Differential susceptibility of cells infected with defective and intact HIV proviruses to killing by obatoclax and other small molecules.

OBJECTIVES: Some drugs that augment cell-intrinsic defenses or modulate cell death/survival pathways have been reported to selectively kill cells infected with HIV or Simian Immunodeficiency Virus (SIV), but comparative studies are lacking. We hypothesized that these drugs may differ in their ability to kill cells infected with intact and defective proviruses. DESIGN: To investigate this hypothesis, drugs were tested ex vivo on peripheral blood mononuclear cells (PBMC) from nine antiretroviral therapy (ART)-suppressed individuals. METHODS: We tested drugs currently in clinical use or human trials, including auranofin (p53 modulator), interferon alpha2A, interferon gamma, acitretin (RIG-I inducer), GS-9620/vesatolimod (TLR7 agonist), nivolumab (PD-1 blocker), obatoclax (Bcl-2 inhibitor), birinapant [inhibitor of apoptosis proteins (IAP) inhibitor], bortezomib (proteasome inhibitor), and INK128/sapanisertib [mammalian target of rapamycin mTOR] [c]1/2 inhibitor). After 6 days of treatment, we measured cell counts/viabilities and quantified levels of total, intact, and defective HIV DNA by droplet digital PCR (Intact Proviral DNA Assay). RESULTS: Obatoclax reduced intact HIV DNA [median = 27-30% of dimethyl sulfoxide control (DMSO)] but not defective or total HIV DNA. Other drugs showed no statistically significant effects. CONCLUSION: Obatoclax and other Bcl-2 inhibitors deserve further study in combination therapies aimed at reducing the intact HIV reservoir in order to achieve a functional cure and/or reduce HIV-associated immune activation.

Increased 90-Day Mortality and Morbidity Among Patients Recovering From Elective Primary Arthroplasty During the COVID-19 Pandemic in New York City.

All elective procedures were stopped in March 2020 because of the coronavirus disease 2019 (COVID-19) pandemic. We report the 90-day mortality and complications of patients who underwent primary arthroplasty before the stopping of elective procedures at a single academic medical center. A retrospective cohort study was conducted including patients who underwent elective primary arthroplasty between December 2019 and mid-March 2020. Their 90-day postoperative mortality and medical complications were statistically compared with those of a historical cohort from the same operative period in 2019. The 2020 and 2019 cohorts included 372 and 410 patients, respectively. Except for the prevalence of diabetes, there was no significant difference between the two cohorts regarding baseline characteristics or preoperative health. The 2020 cohort had statistically significant higher rates of pneumonia (2.7% vs 0.7%; P=.03), readmission (9.1% vs 5.4%; P=.04), pulmonary embolism (1.6% vs 0.2%; P=.04), and 90-day mortality (1.1% vs 0%; P=.04). The 2020 cohort also had a trend for increased rates of deep venous thrombosis (1.1% vs 0.7%; P=.7) and cardiac complications (1.9% vs 0.5%; P=.07) and no change in emergency department visits (14.0% vs 11.7%; P=.3). There were 7 confirmed cases of COVID-19 in the 2020 cohort and 1 death. This study demonstrates that patients who underwent primary arthroplasty procedures at our institution close to the time of the first wave of the COVID-19 pandemic experienced a statistically significant increase in mortality, pneumonia, pulmonary embolism, and readmission compared with a historical cohort. As elective procedures have resumed during the ongoing pandemic, providers and patients should be aware of these increased risks. [Orthopedics. 202x;4x(x):xx-xx.].

Institutional and individual factors associated with functional outcomes in nursing home residents under long-term care insurance in Korea: A multilevel analysis.

OBJECTIVE: The purpose of this study was to analyze the relationship between institutional and individual characteristics that influence changes in the functional outcomes of nursing home residents. METHODS: Long-term Care Insurance claims data with basic information of nursing home(n = 3,263) and long-term care needs assessment data of nursing home residents(n = 34,717) was used. The independent variable was classified into individual (level 1) and nursing home characteristics (level 2). Changes in physical function, cognitive function, and behavioral symptoms were used as dependent variables. RESULTS: The institutional factors of nursing homes contributed relatively little to substantial changes in function of nursing home residents. Ownership, size, and nursing home staff including care worker and physical/occupational therapist were important determinants of functional changes in nursing home residents. CONCLUSION: To improve the quality of nursing homes in Korea, regulations on staffing should be modified, and an disincentive policy should be introduced for low-quality institutions.

