Journal articles: 'Diagnostic Assay Development'

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Pang, Junxiong, Po Ying Chia, David C. Lye, and Yee Sin Leo. "Progress and Challenges towards Point-of-Care Diagnostic Development for Dengue." Journal of Clinical Microbiology 55, no. 12 (September 13, 2017): 3339–49. http://dx.doi.org/10.1128/jcm.00707-17.

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ABSTRACTDengue detection strategies involve viral RNA, antigen, and/or antibody detection. Each strategy has its advantages and disadvantages. Optimal, user-friendly, rapid diagnostic tests based on immunochromatographic assays are pragmatic point-of-care tests (POCTs) in regions where dengue is endemic where there are limited laboratory capabilities and optimal storage conditions. Increasingly, there is a greater public health significance for a multiplexing assay that differentiates dengue from Zika or pathogens with similar clinical presentations. Although there have been many assay/platform developments toward POCTs, independent validation and implementation remain very limited. This review highlights the current key progress and challenges toward the development of a dengue POCT.

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Klosterman, Katja E., Kiersten D. Lenz, Harshini Mukundan, and Jessica Kubicek-Sutherland. "Biophysical Characterization of Human Lipoproteins for Diagnostic Assay Development." Biophysical Journal 120, no. 3 (February 2021): 232a. http://dx.doi.org/10.1016/j.bpj.2020.11.1538.

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Roberts, Christine, and Joel Maslow. "Assay Challenges for Emerging Infectious Diseases: The Zika Experience." Vaccines 6, no. 4 (October 2, 2018): 70. http://dx.doi.org/10.3390/vaccines6040070.

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From the perspective of vaccine development, it is imperative to accurately diagnose target infections in order to exclude subjects with prior exposure from evaluations of vaccine effectiveness, to track incident infection during the course of a clinical trial and to differentiate immune reactions due to natural infections from responses that are vaccine related. When vaccine development is accelerated to a rapid pace in response to emerging infectious disease threats, the challenges to develop such diagnostic tools is even greater. This was observed through the recent expansion of Zika virus infections into the Western Hemisphere in 2014–2017. When initial Zika vaccine clinical trials were being designed and launched in response to the outbreak, there were no standardized sets of viral and immunological assays, and no approved diagnostic tests for Zika virus infection. The diagnosis of Zika virus infection is still an area of active research and development on many fronts. Here we review emerging infectious disease vaccine clinical assay development and trial execution with a special focus on the state of Zika virus clinical assays and diagnostics.

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Widiyanti, Dian, Nobuo Koizumi, Takashi Fukui, Lisa T. Muslich, Takaya Segawa, Sharon Y. A. M. Villanueva, Mitsumasa Saito, Toshiyuki Masuzawa, Nina G. Gloriani, and Shin-ichi Yoshida. "Development of Immunochromatography-Based Methods for Detection of Leptospiral Lipopolysaccharide Antigen in Urine." Clinical and Vaccine Immunology 20, no. 5 (March 6, 2013): 683–90. http://dx.doi.org/10.1128/cvi.00756-12.

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ABSTRACTLeptospirosis is an infectious disease caused by the spirochete bacteriaLeptospiraspp. and is commonly found throughout the world. Diagnosis of leptospirosis performed by culture and microscopic agglutination tests is laborious and time-consuming. Therefore, we aimed to develop a novel immunochromatography (ICG)-based method for detectingLeptospiraantigen in the urine of patients and animals. We used the 1H6 monoclonal antibody (MAb), which is specific to the lipopolysaccharide (LPS) that is common amongLeptospiraspp. The MAb was coupled to 40-nm-diameter colloidal gold, and the amounts of labeled antibody and immobilized antibody were 23 μg and 2 μg per test, respectively. Several strains ofLeptospiraand other bacterial species were used to evaluate the sensitivities and specificities of the assays we developed. The detection limit of the assays was 106cells/ml when disrupted whole bacterial cells were used. The assays wereLeptospiraspecific since they did not cross-react with non-Leptospirabacteria used in the study. Application of diagnostic assays was done on the urine samples of 46Leptospira-infected hamsters, 44 patients with suspected leptospirosis, and 14 healthy individuals. Pretreatment of the urine samples by boiling and centrifugation (for ultrafiltration and concentration) eliminated nonspecific reactions that occurred in the assay. The sensitivity and specificity of the ICG-based lateral flow assay (LFA) were 89% and 87%, respectively, which were higher than those of the dipstick assay, which were 80% and 74%, respectively. In summary, this ICG-based LFA can be used as an alternative diagnostic assay for leptospirosis. Further development is still necessary to improve the assay.

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Liu, Hongna, Kathryn Heflin, Jian Han, Matt Conover, Leslie Wagner, Jeff Bertrand, Phillip Ewing, et al. "Development of the iCubate Molecular Diagnostic Platform Utilizing Amplicon Rescue Multiplex Polymerase Chain Reaction." Journal of Biomedical Nanotechnology 15, no. 7 (July 1, 2019): 1598–608. http://dx.doi.org/10.1166/jbn.2019.2783.

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We utilized Amplicon-Rescue Multiplex PCR (ARM-PCR) and microarray hybridization to develop and validate the iC-GPC Assay, a multiplexed, in vitro diagnostic test that identifies five of the most common gram positive bacteria and three clinically relevant resistance markers associated with bloodstream infections (BSI). The iC-GPC Assay is designed for use with the iC-System™, which automates sample preparation, ARM-PCR, and microarray detection within a closed cassette. Herein, we determined the limit of detection for each of the iC-GPC Assay targets to be between 3.0 × 105–1.7 × 107 CFU/mL, well below clinically relevant bacterial levels for positive blood cultures. Additionally, we tested 106 strains for assay inclusivity and observed a target performance of 99.4%. 95 of 96 non-target organisms tested negative for cross-reactivity, thereby assuring a high level of assay specificity. Overall performance above 99% was observed for iC-GPC Assay reproducibility studies across multiple sites, operators and cassette lots. In conclusion, the iC-GPC Assay is capable of accurately and rapidly identifying bacterial species and resistance determinants present in blood cultures containing gram positive bacteria. Utilizing molecular diagnostics like the iC-GPC Assay will decrease time to treatment, healthcare costs, and BSI-related mortality.

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Al-Yousif, Yousif, Fahad Al-Majhdi, Cindy Chard-Bergstrom, Joe Anderson, and Sanjay Kapil. "Development, Characterization, and Diagnostic Applications of Monoclonal Antibodies against Bovine Rotavirus." Clinical Diagnostic Laboratory Immunology 7, no. 2 (March 1, 2000): 288–92. http://dx.doi.org/10.1128/cdli.7.2.288-292.2000.

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ABSTRACT Hybridomas secreting monoclonal antibodies (MAbs) against the Nebraska calf diarrhea strain of bovine rotavirus (BRV) were characterized. Indirect fluorescent-antibody assay, immunodot assay, and immunoprecipitation were used to select hybridomas that produced anti-BRV MAbs. Seven of the MAbs were shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot assay to be reactive with the BRV outer capsid protein, VP7, which has a molecular mass of 37.5 kDa. None of the seven MAbs were reactive with canine rotavirus, bovine coronavirus, or uninfected Madin-Darby bovine kidney cells. Two clones, 8B4 (immunoglobulin G2a [IgG2a]) and 2B11 (IgG1), were found suitable for use in an antigen capture enzyme-linked immunosorbent assay for detecting BRV in bovine fecal samples. Both were subtype A specific (G6 subtype) but did not react with all isolates of BRV group A.

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Oertelt-Prigione, S., R. Rieger, C. Selmi, P. Tnvertiizzi, M. Podda, and M. E. Gershwin. "[673] DEVELOPMENT OF A NOVEL DIAGNOSTIC ASSAY FOR PRIMARY BILIARY CIRRHOSIS." Journal of Hepatology 46 (April 2007): S255. http://dx.doi.org/10.1016/s0168-8278(07)62271-5.

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Szafranska-Schwarzbach, Anna E., Alex T. Adai, Linda S. Lee, Darwin L. Conwell, and Bernard F. Andruss. "Development of a miRNA-based diagnostic assay for pancreatic ductal adenocarcinoma." Expert Review of Molecular Diagnostics 11, no. 3 (April 2011): 249–57. http://dx.doi.org/10.1586/erm.11.10.

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Phillippy, A. M., K. Ayanbule, N. J. Edwards, and S. L. Salzberg. "Insignia: a DNA signature search web server for diagnostic assay development." Nucleic Acids Research 37, Web Server (May 5, 2009): W229—W234. http://dx.doi.org/10.1093/nar/gkp286.

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Pieck, Michael L., Amy Ruck, Mark L. Farman, Gary L. Peterson, James P. Stack, Barbara Valent, and Kerry F. Pedley. "Genomics-Based Marker Discovery and Diagnostic Assay Development for Wheat Blast." Plant Disease 101, no. 1 (January 2017): 103–9. http://dx.doi.org/10.1094/pdis-04-16-0500-re.

