Excellent Analytical Sensitivity Research Articles

Development of a fully automated chemiluminescent immunoassay for the quantitative and qualitative detection of antibodies against African swine fever virus p72.

African swine fever (ASF), caused by ASF virus (ASFV), is a highly infectious and severe hemorrhagic disease of pigs that causes major economic losses. Currently, no commercial vaccine is available and prevention and control of ASF relies mainly on early diagnosis. Here, a novel automated double antigen sandwich chemiluminescent immunoassay (DAgS-aCLIA) was developed to detect antibodies against ASFV p72 (p72-Ab). For this purpose, recombinant p72 trimer was produced, coupled to magnetic particles as carriers and labeled with acridinium ester as a signal trace. Finally, p72-Ab can be sensitively and rapidly measured on an automated chemiluminescent instrument. For quantitative analysis, a calibration curve was established with a laudable linearity range of 0.21 to 212.0 ng/mL (R2 = 0.9910) and a lower detection limit of 0.15 ng/mL. For qualitative analysis, a cut-off value was set at 1.50 ng/mL with a diagnostic sensitivity of 100.00% and specificity of 98.33%. Furthermore, antibody response to an ASF gene-deleted vaccine candidate can be accurately quantified using this DAgS-aCLIA, as evidenced by early seroconversion as early as 7 days post-immunization and high antibody levels. Compared with available enzyme-linked immunosorbent assays, this DAgS-aCLIA demonstrated a wider linearity range of 4 to 16-fold, and excellent analytical sensitivity and agreement of over 95.60%. In conclusion, our proposed DAgS-aCLIA would be an effective tool to support ASF epidemiological surveillance.IMPORTANCEAfrican swine fever virus (ASFV) is highly contagious in wild boar and domestic pigs. There is currently no vaccine available for ASF, so serological testing is an important diagnostic tool. Traditional enzyme-linked immunosorbent assays provide only qualitative results and are time and resource consuming. This study will develop an automated chemiluminescent immunoassay (CLIA) that can quantitatively and qualitatively detect antibodies to ASFV p72, greatly reducing detection time and labour-intensive operation, and improving detection sensitivity and linearity range. This novel CLIA would serve as a reliable and convenient tool for ASF pandemic surveillance and vaccine development.

Establishment of genomic RNA reference materials for BCR-ABL1 P210 measurement.

Quantitation of BCR-ABL1 with thequantitative reverse transcriptase polymerase chain reaction (RT-PCR) is very important in monitoring chronic myeloid leukemia (CML), which relies on an RNA reference material. A genomic RNA reference material (RM) containing the BCR-ABL1 P210 fusion mutation was developed, and an absolute quantitative method based on one-step reverse transcription digital PCR (RT-dPCR) was established for characterizing the RM. The proposed dPCR method demonstrates high accuracy and excellent analytical sensitivity, as shown by the linear relationship (0.94 < slope < 1.04, R2≧0.99) between the measured and nominal values of b2a2, b3a2, and ABL1-ref within the dynamic range (104-101 copies/reaction). Homogeneity and stability assessment based on dPCR indicated that the RM was homogeneous and stable for 24months at -80°C. The RM was used to evaluate inter-laboratory reproducibility in eight different laboratories, demonstrating that participating laboratories could consistently produce copy concentrations of b3a2 and ABL1-ref, as well as the BCR-ABL1/ABL1 ratio (CV < 2.0%). This work suggests that the RM can be employed in establishing metrological traceability for detecting mutations in the BCR-ABL1 fusion gene, as well as in quality control for testing laboratories.

A simplified molecular tool for detecting the Chagas etiological agent using a vector feces sample in field conditions

Triatomine bugs are vectors of Trypanosoma cruzi, the etiologic agent of Chagas disease in the American continent. Here, we have tested a loop-mediated isothermal amplification (LAMP) test for a direct detection of T. cruzi in feces of Triatoma infestans, the main vector of this parasite in the Southern Cone of America. The analytical evaluation showed positive results with samples of triatomine feces artificially inoculated with DNA from strains of T. cruzi corresponding to each Discrete Typing Units (I-VI), with a sensitivity of up to one parasite per reaction. Conversely, the reaction yielded negative results when tested with DNA from Trypanosoma rangeli and other phylogenetically related and unrelated organisms. In triatomines captured under real field conditions (from urban households), and defined as positive or negative for T. cruzi using the reference microscopy technique, the LAMP test achieved a concordance of 100 %. Our results demonstrate that this LAMP reaction exhibits excellent analytical specificity and sensitivity without interference from the fecal matrix, since all the reactions were conducted without purification steps. This simple molecular diagnostic technique can be easily used by vector control agencies under field conditions.

