Multiple Diagnostic Assays Research Articles

Discordant performance of mpox serological assays

BackgroundSince July 23, 2022, global mpox cases reached 92,546, with over 31,000 in the United States. Asymptomatic carriage is a critical mechanism influencing the global dissemination of mpox. Seroprevalence studies are crucial for determining the epidemic's true burden, but uncertainties persist in serologic assay performance and how smallpox vaccination may influence assay interpretation. ObjectivesOur study aimed to assess the performance of several diagnostic assays among mpox-positive, vaccinated, and pre-outbreak negative control samples. This investigation sought to enhance our understanding and management of future mpox outbreaks. Study designSerum samples from 10 mpox-positive, five vaccinated uninfected, and 137 pre-outbreak controls were obtained for serological testing. The mpox-positive samples were obtained around 100 days post symptom onset, and vaccinated patients were sampled approximately 90 days post-vaccination. Multiple diagnostic assays were employed, including four commercial ELISAs (Abbexa, RayBioTech, FineTest, ProteoGenix) and a multiplex assay (MesoScale Diagnostics (MSD)) measuring five mpox and five smallpox antigens. ResultsThree commercial ELISA kits had low specificity (<50 %). The Proteogenix ELISA targeting the E8L antigen had a 94 % sensitivity and 87 % specificity. The E8L antigen on the MSD assay exhibited the greatest distinction between exposure groups, with 98 % sensitivity and 93 % specificity. ConclusionsNone of the assays could distinguish between mpox-positive and vaccinated samples. The MSD assay targeting the MPXV E8L antigen demonstrated the greatest differentiation between mpox-positive and pre-outbreak negative samples. Our findings underscore the imperative to identify sensitive and specific assays to monitor population-level mpox exposure and infection.

Detection of SARS-CoV-2 Variants by Multiple Diagnostic Assays Second RESUBMISSION JCV-D-21-00675R2.

IntroductionThe emergence of SARS-CoV-2 Variants of Concern (VOC) and Variants Being Monitored (VBM) have presented additional clinical and public health concerns regarding potential virus transmissibility, disease severity, and immune evasion. It is imperative that diagnostic assays can detect all such variants, and since commercial oligo sequences are commonly not available, empirical testing may be necessary to confirm this. To confirm the sensitivity of the SARS-CoV-2 assays used at the Wadsworth Center for the detection of VOC and VBM, relevant specimens were selected from the specimen archive and tested in the various platforms.Materials and MethodsPatient respiratory specimens submitted from clinal laboratories across the state were selected; three samples per variant were chosen to account for inter assay and variant reproducibility. The four molecular diagnostic platforms for SARS-CoV-2 currently in use at our facility were examined.ResultsA total of 64 specimens were tested, representing 2 VOC, 8 VBM and 4 other variants circulating in New York State. For certain samples, original Ct values provided by sample submitters were much higher, or lower, than those obtained from this study. The investigation of submitter testing platforms, with consideration of the assay's viral targets, confirmed the differences in Ct were not variant specific.ConclusionsIt was demonstrated that the diagnostic methods investigated in this study detected all the variants tested. Because of the continual evolution of the virus, it is vital to monitor new variants as they emerge for the ability of molecular diagnostic methods to detect them with acceptable sensitivity.

Conditions for Handling and Optimal Storage of Mycolactone.

The successful isolation of mycolactone in a laboratory or from a clinical sample relies on proper handling and storage of the toxin. Mycolactone is a light-sensitive and an amphiphilic toxin produced by Mycobacterium ulcerans. The biochemistry of the toxin makes it unstable in aqueous matrices such as blood, which causes it to self-aggregate or present in complex with carrier molecules. This biochemistry also impacts the use of the toxin in vitro, in that it tends to aggregate and stick to substrates in an aqueous environment, which alters its physiological presentation and limits its availability in a sample. Glass materials (i.e., tubes, vials, syringes, plates) should be used when possible to avoid loss of mycolactone sticking to plastic surfaces. Dark containers such as amber vials or aluminum-foil wrapped tubes should be used to avoid photodegradation of the toxin upon exposure to light. Sample storage in organic solvents is ideal for mycolactone stability and recovery; however, this is not always amenable as multiple diagnostic assays might be performed on a single sample (such as PCR or ELISA). In these cases, samples can be stored in an aqueous solution containing a small amount of detergent to enhance recovery of the toxin, and in order to avoid aggregation. Therefore, the downstream manipulations should be carefully considered prior to sample collection and storage. Here we present considerations for the optimal handling and storage of mycolactone in order to obtain quality yield of the toxin for various research and diagnostic applications.

