96well-formatted CrfA assay for differentiation of Mycobacterium tuberculosis and Non-tuberculous mycobacteria.

Failure in recognizing non-tuberculous mycobacteria (NTM) leads to misdiagnosis of multidrug-resistant Mycobacterium tuberculosis Complex (MTBC). There is an unmet need for diagnostic tools that can differentiate between NTMs and MTBC, and that are affordable for Low- and Middle-income Countries (LMIC). Earlier we developed a strip-based CrfA assay technology to detect the Carbapenem Resistance Factor A (CrfA) enzyme present only in MTBC. However, the strip-based CrfA assay had low turnaround time and lacked high-throughput capabilities. In this current research, we have developed a 96well-formatted CrfA assay for high-throughput detection of MTBC and differentiation with NTMs. This 96well-formatted CrfA assay displays a low turnaround time of 6-8 h with 100 % specificity and 93.75 % sensitivity on clinical samples. Based on these attributes, this 96well-formatted assay represents a valuable complementary tool to mitigate the misdiagnosis of chronic pulmonary tuberculosis with non-tuberculous mycobacteria in poorer nations.

Detection and differentiation of low virulence and virulent Orthoavulavirus javaense using a molecular beacon with RT-LAMP.

Newcastle disease (ND), an economically important disease in poultry, is caused by virulent strains of the genetically diverse Orthoavulavirus javaense (OAVJ). Laboratories rely on quantitative real-time reverse transcription PCR (qRT-PCR) to detect OAVJ and differentiate between OAVJ pathotypes. This study demonstrates that a fusion cleavage site based molecular beacon with reverse transcription loop mediated isothermal amplification (MB-RT-LAMP) assay can detect and differentiate OAVJ pathotypes in a single assay. Data show that the assay can rapidly identify diverse OAVJ genotypes with sensitivity only one log-fold lower than the current fusion qRT-PCR assay (104 copies), exhibits a high degree of specificity for OAVJ, and the molecular beacon can differentiate mesogenic/velogenic sequences from lentogenic sequences. Further, data show that a two-minute rapid lysis protocol preceding MB-RT-LAMP can detect and differentiate OAVJ RNA from both spiked samples and oropharyngeal swabs without the need for RNA isolation. As the MB-RT-LAMP assay can rapidly detect and discriminate between lentogenic and mesogenic/velogenic sequences of OAVJ within one assay, without the need for RNA isolation, and is adaptable to existing veterinary diagnostic laboratory workflow without additional equipment, this assay could be a rapid primary screening tool before qRT-PCR based validation in resource limited settings.

Recombinant 3AB3 nonstructural protein-based indirect ELISA for detection of foot-and-mouth disease virus infection-elicited antibodies in goat.

Foot-and-mouth disease (FMD) is one of the most important animal diseases of economic significance globally. It is a highly infectious and contagious disease of cloven-hoofed animals including sheep and goat. For sero-diagnosis of FMD, recombinant antigen-based assays are considered as alternatives to conventional approaches such as the liquid phase blocking ELISA (LPBE). The early interventions towards control measures cannot be implemented unless the disease gets promptly diagnosed. It is relatively difficult to clinically diagnose FMD in goat due to the usual milder form or unapparent nature of symptoms. Under such situations where clinical samples are not available, demonstration of infection-specific FMD virus (FMDV) antibodies in serum sample may help identifying the animals exposed to the virus in retrospect. Antibody to 3AB nonstructural protein (NSP) has been considered to be the most reliable indicator for FMD diagnosis. The current study extended the earlier designed recombinant 3AB3 protein-based indirect ELISA originally validated on bovine serum samples to testing serum samples of goat. The performance of the indirect ELISA was validated using internationally accepted PrioCHECK® FMDV NS kit. The overall diagnostic sensitivity (DSn) of the indirect ELISA was estimated to be 95.52% (619/648), while the diagnostic specificity (DSp) on naïve and vaccinated animals varied at 98.06% (557/568) and 94.15% (435/462), respectively. In India, where FMD is prevalent and the goat population is so high, this 'in-house' optimized assay can be considered to be an adjunct in sero-epidemiological investigation of FMD in goat.

