PCR Cycles Research Articles

FRI372 Retinoic Acids Attenuate The Endometriotic Cell Proliferation Through A Cell-specific Receptor Isoform

Abstract Disclosure: M. Izawa: None. Y. Azuma: None. F. Taniguchi: None. [Background] Endometriosis has been accepted as an estrogen-dependent, proliferative and inflammatory disease for over two decades, and the hormonal therapy targeting the reduction of estrogen has been employed as the first choice of treatment to reduce the endometriosis-associated symptoms. However, the limitation of therapy is becoming evident. In the hope of overcoming the limitation, we focused on retinoids, which have been known to elicit antiproliferative activity in breast tumors. Retinoic acid receptor (RAR) expression has been known to correlate with estrogen receptor expression. [Objective] As a first step to test the antiproliferative activity of retinoids in endometriotic lesions, we examined the expression profile of the retinoic acid receptor (RAR) in endometriotic cells. We then examined the antiproliferative activity of retinoids and the effect of estrogen on their activity. [Patients] Institutional Review Boards approved this project. We obtained informed consent from all patients. The chocolate cyst lining in the ovaries of patients with endometriosis was the source of endometriotic tissue. [Methods] Endometriotic cells: Stromal cells were collected from endometriotic tissues and used as endometriotic cells throughout the experiment. RAR gene expression: Single-stranded cDNA was prepared and subjected to PCR. Primer sets were designed using sequences in the UCSF genome browser. At the end of PCR cycles, the reaction mixture was subjected to electrophoresis on agarose gel. As an internal control, β-tubulin mRNA expression was assayed in parallel. Cell proliferation: Endometriotic cells were treated with retinoids in the presence or absence of estradiol (E2). All-trans retinoic acid (ATRA) and selective RAR modulators (SRARMs) were used. The number of viable cells was estimated using WST-8 assay. Ki-67 expression: Ki-67 expression was examined by Western blotting. As an internal control, β-tubulin expression was assayed in parallel. [Results] RAR mRNA expression: Transcripts of wild-type RARα, RARβ, RARγ, and their splice variants were demonstrated. These mRNAs were expressed almost at a comparable level: a truncated form of RARα without a DNA binding domain, a truncated form of RARβ without a transactivation domain at the N-terminal, and a form of RARγ with a distinct transactivation domain at N-terminal region. Effect of Retinoic acids on cell proliferation: The cell proliferation rate and Ki-67 expression were downregulated in the presence of ATRA. Among SRARMs tested, a selective RARγ modulator decreased cell proliferation. The antiproliferative activity of ATRA was attenuated in the presence of E2 in some cells.[Conclusion]We tested the antiproliferative activity of retinoids in endometriotic cells. Although the endometriotic cells responded to retinoids, the present finding suggested the diversity of cell types in endometriotic lesions. Presentation: Friday, June 16, 2023

Correlation of Real-Time PCR Cycle Threshold Values and Clinical Progress, Mortality, and Laboratory Parameters of COVID-19 Patients

Objective: The cycle threshold value was used to predict disease severity, disease progression, determine transmissibility, and differentiate active viral replication from prolonged virus shedding. In this study, we aimed to evaluate the relationship between rRT-PCR cycle threshold and clinical course, mortality and laboratory parameters in COVID-19 patients. Methods: The patients included in the study were grouped according to how they were followed; as outpatients, service and intensive care unit patients. Data of age, gender, length of hospital stay, survival status and comorbidities of the patients were obtained from the hospital information system. In addition, CRP, ferritin, albumin, lactate dehydrogenase, procalcitonin and iron values of inpatients were reviewed retrospectively. Cycle threshold values of the patients were compared with each parameter separately. Results: 1339 patients with positive SARS-CoV-2 rRT-PCR test were included in the study. Of the patients, 774 (58%) were outpatients, 548 (41%) were in the ward, and 17 (1%) were in the intensive care unit. No significant difference was found between any of the groups when the cycle threshold values of the groups were compared statistically. Fifty (3.7%) of the patients included in the study died. Cycle threshold values of deceased patients were found to be significantly lower than those who survived (p=0.019). 306 (22.8%) of the patients had comorbidities. When the cycle threshold values of patients with and without comorbid disease were compared, no significant difference was found between any of the groups (p=0.850). In addition, no significant correlation was found between the length of hospital stay, age, CRP, LDH, ferritin, procalcitonin, albumin and iron values and the cycle threshold value. Conclusion: In conclusion, we did not find a significant difference between the cycle threshold values of outpatients, ward patients and intensive care patients. No significant correlation was found between the laboratory parameters, length of hospital stay and age, and the cycle threshold: however, deceased patients were found to have lower cycle thresholds than surviving patients.