Challenges in diagnosing scrub typhus among hospitalized patients with undifferentiated fever at a national tertiary hospital in northern Vietnam.

BackgroundScrub typhus (ST) is a leading cause of non-malarial febrile illness in Southeast Asia, but evidence of its true disease burden is limited because of difficulties of making the clinical diagnosis and lack of adequate diagnostic tests. To describe the epidemiology and clinical characteristics of ST, we conducted an observational study using multiple diagnostic assays at a national tertiary hospital in Hanoi, Vietnam.Methodology/Principal findingsWe enrolled 1,127 patients hospitalized with documented fever between June 2012 and May 2013. Overall, 33 (2.9%) patients were diagnosed with ST by PCR and/or screening of ELISA for immunoglobulin M (IgM) with confirmatory tests: 14 (42.4%) were confirmed by indirect immunoperoxidase assay (IIP), and 19 (57.6%) were by IIP and PCR. Living by farming, conjunctival injection, eschar, aspartate aminotransferase elevation, and alanine aminotransferase elevation were significantly associated with ST cases (adjusted odds ratios (aORs): 2.8, 3.07, 48.8, 3.51, and 4.13, respectively), and having a comorbidity and neutrophilia were significantly less common in ST cases (aORs: 0.29 and 0.27, respectively). The majority of the ST cases were not clinically diagnosed with rickettsiosis (72.7%). Dominant IIP reactions against a single antigen were identified in 15 ST cases, whereas indistinguishably high reactions against multiple antigens were seen in 11 ST cases. The most frequently observed dominant IIP reaction was against Karp antigen (eight cases) followed by Gilliam (four cases). The highest diagnostic accuracy of IgM ELISA in acute samples was 78%. In a phylogenetic analysis of the 56-kDa type-specific antigen gene, the majority (14 cases) were located in the Karp-related branch followed by the Gilliam-related (two cases), Kato-related (two cases), and TA763-related clades (one case).Conclusions/SignificanceBoth the clinical and laboratory diagnoses of ST remain challenging at a tertiary hospital. Implementation of both serological and nucleic acid amplification assays covering endemic O. tsutsugamushi strains is essential.

Human Cytomegalovirus Cell Tropism and Host Cell Receptors.

In the 1970s–1980s, a striking increase in the number of disseminated human cytomegalovirus (HCMV) infections occurred in immunosuppressed patient populations. Autopsy findings documented the in vivo disseminated infection (besides fibroblasts) of epithelial cells, endothelial cells, and polymorphonuclear leukocytes. As a result, multiple diagnostic assays, such as quantification of HCMV antigenemia (pp65), viremia (infectious virus), and DNAemia (HCMV DNA) in patient blood, were developed. In vitro experiments showed that only low passage or endothelial cell-passaged clinical isolates, and not laboratory-adapted strains, could reproduce both HCMV leuko- and endothelial cell-tropism, which were found through genetic analysis to require the three viral genes UL128, UL130, and UL131 of the HCMV UL128 locus (UL128L). Products of this locus, together with gH/gL, were shown to form the gH/gL/pUL128L pentamer complex (PC) required for infection of epithelial cells/endothelial cells, whereas gH/gL and gO form the gH/gL/gO trimer complex (TC) required for infection of all cell types. In 2016, following previous work, a receptor for the TC that mediates entry into fibroblasts was identified as PDGFRα, while in 2018, a receptor for the PC that mediates entry into endothelial/epithelial cells was identified as neuropilin2 (Nrp2). Furthermore, the olfactory receptor family member OR14I1 was recently identified as a possible additional receptor for the PC in epithelial cells. Thus, current data support two models of viral entry: (i) in fibroblasts, following interaction of PDGFRα with TC, the latter activates gB to fuse the virus envelope with the cell membrane, whereas (ii) in epithelial cells/endothelial cells, interaction of Nrp2 (and OR14I1) with PC promotes endocytosis of virus particles, followed by gB activation by gH/gL/gO (or gH/gL) and final low-pH entry into the cell.

