done at a randomly selected community Well Woman Clinic (WWC) in the district of Sri Lanka. Five women, who were attended WWC for HPV/DNA specimen collection in September, October, and November 2018 were randomly selected for the cost estimation. To estimate the cost incurred in laboratory screening of the HPV/DNA specimen by cobas 4800 HPV/DNA PCR test, a reference laboratory in the district was utilized. Checklists were developed to record all instruments and consumable items used in the clinic and laboratory, while record sheets were developed to record the time taken to complete all activities associated with taking an HPV/DNA specimen by the clinic and laboratory staff. Five HPV/DNA specimen collection procedures were randomly selected at each of three sessions and the average time per procedure was calculated. Categories of staff involved in the procedure and staff time spent on the procedure were measured and recorded on three clinic days. Staff time spent on HPV/DNA specimen screening by an HPV/DNA testing machine in the laboratory was calculated on three working days (One day per month). Results: The total estimated cost of the HPV/DNA specimen collection procedure incurred by the government in the community WWC was Rs.2569.38 (USD 13.81), while the total estimated unit cost in the laboratory procedure for HPV/DNA screening was Rs. 50.57(USD 0.27).Therefore the total estimated cost of the HPV/ DNA specimen screening procedure was Rs. 2619.95(USD 14.08). The estimated unit cost including administrative cost was Rs. 2881.95(USD 15.49). Conclusion: Cobas HPV/DNA test is economically feasible to be incorporated into the national cervical cancer screening programme in Sri Lanka. It is necessary to further attempt alternative methods of cobas 4800 HPV/DNA test (i.e. dry swabs) to determine the most appropriate method for resource limited settings.

Saliva as a Diagnostic Aid in COVID-19: A Boon for Dentistry

Introduction: Coronavirus disease 19 (COVID-19) is a highly contagious and virulent infection caused by severe acute respiratory syndrome coronavirus 2. The symptoms arise within 2 to 14 days after the infection. The specimens are collected from the upper respiratory tract through nasopharyngeal and oropharyngeal swab. This sampling requires close contact with COVID-19 patients and may directly cause transmission to health care professionals. Saliva specimens can be collected easily by asking patients to spit into a sterile container. Materials and Methods: In the current study, the selected patients (n = 20) were those who tested positive for COVID-19 and were admitted to the hospital. After getting approval from ethical committee of the hospital, both nasopharyngeal swab specimens and the salivary samples of each patient were collected and tested using standard procedure of real-time polymerase chain reaction. Results: The mean cycle threshold values obtained from both the sampling techniques were correlated. It was seen that there was no significant difference between them. The advantage of using saliva as a specimen is in the overall specimen collection procedure and its cost and time effectiveness. The diagnosis of COVID-19 through a rapid test procedure using salivary samples could be a boon to dentists. Conclusion: This would help in the detection of positive asymptomatic carriers of the virus among the patients who come to seek dental treatment and reduce the risk of infection in the dental office. The results of this study clearly show that the values obtained from the two sampling techniques are closely related, and hence saliva could definitely be considered as a diagnostic aid for COVID-19.

Bridging the gaps in test interpretation of SARS-CoV-2 through Bayesian network modelling.

Abstract In the absence of an established gold standard, an understanding of the testing cycle from individual exposure to test outcome report is required to guide the correct interpretation of severe acute respiratory syndrome-coronavirus-2 reverse transcriptase real-time polymerase chain reaction (RT-PCR) results and optimise the testing processes. Bayesian network models have been used within healthcare to bring clarity to complex problems. We use this modelling approach to construct a comprehensive framework for understanding the real-world predictive value of individual RT-PCR results. We elicited knowledge from domain experts to describe the test process through a facilitated group workshop. A preliminary model was derived based on the elicited knowledge, then subsequently refined, parameterised and validated with a second workshop and one-on-one discussions. Causal relationships elicited describe the interactions of pre-testing, specimen collection and laboratory procedures and RT-PCR platform factors, and their impact on the presence and quantity of virus and thus the test result and its interpretation. By setting the input variables as ‘evidence’ for a given subject and preliminary parameterisation, four scenarios were simulated to demonstrate potential uses of the model. The core value of this model is a deep understanding of the total testing cycle, bridging the gap between a person's true infection status and their test outcome. This model can be adapted to different settings, testing modalities and pathogens, adding much needed nuance to the interpretations of results.