An approach for integrating droplet generation and detection in digital polymerase chain reaction applications based on a bifunctional microfluidic cross-structure

Digital polymerase chain reaction (dPCR) is an approach for absolute nucleic acid quantification with high sensitivity. Although several successful commercial dPCR devices have been developed to date, further miniaturizing device dimensions, decreasing cross-contamination, and improving automation level are still research highlights. In this study, we developed a fully contamination-free dPCR detection chip with fluorescence flow cytometry and micro droplet approach. A bifunctional cross-structure (BFCS) was designed to realize monodisperse sample droplet generation in forward flow and droplet detection in backward flow with simple pneumatic control and fixed chip position. In order to improve droplet detection efficiency and accuracy, droplets morphology and sequence pattern during microfluidic droplet generation and backward flow droplet detection at the same cross-structure were observed and analyzed under different pneumatic pressures. In addition, during backward flow droplet detection, an optimized declination angle of the chip was applied to increase droplet reflux rates. For the validation of PCR performance, temperature changing processes during PCR cycles were achieved by heating the monodispersed droplet array with a customized PCR amplification device. The fluorescence signal of each droplet right after passing the cross-structure was excitated and detected. The absolute quantification ability of our integrated dPCR microfluidic chip utilizing flow fluorescence cytometry was tested and verified with Influenza A virus gene (from 7.5 copies/μL to 30000 copies/μL). Thus, our platform provides a novel and integrated approach for ddPCR analysis.

B-228 Partition-free Digital Polymerase Chain Reaction: A Novel Approach to Enable Rapid Turnaround Time With Digital Accuracy

Abstract Background Partition-free digital PCR (dPCR) is a new approach developed to provide absolute quantification in less than 10 min. Partition-free eliminates the need for droplets or physical wells that need to be filled and sealed. The technique relies on rapid thermal cycling and microfluidics to limit diffusion and enable the identification of individual amplicon clusters originating from a single target nucleotide template. Rapid heating and cooling were achieved with a thermal cycling engine comprised of a broadband LED with a sapphire block that is both a light guide and heatsink. Sample is distributed through a custom microfluidic cartridge containing 64 microchannels for optical quantification. Methods The feasibility of partition-free dPCR was tested with primers targeting MTHFR c.665. A titration was performed on three 4X serial dilutions of cell line genomic DNA in triplicate. Fluorescent profiles were generated for all 64 microchannels from an image captured after 35 PCR cycles, where amplicon clusters were identified. Fluorescent peaks of the amplicon clusters were counted and quantified via a custom analysis algorithm, which uses Poisson statistics to calculate concentration. Results The quantification results obtained from the partition-free dPCR system were compared to concentration measurements taken via a Bio-Rad QX200 platform. Concentrations of 69.4 ± 0.8, 15.4 ± 1.5, and 3.3 ± 0.6 copies/µL (R2 = 0.9997) were measured on the partition-free system, and concentrations of 67.8 ± 3.6, 17.2 ± 1.1, and 2.8 ± 0.2 copies/µL (R2 = 0.9995) were measured on the Bio-Rad QX200. Conclusion It has been demonstrated that absolute quantification can be achieved in less than 10 min with a partition-free dPCR platform, and shows results comparable to a QX200 droplet dPCR system. The partition-free rapid dPCR technology described herein allows for the exploration of new avenues in molecular diagnostics, with the potential to bring absolute quantification of dPCR to a Point-of-Care setting.

Sensitivity of rapid antigen tests against SARS-CoV-2 Omicron and Delta variants.