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Wheat blast has emerged as a major threat to wheat production in South America. Although originally restricted to Brazil, the disease has since been observed in the neighboring countries of Argentina, Bolivia, and Paraguay and recently the pathogen, Magnaporthe oryzae Triticum pathotype, was isolated from infected wheat in Bangladesh. There is growing concern that the pathogen may continue to spread to other parts of the world, including the United States, where several M. oryzae pathotypes are endemic. M. oryzae pathotypes are morphologically indistinguishable and, therefore, must be characterized genotypically. Symptoms of wheat blast include bleaching of the head, which closely resembles the symptoms of Fusarium head blight, further complicating efforts to monitor for the presence of the pathogen in the field. We used a genomics-based approach to identify molecular markers unique to the Triticum pathotype of M. oryzae. One of these markers, MoT3, was selected for the development of a polymerase chain reaction (PCR)-based diagnostic assay that was evaluated for specificity using DNA from 284 M. oryzae isolates collected from a diverse array of host species. Conventional PCR primers were designed to amplify a 361-bp product, and the protocol consistently amplified from as little as 0.1 ng of purified DNA. The specificity of the MoT3-based assay was also evaluated using Fusarium spp. DNA, from which no amplicons were detected.

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Luster, Douglas G., Michael B. McMahon, Melissa L. Carter, Aaron J. Sechler, Elizabeth E. Rogers, Brenda K. Schroeder, and Timothy D. Murray. "Immunoreagents for development of a diagnostic assay specific for Rathayibacter toxicus." Food and Agricultural Immunology 31, no. 1 (January 1, 2020): 231–42. http://dx.doi.org/10.1080/09540105.2020.1714554.

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Eimanifar, Amin, John Aufderheide, Suzanne Z. Schneider, Henry Krueger, and Sean Gallagher. "Development of an in vitro diagnostic method to determine the genotypic sex of Xenopus laevis." PeerJ 7 (May 1, 2019): e6886. http://dx.doi.org/10.7717/peerj.6886.

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A genotypic sex determination assay provides accurate gender information of individuals with well-developed phenotypic characters as well as those with poorly developed or absent of phenotypic characters. Determination of genetic sex for Xenopus laevis can be used to validate the outcomes of Tier 2 amphibian assays, and is a requirement for conducting the larval amphibian growth and development assay (LAGDA), in the endocrine disruptor screening program (EDSP), test guidelines. The assay we developed uses a dual-labeled TaqMan probe-based real-time polymerase chain reaction (real-time PCR) method to determine the genotypic sex. The reliability of the assay was tested on 37 adult specimens of X. laevis collected from in-house cultures in Eurofins EAG Agroscience, Easton. The newly designed X. laevis-specific primer pair and probe targets the DM domain gene linked-chromosome W as a master female-determining gene. Accuracy of the molecular method was assessed by comparing with phenotypic sex, determined by necropsy and histological examination of gonads for all examined specimens. Genotypic sex assignments were strongly concordant with observed phenotypic sex, confirming that the 19 specimens were male and 18 were female. The results indicate that the TaqMan® assay could be practically used to determine the genetic sex of animals with poorly developed or no phenotypic sex characteristics with 100% precision. Therefore, the TaqMan® assay is confirmed as an efficient and feasible method, providing a diagnostic molecular sex determination approach to be used in the amphibian endocrine disrupting screening programs conducted by regulatory industries. The strength of an EDSP is dependent on a reliable method to determine genetic sex in order to identify reversals of phenotypic sex in animals exposed to endocrine active compounds.

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Song, Yunfeng, Pankaj Singh, Eric Nelson, and Sheela Ramamoorthy. "A Computationally Designed Serological Assay for Porcine Epidemic Diarrhea Virus." Journal of Clinical Microbiology 54, no. 8 (May 25, 2016): 2039–46. http://dx.doi.org/10.1128/jcm.00460-16.

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The periodic emergence of new infectious agents and the genetic and antigenic evolution of existing agents necessitate the improvement of technology for the rapid development of diagnostic assays. The porcine epidemic diarrhea virus (PEDV) emerged in the United States in 2013, causing severe economic damage to the pork industry. The primary goal of this study was to develop methods to reduce the lead time for serological assay development. An approach involving the computational prediction of diagnostic targets, followed by a rapid synthesis of antigens, was adopted to achieve this objective. To avoid cross-reactivity with other closely related swine coronaviruses, the N protein sequences of PEDV were analyzed to identify sequences unique to PEDV. The potential antigenicity of the identified sequence was predicted computationally using the Jameson-Wolf method. A sequence with a high antigenic index was rapidly synthesized using anin vitrotranscription and translation system to yield the diagnostic antigen. The computationally designed enzyme-linked immunosorbent assay (ELISA) was validated using 169 field sera, whose statuses were determined by a PEDV-specific immunofluorescence assay. Comparison of the computationally designed ELISA to a conventionally developed ELISA, using bacterially expressed N protein, and to the immunofluorescence assay showed a high degree of agreement among the three tests (mean kappa statistic, 0.842). The sensitivity and specificity, compared to the conventionally developed assay, were 90.62 and 95.18, respectively. Therefore, the described approach is useful in reducing the development time for serological assays in the face of an infectious disease outbreak.

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Doan, Hung K., Shulu Zhang, and R. Michael Davis. "Development and Evaluation of AmplifyRP Acceler8 Diagnostic Assay for the Detection of Fusarium oxysporum f. sp. vasinfectum Race 4 in Cotton." Plant Health Progress 15, no. 1 (January 2014): 48–52. http://dx.doi.org/10.1094/php-rs-13-0115.

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A rapid and reliable molecular diagnostic assay, AmplifyRP Acceler8, was developed for the direct detection of Fusarium oxysporum f. sp. vasinfectum (FOV) race 4, a virulent genotype of the Fusarium wilt pathogen of cotton (Gossypium spp.), in soil and cotton tissue. Unlike traditional polymerase chain reaction (PCR) assays, the recombinase polymerase amplification based-assay described here utilizes an advanced isothermal technology where the amplification is carried out at a single constant temperature, 39°C, without the need of a thermal cycler. The AmplifyRP Acceler8 diagnostic assay consistently detected FOV race 4 from all infected tissue samples. The test is rapid, simple, and more sensitive than conventional PCR. The AmplifyRP Acceler8 diagnostic assay detected DNA from FOV race 4 at concentrations of 1 ng/μL and above. In addition, it did not amplify DNA from other known FOV races (races 1, 2, 3, 6, and 8). The whole process from sample preparation to reading the results can be completed in as little as 30 min. The test can detect FOV race 4 from cotton taproots, petioles, and stems. Accepted for publication 6 January 2014. Published 15 March 2014.

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Tencza, Sarah Burroughs, Kazi R. Islam, Vandana Kalia, Mohammad S. Nasir, Michael E. Jolley, and Ronald C. Montelaro. "Development of a Fluorescence Polarization-Based Diagnostic Assay for Equine Infectious Anemia Virus." Journal of Clinical Microbiology 38, no. 5 (2000): 1854–59. http://dx.doi.org/10.1128/jcm.38.5.1854-1859.2000.

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The control of equine infectious anemia virus (EIAV) infections of horses has been over the past 20 years based primarily on the identification and elimination of seropositive horses, predominantly by a standardized agar gel immunodiffusion (AGID) assay in centralized reference laboratories. This screening for EIAV-seropositive horses has been to date hindered by the lack of a rapid diagnostic format that can be easily employed in the field. We describe here the development of a rapid solution-phase assay for the presence of serum antibodies to EIAV based on fluorescence polarization (FP) (patent pending). Peptides derived from antigenic regions of EIAV core and envelope proteins were initially screened for their utility as probes in an FP assay to select the best peptide antigen candidates. The FP assay was optimized to detect the presence of EIAV-specific antibodies by a change in the FP of a fluorescein-labeled immunoreactive peptide diagnostic antigen. The most sensitive and specific peptide probe was a peptide corresponding to the immunodominant region of the EIAV transmembrane protein, gp45. This probe was tested for its reactivity in the optimized FP assay with 151 AGID-positive horse sera and 106 AGID-negative serum samples. The results of these studies demonstrated that the FP assay reactivity correlated with reported AGID results in 106 of 106 negative serum samples (100% specificity) and in 135 of 151 positive serum samples (89.4% sensitivity). The FP assay was also found to have a very low background reactivity and to readily detect antibodies produced early in infection (≤3 weeks postinfection). The developed EIAV FP assay is rapid (5 to 20 min) and simple to perform and is equally suitable for use both in the field and in the diagnostic laboratory, thus providing the basis of an improved commercial diagnostic assay for EIAV infection of horses.

