High-throughput Molecular Testing Research Articles

Evaluating Malaria Rapid Diagnostic Tests and Microscopy for Detecting Plasmodium Infection and Status of Plasmodium falciparum Histidine-Rich Protein 2/3 Gene Deletions in Southeastern Nigeria.

Delays in malaria diagnosis increase treatment failures and deaths. In endemic regions, standard diagnostic methods are microscopy and malaria rapid diagnostic tests (mRDTs) detecting Plasmodium falciparum histidine-rich protein 2/3 (PFHRP2/PFHRP3), but gene deletions can allow certain parasites to remain undetected. We enlisted a cohort comprising 207 symptomatic individuals, encompassing both children and adults, at a hospital in Nnewi, Nigeria. The prevalence of parasites was determined using a highly sensitive, species-specific quantitative polymerase chain reaction (SS-qPCR). Within a subset of 132 participants, we assessed the sensitivity and specificity of microscopy and HRP2-mRDTs in comparison to SS-qPCR for the detection of P. falciparum. We also investigated the prevalence of pfhrp2/pfhrp3 gene deletions. Greater sensitivity was achieved with mRDTs (95%) compared with microscopy (77%). Also, mRDTs exhibited greater specificity (68%) than microscopy (44%). The positive predictive value of mRDTs (89%) surpassed that of microscopy (80%), suggesting a greater probability of accurately indicating the presence of infection. The negative predictive value of mRDTs (82%) was far greater than microscopy (39%). Of the 165 P. falciparum-positive samples screened for pfhrp2/pfhrp3 gene deletions, one gene deletion was detected in one sample. Regarding infection prevalence, 84% were positive for Plasmodium spp. (by reverse transcription [RT]-qPCR), with P. falciparum responsible for the majority (97%) of positive cases. Thus, exclusive reliance on microscopy in endemic areas may impede control efforts resulting from false negatives, underscoring the necessity for enhanced training and advocating for high-throughput molecular testing such as RT-qPCR or qPCR at referral centers to address limitations.

Development and evaluation of the automated multipurpose molecular testing system PCRpack for high-throughput SARS-CoV-2 testing

ABSTRACT Increasing the reliable testing capacity for SARS-CoV-2 is important for the diagnosis and control of COVID-19. We developed an automated, customizable, easy-to-implement molecular testing system, named PCRpack, for the high-throughput testing of SARS-CoV-2. PCRpack includes a liquid handling instrument enclosed in a negative-pressure clean booth (Biomek i5), a laboratory information management system (SimpPCR), other equipment, and documents that are needed for testing operation. An in vitro diagnostic assay was employed to detect SARS-CoV-2. System performance was evaluated based on liquid handling accuracy and precision, analytical sensitivity, clinical diagnostic performance, and testing capacity per day. Clinical diagnostic performance was determined against the reference extraction-based reverse transcription-PCR assay using 3,965 upper respiratory samples. Analytical sensitivity analysis showed a lower limit of detection of 1,000 genome copies/mL of sample. The accuracy and precision of sample or reagent dispensing in PCRpack ranged from −2.24% to 0.73% and 0.83% to 4.52%, respectively. In the evaluation of clinical samples, PCRpack showed a positive percent agreement of 96.6% and a negative percent agreement of 100% compared with the reference assay. The average turnaround times per 94 samples and the maximum numbers of tests within an 8-hour shift of one operator were 2 hours and 28 minutes vs 2 hours and 1 minute and 564 vs 376 samples for PCRpack and the manual method, respectively. The developed PCRpack system shows high liquid handling and clinical diagnostic performance and is a promising testing system for increasing the SARS-CoV-2 testing capacity and testing future emerging pathogens. IMPORTANCE Accurate and fast molecular testing is important for the diagnosis and control of COVID-19. During patient surges in the COVID-19 pandemic, laboratories were challenged by a higher demand for molecular testing under skilled staff shortages. We developed an automated multipurpose molecular testing system, named PCRpack, for the rapid, high-throughput testing of infectious pathogens, including SARS-CoV-2. The system is provided in an all-in-one package, including a liquid handling instrument, a laboratory information management system, and other materials needed for testing operation; is highly customizable; and is easily implemented. PCRpack showed robust liquid handling performance, high clinical diagnostic performance, a shorter turn-around time with minimal hands-on time, and a high testing capacity. These features contribute to the rapid implementation of the high-performance and high-throughput molecular testing environment at any phase of the pandemic caused by SARS-CoV-2 or future emerging pathogens.