Rapid antigen tests (RATs) have become an invaluable tool for combating the COVID-19 pandemic. However, concerns have been raised regarding the ability of existing RATs to effectively detect emerging SARS-CoV-2 variants. We compared the performance of 10 commercially available, emergency use authorized RATs against the Delta and Omicron SARS-CoV-2 variants using both individual patient and serially diluted pooled clinical samples. The RATs exhibited lower sensitivity for Omicron samples when using PCR cycle threshold (CT) value (a rough proxy for RNA concentration) as the comparator. Interestingly, however, they exhibited similar sensitivity for Omicron and Delta samples when using quantitative antigen concentration as the comparator. We further found that the Omicron samples had lower ratios of antigen to RNA, which offers a potential explanation for the apparent lower sensitivity of RATs for that variant when using C T value as a reference. Our findings underscore the complexity in assessing RAT performance against emerging variants and highlight the need for ongoing evaluation in the face of changing population immunity and virus evolution.

The clinical significance of Dientamoeba fragilis and Blastocystis in human stool—retrospective cohort study

ObjectivesThe aim of this study was to assess the clinical significance of Dientamoeba fragilis (DF) and Blastocystis species (Bs) in human stool. MethodsObservational study of patients ≥18 years, who were tested by stool multiplex PCR for bacteria and parasites between April 2019 and March 2022. Although DF and Bs are part of the PCR kit, these results are not routinely reported to the patient or the ordering physician. The main outcomes were the incidence of symptoms during 14 days before the referral to stool PCR test, and the incidence of several clinical outcomes during 60 days after the PCR test (symptoms, referrals to further evaluation, prescription of symptomatic, or antibiotic treatment). ResultsA total of 27 918 patients were tested by stool PCR during the 3 study years. A total of 6215 (22.3%) and 5337 (19.2%) were positive for DF and Bs, respectively. The incidence of symptoms before the test was similar in those positive for Bs or DF and those with all-negative PCR (adjusted OR and 95% CI of 0.87 [0.80–0.95] and 0.82 [0.76–0.88] for Bs and DF, respectively), whereas significantly higher (2.47 [2.23–2.73]) in those positive for the other multiplex PCR assay components. During the 60 days after the test, the prevalence of any of the outcomes was similar in those positive for Bs or DF and those with negative PCR (adjusted OR and 95% CI of 0.92 [0.83–1.02] and 0.89 [0.81–0.97] for symptoms, 0.84 [0.75–0.94] and 0.93 [0.85–1.01] for referrals, 0.88 [0.75–1.03] and 0.82 [0.71–0.94] for symptomatic treatment, and 0.88 [0.75–1.02] and 0.86 [0.75–0.98] for antibiotic treatment in the Bs and DF positive individuals, respectively). The PCR cycle threshold was not associated with any of the outcomes. DiscussionPositive stool PCR for DF or Bs was not associated with any of the measured clinical outcomes.

Evaluation of 20 CODIS Core STR Loci For Touch DNA Analysis

The DNA profile is a combination of STR alleles loci as a result of template DNA amplification. The FBI has defined 20 core STR loci for human identification in forensic cases. The aim of this study was to evaluate the twenty CODIS core STR loci to be a reference in DNA touch examination. Nine donors with three shedder statuses, high, intermediate, and low, were holding the sample while tying two types of ropes, porous and non-porous. The touch sample is then sampled using two techniques on each rope: double swab and tape lift. DNA was extracted and quantified by real-time PCR technique to determine the concentration of DNA obtained from each treatment. Twenty-four STR loci of the DNA were amplified, and the fragments were analyzed by capillary electrophoresis. Two PCR cycles were chosen to determine the effect of adding cycles on the concentration of DNA that was not successfully amplified. The results of this study indicate that the quantity of touch DNA has a greater influence on the success rate of STR amplification. Each allele on 20 STR CODIS Loci failed to amplify from touch DNA concentration below 0.0625 ng/µl. It is shown that the longer the target allele at each locus, the easier it is for allelic drop-out to occur. However, the optimal STR locus length for DNA testing is under 200 nucleotides.

