Monoplex Assays Research Articles

Detection of virulence genes and multidrug resistance in Pseudomonas aeruginosa clinical isolates from a public hospital in Lagos, Nigeria

Pseudomonas aeruginosa is a main cause of nosocomial infections and is classified as a priority critical pathogen of global concern. Multidrug resistance and virulence genes attributed to the pathogenicity of the organism has resulted in failed therapies, linked to increased morbidity and mortality in infected patients. The aim of this study was to determine the virulence genes and multidrug resistant P.aeruginosa in clinical specimens from a secondary health care center laboratory in Lagos, Nigeria. MethodologyA total of three hundred and sixty-seven clinical samples were examined with conventional methods and Microbact GNB12A. Forty- seven (47) P. aeruginosa strains were identified. Antimicrobial susceptibility test was performed by the standard disk diffusion method following CLSI guidelines. Identification of P. aeruginosa was done by amplification of oprL and oprI genes. Monoplex and multiplex PCR assays were used for detecting virulence genes (lasB, toxA, exoU and nan1). Descriptive statistical methods were used to analyze the distribution of P. aeruginosa by sample sources, MDR patterns by number of isolates and virulence gene carriage by MDR (≥3classes of antibiotics). ResultsA total of forty-seven (47) isolates were confirmed as P. aeruginosa. The isolates showed pan-resistance to cefuroxime and ceftazidime. An elevated level of resistance was also seen in amoxycillin-clavulanic acid, ceftriaxone, cefixime and meropenem. Susceptibility to Amikacin was 95.7 %. Multidrug resistance was seen in forty (85.1 %) of the isolates. P. aeruginosa (100 %) were confirmed with oprL and 74.5 % oprI genes. All isolates (100 %) had lasB, toxA and exoS virulence genes while nan1 was detected in 19.1 % isolates. ConclusionsDiverse and multiple virulence genes were distributed among the multidrug resistant isolates. Understanding the factors contributing to pathogenicity and antibiotic resistance will aid in the treatment of infection. It will also promote judicious use of antimicrobials.

Optimising multiplex immunofluorescence staining for characterising the tumour immune micro-environment.

Exploring the tumour microenvironment provides insight into the unique interaction between the host and tumour. Ultimately, its study improves understanding of how an individual mounts and achieves an anti-tumour immune response. In the context of colorectal cancer, immune biomarkers within the tumour microenvironment outperform traditional histopathological staging in predicting disease recurrence. Multiplex immunofluorescence enables simultaneous assessment of multiple markers to provide a highly accurate classification of immune cells and their spatial characterisation relative to tumour tissue. Further, automated slide staining provides staining consistency and reduces labour costs. Image acquisition using a non-spectral scanner allows more researchers to utilise multiplexed immunofluorescence for translational research. Herein we describe the optimisation process of conducting automated staining using a five-colour, tyramide signal amplification-based multiplex immunofluorescence panel. Using antibodies against CD3, CD8, CD103 and cytokeratin, the panel characterises T cell populations within human colorectal adenocarcinoma tissue. We provide an overview of primary antibody titration and the development of tyramide signal amplification immunofluorescence monoplex assays. We detail the processes of antibody stripping and the role of exogenous horseradish peroxidase inhibition to facilitate multiplexing. An account of determining the staining sequence and fluorophore assignment is provided. We describe image acquisition using a standard fluorescence microscope slide scanner and the management of spectral crosstalk using this system. Finally, we briefly document the digital image analysis required to characterise cells and determine their spatial distribution within the colorectal tumour microenvironment.

Development and validation of a pentaplex assay for the identification of antibodies against common viral diseases in cattle.