Skin Reactions Following Primary Total Knee Arthroplasty With an Adhesive Superficial Closure System: A Case Series.

BACKGROUND: We investigated a skin adhesive closure device consisting of a self-adhesive polyester mesh placed over the surgical incision, followed by a liquid adhesive that is spread over the mesh and surrounding the skin. It is intended to reduce wound closure times, scarring, and skin complications associated with traditional closure with sutures or staples. The aim of this study was to report on skin reactions in patients who underwent primary total knee arthroplasty (TKA) using the skin adhesive closure system. METHODS: A retrospective review of patients who underwent TKA using adhesive closure between 2016 to 2021 at a single institute was performed. A total of 1,719 cases were analyzed. Patient demographics were collected. The primary outcome was any postoperative skin reaction. Skin reactions were classified as allergic dermatitis, cellulitis, or other. Treatment(s), duration of symptoms, and surgical infections were also collected. RESULTS: A total of 5.0% (86) of patients were found to have any type of skin reaction following their TKA. Of these 86, 39 (2.3%) had symptoms of allergic dermatitis (AD), 23 (1.3%) had symptoms of cellulitis, and 24 (1.4%) had other symptoms. A total of 27 (69%) allergic dermatitis patients were treated with a topical corticosteroid cream only; their symptoms resolved within an average of 25 days. There was only 1 case of superficial infection (<0.001%). No prosthetic joint infections were observed. CONCLUSION: Despite skin reactions appearing in 5.0% of cases, the rate of infection was low. A patient-specific preoperative workup and effective treatment strategies can minimize complications associated with adhesive closure system and increase patient satisfaction following TKA.

Digital Protein Detection in Bulk Solutions.

Quick and accurate molecular diagnostics in protein detection can greatly benefit medicine in disease diagnosis and lead to positive patient outcomes. However, specialized equipment used in clinical laboratories often comes with trade-offs between operation and function serving a single role for very specific needs. For example, to achieve high analytical sensitivity and specificity, instruments such as high-performance liquid chromatography and/or liquid chromatography-mass spectrometry use a complex instrument design and require thorough training of the users. On the other hand, simple tests such as protein detection in urinary tract infection using dip-stick assays provide very quick results but suffer from poor analytical sensitivity. Here, we present an application study for the 3D particle counter technology, which is based on optical confocal detection in order to scan large sample volumes (0.5-3 mL) in glass cuvettes, that aims to close the gap between analytical sensitivity and turnover assay time and simplify protein detection by adopting bead-based immunoassays. Combining the 3D particle counter technology with bead-based immunoassays, a subpicomolar limit of detection-ranging from 119 to 346 fM-was achieved within 3.5-hour assay time for recombinant mouse interleukin 6 detection. As an alternative instrument to a flow cytometer, the 3D particle counter takes advantages of bead-based immunoassays and provides unique accessibility and flexibility for users.

A Protein Microarray-Based Respiratory Viral Antigen Testing Platform for COVID-19 Surveillance.

High-throughput and rapid screening testing is highly desirable to effectively combat the rapidly evolving COVID-19 pandemic co-presents with influenza and seasonal common cold epidemics. Here, we present a general workflow for iterative development and validation of an antibody-based microarray assay for the detection of a respiratory viral panel: (a) antibody screening to quickly identify optimal reagents and assay conditions, (b) immunofluorescence assay design including signal amplification for low viral titers, (c) assay characterization with recombinant proteins, inactivated viral samples and clinical samples, and (d) multiplexing to detect a panel of common respiratory viruses. Using RT-PCR-confirmed SARS-CoV-2 positive and negative pharyngeal swab samples, we demonstrated that the antibody microarray assay exhibited a clinical sensitivity and specificity of 77.2% and 100%, respectively, which are comparable to existing FDA-authorized antigen tests. Moreover, the microarray assay is correlated with RT-PCR cycle threshold (Ct) values and is particularly effective in identifying high viral titers. The multiplexed assay can selectively detect SARS-CoV-2 and influenza virus, which can be used to discriminate these viral infections that share similar symptoms. Such protein microarray technology is amenable for scale-up and automation and can be broadly applied as a both diagnostic and research tool.