Analytical and clinical validation of a personalized, tumor-informed circulating tumor DNA assay for minimal residual disease detection.

e14545 Background: The detection of minimal residual disease (MRD) through circulating tumor DNA (ctDNA) analysis provides an advanced molecular method for early identification of surgical patients at high risk of recurrence. In this study, we present the development of mClear, a personalized, tumor-informed next-generation sequencing (NGS) assay for MRD detection. Furthermore, we report the analytical and clinical validation processes conducted to evaluate the performance of the MRD assay. Methods: mClear is designed to assess MRD in patients with solid tumors. Whole exome sequencing (WES) was performed on tumor tissues and matched peripheral blood samples to detect somatic variants comprehensively. Personalized MRD assays were carefully designed by selecting up to 40 prioritized variants, including single nucleotide variants (SNVs), small insertions and deletions (InDels), and fusions while ensuring coverage of 21 additional key driver genes. For analytical validation, we utilized a pan-cancer MRD ctDNA quality control product (LDT-999, LDT Bioscience), characterized by known variant allele frequency (VAF) gradients of 0%, 0.005%, 0.05%, and 0.5%, confirmed through droplet digital PCR (ddPCR). We conducted 18 replicate tests at VAF gradients of 0.5% and 0.05%, 23 replicates at the 0.005% VAF gradient, and 16 replicates at the 0% VAF gradient. For clinical validation, preoperative and postoperative blood samples from a cohort of 264 pan-cancer patients were analyzed. ctDNA collected for MRD detection underwent ultra-deep next-generation sequencing (100,000× average depth of coverage). The library preparation process commenced with an input of 30ng DNA. Results: At VAF gradients of 0.5% and 0.05%, the assay achieved an excellent sensitivity of 100%, and at VAF gradients of 0.005% and 0%, it demonstrated a sensitivity of 95.7% and a specificity of 100%. Preoperative ctDNA was detected in 86.7% (72/83) of the pan-cancer patients with 71.4% (5/7), 90.9% (10/11), 85.5% (47/55), and 100.0% (10/10) in pathological stage (pStage)I, pStage II, pStage III, and pStage IV, respectively. In the pan-cancer cohort, the positive rate of longitudinal MRD was 35.7% (75/210) with 17.8% (21/118), 50.0% (13/26), 55.1% (27/49), and 82.4% (14/17) in pStage I, II, III, and IV, respectively. In four patients who relapsed, MRD detection during surveillance preceded radiological recurrence by a lead time of 1-8 months. Conclusions: Our personalized MRD assay demonstrated excellent analytical sensitivity and specificity with a limit of detection (LoD) of 0.005% VAF at the sample level. Furthermore, it displayed high clinical sensitivity for the detection of ctDNA in the pan-cancer cohort, highlighting its potential for monitoring of recurrence.

Novel strategy to quantify the viability of oocysts of Cryptosporidium parvum and C. hominis, a risk factor of the waterborne protozoan pathogens of public health concern

While waters might be contaminated by oocysts from >40 Cryptosporidium species, only viable oocysts of C. parvum and C. hominis truly pose the main health risk to the immunocompetent population. Oocyst viability is also an important but often neglected risk factor in monitoring waterborne parasites. However, commonly used methods in water monitoring and surveys cannot distinguish species (microscopic observation) or oocyst viability (PCR), as dead oocysts in water could retain gross structure and DNA content for weeks to months. Here, we report new TaqMan qRT-PCR/qPCR assays for quantitative detection of viable C. parvum and C. hominis oocysts. By targeting a hypothetical protein-encoding gene cgd6_3920 that is highly expressed in oocysts and variable between species, the qRT-PCR/qPCR assays achieve excellent analytical specificity and sensitivity (limit of quantification [LOQ] = 0.25 and 1.0 oocyst/reaction). Using calibration curves, the number and ratio of viable oocysts in specimens could be calculated. Additionally, we also establish a TaqMan-18S qPCR for cost-effective screening of pan-Cryptosporidium-positive specimens (LOQ = 0.1 oocyst/reaction). The assay feasibility is validated using field water (N = 43) and soil (79) specimens from 17 locations in Changchun, China, which detects four Cryptosporidium species from seven locations, including three gp60-subtypes (i.e., IIdA19G1, IIdA17G1 and IIdA24G2) of C. parvum oocysts showing varied viability ratios. These new TaqMan q(RT)-PCR assays supplement current methods in the survey of waters and other samples (e.g., surfaces, foods and beverages), and are applicable to assessing the efficiency of oocyst deactivation protocols.