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Kelly-Cirino, Cassandra, Laura T. Mazzola, Arlene Chua, Christopher J. Oxenford, and Maria D. Van Kerkhove. "An updated roadmap for MERS-CoV research and product development: focus on diagnostics." BMJ Global Health 4, Suppl 2 (January 2019): e001105. http://dx.doi.org/10.1136/bmjgh-2018-001105.

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Diagnostics play a central role in the early detection and control of outbreaks and can enable a more nuanced understanding of the disease kinetics and risk factors for the Middle East respiratory syndrome-coronavirus (MERS-CoV), one of the high-priority pathogens identified by the WHO. In this review we identified sources for molecular and serological diagnostic tests used in MERS-CoV detection, case management and outbreak investigations, as well as surveillance for humans and animals (camels), and summarised the performance of currently available tests, diagnostic needs, and associated challenges for diagnostic test development and implementation. A more detailed understanding of the kinetics of infection of MERS-CoV is needed in order to optimise the use of existing assays. Notably, MERS-CoV point-of-care tests are needed in order to optimise supportive care and to minimise transmission risk. However, for new test development, sourcing clinical material continues to be a major challenge to achieving assay validation. Harmonisation and standardisation of laboratory methods are essential for surveillance and for a rapid and effective international response to emerging diseases. Routine external quality assessment, along with well-characterised and up-to-date proficiency panels, would provide insight into MERS-CoV diagnostic performance worldwide. A defined set of Target Product Profiles for diagnostic technologies will be developed by WHO to address these gaps in MERS-CoV outbreak management.

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Sharypova, D. V., O. V. Kapustina, I. Yu Zhukov, N. N. Vlasova, and A. S. Igolkin. "DEVELOPMENT AND IMPROVEMENT OF ASF SEROLOGICAL DIAGNOSTIC TECHNIQUES." Veterinary Science Today, no. 2 (June 28, 2019): 30–34. http://dx.doi.org/10.29326/2304-196x-2019-2-29-30-34.

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Due to the lack of effective tools of ASF specific prevention it is evident that early diagnosis is one of the most important and resultative ways of the disease control. However, contemporary diagnosis is a complex component of any effective surveillance system. Latest scientific achievements facilitated not only highly specific and sensitive but also rapid methods of laboratory diagnosis. Nevertheless, further development, improvement and expansion of ASF diagnosis techniques including rapid tests is a topical task of a great concern. The research is devoted to development of rapid test methods for rapid detection of antibodies to ASFV in blood sera of infected animals as well as to analysis of their use effectiveness. The following methods were suggested: immunoperoxidase monolayer assay using fixed cell line (ASFV permissive CV-1 cell-line infected with the virus strain ASF/ARRIAH/CV-1) and latex agglutination test using ASFV p30 recombinant protein. The performed research demonstrated the effectiveness of the applied techniques for ASF serological diagnosis. Latex agglutination test and immunoperoxidase monolayer assay give rapid and high quality test results (within 1–2 hours). The advantage of the specified methods as compared to ELISA is their simplicity and the possibility of use in conditions of limited technical support.

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Reddington, Kate, Stefan Schwenk, Nina Tuite, Gareth Platt, Danesh Davar, Helena Coughlan, Yoann Personne, et al. "Comparison of Established Diagnostic Methodologies and a Novel BacterialsmpBReal-Time PCR Assay for Specific Detection of Haemophilus influenzae Isolates Associated with Respiratory Tract Infections." Journal of Clinical Microbiology 53, no. 9 (June 24, 2015): 2854–60. http://dx.doi.org/10.1128/jcm.00777-15.

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Haemophilus influenzaeis a significant causative agent of respiratory tract infections (RTI) worldwide. The development of a rapidH. influenzaediagnostic assay that would allow for the implementation of infection control measures and also improve antimicrobial stewardship for patients is required. A number of nucleic acid diagnostics approaches that detectH. influenzaein RTIs have been described in the literature; however, there are reported specificity and sensitivity limitations for these assays. In this study, a novel real-time PCR diagnostic assay targeting thesmpBgene was designed to detect all serogroups ofH. influenzae. The assay was validated using a panel of well-characterizedHaemophilusspp. Subsequently, 44Haemophilusclinical isolates were collected, and 36 isolates were identified asH. influenzaeusing a gold standard methodology that combined the results of matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) and afucKdiagnostic assay. Using the novelsmpBdiagnostic assay, 100% concordance was observed with the gold standard, demonstrating a sensitivity of 100% (95% confidence interval [CI], 90.26% to 100.00%) and a specificity of 100% (95% CI, 63.06% to 100.00%) when used on clinical isolates. To demonstrate the clinical utility of the diagnostic assay presented, a panel of lower RTI samples (n= 98) were blindly tested with the gold standard andsmpBdiagnostic assays. The results generated were concordant for 94/98 samples tested, demonstrating a sensitivity of 90.91% (95% CI, 78.33% to 97.47%) and a specificity of 100% (95% CI, 93.40% to 100.00%) for the novelsmpBassay when used directly on respiratory specimens.

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Tomlins, S. A., P. Williams, S. Sadis, P. Wyngaard, K. Oades, B. Lee, S. Chattopadhyay, Y. Wang, J. Monforte, and D. Rhodes. "Association of gene expression module biomarkers with clinical and therapeutic endpoints and their use with a universal companion diagnostic assay." Journal of Clinical Oncology 29, no. 27_suppl (September 20, 2011): 228. http://dx.doi.org/10.1200/jco.2011.29.27_suppl.228.

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228 Background: Gene expression patterns are increasingly capable of stratifying patients based on prognosis and response to therapy. Given the limited availability of sample tissue, however, it is not feasible to utilize every test for every patient, suggesting the need for a universal companion diagnostic assay that is informative with respect to multiple clinical and therapeutic endpoints. Key challenges are identification of appropriate gene expression biomarkers, translation of biomarkers to clinical assays, and development of reliable gene expression profiling of formalin-fixed clinical specimens. Here we describe a novel RT-PCR biomarker assay optimized for FFPE clinical samples that has broad prognostic and predictive potential. Methods: A co-expression meta-analysis of 5,339 breast tumors from 56 microarray datasets identified highly co-expressed sets of genes (modules) across multiple datasets. Module biomarkers were tested for their ability to associate with prognostic and predictive targets in published datasets. In addition, each module was reduced from 10–1000 genes to 2-3 genes for use in companion diagnostic assays based on degree of co-expression across the meta-analysis, and validated against an independent panel of tumor samples. Results: This study demonstrates that a single test utilizing multiple module biomarkers is informative with respect to standard parameters such as ER, PR and Her2, and in addition reproduces existing prognostic and predictive genomic signatures. Furthermore, we show that modules of 10-1000 genes can be represented by 2-3 genes for direct use in companion diagnostics development. Conclusions: The molecular heterogeneity of breast cancer can be summarized by discrete gene expression modules that individually represent distinct biological programs, and that collectively can be represented by as few as 96 genes. Modules, together with outlier genes, allow for summation of the entire transcriptional program and provide a universal assay with broad application to companion diagnostics development.

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Bustin, Stephen A., and Reinhold Mueller. "Real-time reverse transcription PCR (qRT-PCR) and its potential use in clinical diagnosis." Clinical Science 109, no. 4 (September 23, 2005): 365–79. http://dx.doi.org/10.1042/cs20050086.

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qRT-PCR (real-time reverse transcription-PCR) has become the benchmark for the detection and quantification of RNA targets and is being utilized increasingly in novel clinical diagnostic assays. Quantitative results obtained by this technology are not only more informative than qualitative data, but simplify assay standardization and quality management. qRT-PCR assays are most established for the detection of viral load and therapy monitoring, and the development of SARS (severe acute respiratory syndrome)-associated coronavirus qRT-PCR assays provide a textbook example of the value of this technology for clinical diagnostics. The widespread use of qRT-PCR assays for diagnosis and the detection of disease-specific prognostic markers in leukaemia patients provide further examples of their usefulness. Their value for the detection of disease-associated mRNA expressed by circulating tumour cells in patients with solid malignancies is far less apparent, and the clinical significance of results obtained from such tests remains unclear. This is because of conceptual reservations as well as technical limitations that can interfere with the diagnostic specificity of qRT-PCR assays. Therefore, although it is evident that qRT-PCR assay has become a useful and important technology in the clinical diagnostic laboratory, it must be used appropriately and it is essential to be aware of its limitations if it is to fulfil its potential.

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Henry Sum, Magdline Sia, Siew Fung Yee, Lily Eng, Evenni Poili, and Julia Lamdin. "Development of an Indirect ELISA and Dot-Blot Assay for Serological Detection of Rice Tungro Disease." BioMed Research International 2017 (2017): 1–7. http://dx.doi.org/10.1155/2017/3608042.