Homogeneous surrogate virus neutralization assay to rapidly assess neutralization activity of anti-SARS-CoV-2 antibodies.

The COVID-19 pandemic triggered the development of numerous diagnostic tools to monitor infection and to determine immune response. Although assays to measure binding antibodies against SARS-CoV-2 are widely available, more specific tests measuring neutralization activities of antibodies are immediately needed to quantify the extent and duration of protection that results from infection or vaccination. We previously developed a 'Serological Assay based on a Tri-part split-NanoLuc® (SATiN)' to detect antibodies that bind to the spike (S) protein of SARS-CoV-2. Here, we expand on our previous work and describe a reconfigured version of the SATiN assay, called Neutralization SATiN (Neu-SATiN), which measures neutralization activity of antibodies directly from convalescent or vaccinated sera. The results obtained with our assay and other neutralization assays are comparable but with significantly shorter preparation and run time for Neu-SATiN. As the assay is modular, we further demonstrate that Neu-SATiN enables rapid assessment of the effectiveness of vaccines and level of protection against existing SARS-CoV-2 variants of concern and can therefore be readily adapted for emerging variants.

Black and Hispanic Patients Do Not Stay Longer After Primary Total Knee Arthroplasty: Results From an Urban Center Serving a Predominantly Minority Cohort.

INTRODUCTION: Previous reports identified minority race/ethnicity to be an independent risk factor for prolonged length of stay (LOS); however, these cohorts consisted of predominantly White patients. This study sought to evaluate minority status as an independent risk factor for prolonged LOS after primary total knee arthroplasty (TKA) in a predominantly Hispanic and Black cohort. METHODS: This was a retrospective study using an institutional database of patients who underwent primary TKA between the years 2016 and 2019. Demographic and socioeconomic data, smoking, body mass index (BMI), medical comorbidities, discharge disposition, and 30-day readmission rates were collected. Patients were first categorized into racial/ethnic groups (Hispanic, Black, or White). An univariate analysis was performed comparing patient characteristics between racial/ethnic groups using the Wilcoxon rank sum, chi-squared, and Fisher exact tests. We then categorized patients into two groups-normal LOS (discharged on postoperative day 1 to 2) and prolonged LOS (discharged after postoperative day 2). An univariate analysis was again performed comparing patient characteristics between LOS groups using Wilcoxon rank sum, chi-squared, and Fisher exact tests. After identifying risk factors markedly associated with LOS, a multivariate logistic regression analysis was performed to identify independent risk factors for prolonged LOS. RESULTS: A total of 3,093 patients were included-47.9% Hispanic and 38.3% Black. Mean LOS was 2.9 ± 1.6 days. An univariate analysis found race/ethnicity, age, low socioeconomic status (SES), discharge disposition, insurance type, weekday of surgery, BMI >40, smoking, increased American Society of Anesthesiologists (ASA)/Charlson Comorbidity Index (CCI) and several medical comorbidities to be associated with prolonged LOS (P < 0.05). A multivariate logistic regression analysis found Black and Hispanic patients were less likely to have prolonged LOS after adjusting for associated risk factors. White race/ethnicity, nonhome discharge, low SES, weekday of surgery, smoking, BMI >40, and increased ASA and CCI were identified as independent risk factors for prolonged LOS (P < 0.05). The overall 30-day readmission rate was 3.6%, with no notable difference between racial/ethnic and LOS groups (P = 0.98 and P = 0.78). CONCLUSION: In contrast to previous reports, our study found that after adjusting for associated risk factors, minority patients do not have prolonged LOS after primary TKA in an urban, socioeconomically disadvantaged, predominantly minority patient cohort. White race/ethnicity, nonhome discharge, low SES, weekday of surgery, smoking, BMI >40, increased CCI, and ASA were all found to be independent risk factors for prolonged LOS. These findings highlight the need to further investigate the role of race/ethnicity on LOS after primary TKA using large-scale, randomized controlled trials with equally represented patient cohorts.

Therapeutic effiacy of T cells expressing chimeric antigen receptor derived from a mesothelin-specific scFv in orthotopic human pancreatic cancer animal models.