Clinical Implementation of a Noninvasive, Multi-Analyte Droplet Digital PCR Test to Screen for Androgen Receptor Alterations

Recent studies have shown that alterations of the androgen receptor (AR) are associated with resistance to AR-directed therapy in prostate cancer. Thus, it is crucial to develop robust detection methods for AR alterations as predictive biomarkers to enable applicability in clinical practice. We designed and validated five multiplex droplet digital PCR (ddPCR) assays for reliable detection of 12 AR targets including AR amplification, AR-V7 and 10 AR hotspot mutations as well as AR and KLK3 gene expression from plasma derived cell-free (cf)DNA and cfRNA.The assays demonstrated excellent analytical sensitivity and specificity ranging from 95% to 100% (95% CI: 75-100%). Intra- and inter-run variation analyses revealed a high level of repeatability and reproducibility. The developed assays were further applied in peripheral blood samples from 77 patients with advanced prostate cancer to assess their feasibility in a real-world scenario. Optimizing the reverse transcription of RNA increased the yield of plasma-derived cfRNA by 30-fold. Among 23 patients with castration-resistant prostate cancer (CRPC), 6 patients (26.1%) had one or a combination of several AR alterations, while only 2 out of 54 patients (3.7%) in the hormone-sensitive stage showed AR alterations. These findings were consistent with other studies and suggest that implementation of a comprehensive AR status detection in clinical practice is feasible and can support the treatment decision-making process.

Performance evaluation of the SMG HHV-6 Q Real-Time PCR Kit for quantitative detection and differentiation of human herpesvirus 6A and 6B.

The aim of this study was to compare the performance of the newly developed SMG HHV-6 Q Real-Time PCR Kit (SMG assay) with the RealStar HHV-6 PCR Kit (RealStar assay). The analytical sensitivity and specificity, linearity, and precision of the SMG assay were evaluated. The clinical performance of the SMG assay was assessed and compared with that of the RealStar assay using 207 clinical specimens (HHV-6A positive, n = 51; HHV-6B positive, n = 64; HHV-6A/B negative, n = 92). The limit of detection of the SMG assay was 2.92 log10 copies/mL for HHV-6A DNA and 2.88 log10 copies/mL for HHV-6B DNA. The linear range was determined to be 3.40-9.00 log10 copies/mL for both viruses. Intra- and inter-assay variability were below 5% at concentrations ranging from 4 to 9 log10 copies/mL. No cross-reactivity was observed with the 25 microorganisms included in the specificity panel. The clinical sensitivity and specificity of the SMG and RealStar assays compared to in-house polymerase chain reaction and sequencing were as follows: SMG assay, 98.0% and 100% for HHV-6A DNA, respectively, and 96.9% and 100% for HHV-6B DNA, respectively; RealStar assay, 98.0% and 100% for HHV-6A DNA, respectively, and 90.6% and 100% for HHV-6B DNA, respectively. The correlation coefficients between viral loads measured by the two assays were 0.948 and 0.975, with mean differences of 0.62 and 0.32 log10 copies/mL for HHV-6A and HHV-6B DNA, respectively. These results demonstrate that the SMG assay is a sensitive and reliable tool for the quantitative detection and differentiation of HHV-6A and HHV-6B DNA.IMPORTANCEQuantitative real-time PCR (qPCR) that can distinguish between HHV-6A and HHV-6B DNA is recommended for diagnosis of active infection. The SMG HHV-6 Q Real-Time PCR Kit (SMG assay) is a newly developed qPCR assay that can differentiate between HHV-6A and HHV-6B DNA; however, little is known about its performance. In this study, we assessed the performance of the SMG assay and compared it with that of a commercially available qPCR assay, the RealStar HHV-6 PCR Kit (RealStar assay). The SMG assay demonstrated excellent analytical sensitivity and specificity, precision, and linearity. Furthermore, the viral loads measured by the SMG assay were highly correlated with those measured by the RealStar assay. Our results suggest that the SMG assay is a useful diagnostic tool for quantitative detection and differentiation of HHV-6A and HHV-6B DNA.