Application of a modified tetra-primer ARMS–PCR assay for rapid Panax species identity authentication in ginseng products

Panax ginseng products can be adulterated with materials from other Panax species. The purpose of this study is to provide a rapid P. ginseng authentication method for simultaneous identification of P. ginseng and detection of adulteration in ginseng products at different processing stages. First, a tetra-primer ARMS–PCR assay was designed based on a single-nucleotide polymorphism (SNP) within the trnL-trnF region and was tested at 28 PCR cycles with DNA extracted from Botanical Reference Materials (BRMs). Next, 5′ end random nucleotide and 3′ terminus phosphorothioates linkage modifications were incorporated into the inner primers to improve sensitivity and specificity at 40 PCR cycles. Finally, the modified assay was validated using characterized market ginseng materials and the detection limit was determined. The modified tetra-primer ARMS–PCR assay can achieve the desired sensitivity and specificity using one set of reaction conditions in ginseng materials at different stages. In validation, it was able to correctly identify target species P. ginseng and differentiate it from closely related species. This study suggests that the modified tetra-primer ARMS–PCR assay can be used for the rapid, species identity authentication of P. ginseng material in ginseng products. This assay can be used to complement chemical analytical methods in quality control, so both species identity and processing attributes of ginseng products can be efficiently addressed.

Comparison of two assays to diagnose herpes simplex virus in patients with central nervous system infections.

Multiplex PCR panels are frequently used for detecting the diagnosis of central nervous system infection, but have demonstrated lower sensitivity for herpes simplex virus (HSV) compared to targeted PCRs. Providers in our institution frequently order both multiplex (BioFire Meningitis/Encephalitis Panel (MEP)) and targeted HSV PCR (Diasorin Simplexa HSV-1/2 Direct), allowing comparison of assay performance in practice and a unique clinical characterization of patients with concordant positive or discordant results. We retrospectively analyzed all HSV testing performed using the two assays simultaneously in a large multi-center institution between 12/10/2018 and 6/6/2022. We described and compared laboratory, diagnostic and therapeutic data between patients with positive results on both assays with patients with discordant results. 1,387 patients were tested with both assays, of which 25 were positive on at least one assay. Eleven (44%) had discordant results; 9 were targeted-PCR-positive/MEP-negative and 2 were targeted-PCR-negative/MEP-positive. Compared with patients with concordant positive results, discordant targeted PCR-positive/MEP-negative patients had a higher PCR cycle threshold value (i.e., >35) and milder illness characterized by lower CSF WBC counts (4vs 152; p=0.032), fewer MRI abnormalities (37.5% vs. 87.5%; p=0.039), HSV meningoencephalitis as primary discharge diagnosis (33% vs. 100%; p<0.0001) and were more often treated with oral medication or none at all. Patients with discordant targeted PCR-positive/MEP-negative results represent a population with milder clinical disease. These findings will help clinicians and laboratorians determine when both targeted HSV and multiplex assays should be performed simultaneously and identify patients for whom a multiplex PCR may suffice, despite lower assay sensitivity.

Diagnostic performance of GenBody COVID-19 rapid antigen test for laboratory and non-laboratory medical professionals in real practice: A retrospective study.

Point-of-care tests for coronavirus disease 2019 (COVID-19) antigen detection have been widely used for rapid diagnosis in various settings. However, research on the diagnostic performance of the COVID-19 antigen test performed by non-laboratory personnel is limited. In this study, we aimed to elucidate the diagnostic performance of GenBody COVID-19 rapid antigen between laboratory professionals and non-laboratory staff. We retrospectively analyzed the data of patients who underwent both GenBody COVID-19 rapid antigen testing and reverse transcription polymerase chain reaction (RT-PCR) between November 01, 2021, and June 30, 2022. The diagnostic performance of the antigen test was compared between laboratory and non-laboratory operators, using RT-PCR as the gold standard. Sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, diagnostic odds ratio, positive predictive value, negative predictive value, and accuracy were calculated and sensitivity analysis was performed based on the PCR cycle threshold (Ct) value. Of the 11,963 patients, 1273 (10.6%) tested positive using real-time RT-PCR. The sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, diagnostic odds ratio, positive predictive value, negative predictive value, and accuracy of the GenBody COVID-19 rapid antigen test with 95% confidence interval were 79.92% (77.26%-82.39%), 99.23% (98.73%-99.57%), 103.25 (62.31-171.11), 0.2 (0.18-0.23), 510.18 (299.81-868.18), 98.11% (96.91%-98.85%), 90.75% (89.64%-91.75%) and 92.76% (91.76%-93.67%), respectively, for non-laboratory staff and 79.80% (74.78%-84.22%), 99.99% (99.94%-100.00%), 6983.92 (983.03-49617.00), 0.2 (0.16-0.25), 34566.45 (4770.30-250474.46) 99.58% (97.09%-99.94%), 99.32% (99.15%-99.46%), and 99.33% (99.13%-99.48%), respectively, for laboratory staff. Notably, when the PCR Ct value exceeded 25, the sensitivity of both the groups decreased to < 40%. The diagnostic performance of GenBody COVID-19 rapid antigen performed by non-laboratory staff was comparable to that of laboratory professionals. However, it should be noted that the sensitivity of the antigen tests decreased when the PCR Ct value exceeded 25. Overall, the GenBody COVID-19 antigen test is a viable option for non-laboratory staff during an epidemic.

