Pooled Specimens Research Articles

SNaPshot minisequencing for SARS CoV 2 Variant Genotyping

Abstract Introduction/Objective The SNaPshot Multiplex System (Thermo Fisher) can investigate up to fifty SNP markers simultaneously by using PCR amplification and subsequent dideoxy single base extension of an unlabeled primer and capillary electrophoresis. After electrophoresis and fluorescence detection, the alleles of a single marker appear as different colored peaks at roughly the same size in the electropherogram plot. The size of the different allele peaks vary slightly due to differences in molecular weight of the dyes. We propose a new set of primers and probes to use this system to differentiate variants of SARs Cov 2 variants. Methods/Case Report SNAPSHOT analysis was performed using the primers designed internally and analysis was done of up to 7 mutation hot spots, which were 417, 4511, 478, 484, 501, 614, and 681. By implementing the algorithm of systems and methods herein with the traces depicted in Figures, true positive and true negative outcomes for the different COVID variants can be performed, based on the highlighted peaks, which are not necessarily the most intense peaks. 10 Positive controls achieved from in stro transcript plasmid was tested then 10 viral RNA obtained through BEI Resources, NIAID was tested after mixed into 30 pooled negative PCR-swab specimens. Genmapper was used for analysis of results. 40 Korean patient samples were tested as well. Results (if a Case Study enter NA) Both In vitro transcript and derived specimens of wild type, alpha, beta, delta, gamma, kappa, omicron, and pirola showed expected results in 78/80 of the specimens. For example,delta B.1.617.2 variant showed 1 = A (green) at Primer 452R-F, 2 = G (blue): Primer 484Q-F, and 3 = A (green) at Primer 614-F. Of note, hetero type was noted in a few of the patient strains but none of the BEI strains and this may be due to a transfromation of the virus and worth investigation. Conclusion The SNaPshot® Multiplex System is a accurate system that may be applied to multiple applications including viral strain anylsis and here we can show how it may be effectively used for SARS-Cov-2 gentotyping. Using this technique, it is possible to analyze more than 50 SNPs distributed throughout the genome in a single multiplex reaction, making it an advantage when compared with traditional sequencing reaction. This is a important study that shows that this technique may be used in multiple field applictions.

A-163 Stability of apixaban in Li-heparin plasma stored refrigerated over mint green-top tube separator gel

Abstract Background Apixaban is a factor Xa inhibitor used as a direct oral anticoagulant (DOAC). The laboratory was asked to provide measurements of plasma apixaban in the context of studies regarding interaction of apixaban with other therapeutic drugs. One study involved isolation of plasma from to-be-discarded specimens, where such specimens were those originally collected in mint green-top tubes (Li-heparin tubes containing plasma separator gel), and that had been centrifuged and stored refrigerated for 5 days. Whereas apixaban concentration is known to be stable in refrigerated plasma specimens for up to two weeks, stability of apixaban concentration in plasma stored refrigerated for prolonged periods over plasma separator gel was unknown. Our objective was to examine stability of apixaban in plasma stored refrigerated for 5 days over plasma separator gel. Methods Pharmacy records were reviewed to identify inpatients who were prescribed apixaban, and for whom a mint green-top tube specimen (Becton-Dickinson BD Vacutainer PST Gel and Lithium Heparin, #367961) had been drawn for routine chemistry tests. From such specimens, 500 uL aliquots of plasma were obtained on day 1 (A) and day 5 (B) of storage under refrigeration (4 °C). Apixaban concentrations for A and B were measured by LC-MS/MS and compared. Additionally, short term (1 day) stability of apixaban was examined by comparison of replicate measurements of apixaban in a single spiked pooled purple-top plasma specimen (Becton-Dickinson BD Vacutainer K2 EDTA, #367856), with (C) or without (D) exposure to mint green-top tube separator gel for a 24h refrigeration period. Data analyses were conducted using R (version 4.0.5). Results A total of 21 specimen pairs (A, B) were examined. [Apixaban] ranged from 30-1000 ng/mL. The ratio B/A was 1.007 ± 0.034, consistent with no effect on plasma [apixaban] of refrigerated storage over separator gel between day 1 and day 5 (by paired t-test: p>0.8). Experiments for replicates of (C, D) specimens (n = 4, average [apixaban] = 410 ng/mL) gave D/C ratio of 1.009 ± 0.019, consistent with no effect on plasma [apixaban] of short term (1 day) refrigerated storage of plasma over separator gel (by paired t-test: p>0.4). Conclusions For plasma specimens collected and centrifuged in mint-green plasma separator tubes, there was no effect on plasma [apixaban] of refrigerated storage over separator gel for periods of 0-5 days.