Animal welfare and economic implications of infectious diseases in cattle demand an efficient surveillance as the foundation for control and eradication programmes. Bovine respiratory syncytial virus (BRSV), Parainfluenza virus type 3 (PI3V), Bovine herpes virus-1 (BoHV-1), Bovine viral diarrhoea virus (BVDV), and Enzootic bovine leukosis virus (EBLV) cause common and often underdiagnosed diseases in cattle that are endemic in most countries [1]. A hallmark of individual exposure to a viral pathogen is the presence of antibodies directed towards that virus. The aim of this study was to develop and validate a pentaplex assay to simultaneously detect and quantify antibodies against BRSV, PI3V, BoHV-1, BVDV and EBLV in serum, as an efficient tool to yield epidemiological data. Monoplex assays were initially developed using either complete BRSV or BoHV-1 viral lysates, or recombinant proteins for BVDV, EBLV or PI3V as capture antigens. In addition, 125 serum samples from unvaccinated cattle, which were classified as positive or negative for each of the viruses by commercial ELISA kits, were used for validation. Conditions established for the Luminex monoplex assays were adopted for the pentaplex assay. The accuracy, determined by the area under the ROC curve, was greater than 0.97, and assay diagnostic sensitivities and specificities were over 95 and 90%, respectively, for all antigens. Intra (r) and interassay (R) coefficients of variation were under 10 and 20 %, respectively. Selectivity towards target viruses was shown by binding inhibition assays where unbound viruses reduced fluorescence intensities. Diagnostic agreement for samples analysed simultaneously in the monoplex and multiplex assays was almost perfect. In conclusion, a highly sensitive pentaplex assay was validated for the simultaneous identification of antibodies directed against BVDV, BoHV-1, PI3V, BRSV and EBLV in serum. The developed pentaplex assay complies with performance characteristics established by international guidelines for diagnostic tests and may be used as a tool for the implementation of epidemiological surveillance.

Simultaneous detection of SARS-CoV-2, influenza A, respiratory syncytial virus, and measles in wastewater by multiplex RT-qPCR

A multiplex quantitative reverse transcription polymerase chain reaction (RT-qPCR)-based method was designed for the simultaneous detection of influenza A, SARS-CoV-2, respiratory syncytial virus, and measles virus. The performance of the multiplex assay was compared to four monoplex assays for relative quantification using standard quantification curves. Results showed that the multiplex assay had comparable linearity and analytical sensitivity to the monoplex assays, and the quantification parameters of both assays demonstrated minimal differences. Viral reporting recommendations for the multiplex method were estimated based on the corresponding limit of quantification (LOQ) and the limit of detection at 95 % confidence interval (LOD) values for each viral target. The LOQ was determined by the lowest nominal RNA concentrations where %CV values were ≤35 %. Corresponding LOD values for each viral target were between 15 and 25 gene copies per reaction (GC/rxn), and LOQ values were within 10 to 15 GC/rxn. The detection performance of a new multiplex assay was validated in the field by collecting composite wastewater samples from a local treatment facility and passive samples from three sewer shed locations. Results indicated that the assay could accurately estimate viral loads from various sample types, with samples collected from passive samplers showing a greater range of detectable viral concentrations than composite wastewater samples. This suggests that the sensitivity of the multiplex method may be improved when paired with more sensitive sampling methods. Laboratory and field results demonstrate the robustness and sensitivity of the multiplex assay and its applicability to detect the relative abundance of four viral targets among wastewater samples. Conventional monoplex RT-qPCR assays are suitable for diagnosing viral infections. However, multiplex analysis using wastewater provides a fast and cost-effective way to monitor viral diseases in a population or environment.

Influenza-associated severe acute respiratory infections among children under five years old in Morocco, September 2017 to March 2019.