Novel CAR T cells targeting mesothelin (MSLN) expressed on pancreatic cancer cells were developed to overcome the limit of the clinical efficacy of CAR T cell therapy for pancreatic cancer patients. Optimal single-chain variable fragments (scFv) binding to MSLN were selected based on the binding activity and the functional effectiveness of various scFv containing CAR-expressing T cells. Engineered MSLN CAR T cells showed successful anti-tumor activity specific to MSLN expression level. Furthermore, MSLN CAR T cells were evaluated for the anti-cancer efficacy in orthotopic mouse models bearing pancreatic cancer cells, MIA Paca-2, MSLN-overexpressed MIA Paca-2 or endogenously MSLN-expressing AsPC-1. Mice were randomized into control, mock treated, MS501 BBz treated, MS501 28z treated or MS501 28BBz treated group. Mice were monitored by weekly IVIS imaging and tumors were harvested and analyzed by immunohistochemical analyses. MSLN CAR T cells produced the therapeutic effect in orthotopic animal models with complete remission in significant number of mice. Histopathological analysis indicated that CD4+ and CD8+ MSLN CAR T cells infiltrated pancreatic tumor tissue and led to cancer cell eradication. Our results demonstrated the anti-tumor efficacy of MSLN CAR T cell therapy against pancreatic cancer, suggesting its therapeutic potential.

Split-enzyme immunoassay to monitor EGFR-HER2 heterodimerization on cell surfaces.

Over 30,000 protein-protein interactions with pathological implications have been identified; yet, discovering and investigating drugs that target these specific interactions is greatly limited by the inability to monitor native protein-protein interactions (PPIs) efficiently. The two most frequently used tools to monitor PPIs, resonance-energy transfer (RET) assays and protein complementation assays (PCA), face significant limitations. RET assays have a narrow working range of 10 to 50 Å, while PCA require permanent attachment of a reporter probe to a protein of interest by chemical conjugation or genetic engineering. We developed a non-invasive assay platform to measure PPIs without modifications to the proteins of interest and is functional at a greater working range than RET assays. We demonstrate our approach by monitoring the EGFR-HER2 heterodimerization on relevant cell surfaces, utilizing various EGFR- and HER2-specific binders (e.g., Fab, DARPin, and VHH) fused with small fragments of a tri-part split-luciferase derived from NanoLuc®. Following independent binding of the binder fusions to their respective targets, the dimerization of EGFR and HER2 induces complementation of the luciferase fragments into a functional native structure, producing glow-type luminescence. We have confirmed the functionality of the platform to monitor EGFR-HER2 dimerization induction and inhibition. STATEMENT OF SIGNIFICANCE: We describe a platform technology for rapid monitoring of protein-protein interactions (PPIs). Our approach is uses a luciferase split into three parts - two short peptide "tags" and a large third fragment. Each of the short peptides can be fused to antibodies which bind to domains of a target antigens which orients the two tags and facilitates refolding of an active enzyme. To our knowledge this is the first example of a split-enzyme used to monitor PPIs without requiring any modification of the target proteins. We demonstrate our approach on the important PPI of HER2 and EGFR. Significantly, we quantify stimulation and inhibition of these partners, opening the possibility of using our approach to assess potential drugs without engineering cells.

Patch-Type Vibration Visualization (PVV) Sensor System Based on Triboelectric Effect.

Self-powered wireless sensor systems have emerged as an important topic for condition monitoring in nuclear power plants. However, commercial wireless sensor systems still cannot be fully self-sustainable due to the high power consumption caused by excessive signal processing in a mini-electronic computing system. In this sense, it is essential not only to integrate the sensor system with energy-harvesting devices but also to develop simple data processing methods for low power schemes. In this paper, we report a patch-type vibration visualization (PVV) sensor system based on the triboelectric effect and a visualization technique for self-sustainable operation. The PVV sensor system composed of a polyethylene terephthalate (PET)/Al/LCD screen directly converts the triboelectric signal into an informative black pattern on the LCD screen without excessive signal processing, enabling extremely low power operation. In addition, a proposed image processing method reconverts the black patterns to frequency and acceleration values through a remote-control camera. With these simple signal-to-pattern conversion and pattern-to-data reconversion techniques, a vibration visualization sensor network has successfully been demonstrated.