Isolation and Genetic Characterization of Genotype VII Velogenic Pathotype Newcastle Disease Virus from Commercial Chicken Farms in Central Ethiopia, Distinct from the Local Vaccine Strains.

Newcastle disease (ND) is caused by virulent strains of avian paramyxovirus type 1, also known as Newcastle disease virus (NDV). Despite vaccination, the frequency of reported outbreaks in Ethiopia has increased. From January to June 2022, an active outbreak investigation was conducted in six commercial chicken farms across areas of central Ethiopia to identify the circulating NDV strains. Thirty pooled tissue specimens were collected from chickens suspected of being infected with NDV. A questionnaire survey of farm owners and veterinarians was also carried out to collect information on the farms and the outbreak status. NDV was isolated using specific-pathogen-free (SPF)-embryonated chicken eggs and detected using haemagglutination and the reverse transcriptase-polymerase chain reaction (RT-PCR). The genotype and virulence of field NDV isolates were determined using phylogenetic analysis of fusion (F) protein gene sequences and the mean death time (MDT) test in SPF-embryonated chicken eggs. The questionnaire results revealed that ND caused morbidity (23.1%), mortality (16.3%), case fatality (70.8%), and significant economic losses. Eleven of thirty tissue specimens tested positive for NDV using haemagglutination and RT-PCR. The MDT testing and sequence analysis revealed the presence of virulent NDV classified as genotype VII of class II velogenic pathotype and distinct from locally used vaccine strains (genotype II). The amino acid sequences of the current virulent NDV fusion protein cleavage site motif revealed 112RRQKR↓F117, unlike the locally used avirulent vaccine strains (112GRQGR↓L117). The epidemiological data, MDT results, cleavage site sequence, and phylogenetic analysis all indicated that the present NDV isolates were virulent. The four NDV sequences were deposited in GenBank with accession numbers F gene (PP726912-15) and M gene (PP726916-19). The genetic difference between avirulent vaccine strains and circulating virulent NDV could explain the low level of protection provided by locally used vaccines. Further studies are needed to better understand the circulating NDV genotypes in different production systems.

Expanding molecular diagnostic coverage for tuberculosis by combining computer-aided chest radiography and sputum specimen pooling: a modeling study from four high-burden countries

BackgroundIn 2022, fewer than half of persons with tuberculosis (TB) had access to molecular diagnostic tests for TB due to their high costs. Studies have found that the use of artificial intelligence (AI) software for chest X-ray (CXR) interpretation and sputum specimen pooling can each reduce the cost of testing. We modeled the combination of both strategies to estimate potential savings in consumables that could be used to expand access to molecular diagnostics.MethodsWe obtained Xpert testing and positivity data segmented into deciles by AI probability scores for TB from the community- and healthcare facility-based active case finding conducted in Bangladesh, Nigeria, Viet Nam, and Zambia. AI scores in the model were based on CAD4TB version 7 (Zambia) and qXR (all other countries). We modeled four ordinal screening and testing approaches involving AI-aided CXR interpretation to indicate individual and pooled testing. Setting a false negative rate of 5%, for each approach we calculated additional and cumulative savings over the baseline of universal Xpert testing, as well as the theoretical expansion in diagnostic coverage.ResultsIn each country, the optimal screening and testing approach was to use AI to rule out testing in deciles with low AI scores and to guide pooled vs individual testing in persons with moderate and high AI scores, respectively. This approach yielded cumulative savings in Xpert tests over baseline ranging from 50.8% in Zambia to 57.5% in Nigeria and 61.5% in Bangladesh and Viet Nam. Using these savings, diagnostic coverage theoretically could be expanded by 34% to 160% across the different approaches and countries.ConclusionsUsing AI software data generated during CXR interpretation to inform a differentiated pooled testing strategy may optimize TB diagnostic test use, and could extend molecular tests to more people who need them. The optimal AI thresholds and pooled testing strategy varied across countries, which suggests that bespoke screening and testing approaches may be needed for differing populations and settings.