The main aim of this research is to investigate the trend of influenza infection among children under 5 years with severe acute respiratory infections (SARI) as well as those who suffer from a high burden of disease. This research is based on a survey conducted from September 2017 to March 2019. During this period nasopharyngeal swabs were collected in a group of 942 children under 5 years with SARI, admitted in pediatric services of 8 sentinel hospitals. The virological surveillance of influenza was carried out at the National influenza Center, located in the National Institute of Hygiene, using a Reverse transcription polymerase chain reaction (qRt-PCR) monoplex assay developed by the Centers for Disease Control and Prevention (CDC; Atlanta, GA). The median age of participants was 11 months, and 40% of them were female. A total of 112 samples were reported positive yielding a frequency of 11.88% (112/942). Among all the influenza confirmed cases, 68.75% (77/112), 15.17% (17/112), 16.04% (18/112) were subtyped as influenza AH1N1pdm09, AH3N2 and influenza B respectively. Meanwhile, the proportion of patients admitted at the intensive care unit was 5,35% (6/112). Out of which 83.33% (5/6) were AH1N1pdm09 and it was reported that just 1.78% (2/112) of the positive cases were vaccinated. The study confirms that influenza affects greatly children with SARI. Thus, the need for influenza vaccines is highly recommended for children under 5 years. Moreover, our findings highlight that influenza virus is not the only cause of SARI among this group of children. Accordingly, special attention should be paid to the non-flu respiratory viruses.

Rapid and specific detection of Mycoplasma gallisepticum and Mycoplasma synoviae infection in poultry using single and duplex PCR assays

Avian mycoplasmosis, mainly caused by Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS), is an economically important disease of poultry industry. The present study was aimed to develop duplex PCR as a rapid, specific and economical method for accurate detection of MG and MS in poultry and its comparison with single (monoplex) MG/MS PCR. During present investigation, a total of 146 poultry flocks having clinical history of respiratory disease were screened. Pooled tissue samples (trachea, lungs and air sacs) from 4-5 birds of each flock were collected during necropsy at disease investigation laboratories, Hisar, Haryana, India. The single and duplex PCR assays were standardized using primers of intergenic spacer region (IGSR; 16S-23S rRNA) for MG and hemagglutinin vlhA gene for MS, with expected amplicon size of 812 bp and 1200 bp products, respectively. In single PCR, 6.85%, 2.74% and 2.74% tissue samples were found positive for MG, MS and both MG and MS, respectively. However, duplex PCR showed, 7.53%, 2.74% and 1.37% positivity for MG, MS and both MG and MS, respectively. Taking the results of monoplex PCR as a gold standard, sensitivity and specificity of the developed duplex PCR was found to be 94.44% and 100%, respectively. Moreover, Cohen's kappa statistic (k = 0.97) measured a ‘perfect’ agreement between monoplex and duplex PCR assays. The positive and negative predictive values of duplex PCR was found to be 1.0 and 0.9922, respectively at 95% confidence interval (CI), as compared to monoplex PCR. The simultaneous use of two genes in a duplex PCR was more rapid and economical than two separate single PCR reactions.

Evaluation of a Trio Toscana Virus Real-Time RT-PCR Assay Targeting Three Genomic Regions within Nucleoprotein Gene.

Toscana virus (TOSV) can cause central nervous system infections in both residents of and travelers to Mediterranean countries. Data mining identified three real-time RT-qPCR assays for detecting TOSV RNA targeting non-overlapping regions in the nucleoprotein gene. Here, they were combined to create a multi-region assay named Trio TOSV RT-qPCR consisting of six primers and three probes. In this study, (i) we evaluated in silico the three RT-qPCR assays available in the literature for TOSV detection, (ii) we combined the three systems to create the Trio TOSV RT-qPCR, (iii) we assessed the specificity and sensitivity of the three monoplex assays versus the Trio TOSV RT-qPCR assay, and (iv) we compared the performance of the Trio TOSV RT-qPCR assay with one of the reference monoplex assays on clinical samples. In conclusion, the Trio TOSV RT-qPCR assay performs equally or better than the three monoplex assays; therefore, it provides a robust assay that can be used for both research and diagnostic purposes.