AIMedGraph: a comprehensive multi-relational knowledge graph for precision medicine.

The development of high-throughput molecular testing techniques has enabled the large-scale exploration of the underlying molecular causes of diseases and the development of targeted treatment for specific genetic alterations. However, knowledge to interpret the impact of genetic variants on disease or treatment is distributed in different databases, scientific literature studies and clinical guidelines. AIMedGraph was designed to comprehensively collect and interrogate standardized information about genes, genetic alterations and their therapeutic and diagnostic relevance and build a multi-relational, evidence-based knowledge graph. Graph database Neo4j was used to represent precision medicine knowledge as nodes and edges in AIMedGraph. Entities in the current release include 30 340 diseases/phenotypes, 26 140 genes, 187 541 genetic variants, 2821 drugs, 15 125 clinical trials and 797 911 supporting literature studies. Edges in this release cover 621 731 drug interactions, 9279 drug susceptibility impacts, 6330 pharmacogenomics effects, 30 339 variant pathogenicity and 1485 drug adverse reactions. The knowledge graph technique enables hidden knowledge inference and provides insight into potential disease or drug molecular mechanisms. Database URL: http://aimedgraph.tongshugene.net:8201.

Clinical evaluation of a fully automated and high-throughput molecular testing system for detection of influenza virus

IntroductionWe investigated the performance of the cobas® 6800 system and cobas SARS-CoV-2 & Influenza A/B, a fully automated molecular testing system for influenza viruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This enabled an assay in a batch of 96 samples in approximately 3 h.MethodsAn assay was performed using the cobas SARS-CoV-2 & Influenza A/B on the cobas 6800 system for samples collected in four facilities between November 2019 and March 2020 in our previous study. The results were compared with those obtained using the reference methods.ResultsOf the 127 samples analyzed, the cobas SARS-CoV-2 & Influenza A/B detected influenza A virus in 75 samples, of which 73 were positive using the reference methods. No false negative results were observed. The overall positive and negative percent agreement for influenza A virus detection were 100.0% and 96.3%, respectively. There were no positive results for the influenza B virus or SARS-CoV-2.ConclusionThe cobas 6800 system and cobas SARS-CoV-2 & Influenza A/B showed high accuracy for influenza A virus detection and can be useful for clinical laboratories, especially those that routinely assay many samples.

Rapid Adaptation of Established High-Throughput Molecular Testing Infrastructure for Monkeypox Virus Detection.

Beginning in May 2022, a rising number of monkeypox cases were reported in non–monkeypox-endemic countries in the Northern Hemisphere. We adapted 2 published quantitative PCRs for use as a dual-target monkeypox virus test on widely used automated high-throughput PCR systems. We determined analytic performance by serial dilutions of monkeypox virus reference material, which we quantified by digital PCR. We found the lower limit of detection for the combined assays was 4.795 (95% CI 3.6–8.6) copies/mL. We compared clinical performance against a commercial manual orthopoxvirus research use only PCR kit by using clinical remnant swab samples. Our assay showed 100% positive (n = 11) and 100% negative (n = 56) agreement. Timely and scalable PCR tests are crucial for limiting further spread of monkeypox. The assay we provide streamlines high-throughput molecular testing for monkeypox virus on existing broadly established platforms used for SARS-CoV-2 diagnostic testing.

Supporting scale-up of COVID-19 RT-PCR testing processes with discrete event simulation.