Rapid detection of monkeypox virus and differentiation of West African and Congo Basin strains using endonuclease restriction-mediated real-time PCR-based testing.

The ongoing multi-country outbreak of monkeypox virus (MPXV) has continuously attracted global attention, highlighting the critical need for timely and accurate methods to detect MPXV and differentiate its clades. Herein, we devised a novel multiplex ET-PCR (endonuclease restriction-mediated real-time PCR) assay that integrates PCR amplification, restriction endonuclease cleavage and real-time fluorescence detection to diagnose MPXV infection and distinguish the Congo Basin and West African MPXV strains. In the MPXV ET-PCR system, three sets of specific primers were designed for MPXV, Congo Basin and West African strains. A short sequence, which could be recognized by restriction endonuclease enzyme BstUI, was added to the 5'end of amplification primers. Then, the modified primers were assigned different reporter dyes and corresponding quenching dyes to each of the three targets, enabling real-time fluorescence reporting of the results and multiplex detection. The designed assay enabled the detection of single or three targets in a single tube, with excellent specificity and analytical sensitivity in terms of plasmid and pseudotyped virus. Moreover, the clinical feasibility of our assay was validated using artificially simulated plasma, nasopharyngeal swab and skin swab samples. In conclusion, the multiplex ET-PCR assay devised here had the advantages of simple primer design, cost-effectiveness, low contamination risk, excellent sensitivity, high specificity and multiplex detection, making it a valuable and dependable tool for curbing the extensive spread of MPXV.

A rapid multiplex loop-mediated isothermal amplification (mLAMP) assay for detection of Entamoeba histolytica and Giardia duodenalis.

We developed a rapid multiplex loop-mediated isothermal amplification (mLAMP) assay for two common intestinal parasites-Entamoeba histolytica and Giardia duodenalis, where early detection may be helpful. The mLAMP assay was optimized for detection of DNA of E. histolytica (18S rRNA gene) and G. duodenalis (Elongation factor 1 alfa gene) from standard strains by using six specific primers (FIP, BIP, F3, B3, FLP, BLP) for each gene target. The amplification time was 16-26 min for E. histolytica and 10-15 min for G. duodenalis, and the parasites could be distinguished based on melting curve analysis for specific annealing temperatures (Tm) of 84-86°C and 88-90°C for E. histolytica and G. duodenalis, respectively. The analytical sensitivity was one fg, and no cross-reactivity with other intestinal pathogens was observed. Thus, the mLAMP assay could detect and clearly distinguish E. histolytica and G. duodenalis with a rapid turnaround time and excellent analytical sensitivity and specificity.

Development of an effective one-step double-antigen sandwich ELISA based on p72 to detect antibodies against African swine fever virus.

African swine fever (ASF), caused by ASF virus (ASFV), is a highly contagious and lethal disease of domestic pigs leading to tremendous economic losses. As there are no vaccines and drugs available. An effective diagnosis to eliminate ASFV-infected pigs is a crucial strategy to prevent and control ASF. To this end, ASFV capsid protein p72 was expressed using Chinese hamster ovary (CHO) cells and subsequently conjugated with horseradish peroxidase (HRP) to develop a one-step double-antigen sandwich enzyme-linked immunosorbent assay (one-step DAgS-ELISA). The performance of this ELISA for detecting ASFV antibodies was evaluated. Overall, a diagnostic sensitivity of 97.96% and specificity of 98.96% was achieved when the cutoff value was set to 0.25. No cross-reaction with healthy pig serum and other swine viruses was observed. The coefficients of variation of the intra-assay and inter-assay were both <10%. Importantly, this ELISA could detect antibodies in standard serum with 12,800-fold dilution, and seroconversion started from the 7th day post-inoculation (dpi), showing excellent analytical sensitivity and great utility. Furthermore, compared to the commercial kit, this ELISA had a good agreement and significantly shorter operation time. Collectively, a novel one-step DAgS-ELISA for detecting antibodies against ASFV is developed, which will be reliable and convenient to monitor ASFV infection.