Dihydropyridine Fluorophores Allow for Specific Detection of Human Antibodies in Serum.

Antigen recognition by antibodies plays an important role in human biology and in the development of diseases. This interaction provides a basis for multiple diagnostic assays and is a guide for treatments. We have developed dihydropyridine-based fluorophores that form stable complexes with double-stranded DNA and upon recognition of the antibodies to DNA (anti-DNA) provide an optical response. The fluorophores described herein have advantageous optical properties compared to those of the currently available dyes making them valuable for research and clinical diagnostics. By studying a series of novel fluorophores, crucial parameters for the design were established, providing the required sensitivity and specificity in the detection of antibodies. Using these DNA–fluorophore complexes in a direct immunofluorescence assay, antibodies to DNA are specifically detected in 80 patients diagnosed with an autoimmune disease, systemic lupus erythematosus. Positivity indicated by emission change of α-(4′-O-methoxyphenyl)-2-furyl dihydropyridine strongly correlates with other disease biomarkers and autoimmune arthritis.

Isolation and characterization of pathogenic leptospires associated with cattle

Pathogenic leptospires colonize the renal tubules of reservoir hosts of infection, including cattle, and are excreted via urine. In order to identify circulating serovars of pathogenic leptospires in beef cattle, and their associated rates of urinary excretion, a cross sectional study was performed. Fifty urine samples were collected one day each month over 12 consecutive months (N = 600), directly from the bladder of beef cattle at a single slaughter facility and assessed for the presence of leptospires by culture and the fluorescent antibody test (FAT). Where possible, a matched serum sample was also collected for the microscopic agglutination test (MAT). Forty-three urine samples were either culture positive or FAT positive, indicating that 7.2% of sampled beef cattle were actively excreting leptospires in urine. Twenty-three urine samples were culture positive. Sequence analysis of 16S ribosomal DNA and secY indicated that all isolates were Leptospira borgpetersenii. Typing by serology indicated that all isolates were serogroup Sejroe. An overall seroprevalence of 20% (MAT ≥ 1:25) was determined; positive bovine sera was most reactive to serogroup Sejroe (serovar Hardjo) (8.1%), and serogroup Australis (serovar Bratislava) (6.7%). There was poor correlation between seroprevalence and excretion of leptospires since 18/43 (41.9%) cattle, which were positive by culture or FAT, were seronegative. The virulence of two selected isolates of L. borgpetersenii was confirmed by experimental infection in small animal models of infection. Results confirm that L. borgpetesenii continues to circulate in beef cattle and that multiple diagnostic assays are required to detect active shedding. These findings also highlight beef cattle as a reservoir host for the potential zoonotic transmission of leptospires.

A Novel Method for Mutation Analysis Using Genomic DNA Obtained from Immunohistochemistry-Stained Sections