A novel syphilis Treponema pallidum lipoprotein peptide antigen diagnostic assay using red cell kodecytes in routine blood centre column agglutination testing platforms.

BACKGROUND AND OBJECTIVES: The detection of treponemal antibodies, which are used to make a diagnosis of syphilis, is important both for diagnostic purposes and as a mandatory blood donor test in most countries. We evaluated the feasibility of using Kode Technology to make syphilis peptide red cell kodecytes for use in column agglutination serologic platforms. MATERIALS AND METHODS: Candidate Kode Technology function-spacer-lipid (FSL) constructs were made for the Treponema pallidum lipoprotein (TmpA) of T. pallidum, using the peptide and FSL selection algorithms, and then used to make kodecytes. Developmental kodecytes were evaluated against a large range of syphilis antibody reactive and non-reactive samples in column agglutination platforms and compared against established methodologies. Overall, 150 reactive and 2072 non-reactive Syphicheck assay (a modified T. pallidum particle agglutination) blood donor samples were used to evaluate the agreement rate of the developed kodecyte assay. RESULTS: From three FSL-peptide candidate constructs, one was found to be the most suitable for diagnostics. Of 150 Syphicheck assay reactive samples, 146 were TmpA-kodecyte reactive (97.3% agreement), compared with 58.0% with the rapid plasmin reagin (RPR) assay for the same samples. Against the 2072 expected syphilis non-reactive samples the agreement rate for TmpA-kodecytes was 98.8%. CONCLUSION: TmpA-kodecytes are viable for use as cost-effective serologic reagent red cells for the detection of treponemal antibodies to diagnose syphilis with a high level of specificity in blood centres. This kodecyte methodology also potentially allows for introduction of the reverse-algorithm testing into low-volume laboratories, by utilizing existing transfusion laboratory infrastructure.

Comparative Bioinformatic Analysis Reveals Conserved Regions in SARS-CoV-2 Genome for RAPID Pandemic Response.

In the face of the SARS-CoV-2 pandemic, characterized by the virus's rapid mutation rates, developing timely and targeted therapeutic and diagnostic interventions presents a significant challenge. This study utilizes bioinformatic analyses to pinpoint conserved genomic regions within SARS-CoV-2, offering a strategic advantage in the fight against this and future pathogens. Our approach has enabled the creation of a diagnostic assay that is not only rapid, reliable, and cost-effective but also possesses a remarkable capacity to detect a wide array of current and prospective variants with unmatched precision. The significance of our findings lies in the demonstration that focusing on these conserved genomic sequences can significantly enhance our preparedness for and response to emerging infectious diseases. By providing a blueprint for the development of versatile diagnostic tools and therapeutics, this research paves the way for a more effective global pandemic response strategy.

Pig Acute Phase Proteins as Non-Antibody Systemic Biomarkers of Intracellular Infections.

As an alternative to traditional serological markers, that is, antibodies, for serum-based specific diagnosis of infections, circulating non-antibody markers may be used to monitor active disease. Acute phase proteins (APPs) are a prominent class of such markers widely used for diagnosing ongoing inflammation and infection. In this chapter, basic theoretical and practical considerations on developing APP assays and using APPs as markers of ongoing infection are presented with a specific focus on intracellular infections in pigs. Examples on APP-based monitoring of infection in pigs with viruses such as porcine respiratory and reproductive syndrome virus (PRRSV), porcine endemic diarrhea virus (PEDV), and influenza A virus (IAV), as well as intracellular bacteria (Lawsonia intracellularis) and the protozoan intracellular parasites Toxoplasma gondii and Cryptosporidium parvum are presented, with an emphasis on major pig APPs C-reactive protein (CRP), haptoglobin, serum amyloid A (SAA), and pig major acute phase protein (pig-MAP). The performance of these APPs as biomarkers in a range of experimental infection studies in pigs is described as examples on their use for estimating the severity of infection, vaccine efficacy, herd health characterization, and differential diagnosis.