Evaluations of modes of pooling specimens for COVID-19 screened by quantitative PCR and droplet digital PCR

Though pooling samples for SARS-CoV-2 detection has effectively met the need for rapid diagnostic and screening tests, many factors can influence the sensitivity of a pooled test. In this study, we conducted a simulation experiment to evaluate modes of pooling specimens and aimed at formulating an optimal pooling strategy. We focussed on the type of swab, their solvent adsorption ability, pool size, pooling volume, and different factors affecting the quality of preserving RNA by different virus solutions. Both quantitative PCR and digital PCR were used to evaluate the sampling performance. In addition, we determined the detection limit by sampling which is simulated from the virus of different titers and evaluated the effect of sample-storage conditions by determining the viral load after storage. We found that flocked swabs were better than fibre swabs. The RNA-preserving ability of the non-inactivating virus solution was slightly better than that of the inactivating virus solution. The optimal pooling strategy was a pool size of 10 samples in a total volume of 9 mL. Storing the collected samples at 4 °C or 25 °C for up to 48 h had little effect on the detection sensitivity. Further, we observed that our optimal pooling strategy performed equally well as the single-tube test did. In clinical applications, we recommend adopting this pooling strategy for low-risk populations to improve screening efficiency and shape future strategies for detecting and managing other respiratory pathogens, thus contributing to preparedness for future public health challenges.

Assessing Accuracy and Precision of Hemoglobin Determination in Venous, Capillary Pool, and Single-Drop Capillary Blood Specimens Using three Different HemoCue® Hb Models: The Multicountry Hemoglobin Measurement (HEME) Study

BackgroundAnemia prevalence estimates reported in population surveys can vary based on the blood specimen source (capillary or venous) and analytic device (hematology autoanalyzers or portable hemoglobinometers) used for hemoglobin (Hb) determination. ObjectivesThis study aimed to compare accuracy and precision of Hb measurement in three blood specimen types on three models of hemoglobinometers against the results from venous blood from the same individuals measured on automated analyzers (AAs). MethodsThis multisite (Cambodia, Ethiopia, Guatemala, Lebanon, Nigeria, and Tanzania) study assessed Hb measurements in paired venous and capillary blood specimens from apparently healthy women (aged 15–49 y) and children (aged 12–59 mo) using three HemoCue® Hb models (201+, 301, and 801). Measurements were compared against reference values: venous blood in hematology AA and adjusted via regression calibration or mean difference in HemoCue® Hb. Venous, capillary pool, and single-drop capillary blood specimens were assessed for accuracy and precision. ResultsVenous blood measured using HemoCue® Hb 301 exhibited a positive mean error, whereas responses in HemoCue® Hb 201+ and 801 were nondirectional compared with the reference. Adjustment with the reference harmonized mean errors for all devices across study sites to <1.0 g/L using venous blood. Precision was highest for venous blood (±5–16 g/L) in all sites, lowest for single-drop capillary (±9–37 g/L), and intermediate (±9–28 g/L) for capillary pool blood specimen. Imprecision differed across sites, especially with both capillary blood specimens, suggesting different levels of personnel skills. ConclusionsFindings suggest that venous blood is needed for accurate and precise Hb determination. Single-drop capillary blood use should be discouraged owing to high measurement variability. Further research should evaluate the viability and reliability of capillary pool blood for this purpose. Accuracy of HemoCue® Hb devices can be improved via standardization against results from venous blood assessed using AA.