Background: Tumor biopsies obtained from patients are often limited in size and availability, and the ability to perform multiple diagnostic assays depends on the quantity and quality of the tissue. Here we describe and evaluate a method for performing DNA-based mutational analyses after immunohistochemistry analysis has been performed, using a single tissue section. Method: Immunohistochemistry analysis was performed on 4-5 μm formalin-fixed paraffin-embedded tumor tissue sections and immunohistochemistry-stained sections were stored for subsequent genomic analysis. DNA was isolated from these immunohistochemistry-stained sections and DNA quality was assessed using a multiplexpolymerase chain reaction method as well as real time quantitative polymerase chain reaction of commonly used reference genes. Subsequently, genomic DNA was pre-amplified and mutations in KRAS, BRAF, NRAS and PIK3CA were detected by validated Taqman assays. Comparisons were made with results from unstained formalinfixed paraffin-embedded sections obtained from the same paraffin block. Results: Our results demonstrate that genomic DNA isolated from immunohistochemistry-stained and unstained formalin-fixed paraffin-embedded tissue sections are comparable in quality and are suitable for down-stream analysis using polymerase chain reaction based assays. We also found that the sensitivity and specificity in detecting hotspot mutations are comparable in both sources of genomic DNA. This study reports 100% concordance in detecting hotspot mutations in KRAS, BRAF, NRAS and PIK3CA using quantitative real-time polymerase chain reaction between stained and unstained formalin-fixed paraffin-embedded sections. Conclusion: We conclude that by using our novel approach, it is possible to perform immunohistochemistry staining followed by genomic analysis using a single 4-5 μm section of formalin-fixed paraffin-embedded tissue.

Paper and toner three-dimensional fluidic devices: programming fluid flow to improve point-of-care diagnostics

We present a new method for fabricating three-dimensional paper-based fluidic devices that uses toner as a thermal adhesive to bond multiple layers of patterned paper together. The fabrication process is rapid, involves minimal equipment (a laser printer and a laminator) and produces complex channel networks with dimensions down to 1 mm. The devices can run multiple diagnostic assays on one or more samples simultaneously, can incorporate positive and negative controls and can be programmed to display the results of the assays in a variety of patterns. The patterns of the results can encode information, which could be used to identify counterfeit devices, identify samples, encrypt the results for patient privacy or monitor patient compliance.

Two general designs for fluidic batteries in paper-based microfluidic devices that provide predictable and tunable sources of power for on-chip assays

Microfluidic devices fabricated out of paper (and paper and tape) have emerged as promising platforms for conducting multiple diagnostic assays simultaneously in resource-limited settings. Certain types of assays in these devices, however, require a source of power to function. Lithium ion, nickel-cadmium, and other types of batteries have been used to power these devices, but these traditional batteries are too expensive and pose too much of a disposal hazard for diagnostic applications in resource-limited settings. To circumvent this problem, we previously designed a “fluidic battery” that is composed of multiple galvanic cells, incorporated directly into a multilayer paper-based microfluidic device. We now show that multiple cells of these fluidic batteries can be connected in series and/or in parallel in a predictable way to obtain desired values of current and potential, and that the batteries can be optimized to last for a short period of time (<1 min) or for up to 10–15 min. This paper also (i) outlines and quantifies the parameters that can be adjusted to maximize the current and potential of fluidic batteries, (ii) describes two general configurations for fluidic batteries, and (iii) provides equations that enable prediction of the current and potential that can be obtained when these two general designs are varied. This work provides the foundation upon which future applications of fluidic batteries will be based.

Abstract 3064: A novel method for mutation analyses using genomic DNA obtained from IHC-stained sections

Abstract Biopsies obtained from patients are often limited in size. The ability to perform multiple diagnostic assays is dependent on the quantity and quality of tissue samples available from patients. Over the last few years, our laboratory has focused our efforts on developing novel methodologies for increased efficiency in use of tissue samples. Here we describe a method for performing immunohistochemistry (IHC) and genotyping using the same tissue section. We isolated genomic DNA (gDNA) from IHC-stained and unstained formalin-fixed paraffin-embedded (FFPE) tissue sections obtained from the same paraffin block. Quality and quantitylity of gDNA obtained from either sections were compared. We demonstrate that quality of gDNA obtained is acceptable for down-stream analysis using PCR techniques. We also show comparable specificity and sensitivity in detecting mutations using either sources of gDNA. This method has proven highly valuable in performing mutation analysis in certain tissue types such as lung cancer where tissue can be extremely limited. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3064. doi:10.1158/1538-7445.AM2011-3064

New globally faces of hepatitis B and C in the world.