Performance evaluation of the Alinity m system for quantifying cytomegalovirus DNA in samples of the respiratory, gastrointestinal, and urinary tract.

Quantitation of cytomegalovirus (CMV) DNA load in specimens other than blood such as bronchoalveolar lavages, intestinal biopsies, or urine has become a common practice as an ancillary tool for the diagnosis of CMV pneumonitis, intestinal disease, or congenital infection, respectively. Nevertheless, most commercially available CMV PCR platforms have not been validated for CMV DNA detection in these specimen types. In this study, a laboratory-developed test based on Alinity m CMV ("Alinity LDT") was evaluated. Reproducibility assessment using spiked bronchial aspirate (BAS) or urine samples showed low standard deviations of 0.08 and 0.27 Log IU/mL, respectively. Evaluating the clinical performance of Alinity LDT in comparison to a laboratory-developed test based on RealTime CMV ("RealTime LDT") showed good concordance across 200 clinical specimens including respiratory specimens, intestinal biopsies, urine, and stool. A high Pearson's correlation coefficient of r = 0.92, a low mean bias of -0.12 Log IU/mL, a good qualitative agreement of 90%, and a Cohen's kappa value of 0.76 (substantial agreement) were observed. In separate analyses of the sample types BAS, tracheal aspirates, bronchoalveolar lavage, biopsies, and urine, the assay results correlated well between the two platforms with r values between 0.88 and 0.99 and a bias <0.5 Log IU/mL. Overall, the fully automated, continuous, random access Alinity LDT yielded good reproducibility, high concordance, and good correlation to RealTime LDT in respiratory, gastrointestinal, and urine samples and may enhance patient management with rapid result reporting.IMPORTANCEIn transplant recipients, a major cause for morbidity and mortality is end-organ disease by primary or secondary CMV infection of the respiratory or gastrointestinal tract. In addition, sensorineural hearing loss and neurodevelopmental abnormalities are frequent sequelae of congenital CMV infections in newborns. Standard of care for highly sensitive detection and quantitation of the CMV DNA load in plasma and whole blood specimens is real-time PCR testing. Beyond that, there is a need for quantitative determination of CMV DNA levels in respiratory, gastrointestinal, and urinary tract specimens using a highly automated, random access CMV PCR assay with a short turnaround time to enable early diagnosis and treatment. In the present study, clinical performance of the fully automated Alinity m analyzer in comparison to the current RealTime LDT assay was evaluated in eight different off-label sample types.

Evaluation of Alinity m CMV assay performance for detecting CMV in plasma, cerebrospinal fluid, and bronchoalveolar lavage specimens.

Accurate detection and quantification of cytomegalovirus (CMV) is crucial to preventing adverse outcomes in immunocompromised individuals. Current assays were developed for use with plasma specimens, but CMV may be present in bronchoalveolar lavage (BAL) fluid and cerebrospinal fluid (CSF). We evaluated the performance of the Abbott Alinity m CMV assay compared to the Abbott RealTime CMV assay for quantification of CMV in plasma, BAL, and CSF specimens. To evaluate clinical performance, 190 plasma, 78 BAL, and 20 CSF specimens were tested with the Alinity m assay and compared to the RealTime assay. The Alinity m CMV assay showed high precision (SD <0.01 to 0.13) for all 3 specimen types. Clincal plasma and BAL specimens with quantifiable CMV DNA demonstrated strong correlation to RealTime CMV assay results (r2 = 0.9779 for plasma, r2 = 0.9373 for BAL). The Alinity m CMV assay may be useful for quantification of CMV in plasma, BAL, and CSF specimens.