Strengthening Bordetella pertussis genomic surveillance by direct sequencing of residual positive specimens.

Whole-genome sequencing (WGS) of microbial pathogens recovered from patients with infectious disease facilitates high-resolution strain characterization and molecular epidemiology. However, increasing reliance on culture-independent methods to diagnose infectious diseases has resulted in few isolates available for WGS. Here, we report a novel culture-independent approach to genome characterization of Bordetella pertussis, the causative agent of pertussis and a paradigm for insufficient genomic surveillance due to limited culture of clinical isolates. Sequencing libraries constructed directly from residual pertussis-positive diagnostic nasopharyngeal specimens were hybridized with biotinylated RNA "baits" targeting B. pertussis fragments within complex mixtures that contained high concentrations of host and microbial background DNA. Recovery of B. pertussis genome sequence data was evaluated with mock and pooled negative clinical specimens spiked with reducing concentrations of either purified DNA or inactivated cells. Targeted enrichment increased the yield of B. pertussis sequencing reads up to 90% while simultaneously decreasing host reads to less than 10%. Filtered sequencing reads provided sufficient genome coverage to perform characterization via whole-genome single nucleotide polymorphisms and whole-genome multilocus sequencing typing. Moreover, these data were concordant with sequenced isolates recovered from the same specimens such that phylogenetic reconstructions from either consistently clustered the same putatively linked cases. The optimized protocol is suitable for nasopharyngeal specimens with diagnostic IS481 Ct < 35 and >10 ng DNA. Routine implementation of these methods could strengthen surveillance and study of pertussis resurgence by capturing additional cases with genomic characterization.

Accurate inference for the Youden index and its associated cutoff point based on the gamma and inverse Gaussian distributed assumption

&lt;p&gt;The Youden index is often used to measure the effectiveness of biomarkers and aids to find the optimal cutoff point. Since pooled specimens have been shown to be an effective cost-cutting technique, we proposed the exact inferential procedures for the Youden index and its associated cutoff point based on the pooled specimens under the gamma or the inverse Gaussian assumption. The generalized confidence intervals (GCIs) were proposed for the Youden index and its associated cutoff point. Monte Carlo simulations were used to assess the performance of the proposed GCIs. The simulation results show that the proposed GCIs outperformed existing methods such as the bootstrap-$ p $ CIs in terms of the coverage probability. Finally, the proposed procedures were illustrated by an example.&lt;/p&gt;

Continuous clinicopathological and molecular recognition of very virulent infectious bursal disease virus in commercial broiler chickens

Gumboro virus is one of the most dangerous immunosuppressant viruses that infect chickens and causes massive financial losses around the world. The purpose of the current study is to conduct a molecular survey of chicken farms for the infectious bursal disease virus (IBDV). On the basis of postmortem (PM) lesions, 125 bursal samples from 25 farms were collected from clinically diseased commercial chicken farms with increased mortality and suspected Gumboro virus infection. Pooled bursal samples from suspected IBD-vaccinated flocks were tested for IBDV by real-time polymerase chain reaction (RT-PCR). Fifteen out of 25 pooled specimens were found positive for IBDV, with a 60% detection rate, and confirmed positive for very virulent IBDV (vvIBDV). Nucleotide phylogenetic analysis of VP1 and VP2 genes was employed to compare the five chosen isolates with strains identified in various localities in 2022. The VP1 and VP2 genes are allotted into groups (I, II). The strains in our study were related to group II and it acquired a new mutation in the VP2 gene that clustered it into new subgroup B. By mutation analysis, the VP2 gene of all strains had a characteristic mutation to vvIBDV. It acquired new mutations in HVRs contrasted with HK64 in Y220F, A222T/V in all strains in our study, and Q221K that was found in IBD-EGY-AH5 and AH2 in the major hydrophilic region peak A in the loop PBC in addition to G254S in all strains in our study and Q249k that found in IBD-EGY-AH1 and AH3 in the minor hydrophilic region 1 in the loop PDE. These mutations are important in the virulency and antigenicity of the virus. The VP1 had 242E and 390L were characteristic of vvIBDV and KpnI restriction enzyme (777GGTAC/C782) in addition to a new mutation (F243Y and N383H) in tIBD-EGY-AH1 and AH4 strains. According to the current study, the strains were distinct from the vaccinal strain; they could be responsible for the most recent IBDV outbreaks observed in flocks instead of received vaccinations. The current study highlighted the importance of regular IBD monitoring to maintain up to date on the type of challenge viruses and the efficacy of commercial vaccinations.