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Rice tungro disease (RTD) is one of the most destructive diseases of rice in South and Southeast Asia. RTD is routinely detected based on visual observation of the plant. However, it is not always easy to identify the disease in the field as it is often confused with other diseases or physiological disorders. Here we report the development of two serological based assays for ease of detection of RTD. In this study we had developed and optimized an indirect ELISA and dot-blot assay for detection of RTD. The efficiency of both assays was evaluated by comparing the specificity and sensitivity of the assays to PCR assay using established primer sets. The indirect ELISA showed 97.5% and 96.6%, while the dot-blot assay showed 97.5% and 86.4% sensitivity and specificity, respectively, when compared to established PCR method. The high sensitivity and specificity of the two assays merit the use of both assays as alternative methods to diagnose RTD. Furthermore, the dot-blot assay is a simple, robust, and rapid diagnostic assay that is suitable for field test for it does not require any specialized equipment. This is a great advantage for diagnosing RTD in paddy fields, especially in the rural areas.

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Azam, Mudsser, Kirti Upmanyu, Ratan Gupta, Karugatharayil Sasi Sruthy, Monika Matlani, Deepali Savargaonkar, and Ruchi Singh. "Development of Two-Tube Loop-Mediated Isothermal Amplification Assay for Differential Diagnosis of Plasmodium falciparum and Plasmodium vivax and Its Comparison with Loopamp™ Malaria." Diagnostics 11, no. 9 (September 16, 2021): 1689. http://dx.doi.org/10.3390/diagnostics11091689.

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To strengthen malaria surveillance, field-appropriate diagnostics requiring limited technical resources are of critical significance. Loop-mediated isothermal amplification (LAMP) based malaria diagnostic assays are potential point-of-care tests with high sensitivity and specificity and have been used in low-resource settings. Plasmodium vivax–specific consensus repeat sequence (CRS)-based and Plasmodium falciparum–specific 18S rRNA primers were designed, and a two-tube LAMP assay was developed. The diagnostic performance of a closed-tube LAMP assay and Loopamp™ Malaria Detection (Pan/Pf, Pv) kit was investigated using nested PCR confirmed mono- and co-infections of P. vivax and P. falciparum positive (n = 149) and negative (n = 67) samples. The closed-tube Pv LAMP assay showed positive amplification in 40 min (limit of detection, LOD 0.7 parasites/µL) and Pf LAMP assay in 30 min (LOD 2 parasites/µL). Pv LAMP and Pf LAMP demonstrated a sensitivity and specificity of 100% (95% CI, 95.96–100% and 89.85–100%, respectively). The LoopampTM Pan/Pf Malaria Detection kit demonstrated a sensitivity and specificity of 100%, whereas LoopampTM Pv showed a sensitivity of 98.36% (95% CI, 91.28–99.71%) and specificity of 100% (95% CI, 87.54–100%). The developed two-tube LAMP assay is highly sensitive (LOD ≤ 2 parasite/µL), demonstrating comparable results with the commercial Loopamp™ Malaria Detection (Pf/pan) kit, and was superior in detecting the P. vivax co-infection that remained undetected by the Loopamp™ Pv kit. The developed indigenous two-tube Pf/Pv malaria detection can reliably be used for mass screening in resource-limited areas endemic for both P. falciparum and P. vivax malaria.

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Pattison, S., L. Oram, M. Crockard, M. M. Tunney, and J. S. Elborn. "85 Development of an in vitro molecular diagnostic assay for CF microbiology." Journal of Cystic Fibrosis 15 (June 2016): S72—S73. http://dx.doi.org/10.1016/s1569-1993(16)30324-1.

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Isaacs, Stephen T., John W. Tessman, Kenneth C. Metchette, John E. Hearst, and George D. Cimino. "Post-PCR sterilization: development and application to an HIV-1 diagnostic assay." Nucleic Acids Research 19, no. 1 (1991): 109–16. http://dx.doi.org/10.1093/nar/19.1.109.

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Gallia, Gary L., Joseph M. Petroziello, Joseph M. Brogan, Ferne K. McCleskey, and Vito G. DelVecchio. "Development of a diagnostic polymerase chain reaction assay for detection ofMycoplasma hominis." Molecular and Cellular Probes 9, no. 6 (December 1995): 415–21. http://dx.doi.org/10.1006/mcpr.1995.0064.

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Vennapusa, Bharathi, Brian Baker, Marcin Kowanetz, Jennifer Boone, Ina Menzl, Jean-Marie Bruey, Gregg Fine, et al. "Development of a PD-L1 Complementary Diagnostic Immunohistochemistry Assay (SP142) for Atezolizumab." Applied Immunohistochemistry & Molecular Morphology 27, no. 2 (February 2019): 92–100. http://dx.doi.org/10.1097/pai.0000000000000594.

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De Los Santos, Rose Ann, Josette William, Debra Ann Hanks, Adam Northrup, Dipeshkumar Jaiswal, Malinka Jansson, Therese Phillips, et al. "Development of a diagnostic PD-L1 immunohistochemical assay for nivolumab therapy in urothelial carcinoma." Journal of Clinical Oncology 35, no. 15_suppl (May 20, 2017): e14585-e14585. http://dx.doi.org/10.1200/jco.2017.35.15_suppl.e14585.

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e14585 Background: PD-L1 IHC 28-8 pharmDx is a qualitative assay developed by Agilent Technologies for the Autostainer Link 48 platform and is based on EnVision FLEX visualization technology and monoclonal rabbit anti-PD-L1, clone 28-8 antibody. The assay has been co-developed with the immunotherapeutic agent nivolumab; initially as an aid in assessing PD-L1 expression in non-squamous non-small cell lung cancer (NSCLC) and melanoma patients. Here we describe the efforts to validate this assay for urothelial carcinoma (UC). Methods: IHC staining was performed on Autostainer Link 48 platform using an automated staining protocol stated per the assay’s instruction for use (IFU). Specimens were coverslipped and interpreted for % PD-L1 positive tumor cells. The assay was analytically validated on commercially acquired formalin-fixed, paraffin-embedded (FFPE) human UC invasive tumor specimens at ≥1% and ≥5% PD-L1 positive tumor cells expression levels. Results: A wide range of % PD-L1 positive tumor cells at all staining intensity levels have been detected. Assay in-house precision was validated for inter-operator, inter-instrument, inter-day, inter-run and intra-run as well as inter-observer and intra-observer agreement. Robustness studies evaluated the assay under multiple conditions for target retrieval pH, temperature and incubation time, slide type as well as tissue section thickness. Assay reproducibility was evaluated at three external sites by testing samples for intra-site/inter-day and inter-site agreement measures. Specimens were also evaluated by an observer at each site, with three reads for each observer to assess intra-observer and inter-observer agreement. All validation studies demonstrated agreement estimates above 85% with values for lower bound 95% confidence intervals calculated above 84%. Conclusions: Results of all conducted studies show high robustness and reproducibility of the assay on UC.

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Emperador, Devy M., Laura T. Mazzola, Betsy Wonderly Trainor, Arlene Chua, and Cassandra Kelly-Cirino. "Diagnostics for filovirus detection: impact of recent outbreaks on the diagnostic landscape." BMJ Global Health 4, Suppl 2 (February 2019): e001112. http://dx.doi.org/10.1136/bmjgh-2018-001112.

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Ebolaviruses and Marburg virus (MARV) both belong to the family Filoviridae and cause severe haemorrhagic fever in humans. Due to high mortality rates and potential for spread from rural to urban regions, they are listed on the WHO R&D blueprint of high-priority pathogens. Recent ebolavirus outbreaks in Western and Central Africa have highlighted the importance of diagnostic testing in epidemic preparedness for these pathogens and led to the rapid development of a number of commercially available benchtop and point-of-care nucleic acid amplification tests as well as serological assays and rapid diagnostic tests. Despite these advancements, challenges still remain. While products approved under emergency use licenses during outbreak periods may continue to be used post-outbreak, a lack of clarity and incentive surrounding the regulatory approval pathway during non-outbreak periods has deterred many manufacturers from seeking full approvals. Waning of funding and poor access to samples after the 2014–2016 outbreak also contributed to cessation of development once the outbreak was declared over. There is a need for tests with improved sensitivity and specificity, and assays that can use alternative sample types could reduce the need for invasive procedures and expensive equipment, making testing in field conditions more feasible. For MARV, availability of diagnostic tests is still limited, restricted to a single ELISA test and assay panels designed to differentiate between multiple pathogens. It may be helpful to extend the target product profile for ebolavirus diagnostics to include MARV, as the viruses have many overlapping characteristics.

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Sharma, Rahul, Pushpendra Singh, Rajiv C. McCoy, Shannon M. Lenz, Kelly Donovan, Maria T. Ochoa, Iris Estrada-Garcia, et al. "Isolation of Mycobacterium lepromatosis and Development of Molecular Diagnostic Assays to Distinguish Mycobacterium leprae and M. lepromatosis." Clinical Infectious Diseases 71, no. 8 (November 16, 2019): e262-e269. http://dx.doi.org/10.1093/cid/ciz1121.