LAMP assay to detect Elsinoë necatrix; an important Eucalyptus shoot and leaf pathogen.

Eucalyptus scab and shoot malformation caused by Elsinoë necatrix is an emerging disease and a serious threat to the global commercial forestry industry. The disease was first discovered in North Sumatra, Indonesia and now requires a simple and effective method for early pathogen detection. In this study, a rapid and sensitive Loop-mediated isothermal amplification (LAMP) assay was developed for E. necatrix. A unique region in a secondary metabolite gene cluster was used as target for the assay. To test robustness of the assay, LAMP amplification was verified in 15 strains of E. necatrix. A specificity test against 23 closely related Elsinoë species and three fungal species commonly isolated on Eucalyptus showed that the LAMP assay exclusively identified E. necatrix isolates. The assay had a high level of sensitivity, able to detect 0.01 ng (approximately 400 target copies) of pure E. necatrix DNA. Furthermore, using a simple DNA extraction method, it was possible to use this assay to detect E. necatrix in infected Eucalyptus leaves.

Occurrence and Multiplex PCR Detection of Citrus Yellow Vein Clearing Virus in Korea.

Citrus yellow vein clearing virus (CYVCV) is a member of the Alphaflexiviridae family that causes yellow vein clearing symptoms on citrus leaves. A total of 118 leaf samples from nine regions of six provinces in Korea were collected from various citrus species in 2020 and 2021. Viral diagnosis using next-generation sequencing and reverse transcription polymerase chain reaction (RT-PCR) identified four viruses: citrus tristeza virus, citrus leaf blotch virus, citrus vein enation virus, and CYVCV. A CYVCV incidence of 9.3% was observed in six host plants, including calamansi, kumquat, Persian lime, and Eureka lemon. Among the citrus infected by CYVCV, only three samples showed a single infection; the other showed a mixed infection with other viruses. Eureka lemon and Persian lime exhibited yellow vein clearing, leaf distortion, and water-soak symptom underside of the leaves, while the other hosts showed only yellowing symptoms on the leaves. The complete genome sequences were obtained from five CYVCV isolates. Comparison of the isolates reported from the different geographical regions and hosts revealed the high sequence identity (95.2% to 98.8%). Phylogenetic analysis indicated that all the five isolates from Korea were clustered into same clade but were not distinctly apart from isolates from China, Pakistan, India, and Türkiye. To develop an efficient diagnosis system for the four viruses, a simultaneous detection method was constructed using multiplex RT-PCR. Sensitivity evaluation, simplex RT-PCR, and stability testing were conducted to verify the multiplex RT-PCR system developed in this study. This information will be useful for developing effective disease management strategies for citrus growers in Korea.

Identification of connective tissue disease autoantibodies and a novel autoantibody anti-annexin A11 in patients with "idiopathic" interstitial lung disease.

BACKGROUND: Autoantibodies are a hallmark feature of Connective Tissue Diseases (CTD). Their presence in patients with idiopathic interstitial lung disease (ILD) may suggest covert CTD. We aimed to determine the prevalence of CTD autoantibodies in patients diagnosed with idiopathic ILD. METHODS: 499 patient sera were analysed: 251 idiopathic pulmonary fibrosis (IPF), 206 idiopathic non-specific interstitial pneumonia (iNSIP) and 42 cryptogenic organising pneumonia (COP). Autoantibody status was determined by immunoprecipitation. RESULTS: 2.4% of IPF sera had a CTD-autoantibody compared to 10.2% of iNSIP and 7.3% of COP. 45% of autoantibodies were anti-synthetases. A novel autoantibody targeting an unknown 56 kDa protein was found in seven IPF patients (2.8%) and two NSIP (1%) patients. This was characterised as anti-annexin A11. CONCLUSION: Specific guidance on autoantibody testing and interpretation in patients with ILD could improve diagnostic accuracy. Further work is required to determine the clinical significance of anti-annexin A11.

CRISPR-Cas12/Cas13: Bibliometric analysis and systematic review of its application in infectious disease detection.