Pooled Pharyngeal, Rectal, and Urine Specimens for the Point-of-Care Detection of Chlamydia trachomatis and Neisseria gonorrhoeae by Lay Providers in Key Population-Led Health Services in Thailand.

Routine testing for Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) in people with heightened risk is lacking in Thailand. This study aimed to assess the performance of the Cepheid Xpert CT/NG assay, conducted by key population (KP) lay providers, for CT and NG detection on single-site and pooled specimens from the pharynx, rectum, and urine. Between August and October 2019, 188 men who have sex with men and 11 transgender women were enrolled. Participants collected urine specimens while trained KP lay providers obtained pharyngeal and rectal swabs. Compared to single-site testing with the Abbott RealTime CT/NG assay by medical technologists, the Xpert assay missed one pharyngeal NG infection out of 199 single-site specimens, giving a 93.3% sensitivity for pharyngeal NG and one missed pharyngeal NG infection out of fifty pooled specimens, giving an 88.9% sensitivity for pharyngeal NG. There was no discrepancy between the two assays for CT detection. The Cohen's Kappa coefficient of pooled specimen testing by the Xpert was 0.93 for NG and 1 for CT when compared to single-site testing by Abbott. Implementing pooled specimen testing by KP lay providers can be a cost-saving strategy to enhance the uptake of CT/NG services for populations facing increased risk.

Metagenomics analysis reveals presence of the Merida-like virus in Georgia.

Arbovirus surveillance is fundamental for the discovery of novel viruses and prevention of febrile vector-borne illnesses. Vector-borne pathogens can rapidly expand and adapt in new geographic and environmental conditions. In this study, metagenomic surveillance was conducted to identify novel viruses in the Country of Georgia. A total of 521 mosquitoes were captured near a military training facility and pooled from species Culex pipiens (Linnaeus) (87%) and Aedes albopictus (Skuse) (13%). We decided to further analyze the Culex pipiens mosquitoes, due to the more extensive number of samples collected. Our approach was to utilize an unbiased total RNA-seq for pathogen discovery in order to explore the mosquito virome. The viral reads from this analysis were mostly aligned to Insect-specific viruses from two main families, the Iflaviridae; a positive-stranded RNA virus and the Rhabdoviridae; a negative- and single-stranded RNA virus. Our pathogen discovery analysis revealed viral reads aligning to the Merida-like virus Turkey (MERDLVT) strain among the Rhabdoviridae. To further validate this result, we conducted a BLAST sequence comparison analysis of our samples with the MERDLVT strain. Our positive samples aligned to the MERDLVT strain with 96-100% sequence identity and 99.7-100% sequence coverage. A bootstrapped maximum-likelihood phylogenetic tree was used to evaluate the evolutionary relationships among these positive pooled specimens with the (MERDLVT) strain. The Georgia samples clustered most closely with two strains from Turkey, the Merida-like virus KE-2017a isolate 139-1-21 and the Merida-like virus Turkey isolate P431. Collectively, these results show the presence of the MERDLVT strain in Georgia.