Mobile Efficient Diagnostics of Infectious Diseases via On-Chip RT-qPCR: MEDIC-PCR.

The COVID-19 outbreak has caused public and global health crises. However, the lack of on-site fast, reliable, sensitive, and low-cost reverse transcription polymerase chain reaction (RT-PCR) testing limits early detection, timely isolation, and epidemic prevention and control. Here, the authors report a rapid mobile efficient diagnostics of infectious diseases via on-chip -RT-quantitative PCR (RT-qPCR): MEDIC-PCR. First, the authors use a roll-to-roll printing process to accomplish low-cost carbon-black-based disposable PCR chips that enable rapid LED-induced photothermal PCR cycles. The MEDIC-PCR can perform RT (3min), and PCR (9min) steps. Further, the cohort of 89 COVID-19 and 103 non-COVID-19 patients testing is completed by the MEDIC-PCR to show excellent diagnostic accuracy of 97%, sensitivity of 94%, and specificity of 98%. This MEDIC-PCR can contribute to the preventive global health in the face of a future pandemic.

Diagnostic Agreement of SARS-CoV-2 Lateral Flow Antigen Assay with the Cycle Threshold Values of RT-PCR

COVID-19 detection via lateral flow antigen assays (LFA) are rapid and economically acquiescent to infrastructure facile healthcare settings. Early, prompt identification of cases to facilitate patient isolation and supportive management is the essence of rapid diagnostic tests. Given the backdrop of post COVID-19 pandemic-molecular testing still remains a costly affair. Additionally, molecular assays are incapable of distinguishing remnant RNA from replication competent viruses. In this scenario, we explore the diagnostic consonance of SARS-CoV-2 LFAs with RT-PCR cycle threshold, in a likelihood that it could be used as a surrogate marker for infection transmissibility. Rapid COVID-19 LFA results were compared with Real-time PCR for detection of SARS-CoV-2 in nasopharyngeal swabs. Two hundred rapid antigen positive nasopharyngeal swabs obtained from COVID-19 suspects/contacts/preoperative/screening patients were subjected to RT-PCR to study the correlation with cycle threshold (CT) values obtained for all the antigen positive cases. 200 Rapid COVID-19 LFA positive samples were analyzed in the present study. Amidst the LFA positive samples included in the study 187 (93.5%) were found to have concordant results when subjected to the gold standard Real-time PCR. Discordant results were documented in 13 (6.5%) COVID-19 LFA positive samples which were found to be negative by RT-PCR. The average Cycle threshold values were found to be 23.75 for E gene, 25.36 for N gene and 24.07 for RdRp gene. The average PCR Cycle threshold of LFA positive cases remained significantly undeterred (p&lt;0.5) throughout the time period of the study stipulating the undaunted viral load across the different waves of the pandemic. Maximum association of LFA positivity with symptom-manifestation was seen during the 1st wave of COVID-19 (September-December 2020 in India). The association of symptoms with LFA test positivity reduced to a significant extent during the 3rd wave of the pandemic in January 2022 (p&lt;0.5) indicating the reduced clinical severity but not infectivity of the SARS-CoV-2 infection during the 3rd wave of the pandemic. Lateral flow assay based diagnostic tests are technically &amp; economically convenient modalities with significant interest concordance in comparison with RT-PCR. Definitive advantage in terms of achieving quick patient triage and thereby patient management can be achieved with the use of these tests.