BACKGROUND: Infectious diseases impose a significant burden on the global public health and economy, resulting in an estimated 15 million deaths out of 57 million annually worldwide. This study examines the current state of CRISPR-Cas12/Cas13 research, focusing on its applications in infectious disease detection and its evolutionary trajectory. METHODS: A bibliometric analysis and systematic review were conducted by retrieving CRISPR-Cas12/Cas13-related articles published between January 1, 2015 to December 31, 2022, from the Web of Science database. The research protocol was registered with International Platform of Registered Systematic Review and Meta-analysis Protocols (INPLASY202380062). RESULTS: Our search identified 1987 articles, of which, 1856 were included in the bibliometric analysis and 445 were used in qualitative analysis. The study reveals a substantial increase in scientific production on CRISPR-Cas12/Cas13, with an annual growth rate of 104.5%. The United States leads in the number of published articles. The systematic review identified 580 different diagnostic assays targeting 170 pathogens, with SARS-CoV-2 dominating with 158 assays. Recombinase polymerase amplification (RPA)/reverse transcription-RPA (RT-RPA) emerged as the predominant amplification method, while lateral flow assay was the most common readout method. Approximately 72% of the diagnostic assays developed are suitable for point-of-care testing. CONCLUSION: The rapid increase in research on CRISPR-Cas12/Cas13 between 2015 and 2022 suggests promising potential for advancements in infectious disease diagnosis. Given the numerous advantages of CRISPR-Cas technology for disease detection over other methods, and the dedicated efforts of scientists from around the world, it is reasonable to anticipate that CRISPR-Cas technology may emerge as a formidable alternative, offering the possibility of expedited point-of-care testing in the not-too-distant future.

Preexisting antibody assays for gene therapy: Considerations on patient selection cutoffs and companion diagnostic requirements.

Recombinant adeno-associated virus (AAV) vectors are the leading delivery vehicle used for in vivo gene therapies. Anti-AAV antibodies (AAV Abs) can interact with the viral capsid component of an AAV-based gene therapy (GT). Therefore, patients with preexisting AAV Abs (seropositive patients) are often excluded from GT trials to prevent treatment of patients who are unlikely to benefit1 or may have a higher risk for adverse events outweighing treatment benefits. On the contrary, unnecessary exclusion of patients with high unmet medical need should be avoided. Instead, a risk-benefit assessment that weighs the potential risks due to seropositivity vs. severity of disease and available treatment options, should drive the decision if patient selection is required. Assays for patient selection must be validated according to their intended use following national regulations/standards for diagnostic assays in appropriate laboratories. In this review, we summarize the current process of patient selection, including assay cutoff criteria and related assay validation approaches. We further provide considerations on regulatory requirements for the development of in vitro diagnostic tests supporting market authorization of a corresponding GT.

Portable, single nucleotide polymorphism-specific duplex assay for virus surveillance in wastewater.

The Argonaute protein from the archaeon Pyrococcus furiosus (PfAgo) is a DNA-guided nuclease that targets DNA with any sequence. We designed a virus detection assay in which the PfAgo enzyme cleaves the reporter probe, thus generating fluorescent signals when amplicons from a reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay contain target sequences. We confirmed that the RT-LAMP-PfAgo assay for the SARS-CoV-2 Delta variant produced significantly higher fluorescent signals (p < 0.001) when a single nucleotide polymorphism (SNP), exclusive to the Delta variant, was present, compared to the samples without the SNP. Additionally, the duplex assay for Pepper mild mottle virus (PMMOV) and SARS-CoV-2 detection produced specific fluorescent signals (FAM or ROX) only when the corresponding sequences were present. Furthermore, the RT-LAMP-PfAgo assay does not require dilution to reduce the impact of environmental inhibitors. The limit of detection of the PMMOV assay, determined with 30 wastewater samples, was 28 gc/µL, with a 95 % confidence interval of [11,103]. Finally, using a point-of-use device, the RT-LAMP-PfAgo assay successfully detected PMMOV in wastewater samples. Based on our findings, we conclude that the RT-LAMP-PfAgo assay can be used as a portable, SNP-specific duplex assay, which will significantly improve virus surveillance in wastewater.