Discontinuation of tyrosine kinase inhibitors based on BCR-ABL1 monitoring by digital droplet PCR in pediatric chronic myeloid leukemia.

Lifelong treatment of pediatric chronic myeloid leukemia (CML) patients with tyrosine kinase inhibitors (TKIs) can affect their growth and development. For these reasons, clinical trials have explored the feasibility of TKI discontinuation in children with a sufficient TKI response. We evaluated the analytical performance of digital droplet PCR (ddPCR) to quantify BCR-ABL1 and compared the results with reverse transcription quantitative polymerase chain reaction (RT-qPCR). We further investigated whether ddPCR could be used to determine TKI discontinuation in a clinical setting. Performance of ddPCR was evaluated using standard materials for BCR-ABL1, and a total of 197 clinical samples from 45 pediatric CML patients was included for comparison with RT-qPCR. ddPCR showed excellent analytical sensitivity with 0.001% international scale (IS) and linearity with R2 > 0.99 in log scale. BCR-ABL1 % IS results correlated well with those of RT-qPCR (R2 = 0.9435), however, they showed a moderate strength for agreement with a Cohen's kappa of 0.41 due to higher sensitivity of ddPCR. Among 45 pediatric CML patients, 42 were treated with first-line TKIs including imatinib (n = 27, 64%) and dasatinib (n = 12, 29%), and three patients that were started with imatinib were switched to dasatinib. When we evaluated whether follow-up samples fulfilled ABL1 copies ≥ 10,000 required for deep molecular response (DMR), all samples were acceptable by ddPCR, whereas 18% by RT-qPCR did not reached acceptable ABL1 copies. Moreover, 52 and 13% reached ABL1 copies ≥ 32,000 required for MR4.5 by ddPCR and RT-qPCR, respectively. Seven patients discontinued TKI and the median TKI treatment duration was 73 months prior to discontinuation. Prior to discontinuation, the median duration of sustained undetected BCR-ABL1 was 60 months. Two patients experienced loss of major MR (MMR) during follow-up and restarted dasatinib 5 months after discontinuation. They achieved MMR again and maintained better than DMR afterward. Results from those patients demonstrated that RT-qPCR did not match the need for adequate ABL1 copies for MR4.5 while majority of ddPCR could. Therefore, ddPCR was technically more acceptable to decide and monitor pediatric CML patients before and after TKI discontinuation.

Development and Clinical Applications of a 5-Plex Real-Time RT-PCR for Swine Enteric Coronaviruses.

A PEDV/PDCoV/TGEV/SADS-CoV/XIPC 5-plex real-time RT-PCR was developed and validated for the simultaneous detection and differentiation of four swine enteric coronaviruses (PEDV, PDCoV, TGEV and SADS-CoV) in one PCR reaction (XIPC serves as an exogenous internal positive control). The 5-plex PCR had excellent analytical specificity, analytical sensitivity, and repeatability based on the testing of various viral and bacterial pathogens, serial dilutions of virus isolates, and in vitro transcribed RNAs. The 5-plex PCR had comparable diagnostic performance to a commercial PEDV/TGEV/PDCoV reference PCR, based on the testing of 219 clinical samples. Subsequently, 1807 clinical samples collected from various U.S. states during 2019–2021 were tested by the 5-plex PCR to investigate the presence of SADS-CoV in U.S. swine and the frequency of detecting swine enteric CoVs. All 1807 samples tested negative for SADS-CoV. Among the samples positive for swine enteric CoVs, there was a low frequency of detecting TGEV, an intermediate frequency of detecting PDCoV, and a high frequency of detecting PEDV. Although there is no evidence of SADS-CoV presence in the U.S. at present, the availability of the 5-plex PCR will enable us to conduct ongoing surveillance to detect and differentiate these viruses in swine samples and other host species samples as some of these coronaviruses can cause cross-species infection.

Diagnostic potential of nanoparticle aided assays for MUC16 and MUC1 glycovariants in ovarian cancer.