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Abstract Background Mycobacterium leprae was thought to be the exclusive causative agent of leprosy until Mycobacterium lepromatosis was identified in a rare form of leprosy known as diffuse lepromatous leprosy (DLL). Methods We isolated M. lepromatosis from a patient with DLL and propagated it in athymic nude mouse footpads. Genomic analysis of this strain (NHDP-385) identified a unique repetitive element, RLPM, on which a specific real-time quantitative polymerase chain reaction assay was developed. The RLPM assay, and a previously developed RLEP quantitative polymerase chain reaction assay for M. leprae, were validated as clinical diagnostic assays according to Clinical Laboratory Improvement Amendments guidelines. We tested DNA from archived histological sections, patient specimens from the United States, Philippines, and Mexico, and US wild armadillos. Results The limit of detection for the RLEP and RLPM assays is 30 M. leprae per specimen (0.76 bacilli per reaction; coefficient of variation, 0.65%–2.44%) and 122 M. lepromatosis per specimen (3.05 bacilli per reaction; 0.84%–2.9%), respectively. In histological sections (n = 10), 1 lepromatous leprosy (LL), 1 DLL, and 3 Lucio reactions contained M. lepromatosis; 2 LL and 2 Lucio reactions contained M. leprae; and 1 LL reaction contained both species. M. lepromatosis was detected in 3 of 218 US biopsy specimens (1.38%). All Philippines specimens (n = 180) were M. lepromatosis negative and M. leprae positive. Conversely, 15 of 47 Mexican specimens (31.91%) were positive for M. lepromatosis, 19 of 47 (40.43%) were positive for M. leprae, and 2 of 47 (4.26%) contained both organisms. All armadillos were M. lepromatosis negative. Conclusions The RLPM and RLEP assays will aid healthcare providers in the clinical diagnosis and surveillance of leprosy.

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Williamson, Charles H. D., David M. Wagner, Paul Keim, and Jason W. Sahl. "Developing Inclusivity and Exclusivity Panels for Testing Diagnostic and Detection Tools Targeting Burkholderia pseudomallei, the Causative Agent of Melioidosis." Journal of AOAC INTERNATIONAL 101, no. 6 (November 1, 2018): 1920–26. http://dx.doi.org/10.5740/jaoacint.18-0014.

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Abstract Background: Diagnostic tools designed to target Burkholderia pseudomallei, the causative agent of melioidosis that was classified as a Tier 1 Select Agent by the U.S. Centers for Disease Control and Prevention, have typically suffered from false-positive and false-negative results because of a lack of understanding of the genomic diversity of B. pseudomallei and its genetic near neighbors. Objective: In this review, we discuss a strategy for using comparative genomics to guide the design of inclusivity and exclusivity panels for the validation of assays as defined by the Standard Method Performance Requirement (SMPR). Methods: Based upon a literature review, comparative genomic analyses, and hands-on experience with diagnostic development and testing, we describe important factors to consider when developing inclusivity and exclusivity panels for testing diagnostic and/or detection tools. Results: The genomic diversity of B. pseudomallei is substantial, with the genome characterized by horizontal gene transfer, including the acquisition of genomic islands from near-neighbor species. This genomic diversity, core genome reduction, and signal erosion can complicate molecular diagnostic tool development and validation. Conclusions: Accurate diagnostic and/or detection tools targeting B. pseudomallei, an important pathogen from a public health and biodefense perspective, are needed for many applications. Utilizing whole genome sequencing data and comparative genomic techniques can guide the development and validation of such tools. Amplicon sequencing assays and assay redundancy can provide improved assay performance. Highlights: When developing and validating diagnostic and/or detection tools targeting B. pseudomallei, it is important to consider genomic diversity, genome reduction, and signal erosion to reduce the effects of typical diagnostic errors.

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Alquicer, Glenda, David Sedlák, Youngjoo Byun, Jiří Pavlíček, Marigo Stathis, Camilo Rojas, Barbara Slusher, Martin G. Pomper, Petr Bartůněk, and Cyril Bařinka. "Development of a High-Throughput Fluorescence Polarization Assay to Identify Novel Ligands of Glutamate Carboxypeptidase II." Journal of Biomolecular Screening 17, no. 8 (June 29, 2012): 1030–40. http://dx.doi.org/10.1177/1087057112451924.

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Glutamate carboxypeptidase II (GCPII) is an important target for therapeutic and diagnostic interventions aimed at prostate cancer and neurologic disorders. Here we describe the development and optimization of a high-throughput screening (HTS) assay based on fluorescence polarization (FP) that facilitates the identification of novel scaffolds inhibiting GCPII. First, we designed and synthesized a fluorescence probe based on a urea-based inhibitory scaffold covalently linked to a Bodipy TMR fluorophore (TMRGlu). Next, we established and optimized conditions suitable for HTS and evaluated the assay robustness by testing the influence of a variety of physicochemical parameters (e.g., pH, temperature, time) and additives. Using known GCPII inhibitors, the FP assay was shown to be comparable to benchmark assays established in the field. Finally, we evaluated the FP assay by HTS of a 20 000–compound library. The novel assay presented here is robust, highly reproducible (Z′ = 0.82), inexpensive, and suitable for automation, thus providing an excellent platform for HTS of small-molecule libraries targeting GCPII.

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Thirion, Laurence, Laura Pezzi, Iban Corcostegui, Audrey Dubot-Pérès, Alessandra Falchi, Xavier de Lamballerie, and Remi N. Charrel. "Development and Evaluation of a Duo Chikungunya Virus Real-Time RT-PCR Assay Targeting Two Regions within the Genome." Viruses 11, no. 8 (August 15, 2019): 755. http://dx.doi.org/10.3390/v11080755.

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Chikungunya virus (CHIKV) re-emerged as a globalized health threat fifteen years ago. There are dozens of RT-PCR assays published. An inventory of the latter was made, and after in silico analysis, two assays were selected for their ability to detect strains belonging to the five CHIKV genetic lineages. They were combined in order to provide a robust assay not affected by genetic point mutations and the resulting Duo CHIKV real-time RT-PCR assay was compared to the two parental single-plex tests against five strains belonging to the five genetic lineages. The Duo CHIKV assay performed equally, or better, in terms of sensitivity, specificity, linearity and signal intensity. Dual-target assays are better suited for viruses having the propensity to evolve into new variants via point mutations or major sequence deletions/insertions. Here, we demonstrated that combining two single systems into a dual-target assay did not impair sensitivity and specificity, and proved a potent diagnostic tool to face a potential emergence of CHIKV variants by newly evolving mutations.

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Pezzi, Laura, Remi N. Charrel, Laetitia Ninove, Antoine Nougairede, Gregory Molle, Bruno Coutard, Guillaume Durand, Isabelle Leparc-Goffart, Xavier de Lamballerie, and Laurence Thirion. "Development and Evaluation of a duo SARS-CoV-2 RT-qPCR Assay Combining Two Assays Approved by the World Health Organization Targeting the Envelope and the RNA-Dependant RNA Polymerase (RdRp) Coding Regions." Viruses 12, no. 6 (June 25, 2020): 686. http://dx.doi.org/10.3390/v12060686.

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The recent emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) worldwide has highlighted the importance of reliable and rapid diagnostic testing to prevent and control virus circulation. Dozens of monoplex in-house RT-qPCR assays are already available; however, the development of dual-target assays is suited to avoid false-negative results caused by polymorphisms or point mutations, that can compromise the accuracy of diagnostic and screening tests. In this study, two mono-target assays recommended by WHO (E-Sarbeco (enveloppe gene, Charite University, Berlin, Germany) and RdRp-IP4 (RdRp, Institut Pasteur, Paris, France)) were selected and combined in a unique robust test; the resulting duo SARS-CoV-2 RT-qPCR assay was compared to the two parental monoplex tests. The duo SARS-CoV-2 assay performed equally, or better, in terms of sensitivity, specificity, linearity and signal intensity. We demonstrated that combining two single systems into a dual-target assay (with or without an MS2-based internal control) did not impair performances, providing a potent tool adapted for routine molecular diagnosis in clinical microbiology laboratories.

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De-Simone, Salvatore Giovanni, Larissa Rodrigues Gomes, Paloma Napoleão-Pêgo, Guilherme Curty Lechuga, Jorge Soares de Pina, and Flavio Rocha da Silva. "Epitope Mapping of the Diphtheria Toxin and Development of an ELISA-Specific Diagnostic Assay." Vaccines 9, no. 4 (March 26, 2021): 313. http://dx.doi.org/10.3390/vaccines9040313.