Multiplex assays for the identification of serological signatures of SARS-CoV-2 infection: an antibody-based diagnostic and machine learning study

SummaryBackgroundInfection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces an antibody response targeting multiple antigens that changes over time. This study aims to take advantage of this complexity to develop more accurate serological diagnostics.MethodsA multiplex serological assay was developed to measure IgG and IgM antibody responses to seven SARS-CoV-2 spike or nucleoprotein antigens, two antigens for the nucleoproteins of the 229E and NL63 seasonal coronaviruses, and three non-coronavirus antigens. Antibodies were measured in serum samples collected up to 39 days after symptom onset from 215 adults in four French hospitals (53 patients and 162 health-care workers) with quantitative RT-PCR-confirmed SARS-CoV-2 infection, and negative control serum samples collected from healthy adult blood donors before the start of the SARS-CoV-2 epidemic (335 samples from France, Thailand, and Peru). Machine learning classifiers were trained with the multiplex data to classify individuals with previous SARS-CoV-2 infection, with the best classification performance displayed by a random forests algorithm. A Bayesian mathematical model of antibody kinetics informed by prior information from other coronaviruses was used to estimate time-varying antibody responses and assess the sensitivity and classification performance of serological diagnostics during the first year following symptom onset. A statistical estimator is presented that can provide estimates of seroprevalence in very low-transmission settings.FindingsIgG antibody responses to trimeric spike protein (Stri) identified individuals with previous SARS-CoV-2 infection with 91·6% (95% CI 87·5–94·5) sensitivity and 99·1% (97·4–99·7) specificity. Using a serological signature of IgG and IgM to multiple antigens, it was possible to identify infected individuals with 98·8% (96·5–99·6) sensitivity and 99·3% (97·6–99·8) specificity. Informed by existing data from other coronaviruses, we estimate that 1 year after infection, a monoplex assay with optimal anti-Stri IgG cutoff has 88·7% (95% credible interval 63·4–97·4) sensitivity and that a four-antigen multiplex assay can increase sensitivity to 96·4% (80·9–100·0). When applied to population-level serological surveys, statistical analysis of multiplex data allows estimation of seroprevalence levels less than 2%, below the false-positivity rate of many other assays.InterpretationSerological signatures based on antibody responses to multiple antigens can provide accurate and robust serological classification of individuals with previous SARS-CoV-2 infection. This provides potential solutions to two pressing challenges for SARS-CoV-2 serological surveillance: classifying individuals who were infected more than 6 months ago and measuring seroprevalence in serological surveys in very low-transmission settings.FundingEuropean Research Council. Fondation pour la Recherche Médicale. Institut Pasteur Task Force COVID-19.

Abstract 4918: Artificial intelligence-assisted macrophage identification in tumor biopsies

Abstract Understanding response to immunotherapy requires accurate and complete characterization of tumor-associated immune cells in order to fully contextualize the immuno-oncology biomarker expression. Current standard practices surrounding enumeration of biomarker-positive immune cells using image analysis necessitate a dual-labeling approach combining the biomarker of interest and immune cell identification assays. Machine Learning (ML) may be used to distinguish different tissue types in a biopsy (e.g. tumor vs non-tumor), or to identify different cell types (e.g. macrophages vs other cells). A machine learning algorithm obtains statistics for a specific tissue class or cell type based on a training set, given by “ground truth” examples. The algorithm then generalizes from the given examples to “learn” the ability to find the tissue or cell type on the rest of the digital scan of the tissue slide, or other slide scans. Here we specifically describe ML methods for macrophage identification in digital scans of cancer tissue slides. The described methods can be used independently or in combination and are both based upon the use of artificial intelligence-based computational tissue analysis (cTA®) algorithms. These technologies can recognize macrophages independently of traditional IHC or IF dual-labeling identification methods. This same methodology may also be applicable for other specific subsets of morphologically distinct immune cells. The first strategy consists of training the cTA algorithm according to pathologist identification of macrophages, without a macrophage-specific staining (e.g., CD68). In this case, the slides are stained only with a PD-L1 assay, and pathologists establish the “ground truth” to teach the ML classifier. The second strategy consists of using CD68 labeling to identify macrophages to teach the ML classifier. Upon successful training and performance testing, the developed macrophage classifier can potentially be applied to new specimens without relying on pathologist annotation or IHC or IF dual-labeling for macrophage recognition. In summary, these two novel approaches demonstrate how ML could be used for characterization of critical immune cells such as macrophages in immuno-oncology tissue evaluations, without the reliance on additional biomarkers to specifically identify the cells. In this study, we evaluated the approach in the context of standard PD-L1 IHC as is used in the Companion Diagnostic (CDx) setting. Successful ML cTA strategies may be applied to other CDx assays to gain additional information about immune cell identification and quantification while only relying on simple monoplex assays. Importantly, these ML strategies would not require a change to the existing clinical practices where higher-level multiplexing would be needed to achieve similar outcomes. Citation Format: Kelsey Weigel, Will Paces, Elliott Ergon, Jeni Caldara, Kile McFadden, Cris Luengo, Roberto Gianani, Bharathi Vennapusa. Artificial intelligence-assisted macrophage identification in tumor biopsies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4918.