Reexamining the Minimum Sweat Rate Requirement for Sweat Chloride Testing.

Guidelines for sweat chloride testing endorse a minimum sweat rate for reporting results. Bilateral sweat collection is recommended, but if both sites fail to meet the minimum rate (quantity not sufficient, QNS), the test should be repeated. In this study, we examine the correlation between sweat rate and sweat chloride concentration ([Cl-]), assess the accuracy of specimens collected at suboptimal rates, and investigate the use of pooled bilateral specimens for chloride measurement. Pearson correlation was employed to analyze the relationship between sweat rate and chloride concentration, [Cl-], in 674 macroduct collections. Weighted kappa was evaluated to determine cystic fibrosis (CF) diagnostic classification concordance for 18 tests with paired arms above vs below the minimum sweat rate. Deming regression was applied to compare [Cl-] from pooled bilateral specimens vs neat specimens in 27 collections with residual volume available after clinical testing. Pearson correlation of sweat rate vs [Cl-] was minimal (r = -0.0735) across specimens with varying rates and [Cl-]. There was substantial agreement in CF diagnostic classification between arms for bilateral collections with discordant sweat rates. Regression analysis of [Cl-] in pooled vs nonpooled specimens revealed a slope of 0.984 and an intercept of 0.796. Negligible correlation of sweat rate and [Cl-] suggests the minimum sweat rate for macroduct collectors may be overly stringent. Reporting of [Cl-] in specimens with ≥10 µL (rate ≥0.3 µL/min) may reduce QNS rates without compromising diagnostic accuracy. Preliminary data suggests pooling of bilateral collections may be a feasible option to achieve the required volume for testing.

A-332 Sweat rate does not influence sweat chloride concentration

Abstract Introduction The recommendations for sweat chloride ([Cl-]) testing from the Clinical and Laboratory Standards Institute (CLSI C34-A4 2019) endorse a minimum sweat rate of 1g/m2/min to validate efficient sweat stimulation. For labs that use macroduct ® collection devices, this rate corresponds to a sweat volume ≥15 µL collected within a 30-minute period. Bilateral collection and testing is also advised to improve the odds of obtaining a sufficient specimen from at least 1 site. When both sites fail to meet the minimum rate, the test is invalid (QNS) and should be repeated, potentially delaying Cystic Fibrosis (CF) diagnosis. In this study, we evaluate the premise for the minimum sweat rate by investigating the relationship between sweat rate and [Cl-]. In a sub-analysis, residual sweat available after clinical testing is used to examine the effect of specimen pooling on [Cl-]. This study has the potential to inform best practices for future sweat testing guidelines, decrease QNS rates, and reduce time to CF diagnosis. Methods Thirteen months of data from sweat tests performed at St. Louis Children's Hospital were reviewed. All collections were performed using macroduct devices following CLSI 34-A4 guidelines (results from QNS specimens were not reported clinically). Pearson correlation was calculated to examine the relationship between sweat rate and [Cl-] (n = 354). Deming regression was applied to evaluate the effect of specimen pooling, versus results from individual sites, in 19 collections with residual volume after clinical testing. Results Figure 1 Conclusion Negligible correlation between rate and [Cl-] across a wide range of values suggests the current minimum volume for macroduct collections (15 µL) may be overly stringent. Reporting of [Cl-] in specimens with ≥10 µL sweat may decrease QNS rates without compromising accuracy. Preliminary data suggests pooling of bilateral collections may be a feasible option to achieve the minimum volume required for testing (10 µL).

Effect of swab pooling on the Accula point-of-care RT-PCR for SARS-CoV-2 detection.