Testing is critical to mitigating the COVID-19 pandemic, but testing capacity has fallen short of the need in the United States and elsewhere, and long wait times have impeded rapid isolation of cases. Operational challenges such as supply problems and personnel shortages have led to these bottlenecks and inhibited the scale-up of testing to needed levels. This paper uses operational simulations to facilitate rapid scale-up of testing capacity during this public health emergency. Specifically, discrete event simulation models were developed to represent the RT-PCR testing process in a large University of Maryland testing center, which retrofitted high-throughput molecular testing capacity to meet pandemic demands in a partnership with the State of Maryland. The simulation models support analyses that identify process steps which create bottlenecks, and evaluate “what-if” scenarios for process changes that could expand testing capacity. This enables virtual experimentation to understand the trade-offs associated with different interventions that increase testing capacity, allowing the identification of solutions that have high leverage at a feasible and acceptable cost. For example, using a virucidal collection medium which enables safe discarding of swabs at the point of collection removed a time-consuming “deswabbing” step (a primary bottleneck in this laboratory) and nearly doubled the testing capacity. The models are also used to estimate the impact of demand variability on laboratory performance and the minimum equipment and personnel required to meet various target capacities, assisting in scale-up for any laboratories following the same process steps. In sum, the results demonstrate that by using simulation modeling of the operations of SARS-CoV-2 RT-PCR testing, preparedness planners are able to identify high-leverage process changes to increase testing capacity.

Abstract P21: Saliva as a testing specimen with or without pooling for SARS-CoV-2 detection by multiplex RT-PCR test

Abstract Objectives Sensitive and high throughput molecular testing availability is essential during the COVID-19 pandemic. The vast majority of the SARS-CoV-2 molecular assays use nasopharyngeal or oropharyngeal swab specimens collected from suspected individuals. However, collecting these specimens has apparent drawbacks, including discomfort to patients and exposure risk to healthcare workers. Methods We developed and validated of QuantiVirus™ SARS-CoV-2 multiplex test using saliva as the testing specimens with pooling. Results The analytical sensitivity (LOD) was confirmed to be 100-200 copies/mL. For clinical evaluation, 85 known positive and 90 knowns negative NPS specimens were showed a positive predictive agreement of 100% and a negative predictive agreement of 98.9%. Twenty paired NPS and saliva samples were tested and showed overall 80% concordance rate without significant difference between NPS and saliva specimens by Wilcoxon signed-rank test (p=0.13). On a large scale of saliva-based population screening, the positive test rate was 1.79% among 389 saliva specimens. Furthermore, saliva sample pooling up to 6 samples for SARS-CoV-2 detection is feasible with sensitivity of 94.8% and specificity of 100%. Conclusions These results demonstrated that the clinical performance of saliva-based testing is comparable to that of NPS-based testing, and that pooling of saliva specimens for SARS-CoV-2 detection is feasible. Citation Format: Qing Sun, Jonathan Li, Hui Ren, Larry Pastor, Yulia Loginova, Joseph Wong, Zhao Zhang, Aiguo Zhang, Chuanyi Mark Lu, Michael Sha. Saliva as a testing specimen with or without pooling for SARS-CoV-2 detection by multiplex RT-PCR test [abstract]. In: Proceedings of the AACR Virtual Meeting: COVID-19 and Cancer; 2021 Feb 3-5. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(6_Suppl):Abstract nr P21.

Whole Locus Sequencing Identifies a Prevalent Founder Deep Intronic RPGRIP1 Pathologic Variant in the French Leber Congenital Amaurosis Cohort.

Leber congenital amaurosis (LCA) encompasses the earliest and most severe retinal dystrophies and can occur as a non-syndromic or a syndromic disease. Molecular diagnosis in LCA is of particular importance in clinical decision-making and patient care since it can provide ocular and extraocular prognostics and identify patients eligible to develop gene-specific therapies. Routine high-throughput molecular testing in LCA yields 70%–80% of genetic diagnosis. In this study, we aimed to investigate the non-coding regions of one non-syndromic LCA gene, RPGRIP1, in a series of six families displaying one single disease allele after a gene-panel screening of 722 LCA families which identified 26 biallelic RPGRIP1 families. Using trio-based high-throughput whole locus sequencing (WLS) for second disease alleles, we identified a founder deep intronic mutation (NM_020366.3:c.1468-128T>G) in 3/6 families. We employed Sanger sequencing to search for the pathologic variant in unresolved LCA cases (106/722) and identified three additional families (two homozygous and one compound heterozygous with the NM_020366.3:c.930+77A>G deep intronic change). This makes the c.1468-128T>G the most frequent RPGRIP1 disease allele (8/60, 13%) in our cohort. Studying patient lymphoblasts, we show that the pathologic variant creates a donor splice-site and leads to the insertion of the pseudo-exon in the mRNA, which we were able to hamper using splice-switching antisense oligonucleotides (AONs), paving the way to therapies.