Worldwide, infections with hepatitis B virus (HBV) and hepatitis C virus (HCV) have become a major cause of chronic hepatitis, cirrhosis and hepatocellular carcinoma (HCC) and the major global problems for public health in the world and Iran (1–3). HBV infection is the main cause of chronic liver disease in Iran (4) and the seroprevalence of HBV infection in Iran has been decreased during the last two decades (5). The main risk factor for HBV transmission was related to maternal-to-infantile transmission and the infantile vaccination with high coverage is the main cause for changes in epidemiology of HBV infection. But today, we have more risk factors for transmission in adulthood. The community contacts more in adolescence and the health policy makers should attend more for control of HBV infection. People awareness, risk factors modifications, harm reduction programmes in young addicted persons, vaccination against HBV in adults should be consider for control of HBV infection in future. We have less new cases, but we will have more cirrhotic cases and we need to consider the best therapy for prevention of HBV infection to end-stage phases. Therapy of HBV infection in which phase and which drugs are the debate. The goal of therapy in chronic hepatitis B (CHB) patients is sustained suppression of HBV viremia and decrease progression of liver disease to cirrhosis and HCC with the ultimate goal of improving survival. This can be pursued by maintaining constant inhibition of viral replication through a long term treatment with nucleos(t)ide analogs or by inducing, through the combined antiviral and immunomodulatory effects of interferon, a sustained immune response (6). Standard interferon is the oldest approved drug in therapy of HBV infection, but it has a limited response rate. Recently new published data have shown the better response with pegylated interferon in therapy of chronic HBV infection (7, 8). PEG-IFNα-2a has been licensed for the treatment of both HBeAg-positive and HBeAg-negative chronic HBV as a 48-week course, given by subcutaneous injection once weekly in a dosage of 180 mg. The main side effects of therapy with nucleos(t)ide analogs such as lamivudine, adefovir and entecavir, is emergence of resistance (9, 10). These events will decrease benefit of therapy with anti-viral agents. HBs Ag level in prediction of therapy response The main question was and is regarding how and when we can conclude the therapy in HBV infection is effective and we should continue or it is ineffective and we should discontinue it. During recent years serum HBsAg is hypothesized to be a marker for immunological response to therapy, independent of virological response as measured using HBV DNA levels, because of its correlation with intrahepatic HBV covalently closed circular (ccc) DNA, the main replicative template of HBV (11). HBsAg levels appear to differentiate patients in the inactive carrier state from those with active disease or from inactive carriers with a high probability of subsequent relapse. Furthermore, recent studies have shown that on- treatment HBsAg levels are predictive of a durable off-treatment response to peginterferon (PEG-IFN), and can accurately identify non-responders early during therapy, both in HBeAg-positive, and HBeAg-negative patients. Among those who underwent HBsAg loss within 6 years of follow-up, serum HBsAg levels were a better predictor than HBV DNA levels. Low serum levels of HBsAg, alone or in combination with HBV DNA levels, 1 year after HBeAg seroconversion can predict HBsAg loss in patients with HBV infection (12). Currently, multiple diagnostic assays are available for quantification of HBsAg. That we should use the validate method for diagnosis.

A novel method for mutation analyses using genomic DNA obtained from IHC-stained sections

Abstract Biopsies obtained from patients are often limited in size. The ability to perform multiple diagnostic assays is dependent on the quantity and quality of tissue samples available from patients. Over the last few years, our laboratory has focused our efforts on developing novel methodologies for increased efficiency in use of tissue samples. Here we describe a method for performing immunohistochemistry (IHC) and genotyping using the same tissue section. We isolated genomic DNA (gDNA) from IHC-stained and unstained formalin-fixed paraffin-embedded (FFPE) tissue sections obtained from the same paraffin block. Quality and quantity of gDNA obtained from either section were compared. We demonstrate that quality of gDNA obtained is acceptable for down-stream analysis using PCR techniques. We also show comparable specificity and sensitivity in detecting mutations using either sources of gDNA. This method has proven highly valuable in performing mutation analysis in certain tissue types such as lung cancer where tissue can be extremely limited.