Swab pooling may allow for more efficient use of point-of-care assays for SARS-CoV-2 detection in settings where widespread testing is warranted, but the effects of pooling on assay performance are not well described. We tested the Thermo-Fisher Accula rapid point-of-care RT-PCR platform with contrived pooled nasal swab specimens. We observed a higher limit of detection of 3,750 copies/swab in pooled specimens compared to 2,250 copies/swab in individual specimens. Assay performance appeared worse in a specimen with visible nasal mucous and debris, although performance was improved when using a standard laboratory mechanical pipette compared to the transfer pipette included in the assay kit. Clinicians and public health officials overseeing mass testing efforts must understand limitations and benefits of swab or sample pooling, including reduced assay performance from pooled specimens. We conclude that the Accula RT-PCR platform remains an attractive candidate assay for pooling strategies owing to the superior analytical sensitivity compared to most home use and point-of-care tests despite the inhibitory effects of pooled specimens we characterized.

Increasing SARS-CoV-2 testing capacity through specimen pooling: An acute care center experience.

Innovation in laboratory testing algorithms to address seemingly uncontrollable global supply chain shortages in plastics and other consumables during emergencies such as the current COVID-19 pandemic have been urgently needed. We report our experience with specimen pooling on SARS-CoV-2 testing in an acute care hospital microbiology laboratory during a high testing demand period that exceeded available processing capacity. A fully automated four-in-one pooling algorithm was designed and validated. Correlation and agreement were calculated. A custom Microsoft Excel tool was designed for use by the technologists to aid interpretation, verification and result entry. Cost-per-test impact for pooling was measured in reference to the consumable cost and was denoted as the percentage reduction of cost versus the baseline cost-per-test of testing specimens individually. Validation showed a strong correlation between the signals observed when testing specimens individually versus those that were pooled. Average crossing point difference was 1.352 cycles (95% confidence interval of -0.235 and 2.940). Overall agreement observed between individually and pooled tested specimens was 96.8%. Stratified agreement showed an expected decreased performance of pooling for weakly positive specimens dropping below 60% after a crossing point of 35. Post-implementation data showed the consumable cost-savings achieved through this algorithm was 85.5% after 8 months, creating both testing and resource capacity. Pooling is an effective method to be used for SARS-CoV-2 testing during the current pandemic to address resource shortages and provide quick turnaround times for high test volumes without compromising performance.

Evaluation of SARS-CoV-2 Detection Systems Using Clinical Samples and Standard Material: A Comparative Study.

Due to the decreasing trends in daily confirmed COVID-19 cases and daily confirmed tests, there is a need for a new testing system capable of quickly and efficiently testing small amounts of samples. Therefore, we compared and evaluated the testing performance of the Aptima SARS-CoV-2 assay, an automated testing system that allows continuous loading of samples, and the Real-Q Direct SARS-CoV-2 detection kit that is currently being used in our laboratory. We compared the results of the two testing systems using 259 residual individual nasopharyngeal specimens and 91 residual pooled nasopharyngeal specimens that were submitted for COVID-19 testing in January and February 2023. The 95% limit of detection (LoD) for the Aptima SARS-CoV-2 assay determined using reference material for SARS-CoV-2 nucleic acid was confirmed to be 17.793 copies/mL, while the LoD for the Real-Q Direct SARS-CoV-2 detection kit was determined to be 131.842 copies/mL for the RdRP gene and 241.77 copies/mL for the E gene. The comparative study using clinical specimens showed almost perfect agreement. Our data showed that the Aptima SARS-CoV-2 assay has a very low LoD. In addition, the Aptima SARS-CoV-2 assay and Real-Q Direct detection kit have comparable clinical performance for SARS-CoV-2 for individual and pooled samples.