693. Shaking Things Up: Direct-to-PCR Viral Detection off Swabs Using Shaker-Mill Homogenization

BackgroundAs the number of viral diseases are on the rise, it is critical to continue to innovate and advance diagnostic, treatment, and surveillance methods surrounding viral infections. Currently, one of the most reliable methods for viral infection detection are polymerase chain reaction (PCR) based assays. These assays often involve procedures of swabbing a patient, processing the sample to lyse the virus, extract, and purify it’s nucleotides, and then run the purified genetic material via PCR for detection of a gene product needed to confirm the patient’s suspected diagnosis. This process requires time to complete and is dependent on the availability of the reagents and plastics required to complete the lysis, extraction, purification, and amplification procedures. Herein, we have developed a method to detect virus off a swab using solely shaker-mill based mechanical lysis and the transfer of the viral lysate directly to a PCR based assay, bypassing the reagent heavy and time consuming extraction and purification steps.MethodsUsing Human Coronavirus 229E (HCoV-229E) as a model system, we spiked swabs with clinically relevant levels of the virus for proof-of-concept testing. Swabs were spiked in serial dilutions from 1.2e7 copies/mL to 1.2e1 copies/mL. The swabs were then placed in 2mL tubes with viral transport media (VTM) to mimic the specimen collection procedures in the clinic prior to processing via shaker-mill homogenization. After homogenization, 1 uL of viral lysate was run in RT-qPCR for amplification of the nucleocapsid (N) gene, qualifying viral detection from the sample.ResultsHCoV-229E spiked swabs were run through the two-step process of homogenization direct to RT-qPCR for viral detection. After running 54 swabs, we confidently determined our limit of detection to be 1.2e3 viral copies/mL with 96.30% sensitivity in vitro.ConclusionWe have successfully proven that shaker-mill homogenization provides sufficient viral lysis off swabs, where the resulting lysate can be used directly in PCR based assays for the detection of virus. This finding allows for decreased run time in traditional PCR based diagnostics and reduces the reagents and plastics required for each sample, ultimately reducing the cost and time of each viral test when compared to traditional PCR based methods.DisclosuresZachary P. Morehouse, MS, OMS-III, Omni International Inc (Consultant) Caleb Proctor, BS, Omni International Inc (Employee) Gabriella Ryan, BS, Omni International Inc (Employee) Rodney J. Nash, PhD, Omni International Inc (Employee)

Willingness to Use Home Collection Methods to Provide Specimens for SARS-CoV-2/COVID-19 Research: Survey Study.

BackgroundInnovative laboratory testing approaches for SARS-CoV-2 infection and immune response are needed to conduct research to establish estimates of prevalence and incidence. Self-specimen collection methods have been successfully used in HIV and sexually transmitted infection research and can provide a feasible opportunity to scale up SARS-CoV-2 testing for research purposes.ObjectiveThe aim of this study was to assess the willingness of adults to use different specimen collection modalities for themselves and children as part of a COVID-19 research study.MethodsBetween March 27 and April 1, 2020, we recruited 1435 adults aged 18 years or older though social media advertisements. Participants completed a survey that included 5-point Likert scale items stating how willing they were to use the following specimen collection testing modalities as part of a research study: home collection of a saliva sample, home collection of a throat swab, home finger-prick blood collection, drive-through site throat swab, clinic throat swab, and clinic blood collection. Additionally, participants indicated how the availability of home-based collection methods would impact their willingness to participate compared to drive-through and clinic-based specimen collection. We used Kruskal-Wallis tests and Spearman rank correlations to assess if willingness to use each testing modality differed by demographic variables and characteristics of interest. We compared the overall willingness to use each testing modality and estimated effect sizes with Cohen d.ResultsWe analyzed responses from 1435 participants with a median age of 40.0 (SD=18.2) years and over half of which were female (761/1435, 53.0%). Most participants agreed or strongly agreed that they would be willing to use specimens self-collected at home to participate in research, including willingness to collect a saliva sample (1259/1435, 87.7%) or a throat swab (1191/1435, 83.1%). Willingness to collect a throat swab sample was lower in both a drive-through setting (64%) and clinic setting (53%). Overall, 69.0% (990/1435) of participants said they would be more likely to participate in a research study if they could provide a saliva sample or throat swab at home compared to going to a drive-through site; only 4.4% (63/1435) of participants said they would be less likely to participate using self-collected samples. For each specimen collection modality, willingness to collect specimens from children for research was lower than willingness to use on oneself, but the ranked order of modalities was similar.ConclusionsMost participants were willing to participate in a COVID-19 research study that involves laboratory testing; however, there was a strong preference for home specimen collection procedures over drive-through or clinic-based testing. To increase participation and minimize bias, epidemiologic research studies of SARS-CoV-2 infection and immune response should consider home specimen collection methods.