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Background: The diphtheria toxoid antigen is a major component in pediatric and booster combination vaccines and is known to raise a protective humoral immune response upon vaccination. Although antibodies are considered critical for diphtheria protection, little is known about the antigenic determinants that maintain humoral immunity. Methods: One-hundred and twelve 15 mer peptides covering the entire sequence of diphtheria toxin (DTx) protein were prepared by SPOT synthesis. The immunoreactivity of membrane-bound peptides with sera from mice immunized with a triple DTP vaccine allowed mapping of continuous B-cell epitopes, topological studies, multiantigen peptide (MAP) synthesis, and Enzyme-Linked Immunosorbent Assay (ELISA) development. Results: Twenty epitopes were identified, with two being in the signal peptide, five in the catalytic domain (CD), seven in the HBFT domain, and five in the receptor-binding domain (RBD). Two 17 mer (CB/Tx-2/12 and CB/DTx-4–13) derived biepitope peptides linked by a Gly-Gly spacer were chemically synthesized. The peptides were used as antigens to coat ELISA plates and assayed with human (huVS) and mice vaccinated sera (miVS) for in vitro diagnosis of diphtheria. The assay proved to be highly sensitive (99.96%) and specific (100%) for huVS and miVS and, when compared with a commercial ELISA test, demonstrated a high performance. Conclusions: Our work displayed the complete picture of the linear B cell IgG response epitope of the DTx responsible for the protective effect and demonstrated sufficient specificity and eligibility for phase IIB studies of some epitopes to develop new and fast diagnostic assays.

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Anahtar, Melis N., Bennett M. Shaw, Damien Slater, Elizabeth H. Byrne, Yolanda Botti-Lodovico, Gordon Adams, Stephen F. Schaffner, et al. "Development of a qualitative real-time RT-PCR assay for the detection of SARS-CoV-2: a guide and case study in setting up an emergency-use, laboratory-developed molecular microbiological assay." Journal of Clinical Pathology 74, no. 8 (May 28, 2021): 496–503. http://dx.doi.org/10.1136/jclinpath-2020-207128.

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Developing and deploying new diagnostic tests are difficult, but the need to do so in response to a rapidly emerging pandemic such as COVID-19 is crucially important. During a pandemic, laboratories play a key role in helping healthcare providers and public health authorities detect active infection, a task most commonly achieved using nucleic acid-based assays. While the landscape of diagnostics is rapidly evolving, PCR remains the gold-standard of nucleic acid-based diagnostic assays, in part due to its reliability, flexibility and wide deployment. To address a critical local shortage of testing capacity persisting during the COVID-19 outbreak, our hospital set up a molecular-based laboratory developed test (LDT) to accurately and safely diagnose SARS-CoV-2. We describe here the process of developing an emergency-use LDT, in the hope that our experience will be useful to other laboratories in future outbreaks and will help to lower barriers to establishing fast and accurate diagnostic testing in crisis conditions.

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Pressman, Melissa A., Elizabeth Wuitschick, and Suzette C. Chance. "Development and Performance of the GTI ADAMTS-13 Activity Assay." Blood 106, no. 11 (November 16, 2005): 4097. http://dx.doi.org/10.1182/blood.v106.11.4097.4097.

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Abstract Thrombotic Thrombocytopenic Purpura (TTP) is a disease characterized by the sudden onset of a classical pentad of symptoms: thrombocytopenia, fever, renal insufficiency, neurologic deficit, and microangiopathic hemolytic anemia. It was recently discovered that ADAMTS-13 is the protease responsible for cleaving von Willebrand Factor; deficiency of ADAMTS-13 activity has been demonstrated in the plasma of TTP patients. The lack of ADAMTS-13 activity results in the accumulation of multimers of vWF in the plasma and ultimately intravascular platelet aggregation resulting in the clinical symptoms associated with TTP. Currently there are no commercially available diagnostic assays for TTP. Several laboratories have developed “in house” assays that measure the activity of the ADAMTS-13 protease. However, these assays are cumbersome, complex, difficult to perform, and there is a long turn around time, as they require long incubations. The results are difficult to interpret and are not easily quantitated. In addition, there is no standardization of results from laboratory to laboratory, mainly because the assays use various technologies. Determining the extent to which the ADAMTS-13 enzyme activity is decreased is believed to be important in distinguishing a patient with TTP from those presenting with similar clinical symptoms. Until ADAMTS-13 assays are more quantitative, there is not a good method for studying patients with severe decreases in ADAMTS-13 levels versus those who have some intermediate but less than normal level of enzyme activity. Furthermore, the lack of standardized results makes the assays less useful in monitoring the effectiveness of treatment of the TTP patients. GTI has developed a rapid, quantitative assay for ADAMTS-13 activity that is based on the cleavage of a substrate which consists of a peptide fragment of von Willebrand Factor and subsequent fluorescent detection. An initial study using plasma (obtained from the Blood Center of Wisconsin) from 5 TTP patients demonstrated good correlation between the GTI assay and the collagen binding assay, thus demonstrating feasibility of the assay. A more extensive evaluation using 40 normal samples and 55 samples from suspected TTP patients was initiated, the results of which will be reported. Assay sensitivity/limit of detection (based on dilution studies) was 3% normal ADAMTS-13 activity. Assay precision using 3 samples (mean values of 96%, 55%, and 9% normal ADAMTS-13 activity) run in duplicate over 8 days was evaluated. Assay imprecision was less than 10% cv for all samples tested.

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Soltani, Nourolah, Kristian A. Stevens, Vicki Klaassen, Min-Sook Hwang, Deborah A. Golino, and Maher Al Rwahnih. "Quality Assessment and Validation of High-Throughput Sequencing for Grapevine Virus Diagnostics." Viruses 13, no. 6 (June 11, 2021): 1130. http://dx.doi.org/10.3390/v13061130.

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Development of High-Throughput Sequencing (HTS), also known as next generation sequencing, revolutionized diagnostic research of plant viruses. HTS outperforms bioassays and molecular diagnostic assays that are used to screen domestic and quarantine grapevine materials in data throughput, cost, scalability, and detection of novel and highly variant virus species. However, before HTS-based assays can be routinely used for plant virus diagnostics, performance specifications need to be developed and assessed. In this study, we selected 18 virus-infected grapevines as a test panel for measuring performance characteristics of an HTS-based diagnostic assay. Total nucleic acid (TNA) was extracted from petioles and dormant canes of individual samples and constructed libraries were run on Illumina NextSeq 500 instrument using a 75-bp single-end read platform. Sensitivity was 98% measured over 264 distinct virus and viroid infections with a false discovery rate (FDR) of approximately 1 in 5 positives. The results also showed that combining a spring petiole test with a fall cane test increased sensitivity to 100% for this TNA HTS assay. To evaluate extraction methodology, these results were compared to parallel dsRNA extractions. In addition, in a more detailed dilution study, the TNA HTS assay described here consistently performed well down to a dilution of 5%. In that range, sensitivity was 98% with a corresponding FDR of approximately 1 in 5. Repeatability and reproducibility were assessed at 99% and 93%, respectively. The protocol, criteria, and performance levels described here may help to standardize HTS for quality assurance and accreditation purposes in plant quarantine or certification programs.

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Bluett, James, Isabel Riba-Garcia, Suzanne M. M. Verstappen, Thierry Wendling, Kayode Ogungbenro, Richard D. Unwin, and Anne Barton. "Development and validation of a methotrexate adherence assay." Annals of the Rheumatic Diseases 78, no. 9 (June 5, 2019): 1192–97. http://dx.doi.org/10.1136/annrheumdis-2019-215446.

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BackgroundThe first-line therapy for rheumatoid arthritis (RA) is weekly oral methotrexate (MTX) at low dosages (7.5–25 mg/week). However, ~40% of patients are non-adherent which may explain why some do not respond and need to start more expensive biological therapies. To monitor adherence more accurately and develop strategies to improve it, a validated objective MTX adherence test is required.ObjectiveTo develop and validate the diagnostic accuracy of a novel MTX adherence assay using high-performance liquid chromatography–selected reaction monitoring– mass spectrometry (HPLC-SRM-MS) based biochemical analysis of drug levels.Methods20 patients with RA underwent MTX pharmacokinetic assessment using HPLC-SRM-MS MTX plasma concentration analysis over a 6-day period. Directly observed therapy was the reference standard. Pharmacokinetic model validation was performed using independent plasma samples from real-world patients (n=50) with self-reported times of drug administration. Following assay optimisation, the sensitivity of the assay to detect adherence was established using samples from an observational cohort study (n=138).ResultsA two-compartment pharmacokinetic model was developed and validated. Simulations described the sensitivity required for MTX detection over 7 days; subsequent assay optimisation and retesting of samples confirmed that all patients were correctly identified as MTX adherers. Using real-world samples, the assays sensitivity was 95%.ConclusionNon-adherence to MTX is common and can have a significant effect on disease activity. HPLC-SRM-MS plasma analysis accurately detects MTX adherence. The validated objective test could easily be used in clinic to identify patients requiring adherence support.