A systematic approach to improve downstream single-cell analysis for the DEPArray™ technology.

Most commercially available STR amplification kits have never been fully validated for low template DNA analysis, highlighting the need for testing different PCR kits and conditions for improving single-cell profiling. Here, current strategies rely mainly on adjusting PCR cycle number and analytical threshold settings, with a strong preference for using 30 amplification cycles and thresholds at 30-150 RFU for allele detection. This study aimed to (1) determine appropriate conditions for obtaining informative profiles utilizing a dilution series, and (2) test the outcome on single cells using the DEPArray™ technology. Four routinely applied forensic STR kits were compared by using three different amplification volumes and DNA dilutions down to 3.0 pg, while two well-performing kits were used for single/pooled leucocyte and sperm cell genotyping. Besides reduced costs, the results demonstrate that a 50%-75% PCR volume reduction was beneficial for peak height evaluation. However, this was counteracted by an increased artifact generation in diluted DNA volumes. Regarding profile completeness, the advantage of volume reduction was only prominent in samples processed with Fusion 6C. For single and pooled cells, ESIFast and NGMDetect provided a solid basis for consensus profiling regarding locus failure, although locus dropouts were generally observed as stochastic events. Amplification volume of 12.5 μL was confirmed as appropriate in terms of peak heights and stutter frequencies, with increased stutter peaks being the main artifact in single-cell profiles. Limitations associated with these analyses are discussed, providing a solid foundation for further studies on low template DNA.

Haplotype-level metabarcoding of freshwater macroinvertebrate species: A prospective tool for population genetic analysis.

Metabarcoding is a molecular-based tool capable of large quantity high-throughput species identification from bulk samples that is a faster and more cost-effective alternative to conventional DNA-sequencing approaches. Still, further exploration and assessment of the laboratory and bioinformatics strategies are required to unlock the potential of metabarcoding-based inference of haplotype information. In this study, we assessed the inference of freshwater macroinvertebrate haplotypes from metabarcoding data in a mock sample. We also examined the influence of DNA template concentration and PCR cycle on detecting true and spurious haplotypes. We tested this strategy on a mock sample containing twenty individuals from four species with known haplotypes based on the 658-bp Folmer region of the mitochondrial cytochrome c oxidase gene. We recovered fourteen zero-radius operational taxonomic units (zOTUs) of 421-bp length, with twelve zOTUs having a 100% match with the Sanger haplotype sequences. High-quality reads relatively increased with increasing PCR cycles, and the relative abundance of each zOTU was consistent for each cycle. This suggests that increasing the PCR cycles from 24 to 64 did not affect the relative abundance of each zOTU. As metabarcoding becomes more established and laboratory protocols and bioinformatic pipelines are continuously being developed, our study demonstrated the method's ability to infer intraspecific variability while highlighting the challenges that must be addressed before its eventual application for population genetic studies.

16S rRNA Gene PCR/Sequencing of Heart Valves for Diagnosis of Infective Endocarditis in Routine Clinical Practice.

Sequencing is increasingly used for infective endocarditis (IE) diagnosis. Here, the performance of 16S rRNA gene PCR/sequencing of heart valves utilized in routine clinical practice was compared with conventional IE diagnostics. Subjects whose heart valves were sent to the clinical microbiology laboratory for 16S rRNA gene PCR/sequencing from August 2020 through February 2022 were studied. A PCR assay targeting V1 to V3 regions of the 16S rRNA gene was performed, followed by Sanger and/or next-generation sequencing (NGS) (using an Illumina MiSeq), or reported as negative, depending on an algorithm that included the PCR cycle threshold value. Fifty-four subjects, including 40 with IE, three with cured IE, and 11 with noninfective valvular disease, were studied. Thirty-one positive results, 11 from NGS and 20 from Sanger sequencing, were generated from analysis of 16S rRNA gene sequence(s). Positivity rates of blood cultures and 16S rRNA gene PCR/sequencing of valves were 55% and 75%, respectively (P = 0.06). In those with prior antibiotic exposure, positivity rates of blood cultures and 16S rRNA gene PCR/sequencing of valves were 11% and 76%, respectively (P < 0.001). Overall, 61% of blood culture-negative IE subjects had positive valve 16S rRNA gene PCR/sequencing results. 16S rRNA gene-based PCR/sequencing of heart valves is a useful diagnostic tool for pathogen identification in patients with blood culture-negative IE undergoing valve surgery in routine clinical practice.