Our study reports the discovery and evaluation of nanoparticle aided sensitive assays for glycovariants of MUC16 and MUC1 in a unique collection of paired ovarian cyst fluids and serum samples obtained at or prior to surgery for ovarian carcinoma suspicion. Selected glycovariants and the immunoassays for CA125, CA15‐3 and HE4 were compared and validated in 347 cyst fluid and serum samples. Whereas CA125 and CA15‐3 performed poorly in cyst fluid to separate carcinoma and controls, four glycovariants including MUC16MGL, MUC16STn, MUC1STn and MUC1Tn provided highly improved separations. In serum, the two STn glycovariants outperformed conventional CA125, CA15‐3 and HE4 assays in all subcategories analyzed with main benefits obtained at high specificities and at postmenopausal and early‐stage disease. Serum MUC16STn performed best at high specificity (90%‐99%), but sensitivity was also improved by the other glycovariants and CA15‐3. The highly improved specificity, excellent analytical sensitivity and robustness of the nanoparticle assisted glycovariant assays carry great promise for improved identification and early detection of ovarian carcinoma in routine differential diagnostics.

Evaluation of accuracy, exclusivity, limit-of-detection and ease-of-use of LumiraDx\u2122: An antigen-detecting point-of-care device for SARS-CoV-2

PurposeRapid antigen-detecting tests (Ag-RDTs) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can transform pandemic control. Thus far, sensitivity (≤ 85%) of lateral-flow assays has limited scale-up. Conceivably, microfluidic immunofluorescence Ag-RDTs could increase sensitivity for SARS-CoV-2 detection.MethodsThis multi-centre diagnostic accuracy study investigated performance of the microfluidic immunofluorescence LumiraDx™ assay, enrolling symptomatic and asymptomatic participants with suspected SARS-CoV-2 infection. Participants collected a supervised nasal mid-turbinate (NMT) self-swab for Ag-RDT testing, in addition to a professionally collected nasopharyngeal (NP) swab for routine testing with reverse transcriptase polymerase chain reaction (RT-PCR). Results were compared to calculate sensitivity and specificity. Sub-analyses investigated the results by viral load, symptom presence and duration. An analytical study assessed exclusivity and limit-of-detection (LOD). In addition, we evaluated ease-of-use.ResultsThe study was conducted between November 2nd 2020 and 4th of December 2020. 761 participants were enrolled, with 486 participants reporting symptoms on testing day. 120 out of 146 RT-PCR positive cases were detected positive by LumiraDx™, resulting in a sensitivity of 82.2% (95% CI 75.2–87.5%). Specificity was 99.3% (CI 98.3–99.7%). Sensitivity was increased in individuals with viral load ≥ 7 log10 SARS-CoV2 RNA copies/ml (93.8%; CI 86.2–97.3%). Testing against common respiratory commensals and pathogens showed no cross-reactivity and LOD was estimated to be 2–56 PFU/mL. The ease-of-use-assessment was favourable for lower throughput settings.ConclusionThe LumiraDx™ assay showed excellent analytical sensitivity, exclusivity and clinical specificity with good clinical sensitivity using supervised NMT self-sampling.Trial registration number and registration dateDRKS00021220 and 01.04.2020

An enzyme-free three-dimensional DNA walker powered by catalytic hairpin assembly for H5N1 DNA ratiometric detection

The reliable and quantitative detection of virus DNA is highly desirable for the early diagnosis and clinical treatment of diseases. In this work, a three-dimensional (3D) DNA walker based on a non-enzyme-mediated nucleic acid cascade amplification reaction was constructed for the ratiometric detection of H5N1 DNA. When the DNA walker was developed by the surface modification of aminated silica microsphere (SiO2-NH2) with H1 hairpin structures were activated in the presence of target DNA, the fluorescence of the H1-conjugated Thioflavin T (ThT) fluorophore (495 nm) was turned on, while the fluorescence of 2-aminopurine (2-AP) (363 nm) conjugated to the H2 hairpin remained unchanged. This strategy combines DNA walker powered by catalyzed hairpin assembly (CHA) reaction with proportional fluorescence signal output methods, which can effectively reduce the risk of generating false-positive signals. The developed DNA walker demonstrated excellent analytical sensitivity and specificity, with a detection limit of 60 pM. The DNA walker was also capable of selectively detecting H5N1 DNA in clinical blood serum samples, which demonstrated its potential for use in various clinical applications.