Lack of interference of metronidazole with Roche cobas c502 and c702 chemistry tests

ObjectiveThe antibiotic metronidazole has been suggested to absorb light at a wavelength range commonly used in spectrophotometric assays. We sought to determine if any of the spectrophotometric assays used in our core laboratory would be susceptible to clinically significant interference from metronidazole in blood-based patient specimens. MethodsFollowing characterization of the absorbance spectrum for metronidazole, spectrophotometric assays involving either main or subtraction wavelengths that might be susceptible to interference from metronidazole were identified. A total of 24 chemistry tests performed on Roche cobas c502 and/or c702 instruments were evaluated for interference from metronidazole. For each assay, two pools of leftover patient serum, plasma, or whole blood specimens containing the analyte of interest at clinically relevant concentrations were prepared. Each pool was spiked with metronidazole at a final concentration of 200 mg/L (1169 µmol/L) or 10 mg/L (58 µmol/L) or the same volume of water as a control, with triplicate samples for each group. The difference in the measured analyte concentration between experimental and control groups was then compared against the total allowable error for each assay to determine whether clinically significant interference had occurred. ResultsThere was no significant interference observed with Roche chemistry tests due to the presence of metronidazole. ConclusionThis study provides reassurance that metronidazole is not interfering with the chemistry assays used in our core laboratory. Interference from metronidazole may be a historical problem and current spectrophotometric assays may not be susceptible due to improvements in assay design.

Estimating the prevalence of two or more diseases using outcomes from multiplex group testing.

When screening a population for infectious diseases, pooling individual specimens (e.g., blood, swabs, urine, etc.) can provide enormous cost savings when compared to testing specimens individually. In the biostatistics literature, testing pools of specimens is commonly known as group testing or pooled testing. Although estimating a population-level prevalence with group testing data has received a large amount of attention, most of this work has focused on applications involving a single disease, such as human immunodeficiency virus. Modern methods of screening now involve testing pools and individuals for multiple diseases simultaneously through the use of multiplex assays. Hou etal. (2017, Biometrics, 73, 656-665) and Hou etal. (2020, Biostatistics, 21, 417-431) recently proposed group testing protocols for multiplex assays and derived relevant case identification characteristics, including the expected number of tests and those which quantify classification accuracy. In this article, we describe Bayesian methods to estimate population-level disease probabilities from implementing these protocols or any other multiplex group testing protocol which might be carried out in practice. Our estimation methods can be used with multiplex assays for two or more diseases while incorporating the possibility of test misclassification for each disease. We use chlamydia and gonorrhea testing data collected at the State Hygienic Laboratory at the University of Iowa to illustrate our work. We also provide an online R resource practitioners can use to implement the methods in thisarticle.

Wastewater monitoring of SARS-CoV-2 RNA at K-12 schools: comparison to pooled clinical testing data.

Wastewater measurements of SARS-CoV-2 RNA have been extensively used to supplement clinical data on COVID-19. Most examples in the literature that describe wastewater monitoring for SARS-CoV-2 RNA use samples from wastewater treatment plants and individual buildings that serve as the primary residence of community members. However, wastewater surveillance can be an attractive supplement to clinical testing in K-12 schools where individuals only spend a portion of their time but interact with others in close proximity, increasing risk of potential transmission of disease. Wastewater samples were collected from two K-12 schools in California and divided into solid and liquid fractions to be processed for detection of SARS-CoV-2. The resulting detection rate in each wastewater fraction was compared to each other and the detection rate in pooled clinical specimens. Most wastewater samples were positive for SARS-CoV-2 RNA when clinical testing was positive (75% for solid samples and 100% for liquid samples). Wastewater samples continued to test positive for SARS-CoV-2 RNA when clinical testing was negative or in absence of clinical testing (83% for both solid and liquid samples), indicating presence of infected individuals in the schools. Wastewater solids had a higher concentration of SARS-CoV-2 than wastewater liquids on an equivalent mass basis by three orders of magnitude.

Using pooled urogenital, anorectal and oropharyngeal specimens to detect Chlamydia trachomatis and Neisseria gonorrhoeae among men who have sex with men in China: a multisite diagnostic accuracy study

ObjectivesScreening for Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) at both urogenital and extragenital sites has been recommended in many countries. Testing of the infections using pooled specimens from urogenital...