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Gotham, Dzintars, Lindsay McKenna, Stijn Deborggraeve, Suraj Madoori, and David Branigan. "Public investments in the development of GeneXpert molecular diagnostic technology." PLOS ONE 16, no. 8 (August 31, 2021): e0256883. http://dx.doi.org/10.1371/journal.pone.0256883.

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Background The GeneXpert diagnostic platform from the US based company Cepheid is an automated molecular diagnostic device that performs sample preparation and pathogen detection within a single cartridge-based assay. GeneXpert devices can enable diagnosis at the district level without the need for fully equipped clinical laboratories, are simple to use, and offer rapid results. Due to these characteristics, the platform is now widely used in low- and middle-income countries for diagnosis of diseases such as TB and HIV. Assays for SARS-CoV-2 are also being rolled out. We aimed to quantify public sector investments in the development of the GeneXpert platform and Cepheid’s suite of cartridge-based assays. Methods Public funding data were collected from the proprietor company’s financial filings, grant databases, review of historical literature concerning key laboratories and researchers, and contacting key public sector entities involved in the technology’s development. The value of research and development (R&D) tax credits was estimated based on financial filings. Results Total public investments in the development of the GeneXpert technology were estimated to be $252 million, including >$11 million in funding for work in public laboratories leading to the first commercial product, $56 million in grants from the National Institutes of Health, $73 million from other U.S. government departments, $67 million in R&D tax credits, $38 million in funding from non-profit and philanthropic organizations, and $9.6 million in small business ‘springboard’ grants. Conclusion The public sector has invested over $250 million in the development of both the underlying technologies and the GeneXpert diagnostic platform and assays, and has made additional investments in rolling out the technology in countries with high burdens of TB. The key role played by the public sector in R&D and roll-out stands in contrast to the lack of public sector ability to secure affordable pricing and maintenance agreements.

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Porschewski, Peter, Mira A. M. Grättinger, Kerstin Klenzke, Anja Erpenbach, Michael R. Blind, and Frank Schäfer. "Using Aptamers as Capture Reagents in Bead-Based Assay Systems for Diagnostics and Hit Identification." Journal of Biomolecular Screening 11, no. 7 (September 14, 2006): 773–81. http://dx.doi.org/10.1177/1087057106292138.

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Most applications of xMAP™ (Luminex®) bead-based assay technology in diagnostics and drug discovery use immobilized antigens or antibodies. Here the authors describe the development of novel assay systems in which synthetic oligonucleotides that specifically bind and inhibit other biomolecules—so-called aptamers—are directly immobilized on beads. The robustness, specificity, and sensitivity of aptamer-based assays were demonstrated in a test system that detected human α-thrombin in serum samples. xMAP technology was also adapted to competitive screening formats where an aptamer/protein complex was disrupted by a functionally analogous competitor. The results indicate that such assays are excellently suited for diagnostic applications or drug screening, where aptamers serve as competitive binding probes for the identification of small-molecule hits. These methods should be transferable to a large number of applications because specific aptamers can be rapidly generated for almost any protein target.

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Hembrough, Todd A., Wei-Li Liao, Sheeno Thyparambil, Heidi S. Erickson, George Carey, Thomas Guiel, Robert B. Heaton, et al. "Development and clinical validation of an adeno/squamous multiplexed diagnostic assay for NSCLC." Journal of Clinical Oncology 31, no. 15_suppl (May 20, 2013): e19057-e19057. http://dx.doi.org/10.1200/jco.2013.31.15_suppl.e19057.

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e19057 Background: The diagnosis of adenocarcinoma (ADC) vs. squamous cell carcinoma (SCC) in NSCLC is critical since new therapies (e.g. pemetrexed) are restricted to non-squamous NSCLC. Most diagnoses are made based on histopathology, though IHC is often performed as well. Since IHC is not quantitative, interpretation of intensity is subjective, and reproducibility can be problematic. Using the Liquid Tissue-SRM mass spectrometry (MS) platform, we have developed a quantitative, multiplexed clinical diagnostic assay to measure the adeno/squamous markers TTF-1/CK7 (ADC) and K5 and p63/40 (SCC) in FFPE tissue. This proteomic assay is epitope independent, and highly reproducible. Methods: Recombinant K5, K7, TTF-1 and p63/p40, were subjected to trypsin digestion mapping to identify peptides for MS analysis. Heavy isotope labeled peptides were synthesized as positive control peptides for quantitation. The multiplexed assay was technically validated on cell lines, and human tumor tissues. Results: ADC/SCC proteomic analysis was performed on a training set of 39 NSCLC tissues from U. of Chicago (20 ADC and 19 SCC). Principal Component Analysis was used to define optimal quantitative cutoffs for each of the four markers for the two tumor subtypes. The ADC diagnosis was unequivocal for all 20 ADC samples, however within the 19 SCC’s, one sample appeared to be ADC and two samples had markers for both ADC and SCC. Proteomic analysis of a test set of 194 NSCLC tumors (98 ADC and 98 SCC based on pathology reports) from MD Anderson Cancer Center was consistent with the observations in the training set. 93/98 ADC cases were confirmed by proteomic analysis, four ADC cases showed mixed or SCC phenotypes. Proteomic analysis confirmed 58/98 SCC cases but identified 27 cases as mixed ADC SCC or ADC based on TTF1/K5 expression. Further studies are ongoing to determine the clinical utility of these findings. Conclusions: These data demonstrate that quantitative proteomic analysis can define cut offs for ADC and SCC Biomarkers profiles in NSCLC tumors. These profiles can determine ADC, SCC and mixed phenotypes and provide physicians with accurate information to help them stratify their patients to the right evidence based treatment therapy.

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Zhang, Jia, Bo Meng, Duanfang Liao, Lvyi Zhou, Xu Zhang, Linling Chen, Zifen Guo, et al. "De Novo Synthesis of PCR Templates for the Development of SARS Diagnostic Assay." Molecular Biotechnology 25, no. 2 (2003): 107–12. http://dx.doi.org/10.1385/mb:25:2:107.

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Yang, Chunjiao, Zhongfeng Sun, Guojun Zhang, Lijuan Wang, Jie Zhang, and Xin Zhang. "Development of a novel parallel determination platform: a feasibility study tested on a chemiluminescence device." Analytical Methods 10, no. 3 (2018): 298–307. http://dx.doi.org/10.1039/c7ay02394d.

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McKerrell, Thomas, Thaidy Moreno, Hannes Ponstingl, Niccolo Bolli, João M. L. Dias, German Tischler, Vincenza Colonna, et al. "Development and validation of a comprehensive genomic diagnostic tool for myeloid malignancies." Blood 128, no. 1 (July 7, 2016): e1-e9. http://dx.doi.org/10.1182/blood-2015-11-683334.

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Key Points We develop and validate Karyogene, a comprehensive one-stop diagnostic platform for the genomic analysis of myeloid malignancies. Karyogene simultaneously detects substitutions, insertions/deletions, translocations, copy number and zygosity changes in a single assay.

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Fonong, T., S. M. Evans, and H. A. Homburger. "Development and comparative evaluation of immunoblot assays for detecting autoantibodies to Scl 70 and Jo 1 antigens in serum." Clinical Chemistry 36, no. 12 (December 1, 1990): 2053–56. http://dx.doi.org/10.1093/clinchem/36.12.2053.

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Abstract We developed rapid 24-h immunoblot assays for detecting autoantibodies to Scl 70 and Jo 1 antigens in serum. In comparative studies, we evaluated the analytical sensitivity of the immunoblot assays and commercial immunodiffusion assays for anti-Scl 70 and anti-Jo 1 autoantibodies with the use of positive control sera, and compared the frequencies of positive and negative results in a group of 116 sera, including specimens from 34 healthy controls and 82 patients with various connective-tissue diseases. The immunoblot assays were greater than 100-fold more sensitive than immunodiffusion for detecting both autoantibodies. Despite greater analytical sensitivity, there were no false-positive results by the immunoblot assay for anti-Scl 70 or anti-Jo 1 autoantibodies in sera from either the controls or the patients. The diagnostic sensitivity of the immunoblot assay for anti-Scl 70 autoantibodies in patients with scleroderma was greater than that of the immunodiffusion assay, 70% vs 20%, and was equivalent in patients with polymyositis, 43%. We conclude that rapid immunoblot assays for anti-Scl 70 and anti-Jo 1 autoantibodies are superior to immunodiffusion assays for clinical use and are suitable for routine use in the clinical laboratory.

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Nakabashi, Cláudia C. D., Rosa Paula M. Biscolla, Teresa S. Kasamatsu, Teresinha T. Tachibana, Rafaela N. Barcelos, Eduardo Z. Malouf, Danielle M. Andreoni, Rui M. B. Maciel, and José Gilberto H. Vieira. "Development, characterization and clinical validation of new sensitive immunofluorometric assay for the measurement of serum thyroglobulin." Arquivos Brasileiros de Endocrinologia & Metabologia 56, no. 9 (December 2012): 658–65. http://dx.doi.org/10.1590/s0004-27302012000900010.