Development and Evaluation of a Multiplexed Immunoassay for Simultaneous Detection of Serum IgG Antibodies to Six Human Coronaviruses

Known human coronaviruses (hCoV) usually cause mild to moderate upper-respiratory tract illnesses, except SARS-CoV and MERS-CoV, which, in addition to mild illness can also be associated with severe respiratory diseases and high mortality rates. Well-characterized multiplexed serologic assays are needed to aid in rapid detection and surveillance of hCoVs. The present study describes development and evaluation of a multiplexed magnetic microsphere immunoassay (MMIA) to simultaneously detect immunoglobulin G (IgG) antibodies specific for recombinant nucleocapsid proteins (recN) from hCoVs 229E, NL63, OC43, HKU1, SARS-CoV, and MERS-CoV. We used paired human sera to screen for IgG with reactivity against six hCoVs to determine assay sensitivity, specificity and reproducibility. We found no signal interference between monoplex and multiplex assay formats (R2 range = 0.87–0.97). Screening of paired human sera using MMIA, resulted in 92 of 106 (sensitivity: 86%) as positive and 68 of 80 (specificity: 84%) as negative. This study serves as a proof of concept that it is feasible to develop and use a multiplexed microsphere immunoassay as a next generation screening tool for use in large scale seroprevalence studies of hCoVs.

Validation of monoplex assays detecting antibodies against Corynebacterium diphtheriae and Clostridium tetani toxins, rubella virus and parvovirus B19 for incorporation into Multiplex Serology.

Serological assays detecting antibodies in serum or plasma samples are useful and versatile instruments to investigate an individual's infection and vaccination history, e.g. for clinical diagnosis, personal risk evaluation, and seroepidemiological studies. Multiplex Serology is a suspension bead array-based high-throughput methodology for simultaneous measurement of antibodies against multiple pathogens in a single reaction vessel, thus economizing sample volume, measurement time, and costs. We developed and validated bead-based pathogen-specific Monoplex Serology assays, i.e. assays including only antigens for the respective pathogen, to detect antibodies against Corynebacterium diphtheriae and Clostridium tetani toxins, rubella virus and parvovirus B19. The developed assays expand the portfolio of existing pathogen-specific bead-based serology assays and can be efficiently incorporated into larger Multiplex Serology panels. The newly developed Monoplex Serology assays consist of only one antigen per infectious agent, expressed as Glutathione S-transferase-fusion proteins in E. coli. Specificity, sensitivity and Cohen's kappa statistics in comparison with routine clinical diagnostic assays were calculated for serum dilutions 1:100 and 1:1000. All pathogen-specific assays were successfully validated at both serum dilutions with the exception of rubella Monoplex Serology which showed impaired sensitivity (57.6%) at dilution 1:1000. Specificities of successfully validated Monoplex Serology assays ranged from 85.6% to 100.0% (median: 91.7%), and sensitivities from 81.3% to 95.8% (median: 90.9%); agreement with the reference assays ranged from substantial to almost perfect (kappa: 0.66-0.86, median: 0.78). Statistical performance and slim assay design enable efficient incorporation of the developed assays into Multiplex Serology.