Group Testing for Severe Acute Respiratory Syndrome- Coronavirus 2 to Enable Rapid Scale-up of Testing and Real-Time Surveillance of Incidence.

High-throughput molecular testing for severe acute respiratory syndrome–coronavirus 2 (SARS-CoV-2) may be enabled by group testing in which pools of specimens are screened, and individual specimens tested only after a pool tests positive. Several laboratories have recently published examples of pooling strategies applied to SARS-CoV-2 specimens, but overall guidance on efficient pooling strategies is lacking. Therefore we developed a model of the efficiency and accuracy of specimen pooling algorithms based on available data on SAR-CoV-2 viral dynamics. For a fixed number of tests, we estimate that programs using group testing could screen 2–20 times as many specimens compared with individual testing, increase the total number of true positive infections identified, and improve the positive predictive value of results. We compare outcomes that may be expected in different testing situations and provide general recommendations for group testing implementation. A free, publicly-available Web calculator is provided to help inform laboratory decisions on SARS-CoV-2 pooling algorithms.

Determining an optimal pool size for testing beef herds for Johne's disease in Australia.

Bovine Johne’s disease (JD) is a chronic debilitating disease affecting cattle breeds worldwide. Pooled faecal samples are routinely tested by culture to detect Mycobacterium avium subsp. paratuberculosis (Mptb) infection. More recently, a direct high throughput molecular test has been introduced in Australia for the detection of Mptb in faeces to circumvent the long culture times, however, the optimal pool size for beef cattle faeces is not known. This study aimed to determine the optimal pool size to achieve the highest test sensitivity and specificity for beef cattle. Individual archived faecal samples with low, medium and high quantities of Mptb (n = 30) were pooled with faecal samples from confirmed JD negative animals to create pool sizes of 5, 10, 15 and 20, to assess the diagnostic sensitivity relative to individual faecal qPCR. Samples from JD-free cattle (n = 10) were similarly evaluated for diagnostic specificity. Overall, 160 pools were created, with Mptb DNA extracted using magnetic bead isolation method prior to Mptb-specific IS900 quantitative PCR (qPCR). The pool size of 10 yielded the highest sensitivity 73% (95% CI: 54–88%), regardless of the quantity of Mptb DNA present in the faeces. There was no significant differences between the four different pool sizes for positive pool detection, however, there was statistical significance between low, medium and high quantities of Mptb. Diagnostic specificity was determined to be 100%. The increase in pool size greater than 10 increased the chances of PCR inhibition, which was successfully relieved with the process of DNA dilution. The results of this study demonstrate that the pool size of 10 performed optimally in the direct faecal qPCR. The results from this study can be applied in future simulation modelling studies to provide suggestions on the cost-effective testing for JD in beef cattle.

The development and application of nucleic acid detection techniques of infectious microorganisms in pediatrics

The advance of molecular diagnosis has evolved the field of detection of pathogens in pediatric disease towards the high-throughput ultra-sensitive molecular testing. This review provides an overview and comparison of current molecular tools on the market, including qPCR, multiplex PCR, digital PCR, isothermal amplification, and microfluidic chip, highlighting the future trends of integrated high-throughput molecular testing and automation. These evolutions will maximize the utility of limit patient samples from children and contribute significantly on the diagnosis of infectious disease. Key words: Communicable diseases; High-throughput nucleotide sequencing; Child

Histopathological and novel high-throughput molecular monitoring data from farmed salmon (Salmo salar and Oncorhynchus spp.) in British Columbia, Canada, from 2011–2013