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OBJECTIVE: In the last decade, data published stressed the role of highly-sensitive thyroglobulin (Tg) assays in the follow-up of differentiated thyroid carcinoma (DTC) patients. The present study describes a new, highly-sensitive Tg assay, compares it with an available commercial assay, and validates it in the follow-up of DTC patients. SUBJECTS AND METHODS: The immunofluorometric high-sensitivity Tg assay is based on monoclonal and polyclonal antibodies produced at our laboratories. It was validated in 100 samples of 87 patients with DTC submitted to total thyroidectomy, 87% of whom also received radioiodine. For correlation, all samples were also tested using a commercial Tg assay (Beckman Access) with functional sensitivity (FS) of 0.1 ng/mL. RESULTS: The new method showed FS of 0.3 ng/mL. The correlation between the two methods was good (r = 0.74; p < 0.0001). The diagnostic sensitivity was 88.9%, and it was increased to 100% when combined with neck US. CONCLUSION: This new, high-sensitivity Tg assay presented a good correlation with Beckman Access assay and with the clinical outcome of the patients. The continuous availability of a validated assay is an additional advantage for long term follow-up of DTC patients. Arq Bras Endocrinol Metab. 2012;56(9):658-65

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Rojas, J. Alejandro, Timothy D. Miles, Michael D. Coffey, Frank N. Martin, and Martin I. Chilvers. "Development and Application of qPCR and RPA Genus- and Species-Specific Detection of Phytophthora sojae and P. sansomeana Root Rot Pathogens of Soybean." Plant Disease 101, no. 7 (July 2017): 1171–81. http://dx.doi.org/10.1094/pdis-09-16-1225-re.

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Phytophthora root rot of soybean, caused by Phytophthora sojae, is one of the most important diseases in the Midwestern United States, and is estimated to cause losses of up to 1.2 million metric tons per year. Disease may also be caused by P. sansomeana; however, the prevalence and damage caused by this species is not well known, partly due to limitations of current diagnostic tools. Efficient, accurate, and sensitive detection of pathogens is crucial for management. Thus, multiplex qPCR and isothermal RPA (recombinase polymerase amplification) assays were developed using a hierarchical approach to detect these Phytophthora spp. The assays consist of a genus-specific probe and two species-specific probes that target the atp9-nad9 region of the mitochondrial genome that is highly specific for the genus Phytophthora. The qPCR approach multiplexes the three probes and a plant internal control. The RPA assays run each probe independently with a plant internal control multiplexed in one amplification, obtaining a result in as little as 20 mins. The multicopy mitochondrial genome provides sensitivity with sufficient variability to discern among different Phytophthora spp. The assays were highly specific when tested against a panel of 100 Phytophthora taxa and range of Pythium spp. The consistent detection level of the assay was 100 fg for the qPCR assay and 10 pg for the RPA assay. The assays were validated on symptomatic plants collected from Michigan (U.S.) and Ontario (Canada) during the 2013 field season, showing correlation with isolation. In 2014, the assays were validated with samples from nine soybean producing states in the U.S. The assays are valuable diagnostic tools for detection of Phytophthora spp. affecting soybean.

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Perkins, Julie, Satya Parida, and Alfonso Clavijo. "Use of a Standardized Bovine Serum Panel To Evaluate a Multiplexed Nonstructural Protein Antibody Assay for Serological Surveillance of Foot-and-Mouth Disease." Clinical and Vaccine Immunology 14, no. 11 (October 3, 2007): 1472–82. http://dx.doi.org/10.1128/cvi.00227-07.

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ABSTRACT Liquid array technology has previously been used to show proof of principle of a multiplexed nonstructural protein serological assay to differentiate foot-and-mouth disease virus-infected and vaccinated animals. The current multiplexed assay consists of synthetically produced peptide signatures 3A, 3B, and 3D and the recombinant protein signature 3ABC in combination with four controls. To determine the diagnostic specificity of each signature in the multiplex, the assay was evaluated against a naive population (n = 104) and a vaccinated population (n = 94). Subsequently, the multiplexed assay was assessed by using a panel of bovine sera generated by the World Reference Laboratory for foot-and-mouth disease in Pirbright, United Kingdom. This serum panel has been used to assess the performance of other singleplex enzyme-linked immunosorbent assay (ELISA)-based nonstructural protein antibody assays. The 3ABC signature in the multiplexed assay showed performance comparable to that of a commercially available nonstructural protein 3ABC ELISA (Cedi test), and additional information pertaining to the relative diagnostic sensitivity of each signature in the multiplex was acquired in one experiment. The encouraging results of the evaluation of the multiplexed assay against a panel of diagnostically relevant samples promote further assay development and optimization to generate an assay for routine use in foot-and-mouth disease serological surveillance.

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Lim, Ho Jae, Eun-Rim Kang, Min Young Park, Bo Kyung Kim, Min Jin Kim, Sunkyung Jung, Kyoung Ho Roh, et al. "Development of a multiplex real-time PCR assay for the simultaneous detection of four bacterial pathogens causing pneumonia." PLOS ONE 16, no. 6 (June 17, 2021): e0253402. http://dx.doi.org/10.1371/journal.pone.0253402.

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Classification of clinical symptoms and diagnostic microbiology are essential to effectively employ antimicrobial therapy for lower respiratory tract infections (LRTIs) in a timely manner. Empirical antibiotic treatment without microbial identification hinders the selective use of narrow-spectrum antibiotics and effective patient treatment. Thus, the development of rapid and accurate diagnostic procedures that can be readily adopted by the clinic is necessary to minimize non-essential or excessive use of antibiotics and accelerate patient recovery from LRTI-induced damage. We developed and validated a multiplex real-time polymerase chain reaction (mRT-PCR) assay with good analytical performance and high specificity to simultaneously detect four bacterial pathogens causing pneumonia: Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, and Moraxella catarrhalis. The analytical performance of mRT-PCR against target pathogens was evaluated by the limit of detection (LOD), specificity, and repeatability. Two hundred and ten clinical specimens from pneumonia patients were processed using an automatic nucleic acid extraction system for the “respiratory bacteria four” (RB4) mRT-PCR assay, and the results were directly compared to references from bacterial culture and/or Sanger sequencing. The RB4 mRT-PCR assay detected all target pathogens from sputum specimens with a coefficient of variation ranging from 0.29 to 1.71 and conservative LOD of DNA corresponding to 5 × 102 copies/reaction. The concordance of the assay with reference-positive specimens was 100%, and additional bacterial infections were detected from reference-negative specimens. Overall, the RB4 mRT-PCR assay showed a more rapid turnaround time and higher performance that those of reference assays. The RB4 mRT-PCR assay is a high-throughput and reliable tool that assists decision-making assessment and outperforms other standard methods. This tool supports patient management by considerably reducing the inappropriate use of antibiotics.

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Stephan, Carsten, Moritz Klaas, Christian Müller, Dietmar Schnorr, Stefan A. Loening, and Klaus Jung. "Interchangeability of Measurements of Total and Free Prostate-Specific Antigen in Serum with 5 Frequently Used Assay Combinations: An Update." Clinical Chemistry 52, no. 1 (January 1, 2006): 59–64. http://dx.doi.org/10.1373/clinchem.2005.059170.

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Abstract Background: The comparability of total and free prostate-specific antigen (tPSA and fPSA) results among commercial PSA assays has been suggested to be improved by calibration to WHO PSA reference materials and the development of equimolar-response assays. To characterize the current situation, we assessed 5 frequently used commercial assay combinations for tPSA and fPSA regarding the interchangeability of the PSA values and the ratio of fPSA to tPSA (%fPSA), equimolar characteristics, and diagnostic accuracy. Methods: Sera from 314 patients with prostate cancer (PCa) and 282 men with no evidence of prostate cancer (NPCa) were measured with tPSA and fPSA assays from Abbott (AxSYM), Beckman Coulter (Access), Diagnostic Products Corporation (Immulite 2000), and Roche (Elecsys 2010) and with tPSA and complexed PSA (cPSA) assays from Bayer (ADVIA Centaur). Results: Method comparisons (Passing and Bablok regressions; Bland–Altman plots) showed assay-dependent results for tPSA, fPSA, and %fPSA. With the Access tPSA values taken as 100%, tPSA concentrations varied from 87% (AxSYM and ADVIA Centaur) to 115% (Immulite), leading to different numbers of patients classified according to the commonly recommended tPSA cutoffs for performing a biopsy. Different %fPSA values also led to assay-dependent ROC analysis results, a finding that shows the importance for the diagnostic accuracy. Conclusion: Interchangeability of tPSA, fPSA, and %fPSA values obtained by commercial PSA assays remains inadequate, but attention to this issue may minimize the misinterpretation of PSA results obtained by different assays.

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Journal articles on the topic 'Diagnostic Assay Development'

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