Validation of Multiplex Serology for human hepatitis viruses B and C, human T-lymphotropic virus 1 and Toxoplasma gondii.

Multiplex Serology is a high-throughput technology developed to simultaneously measure specific serum antibodies against multiple pathogens in one reaction vessel. Serological assays for hepatitis B (HBV) and C (HCV) viruses, human T-lymphotropic virus 1 (HTLV-1) and the protozoan parasite Toxoplasma gondii (T. gondii) were developed and validated against established reference assays. For each pathogen, between 3 and 5 specific antigens were recombinantly expressed as GST-tag fusion proteins in Escherichia coli and tested in Monoplex Serology, i.e. assays restricted to the antigens from one particular pathogen. For each of the four pathogen-specific Monoplex assays, overall seropositivity was defined using two pathogen-specific antigens. In the case of HBV Monoplex Serology, the detection of past natural HBV infection was validated based on two independent reference panels resulting in sensitivities of 92.3% and 93.0%, and specificities of 100% in both panels. Validation of HCV and HTLV-1 Monoplex Serology resulted in sensitivities of 98.0% and 95.0%, and specificities of 96.2% and 100.0%, respectively. The Monoplex Serology assay for T. gondii was validated with a sensitivity of 91.2% and specificity of 92.0%. The developed Monoplex Serology assays largely retained their characteristics when they were included in a multiplex panel (i.e. Multiplex Serology), containing additional antigens from a broad range of other pathogens. Thus HBV, HCV, HTLV-1 and T. gondii Monoplex Serology assays can efficiently be incorporated into Multiplex Serology panels tailored for application in seroepidemiological studies.

Development and evaluation of a multiplex reverse-transcription real-time PCR assay for detection of equine respiratory disease viruses

We developed a multiplex reverse-transcription real-time PCR (RT-rtPCR) assay for the simultaneous detection of the main equine respiratory viruses: equid alphaherpesviruses 1 and 4 (EHV-1, -4) and equine influenza virus (EIV; species Influenza A virus). The primers and probes amplified only the targeted viruses, and there were no inter-assay cross-amplifications or nonspecific interactions. The multiplex assay efficiencies were 92.5%, 97%, and 90% for EHV-1, EHV-4, and EIV, respectively. The R2 values of the monoplex and multiplex assays were ⩾0.990, and the slopes were -3.37 to -3.59. The performance of the assay was evaluated by analyzing 152 samples from clinically infected horses. EHV-1 DNA was detected in 12 samples, EHV-4 DNA in 9 samples, and both EHV-1 and EHV-4 in 4 samples. The accuracy of the assay was confirmed by comparing these results using commercial rtPCR and RT-rtPCR kits. Our multiplex RT-rtPCR was a sensitive, specific, accurate, and cost-effective method for the detection of the target viruses whether they occur alone or as part of coinfections.

Detection of Zika, dengue and chikungunya viruses using single-reaction multiplex real-time RT-PCR

Zika (ZIKV), Dengue (DENV) and Chikungunya viruses (CHIKV) co-circulate in the same geographical areas during the same seasonal period through the same biting arthropods.Therefore a rapid sensitive and specific molecular assay for these viruses would be a considerable help in the disease management and the epidemiological survey.We developed a one-step multiplex real-time PCR for the simultaneous detection of these viruses.Intra and inter-reproducibilities varied from 0.41% to 3.29% and from 1.13% to 4.93% for each virus respectively. The specificity was 100%. Whole blood, plasma and urines were used for comparison with commercially available monoplex assays (RealStar® kits, Altona Diagnostics GmbH, Hamburg, Germany). The concordance was 96%, 92.9% and 95.7% for ZIKV, DENV and CHIKV respectively. No cross reaction and no PCR inhibition were observed for any of the clinical samples.This test can thus be used as a rapid molecular assay for ZIKV, DENV1–4 and CHIKV infections.