Evaluation of rRNA depletion methods for capturing the RNA virome from environmental surfaces

ObjectiveMetatranscriptomic analysis of RNA viromes on built-environment surfaces is hampered by low RNA yields and high abundance of rRNA. Therefore, we evaluated the quality of libraries, efficiency of rRNA depletion, and viral detection sensitivity using a mock community and a melamine-coated table surface RNA with levels below those required (< 5 ng) with a library preparation kit (NEBNext Ultra II Directional RNA Library Prep Kit).ResultsGood-quality RNA libraries were obtained from 0.1 ng of mock community and table surface RNA by changing the adapter concentration and number of PCR cycles. Differences in the target species of the rRNA depletion method affected the community composition and sensitivity of virus detection. The percentage of viral occupancy in two replicates was 0.259 and 0.290% in both human and bacterial rRNA-depleted samples, a 3.4 and 3.8-fold increase compared with that for only bacterial rRNA-depleted samples. Comparison of SARS-CoV-2 spiked-in human rRNA and bacterial rRNA-depleted samples suggested that more SARS-CoV-2 reads were detected in bacterial rRNA-depleted samples. We demonstrated that metatranscriptome analysis of RNA viromes is possible from RNA isolated from an indoor surface (representing a built-environment surface) using a standard library preparation kit.

Mitochondrial DNA control region typing from highly degraded skeletal remains by single-multiplex next-generation sequencing.

Poor nuclear DNA preservation from highly degraded skeletal remains is the most limiting factor for the genetic identification of individuals. Mitochondrial DNA (mtDNA) typing, and especially of the control region (CR), using next-generation sequencing (NGS), enables retrieval of valuable genetic information in forensic contexts where highly degraded human skeletal remains are the only source of genetic material. Currently, NGS commercial kits can type all mtDNA-CR in fewer steps than the conventional Sanger technique. The PowerSeq CRM Nested System kit (Promega Corporation) employs a nested multiplex-polymerase chain reaction (PCR) strategy to amplify and index all mtDNA-CR in a single reaction. Our study analyzes the success of mtDNA-CR typing of highly degraded human skeletons using the PowerSeq CRM Nested System kit. We used samples from 41 individuals from different time periods to test three protocols (M1, M2, and M3) based on modifications of PCR conditions. To analyze the detected variants, two bioinformatic procedures were compared: an in-house pipeline and the GeneMarker HTS software. The results showed that many samples were not analyzed when the standard protocol (M1) was used. In contrast, the M3 protocol, which includes 35 PCR cycles and longer denaturation and extension steps, successfully recovered the mtDNA-CR from highly degraded skeletal samples. Mixed base profiles and the percentage of damaged reads were both indicators of possible contamination and can provide better results if used together. Furthermore, our freely available in-house pipeline can provide variants concordant with the forensic software.

Development of an amplicon-based sequencing approach in response to the global emergence of mpox.