The purpose of the study was to describe spatial and time-at-sea patterns of endemic and new infectious agents and histopathologically-identified lesions in dead-and-dying farmed non-native Atlantic (AS) and native Pacific (PS) salmon in BC, between 2011 and 2013. Novel high-throughput molecular testing and blinded histopathological examination of tissues were used to evaluate these patterns in fish-level analyses. Twenty-five of 45 infectious agents were detected, and 87% of 897 total fish tested had mixed detections, with up to nine agents in a single fish, and a higher agent diversity in PS than AS. Most frequently detected agents were the parasite Desmozoon lepeophtherii (D.lep) in farmed AS (88%), and the bacterium Candidatus Branchiomonas cysticola (Ca.B.cys) in farmed PS (89%). Overall, 92% of AS and 88% of PS had some histopathological change, mostly of mild to moderate severity, with renal interstitial hyperplasia as the most frequent change (AS: 33%; PS: 48%). Spatial patterns were statistically significant for five agents in PS versus the same agents in AS for Fish Health Zones in southwest Vancouver Island, Sunshine Coast, and Discovery Islands. Statistically significant time-at-sea patterns were detected for three agents each in AS (piscine reovirus, Piscirickettsia salmonis, and D.lep) and PS (Ca.B.cys, Renibacterium salmoninarum, and D.lep). Importantly, infectious salmon anemia virus, salmonid herpesvirus, salmon alphavirus, and infectious pancreatic necrosis virus were not detected. Furthermore, while the agents associated with proliferative gill disease (D.lep, Ca.B.cys, and gill chlamydia) were all detected, few fish showed evidence of lesions associated with this multifactorial disease. The majority of agents detected on BC salmon farms were known to be endemic, but new findings include the marine detections of some infectious agents reported to only cause freshwater or hatchery-based diseases (Flavobacterium psychrophilum and Ichthyophthirius multifiliis). The results of this descriptive study provide the proportion of positive test results in sampled dead-and-dying farmed AS and PS, as well as spatial and time-at-sea information on both agent and lesion detection, targeting areas of interest and concern to researchers, regulators, and aquaculture industry veterinarians for future population-based analyses.

Genomic Profiling of Metastatic Gastroenteropancreatic Neuroendocrine Tumor (GEP-NET) Patients in the Personalized-Medicine Era.

Background: We have conducted molecular profiling through a high-throughput molecular test as part of our clinical practice for patients with advanced gastrointestinal (GI) cancer or rare cancers including gastroenteropancreatic neuroendocrine tumors (GEP-NETs). Herein, we report on the molecular characterization of 14 metastatic GEP-NET patients.Methods: We conducted the Ion AmpliSeq Cancer Hotspot Panel v2 (detecting 2,855 oncogenic mutations in 50 commonly mutated genes) and nCounter Copy Number Variation Assay, which was designed with 21 genes based on available targeted agents, as a high throughput genomic platform in 14 patients with metastatic GEP-NETs.Results: Among the 14 GEP-NET patients analyzed in this study, 8 patients had grade III neuroendocrine carcinoma (NEC) and 6 had grade I/II NET. Primary sites included pancreas (n=3), small intestine and ascending colon (n=3), distal colon and rectum (n=5), and unknown primary origin (n=3). The most common metastatic site was the liver. Of 14 GEP-NET patients available for mutational profiling, 7 (50.0%) patients had one or more aberrations detected. Common aberrations were as follows: SMARCB1 mutation (n=2), TP53 mutation (n=2), STK11 mutation (n=1), RET mutation (n=1), and BRAF mutation (n=1). Gene amplification by nCounter was detected in only 1 patient, showing CCNE1 amplification, and this patient also had a TP53 mutation.Conclusions: This high throughput genomic test may be useful to identify new drug targets in metastatic GEP-NET patients. Currently, we plan to conduct further genomic analysis to develop predictive and prognostic biomarkers in a larger number of GEP-NET patients.

Reducing the pre-analysis time in the anatomicalpathological diagnosis of breast conditions, utopia or reality?

Surgical pathology diagnosis of a breast lesion is an important stage in the diagnostic process of breast diseases. Quality of the pre-analytical phase is essential to ensure reliable and reproducible diagnoses, and is based on neutral-buffered formalin fixation of tissue specimens. It is important to respect aminimum fixation timewithformalin inorder to guarantee the quality of subsequent morphological, immunohistochemical and cytogenetic studies. We present here an alternative to formalin fixation, based on tissue dehydration, enabling the shortening of technical stages and sign out. Furthermore, this method ensures better nucleic acid preservation, which opens the way for the development of high-throughput molecular testing in breast cancer.