Enhanced Single-tube Multiplex PCR Assay for Detection and Identification of Six Arcobacter Species.

A single-tube multiplex PCR (mPCR) assay was developed for rapid, sensitive and simultaneous detection and identification of six Arcobacter species including two new species, A. lanthieri and A. faecis, along with A. butzleri, A. cibarius, A. cryaerophilus and A. skirrowii on the basis of differences in the lengths of their PCR products. Previously designed monoplex, mPCR and RFLP assays do not detect or differentiate A. faecis and A. lanthieri from other closely related known Arcobacter spp. Primer pairs for each target species (except A. skirrowii) and mPCR protocol were newly designed and optimized using variable regions of housekeeping including cpn60, gyrA, gyrB and rpoB genes. The accuracy and specificity of the mPCR assay was assessed using DNA templates from six targets and 11 other Arcobacter spp. as well as 50 other bacterial reference species and strains. Tests on the DNA templates of target Arcobacter spp. were appropriately identified, whereas all 61 other DNA templates from other bacterial species and strains were not amplified. Sensitivity and specificity of the mPCR assay was 10pgμl-1 of DNA concentration per target species. The optimized assay was further evaluated, validated and compared with other mPCR assays by testing Arcobacter cultures isolated from various faecal and water sources. Study results confirm that the newly developed mPCR assay is rapid, accurate, reliable, simple, and valuable for the simultaneous detection and routine diagnosis of six human- and animal-associated Arcobacter spp. The new mPCR assay is useful not only for pure but also mixed cultures. Moreover, it has the ability to rapidly detect six species which enhances the value of this technology for aetiological and epidemiological studies.

Rapid Molecular Panels: What Is in the Best Interest of the Patient? A Review of Patient Outcome Studies for Multiplex Panels Used in Bloodstream, Respiratory, and Neurological Infections

In the clinical microbiology laboratory, the focus when choosing new tests is often on performance, turnaround time, and labor needs. This review examines available rapid, multiplexed tests from a different perspective: that of the patient. It considers whether published evidence supports the notion that use of rapid, on-demand tests (as opposed to batched testing) leads to better patient outcomes and whether broad, syndrome-based, multiplexed panels translate into better patient care than narrower monoplex or duplex assays. Finally, we examine how synergy between the clinical microbiology and antimicrobial stewardship programs is necessary to ensure that rapid tests, if implemented, impact the patients they are designed to support.

Abstract 3832: Optimization strategy for fluorescent multiplex immunohistochemistry tissue staining

Abstract Introduction: Recent insights into the tumor microenvironment have fueled the need for additional information beyond the one or two phenotypes provided by traditional immunohistochemistry (IHC). As a response, manual and automated fluorescent multiplex immunohistochemistry (fIHC) techniques have been recently developed and accepted by the immuno-oncology space. Fluorescent multiplex immunohistochemistry (fIHC) assays are designed to simultaneously measure multiple biomarkers in tissue sections with visual context that is lost in other methods, such as flow cytometry. Here we describe a novel optimization strategy to achieve quantitative, robust, and specific multiplex fIHC staining results with both manual and automated multiple-color Opal procedures. Methods: Formalin-fixed paraffin-embedded samples of primary tumors were immunostained using Opal™ reagents both manually and on a fully automated Leica BOND RX™ stainer. The impact of different reagent concentrations and quantities were analyzed in respect to signal specificity and robustness, stripping efficiency, signal co-localization, signal to noise, and color separation. Images were acquired on a Vectra 3.0® automated imaging system, and analyzed with inForm® software. Results: The goals of this study were two-fold: -to understand the impact of Opal reagent concentrations/quantities on fluorescent signal intensities acquired from cells within the context of tissue. -to develop an Opal reagent optimization method that yields more consistent, quantitative results from separated and co-localized fluorescent signals. We’ve applied this novel fIHC experimental approach and optimization strategy to Opal monoplex and multiplex assays and explored staining robustness, contextual specificity, staining order independence, and co-localized signal separation. Using the novel optimization strategy, we have achieved optimal staining patterns with improved confidence in the quantitative characteristics of the assay. Tissue sections stained after optimization have exhibited staining order independence and closely align with traditional IHC patterns. Conclusion: This novel optimization strategy, developed for Opal fIHC assays, is more quantitative, improves staining results, and minimizes interference between co-localized biomarkers. Citation Format: Yi zheng, Carla Coltharp, Ryan Dilworth, Linying Liu, Darryn Unfricht, Cliff Hoyt, Milind Rajopadhye, Peter Miller. Optimization strategy for fluorescent multiplex immunohistochemistry tissue staining [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3832. doi:10.1158/1538-7445.AM2017-3832