A rapid near-patient detection system for SARS-CoV-2 using saliva

The highly infectious nature of SARS-CoV-2 necessitates the use of widespread testing to control the spread of the virus. Presently, the standard molecular testing method (reverse transcriptase-polymerase chain reaction, RT-PCR) is restricted to the laboratory, time-consuming, and costly. This increases the turnaround time for getting test results. This study sought to develop a rapid, near-patient saliva-based test for COVID-19 (Saliva-Dry LAMP) with similar accuracy to that of standard RT-PCR tests. A lyophilized dual-target reverse transcription-loop-mediated isothermal amplification (RT-LAMP) test with fluorometric detection by the naked eye was developed. The assay relies on dry reagents that are room temperature stable. A device containing a centrifuge, heat block, and blue LED light system was manufactured to reduce the cost of performing the assay. This test has a limit of detection of 1 copy/µL and achieved a positive percent agreement of 100% [95% CI 88.43% to 100.0%] and a negative percent agreement of 96.7% [95% CI 82.78–99.92%] relative to a reference standard test. Saliva-Dry LAMP can be completed in 105 min. Precision, cross-reactivity, and interfering substances analysis met international regulatory standards. The combination of ease of sample collection, dry reagents, visual detection, low capital equipment cost, and excellent analytical sensitivity make Saliva-Dry LAMP particularly useful for resource-limited settings.

Comparative Analysis of the Analytical Sensitivity of ELISA Test System DIA®-SARS-CoV-2-Ag-R with Rapid Tests for Viral Antigen SARS-CoV-2 Detection

A novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease (COVID)-19, that emerged as a major pandemic. SARS-CoV-2 was identified as a betacoronavirus. Nucleocapsid protein (NP) is evolutionary high protein homologies and solid structure protein for SARS-CoV-2 detection as opposed to other proteins, that aren`t reliable as a single viral antigen during diagnostics methods. The viral RNA can be detected from nasal and pharyngeal swabs and bronchoalveolar lavage samples by PCR assay. However, the wrong collection of samples can lead to false-negative diagnosis and have dangerous consequences at this stage of pandemic. One of efficient and accurate methodological approaches for the screening of pathogens are serological assays. Aim. Evaluation and comparison of the sensitivity of invented DIA®-SARS-CoV-2-Ag-R enzyme-linked immunosorbent assay (ELISA)-based test system and commercial rapid tests, which detect the viral antigen of SARS-CoV-2. Methods. We carried out a comparison of DIA®-SARS-CoV-2-Ag-R and existed commercial test systems, which are used to detect the antigen of SARS-CoV-2 virus. Rapid tests are intended for nasopharyngeal swabs, but we proposed a protein of our own manufacture – recombinant NP as a calibrator. The detection limit was calibrated by standard CFAR #100982 NIBSC, UK. We had determined levels of NP (1400, 900, 750, 640 and 280 pg/mL) that we used as a sample for the rapid tests. The COVID-19 Ag Rapid Tests were performed according to the manufactures instructions at room temperature. Results. DIA®-SARS-CoV-2-Ag-R detected 10 pg/mL of in-house standard of recombinant SARS-CoV-2 NP. The positive results were observed using 1400, 900, 750 pg/mL, while 640 and 280 pg/mL samples were performed as negative in ABBOTT-PanBio test. The rapid tests manufactured by МBU, BIOTIME, Core Technology, SD BIOSENSOR and Turklab showed positive results only in 1400 pg/mL concentration. NP of lower levels was detected as a negative sample. The LEPU MEDICAL test was evaluated as positive sample using 900 pg/mL. The rapid test manufactured by Green Cross Medical Science Corp. showed negative results for all levels of NP. It can mean that the sensitivity of test is lower and demands higher level of antigen to detect the presence of SARS-CoV-2. Conclusions. The study presented an excellent analytical sensitivity of DIA®-SARS-CoV-2-Ag-R against commercial Antigen rapid tests. Thus, invented ELISA test system can be recommended for widespread usage for detection and confirmation of acute stage of SARS-CoV-2 infection.

Analytical Evaluation of Visby Medical RT-PCR Portable Device for Rapid Detection of SARS-CoV-2.