A novel two-step, direct-to-PCR method for virus detection off swabs using human coronavirus 229E

BackgroundCurrently, one of the most reliable methods for viral infection detection are polymerase chain reaction (PCR) based assays. This process is time and resource heavy, requiring multiple steps of lysis, extraction, purification, and amplification procedures. Herein, we have developed a method to detect virus off swabs using solely shaker-mill based mechanical lysis and the transfer of the viral lysate directly to a PCR assay for virus detection, bypassing the substantial reagent and time investments required for extraction and purification steps.MethodsUsing Human Coronavirus 229E (HCoV-229E) as a model system, we spiked swabs in vitro for proof-of-concept testing. Swabs were spiked in serial dilutions from 1.2 × 106 to 1.2 × 101 copies/mL and then placed in 2 mL tubes with viral transport media (VTM) to mimic the specimen collection procedures in the clinic prior to processing via shaker-mill homogenization. After homogenization, 1 μL of lysate was processed using RT-qPCR for amplification of the nucleocapsid (N) gene, qualifying viral detection.ResultsHCoV-229E in vitro spiked swabs were processed in a novel two-step, direct-to-PCR methodology for viral detection. After running 54 swabs, we confidently determined our limit of detection to be 1.2 × 103 viral copies/mL with 96.30% sensitivity.ConclusionWe have proven that the shaker-mill homogenization-based two-step, direct-to-PCR procedures provides sufficient viral lysis off swabs, where the resulting lysate can be used directly in PCR for the detection of HCoV-229E. This finding allows for reductions in the time and resources required for PCR based virus detection in comparison to the traditional extraction-to-PCR methodology.

Abstract 3101: Utility of a new targeted next generation sequencing test for liquid biopsy samples from patients with NSCLC

Abstract Blood-based methods for profiling tumor-sourced nucleic acids have become increasingly important in the diagnostic workup for cancer patients, particularly in non-small cell lung cancer (NSCLC). Blood-based assays address several limitations of tissue-based testing approaches such as inaccessible lesions, limited tissue, extended time to results, and invasive specimen collection procedures. Broad-profiling next-generation sequencing (NGS) methods are used to detect rare variants in blood samples. Here we describe the utility of a newly developed 52 gene NGS test, which uses the GeneStudio S5 Prime platform. We report on performance verification of the assay using contrived specimens as well as studies which evaluated clinical specimens at diagnosis and during monitoring. Thirty-eight cell-free nucleic acid (cfNA) specimens were evaluated from donors previously diagnosed with NSCLC. Specimens were collected in blood collection tubes containing preservatives (Streck cfDNA BCT) and shipped at ambient temperature to our central testing Laboratory. cfNA was isolated from plasma and reverse transcribed. The sequencing metrics for these studies included an average of 15 million mapped reads, 94.4% of on target reads, and a mean sequencing depth of 52,000. Additionally, the average molecular coverage was 2400 reads per amplicon with 99.9% uniformity at 500x coverage. Detected variants and their allelic frequencies were compared with reference results from a validated ddPCR method. Eleven variants (6 EGFR and 5 KRAS) were detected in the cohort with allele frequencies ranging from 0.90 - 96.16%. Results were highly concordant between the two methods (R2=0.992) with 90.3% sensitivity and 100% specificity. In addition, we present a case study to further demonstrate clinical utility of NGS for longitudinal monitoring over 540 days for clearance followed by re-emergence of EGFR positive cfNA harboring the EGFR C797S resistance mutation. These studies add to the growing body of knowledge supporting the utility of molecular detection for actionable and emerging biomarkers in liquid biopsy. Citation Format: Jordan Reese, Victoria Maxwell, Leisa Jackson, Gary Pestano, Hestia S. Mellert. Utility of a new targeted next generation sequencing test for liquid biopsy samples from patients with NSCLC [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3101.