Comparison of monoplex and duplex RT-PCR assays for the detection of measles virus

Rapid and accurate detection of measles virus is important for case diagnosis and public health management. This study compared the performance of two monoplex RT-PCR reactions targeting the H and N genes to a duplex RT-PCR targeting both genes simultaneously. The duplex simplified processing without compromising assay performance characteristic.

Laboratory Diagnosis of Central Nervous System Infection.

Central nervous system (CNS) infections are potentially life threatening if not diagnosed and treated early. The initial clinical presentations of many CNS infections are non-specific, making a definitive etiologic diagnosis challenging. Nucleic acid in vitro amplification-based molecular methods are increasingly being applied for routine microbial detection. These methods are a vast improvement over conventional techniques with the advantage of rapid turnaround and higher sensitivity and specificity. Additionally, molecular methods performed on cerebrospinal fluid samples are considered the new gold standard for diagnosis of CNS infection caused by pathogens, which are otherwise difficult to detect. Commercial diagnostic platforms offer various monoplex and multiplex PCR assays for convenient testing of targets that cause similar clinical illness. Pan-omic molecular platforms possess potential for use in this area. Although molecular methods are predicted to be widely used in diagnosing and monitoring CNS infections, results generated by these methods need to be carefully interpreted in combination with clinical findings. This review summarizes the currently available armamentarium of molecular assays for diagnosis of central nervous system infections, their application, and future approaches.

Implementation and application of a multiplex assay to detect malaria-specific antibodies: a promising tool for assessing malaria transmission in Southeast Asian pre-elimination areas

BackgroundEpidemiological surveillance is a key activity in malaria control and elimination in low-transmission and pre-elimination settings. Hence, sensitive tools for estimating malaria force of infection are crucial. Serological markers might provide additional information in estimating force of infection in low-endemic areas along with classical parasite detection methods. Serological markers can be used to estimate recent, past or present malaria exposure, depending on the used markers and their half-life.MethodsAn assay based on 14 Plasmodium-specific peptides, one peptide specific for Anopheles gambiae saliva protein and five Plasmodium-specific recombinant proteins was developed for the MAGPIX system, assessed for its performance, and applied on blood spots from 2000 individuals collected in the Ratanakiri Province, Cambodia.ResultsA significant correlation for the use of 1000 and 2000 beads/antigen/well as well as for the monoplex versus multiplex assay was observed for all antigens (p < 0.05). For the majority of antigens, antigen-coupled beads were stable for at least 2 months. The assay was very reproducible with limited intercoupling, interplate and intraplate variability (mean RSD <15 %). Estimating seroconversion and seroreversion per antigen using reversible catalytic models and models allowing two seroconversion rates showed higher seroconversion rates in adults.ConclusionThe multiplex bead-based immunoassay was successfully implemented and analysis of field blood samples shows that changes detected in force of malaria infection vary according to the serological markers used. Multivariate analysis of the antibody responses and insights into the half-life of antibodies are crucial for improving the interpretation of these results and for identifying the most useful serological markers of past and recent malaria infection.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-0868-z) contains supplementary material, which is available to authorized users.