The 2022 multicountry mpox outbreak concurrent with the ongoing Coronavirus Disease 2019 (COVID-19) pandemic further highlighted the need for genomic surveillance and rapid pathogen whole-genome sequencing. While metagenomic sequencing approaches have been used to sequence many of the early mpox infections, these methods are resource intensive and require samples with high viral DNA concentrations. Given the atypical clinical presentation of cases associated with the outbreak and uncertainty regarding viral load across both the course of infection and anatomical body sites, there was an urgent need for a more sensitive and broadly applicable sequencing approach. Highly multiplexed amplicon-based sequencing (PrimalSeq) was initially developed for sequencing of Zika virus, and later adapted as the main sequencing approach for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Here, we used PrimalScheme to develop a primer scheme for human monkeypox virus that can be used with many sequencing and bioinformatics pipelines implemented in public health laboratories during the COVID-19 pandemic. We sequenced clinical specimens that tested presumptively positive for human monkeypox virus with amplicon-based and metagenomic sequencing approaches. We found notably higher genome coverage across the virus genome, with minimal amplicon drop-outs, in using the amplicon-based sequencing approach, particularly in higher PCR cycle threshold (Ct) (lower DNA titer) samples. Further testing demonstrated that Ct value correlated with the number of sequencing reads and influenced the percent genome coverage. To maximize genome coverage when resources are limited, we recommend selecting samples with a PCR Ct below 31 Ct and generating 1 million sequencing reads per sample. To support national and international public health genomic surveillance efforts, we sent out primer pool aliquots to 10 laboratories across the United States, United Kingdom, Brazil, and Portugal. These public health laboratories successfully implemented the human monkeypox virus primer scheme in various amplicon sequencing workflows and with different sample types across a range of Ct values. Thus, we show that amplicon-based sequencing can provide a rapidly deployable, cost-effective, and flexible approach to pathogen whole-genome sequencing in response to newly emerging pathogens. Importantly, through the implementation of our primer scheme into existing SARS-CoV-2 workflows and across a range of sample types and sequencing platforms, we further demonstrate the potential of this approach for rapid outbreak response.

SNP analysis of challenging bone DNA samples using the HID-Ion AmpliSeq™ Identity Panel: facts and artefacts

PCR-MPS is an emerging tool for the analysis of low-quality DNA samples. In this study, we used PCR-MPS to analyse 32 challenging bone DNA samples from three Second World War victims, which previously yielded no results in conventional STR PCR-CE typing. The Identity Panel was used with 27 cycles of PCR. Despite that we only had an average of 6.8 pg of degraded DNA as template, 30 out of 32 libraries (93.8%) produced sequencing data for about 63/90 autosomal markers per sample. Out of the 30 libraries, 14 (46.7%) yielded single source genetic profiles in agreement with the biological identity of the donor, whereas 12 cases (40.0%) resulted in SNP profiles that did not match or were mixed. The misleading outcomes for those 12 cases were likely due to hidden exogenous human contamination, as shown by the higher frequencies of allelic imbalance, unusual high frequencies of allelic drop-ins, high heterozygosity levels in the consensus profiles generated from challenging samples, and traces of amplified molecular products in four out of eight extraction negative controls. Even if the source and the time of the contamination were not identified, it is likely that it occurred along the multi-step bone processing workflow. Our results suggest that only positive identification by statistical tools (e.g. likelihood ratio) should be accepted as reliable; oppositely, the results leading to exclusion should be treated as inconclusive because of potential contamination issues. Finally, strategies are discussed for monitoring the workflow of extremely challenging bone samples in PCR-MPS experiments with an increased number of PCR cycles.

Toxoplasma Lymphadenopathy: A Comparative Diagnostic Assessment of Clinical, Serological and Histopathological Findings.

Toxoplasma Gondii (TG) is a zoonotic protozoan with extensive symptomatology. Toxoplasmic lymphadenopathy is considered an affirmative sign and is proved by a biopsy of the enlarged nodule. This study was conducted to compare the clinical, serological, and histopathological findings for the diagnosis of toxoplasmic lymphadenopathy. This study involved biopsy examinations from twelve cases with TG lymphadenopathy. ELISA serological tests were performed for TG specific IgM and IgG immunoglobulins. PCR was done to ratify the results obtained by ELISA. The ages of the patients ranged from 15 to 48 years (mean=27.8). Most of the cases are male n=8(66.7%), while female n=4(33.3%). The asthenia was not only the most frequent clinical presentation (83.3%), but it also last longer. All cases had a positive biopsy. Eight (67.7%) cases revealed seropositivity. Two of them had positive PCR in those who were positive IgM, suggesting that the infection was acute. Six (50%) cases revealed positive IgG tests, while those with negative serology were 4(33.3%). The site of lymph nodes involvement had been assessed and mostly cervical (91.6%). The histopathological results yielded 100% positive findings, thus biopsy was very important in the diagnosis and differential diagnosis of lymph nodes enlargement. The chronic phase of toxoplasmosis does not show the protozoa in the blood causing an absent DNA band for amplification of the PCR cycles, which could explain the lack of bands particular for TG. A negative serological test does not exclude toxoplasmic lymphadenitis, especially in immune-compromised patients.