Principles of Genetic Counseling

AbstractPurpose To present the genetic counselling needs of families with inherited eye disease and examine the ethical and emotional issues around genetic testingMethods The presentation will focus on the psychosocial impact of inherited eye disease and the dilemmas in counselling and testing using case discussions and data from qualitative studies.Results Genetic counselling is a process of communication to provide information about a genetic condition, inheritance and support decision making and adjustment in families. Genetic testing for inherited eye diseases is rapidly advancing with massive improvements in high throughput molecular testing. While this can allow accurate diagnosis and information, the issues of genetic heterogeneity, variable penetrance and overlapping phenotypes mean that the provision of accurate information particularly challenging for genetic eye disease. These scientific advances have also led to increased patient demand and expectations. Complex cases presenting to the multi‐disciplinary genetic eye clinic in Manchester will be presented to highlight the needs of families requesting genetic counselling including approaches to complex situations such as pre‐symptomatic testing, childhood testing and pre‐natal diagnosis. Qualitative data involving in‐depth interviews with families with inherited retinal dystrophy describes the burden of living with the risk of blindness and decision making around genetic tests. Research evidence also demonstrates that families feel their needs are not met by current services, suggesting a need for improvements in evidenced‐based practice.Conclusion Families with inherited eye disease have complex genetic counselling needs requiring multidisciplinary services to provide accurate diagnosis, information, genetic testing, decision‐making, support and follow‐up.

Molecular malaria epidemiology: mapping and burden estimates for the Democratic Republic of the Congo, 2007.

BackgroundEpidemiologic data on malaria are scant in many high-burden countries including the Democratic Republic of the Congo (DRC), which suffers the second-highest global burden of malaria. Malaria control efforts in regions with challenging infrastructure require reproducible and efficient surveillance. We employed new high-throughput molecular testing to characterize the state of malaria control in the DRC and estimate childhood mortality attributable to excess malaria transmission.Methods and FindingsThe Demographic and Health Survey was a cross-sectional, population-based cluster household survey of adults aged 15–59 years in 2007 employing structured questionnaires and dried blood spot collection. Parasitemia was detected by real-time PCR, and survey responses measured adoption of malaria control measures and under-5 health indices. The response rate was 99% at the household level, and 8,886 households were surveyed in 300 clusters; from 8,838 respondents molecular results were available. The overall prevalence of parasitemia was 33.5% (95% confidence interval [C.I.] 32–34.9); P. falciparum was the most prevalent species, either as monoinfection (90.4%; 95% C.I. 88.8–92.1) or combined with P. malariae (4.9%; 95% C.I. 3.7–5.9) or P. ovale (0.6%; 95% C.I. 0.1–0.9). Only 7.7% (95% CI 6.8–8.6) of households with children under 5 owned an insecticide-treated bednet (ITN), and only 6.8% (95% CI 6.1–7.5) of under-fives slept under an ITN the preceding night. The overall under-5 mortality rate was 147 deaths per 1,000 live births (95% C.I. 141–153) and between clusters was associated with increased P. falciparum prevalence; based on the population attributable fraction, 26,488 yearly under-5 deaths were attributable to excess malaria transmission.ConclusionsAdult P. falciparum prevalence is substantial in the DRC and is associated with under-5 mortality. Molecular testing offers a new, generalizable, and efficient approach to characterizing malaria endemicity in underserved countries.

Molecular Diagnostic Assays for Detection of Viral Respiratory Pathogens in Institutional Outbreaks

Outbreaks of viral respiratory disease in institutions may be associated with high morbidity and mortality, depending upon the viral etiology and the age and immune status of the affected patients. Control of outbreaks may include isolation and/or cohorting, and prohylaxis or treatment with specific antiviral agents may be indicated, all dependent upon the specific cause of the outbreak. Conventional methods of viral diagnosis detect only a limited number of the viruses that are known to cause outbreaks. The availability of sensitive and specific molecular assays has facilitated rapid diagnosis of a wider range of viruses from respiratory outbreaks. Molecular methods have distinct advantages over conventional methods, including the ability to rapidly develop assays for emerging viruses and new variants of existing viruses. In addition, molecular testing allows rapid detection of resistance to antiviral agents or mutations leading to increased virulence. However, high-throughput molecular testing requires batch processes that may compromise the ability to respond quickly to urgent testing demands.