Extended community testing constitutes one of the main strategic pillars in controlling the COVID-19 pandemic. Reverse transcription PCR (RT-PCR) targeting the SARS-CoV-2 genome on nasopharyngeal swab samples is currently the reference test. While displaying excellent analytical sensitivity and specificity, this test is costly, often requires a substantial turnaround time, and, more importantly, is subject to reagent and other material shortages. To complement this technology, rapid antigen tests have been developed and made available worldwide, allowing cheap, quick, and decentralized SARS-CoV-2 testing. The main drawback of these tests is the reduced sensitivity when RT-PCR is the gold standard. In this study, we evaluate Visby an innovative, portable, easy-to-use RT-PCR point-of-care (POC) diagnostic device. Our retrospective analysis shows that overall, compared to the Cobas 6800 RT-qPCR assay (Roche), this RT-PCR POC technology detects SARS-CoV-2 RNA with 95% sensitivity (95%CI = 86.3–99%) and 100% specificity (95% CI = 80.5–100%). For samples with cycle-threshold values below 31, we observed 100% sensitivity (95% CI = 66.4–100%). While showing an analytical sensitivity slightly below that of a standard RT-qPCR system, the evaluated Visby RT-PCR POC device may prove to be an interesting diagnostic alternative in the COVID-19 pandemic, potentially combining the practical advantages of rapid antigen tests and the robust analytical performances of nucleic acid detection systems.

Clinical Performance and Analytical Sensitivity of Three SARS-CoV-2 Nucleic Acid Diagnostic Tests.

ABSTRACTHundreds of RT-qPCR kits are available in the market for SARS-CoV-2 diagnosis, some of them with emergency use authorization (EUA) by the Food Drug Administration (FDA) or their country of origin agency, but also many of them without any independent clinical performance evaluation. We performed a clinical evaluation for two Chinese SARS-CoV-2 RT-PCR kits available in South America, COVID-19 Nucleic Acid Test Kit (eDiagnosis Biomedicine, Wuhan, China) and 2019-nCoV Nucleic Acid Diagnostic Kit (Sansure Biotech, Changsha, China), for RT-qPCR SARS-CoV-2 diagnosis using the FDA EUA 2019-nCoV CDC kit (IDT, Coralville, IA) as gold standard. We found an excellent clinical performance and analytical sensitivity for both kits with sensitivity values of 100% and 95.3% and estimated limit of detection of 500 copies/mL and 1,000 copies/mL, for eDiagnosis and Sansure Biotech kits, respectively. COVID-19 Nucleic Acid Test Kit (eDiagnosis) and 2019-nCoV Nucleic Acid Diagnostic Kit (Sansure Biotech) are both made in China and hold EUA by the Chinese CDC. Also, Sansure Biotech kit has EUA by the FDA. In conclusion, our results endorse the use of these two commercially available kits imported to Ecuador for SARS-CoV-2 diagnosis, as they had the similar clinical performance as the gold standard from the CDC.

Evaluation of a commercial microbial enrichment kit used prior DNA extraction to improve the molecular detection of vector-borne pathogens from naturally infected dogs

Accurate detection of vector-borne pathogens (VBPs) is extremely important as the number of reported cases in humans and animals continues to rise in the US and abroad. Validated PCR assays are currently the cornerstone of molecular diagnostics and can achieve excellent analytical sensitivity and specificity. However, the detection of pathogens at low parasitemia still presents a challenge for VBP diagnosis, especially given the very low volume of specimens tested by molecular methods. The objective of this study is to determine if a commercially available microbial enrichment kit, used prior DNA extraction, is capable of expanding the overall microbial community and increasing detectable levels of VBPs in canine blood samples through host DNA depletion. This study used EDTA-whole blood samples from dogs naturally infected with varying parasitemia levels of either Anaplasma phagocytophilum, Babesia gibsoni, or Ehrlichia ewingii. For two VBPs, EDTA-blood samples were diluted to determine the effect of microbial concentration at low parasitemia. Paired EDTA-blood samples from each dog were subjected to traditional, automated DNA extraction with or without the microbial concentrating kit (MolYsis®) prior DNA extraction. Relative amounts of pathogen DNA in paired samples were determined by real-time PCR and Next-Generation Sequencing targeting conserved regions of 16S rRNA (for bacteria) and 18S rRNA (for protozoa). Results from the three molecular methods suggest that the microbial concentrating kit did not improve the detection of VBPs, although significantly reduced the presence of host DNA. Alternative methods for VBP enrichment in clinical samples prior to molecular testing should continue to be investigated, as it may significantly improve clinical sensitivity and reduce the number of false-negative results.