Combination of Biomarkers-IGM by Logistic Regression Improves Diagnostic Accuracy in Hepatocellular Carcinoma

Background and aim Circulating immune complexes of alpha-fetoprotein (AFP) and SCCA (squamous cell carcinoma antigen) with IgMs (AFP-IgM and SCCA-IgM, respectively) represent a promising new class of serum markers with diagnostic and prognostic value in the management of patients with hepatocellular carcinoma (HCC). The enhancement of diagnostic indexes for cancer detection achieved with combination of biomarkers by linear logistic regression has been demonstrated in several simulation studies on multiple diagnostic assays. The aim of the study was to evaluate the improvement of the diagnostic accuracy by combination of SCCA-IgM and AFP-IgM compared with the diagnostic accuracy of free AFP in sera of patients with HCC and cirrhosis. Patients and methods Serum samples from 81 patients with HCC (mean age ± SD: 63 ± 8 years; male patients 73%; HCV infected 52%; without any therapeutic treatment 50%) and 82 patients with cirrhosis (mean age ± SD: 53 ± 9 years; male patients 66%; HCV infected 68%) were collected. The logistic regression parameters were calculated on a previously published data set of 50 patients with HCC and 50 patients with cirrhosis, where the distribution of serum levels of SCCA-IgM and AFP-IgM in HCC patients was significantly different from that in patients with cirrhosis (Mann-Whitney U test, p&lt;0.01). Serum levels of SCCA-IgM and AFP-IgM were assessed in parallel using the Hepa-IC and Hepa AFP-IC kits and AFP was determined by an automatic analyzer (ADVIA Centaur®, Siemens Diagnostics, Italy). Results The patients were stratified according to sex (male), HCV infection (positive) and therapeutic treatment (negative) to apply a linear logistic regression model using regression parameters calculated from a data set of a reference population. A subgroup of 30 patients with HCC and 41 patients with cirrhosis was analyzed. The diagnostic accuracy measured as the area under the ROC curve (AUC) for the SCCA-IgM and AFP-IgM assays was roughly the same, with a weak supremacy of AFP-IgM (AUC = 0.62), and was comparable with that of free AFP (AUC = 0.64). The gain in diagnostic accuracy achieved with the combination of SCCA-IgM and AFP-IgM levels by linear logistic regression was 14% (AUC = 0.71) compared with the diagnostic accuracy of AFP-IgM and 11% compared with that of free AFP. With the estimation of the partial AUC (pAUC0.3), an alternative measurement of AUC that takes into consideration only specificity rates with clinical relevance (specificity &gt;70%), the highest improvement in accuracy for HCC detection was obtained, with an increase of up to 62% compared to pAUC0.3 of AFP-IgM and up to 23% compared to pAUC0.3 of free AFP. Conclusion The results demonstrate that the combination by linear logistic regression of biomarkers-IgM immune complexes improves the diagnostic accuracy for HCC detection using regression parameters calculated in a reference population with defined clinical characteristics. These results support the usefulness of devices based on a panel of non-overlapping biomarkers-IgM complexes, such as multi-marker biochips, increasing the sensitivity and maintaining a high specificity for HCC diagnosis compared with conventional single-marker assays.

Combining SCCA-IgM and AFP-IgM levels increases accuracy of hepatocellular carcinoma detection

Background and AIm. The assessment of serum levels of SCCA-IgM or AFP-IgM immune complexes may improve diagnosis of HCC and may also predict the progression of cirrhosis to HCC. Simulation studies on multiple diagnostic assays of cancer biomarkers suggest to combine biomarkers by linear logistic regression in order to optimize diagnostic accuracy. Aim of the study was to evaluate the diagnostic accuracy improvement in HCC by SCCA-IgM and AFP-IgM combination in sera of HCC and cirrhotic patients.

Patterned Paper as a Platform for Inexpensive, Low‐Volume, Portable Bioassays

Patterned Paper as a Platform for Inexpensive, Low‐Volume, Portable Bioassays