Bacterial Leaf Spot Susceptibility Screening of Chili Pepper Cultivars Using qPCR Determination of Xanthomonas euvesicatoria pv. euvesicatoria Titers.

Bacterial leaf spot is a serious disease of chili pepper (Capsicum spp.) caused by Xanthomonas euvesicatoria pv. euvesicatoria. Conventional resistance screening is time and resource intensive. It was considered that a quick and simple determination of cultivar susceptibility could be achieved through estimating bacterial titers of inoculated plants. A SYBR quantitative polymerase chain reaction (qPCR)-based assay was compared with conventional PCR, then used to detect and enumerate pathogen titers in serial dilutions and DNA extracted from infected plant leaves. The qPCR detection limit was approximately 1 CFU µl-1, 10 times more sensitive than conventional PCR. A linear correlation (R2 = 0.994) was obtained from the standard curve comparing plate-truthed serial dilutions of the pathogen with the qPCR cycle threshold. Six strains were used to inoculate cultivars Hugo and Warlock. One strain, X. euvesicatoria pv. euvesicatoria BRIP62403, was consistently the most virulent based on visual symptoms and pathogen titers in planta inferred by qPCR performed on DNA extracted from infected leaves 2 and 6 weeks postinoculation. Visual observations 6 weeks after inoculation were highly correlated (R2 = 0.8254) to pathogen titers. The qPCR method was used to categorize 20 chili pepper cultivars 2 weeks after inoculation. A high positive correlation (R2 = 0.6826) was observed between visual scoring and pathogen titers from 20 chili pepper cultivars, facilitating categorization of susceptible, intermediate, and resistant cultivars. The qPCR approach developed here facilitates susceptibility screening of chili pepper cultivars at an early stage of selection and could be readily adapted to a range of other pathosystems.

Utilizing Electrochemical Biosensors as an Innovative Platform for the Rapid and On-Site Detection of Animal Viruses.

Animal viruses are a significant threat to animal health and are easily spread across the globe with the rise of globalization. The limitations in diagnosing and treating animal virus infections have made the transmission of diseases and animal deaths unpredictable. Therefore, early diagnosis of animal virus infections is crucial to prevent the spread of diseases and reduce economic losses. To address the need for rapid diagnosis, electrochemical sensors have emerged as promising tools. Electrochemical methods present numerous benefits, including heightened sensitivity and selectivity, affordability, ease of use, portability, and rapid analysis, making them suitable for real-time virus detection. This paper focuses on the construction of electrochemical biosensors, as well as promising biosensor models, and expounds its advantages in virus detection, which is a promising research direction.

Nomenclature for standardized designation of diploid genotypes in genetically modified laboratory animals.

Information about the diploid genotype of a gene-modified or mutant laboratory animal is essential for breeding and experimental planning. It is also required for the exchange of animals between different research groups or for communication with professional genotyping service providers. While there are detailed, standardized rules for creating an allele name of a genome modification or mutation, the notation of the diploid genotype after biopsy and genotyping has not been standardized yet. Therefore, a uniform, generally understandable nomenclature for the diploid genotype of gene-modified laboratory animals is needed. With the here-proposed nomenclature recommendations from the Committee on Genetics and Breeding of Laboratory Animals of the German Society for Laboratory Animal Science (GV-SOLAS), we provide a practical, standardized representation of the genotype of gene-modified animals. It is intended to serve as a compact guide for animal care and scientific personnel in animal research facilities and to simplify data exchange between groups and with external service providers.

Multicenter performance evaluation of the Alinity m CMV assay for quantifying cytomegalovirus DNA in plasma samples.

Monitoring of cytomegalovirus (CMV) viral load is critical for informing treatment decisions in order to prevent the severe health consequences of CMV infection or reactivation of latent CMV in immunocompromised individuals. This first field evaluation examined the analytical and clinical performance of the Alinity m CMV assay. Analytical performance was assessed with a commercially available six-member panel, while the clinical performance evaluation compared the Alinity m CMV assay to the RealTime CMV assay and a laboratory-developed test (LDT) as the test of record at three large hospital-based clinical laboratories. Precision of the Alinity m CMV assay was demonstrated with total standard deviation (SD) between 0.08 and 0.28 Log IU/mL. A total of 457 plasma specimens were tested on the Alinity m CMV assay and compared to the test of record at each site (n = 304 with RealTime CMV and n = 153 with LDT CMV). The Alinity m CMV assay had excellent correlation (correlation coefficient r ≥0.942) in comparison to the RealTime CMV or LDT CMV assays. The mean observed bias ranged from -0.03 to 0.34 Log IU/mL. Median onboard turnaround time of Alinity m CMV was less than 3 h. When the CMV assay is run on the Alinity m system, it has the capacity to shorten time to result and, therefore, to therapy.

Multi-site performance evaluation of the Alinity m Molecular assay for quantifying Epstein-Barr virus DNA in plasma samples.

Detection and monitoring of acute infection or reactivation of Epstein-Barr virus (EBV) are critical for treatment decision-making and to reduce the risk of EBV-related malignancies and other associated diseases in immunocompromised individuals. The analytical and clinical performance of the Alinity m EBV assay was evaluated at two independent study sites; analytical performance was assessed by evaluating precision with a commercially available 5-member EBV verification panel, while the clinical performance of the Alinity m EBV assay was compared to the RealTime EBV assay and a laboratory-developed test (LDT) as the routine test of record (TOR). Analytical analysis demonstrated standard deviation (SD) between 0.08 and 0.13 Log IU/mL. A total of 300 remnant plasma specimens were retested with the Alinity m EBV assay, and results were compared to those of the TOR at the respective study sites (n = 148 with the RealTime EBV assay and n = 152 with the LDT EBV assay). Agreement between Alinity m EBV and RealTime EBV or LDT EBV assays had kappa values of 0.88 and 0.84, respectively, with correlation coefficients r of 0.956 and 0.912, while the corresponding observed mean bias was -0.02 and -0.19 Log IU/mL. The Alinity m EBV assay had a short median onboard turnaround time of 2:40 h. Thus, the Alinity m system can shorten the time to results and, therefore, to therapy.

A strip-based assay for detection of CrfA enzyme activity to differentiate Mycobacterium tuberculosis and non-tuberculous mycobacteria.

There is an unmet need for tools that permit diagnosis of Tuberculosis (TB) that are affordable, low-tech, and can differentiate Mycobacterium tuberculosis (M.tb) from non-tuberculous mycobacteria (NTM). In this study, we have developed a strip-based assay to detect the activity of a unique Carbapenem Resistance Factor A (CrfA) enzyme present only in M.tb. The strip comprises of PVDF (Polyvinylidene fluoride) membrane that has an immobilized anti-CrfA antibody to capture the CrfA enzyme from M.tb lysate. Lysate of mycobacteria is applied to the strip, washed, and incubated in the presence of chromogenic reporter dye which is a substrate for CrfA. A change in the color of the dye that is readily visible to the naked eye is the readout. We evaluated lysates from M.tb and various NTMs namely, M. abscessus, M. chelonae, M. avium, M. obuense, M. paraintracellulare, M. kansasi, including the patient-derived sputum samples. The strip assay selectively identified only those samples containing M.tb. Based on this evidence, this new assay enables the identification and differentiation of M.tb from NTMs in patient sputum samples. As this tool can be simple to use, therefore has the potential to serve the unmet need for diagnosis of TB and NTM infections in resource-limited settings.