Homogeneous Immunoassay Using a Tri-Part Split-Luciferase for Rapid Quantification of Anti-TNF Therapeutic Antibodies.

Anti-TNF therapeutics bind and sequester tumor necrosis factor (TNF) to prevent downstream signaling and are clinically important in the treatment of several autoimmune diseases. Effective treatment with these drugs requires frequent therapeutic drug monitoring (TDM). Current analytical methods, including reporter gene assay (RGA), enzyme-linked immunosorbent assay (ELISA), and mobility shift assay (MSA), can be technically rigorous, slow, and expensive. These qualities prevent the implementation of point-of-care testing and ultimately limit the frequency and utility of monitoring. An assay simple enough to be performed in the clinic would enable increased TDM frequency, more accurate dosing, and improved patient outcomes. Toward this end, we developed a homogeneous immunoassay based on a tri-part split-luciferase system for "add-and-read" detection of anti-TNF therapeutics. In our platform, two small fragments of the split-luciferase, called ß9 and ß10, are each fused to a different interacting protein. The binding of each of these proteins to anti-TNF antibodies forces the split-luciferase components into proximity where they reform the active luciferase. We identified the fusion proteins, ß9-protein A (ß9-A) and ß10-TNF, as promising binding pairs. We systematically adjusted assay conditions to optimize the signal/background (S/B) ratio, limit of detection (LOD), and percent recovery. The assay has a large dynamic range (0.5-32 µg/mL) and is sensitive enough to monitor both subtherapeutic and supratherapeutic serum concentrations of anti-TNF antibodies, as demonstrated in clinical samples.

A homogeneous split-luciferase assay for rapid and sensitive detection of anti-SARS CoV-2 antibodies.

Better diagnostic tools are needed to combat the ongoing COVID-19 pandemic. Here, to meet this urgent demand, we report a homogeneous immunoassay to detect IgG antibodies against SARS-CoV-2. This serological assay, called SATiN, is based on a tri-part Nanoluciferase (tNLuc) approach, in which the spike protein of SARS-CoV-2 and protein G, fused respectively to two different tNLuc tags, are used as antibody probes. Target engagement of the probes allows reconstitution of a functional luciferase in the presence of the third tNLuc component. The assay is performed directly in the liquid phase of patient sera and enables rapid, quantitative and low-cost detection. We show that SATiN has a similar sensitivity to ELISA, and its readouts are consistent with various neutralizing antibody assays. This proof-of-principle study suggests potential applications in diagnostics, as well as disease and vaccination management.

CD8+ T Cells Directed Against a Peptide Epitope Derived From Peptidoglycan-Associated Lipoprotein of Legionella pneumophila Confer Disease Protection.

Legionella pneumophila, an intracellular bacterium, may cause life-threatening pneumonia in immunocompromised individuals. Mononuclear cells and antibodies have been reported to be associated with the host defense response against L. pneumophila. This study is to determine whether Legionella peptidoglycan-associated lipoprotein (PAL)-specific CD8+ T cells are directly associated with protection against L. pneumophila, with a focus on potential epitopes. Synthetic peptides derived from PAL of L. pneumophila were obtained and tested through in vitro and in vivo cytotoxic T lymphocyte (CTL) assays for immunogenicity. PAL DNA vaccines or a peptide epitope with or without CpG-oligodeoxynucleotides (ODN) was evaluated for protection against L. pneumophila infection in animal models. When mice were immunized with DNA vaccines expressing the PAL of L. pneumophila, they were significantly protected against a lethal challenge with L. pneumophila through induction of antigen-specific CD8+ CTLs. Of the 13 PAL peptides tested, PAL92-100 (EYLKTHPGA) was the most immunogenic and induced the strongest CTL responses. When mice were immunized with the PAL92-100 peptide plus CpG-ODN, they were protected against the lethal challenge, while control mice died within 3-6 days after the challenge. Consistent with lung tissue histological data, bacterial counts in the lungs of immunized mice were significantly lower than those in control mice. Also, the amino acid sequence of PAL92-100 peptides is conserved among various Legionella species. To our knowledge, this study is the first to demonstrate that PAL92-100-specific CD8+ T cells play a central role in the host defense response against L. pneumophila.

Assignment of opsonic values to pneumococcal reference serum 007sp and a second pneumococcal OPA calibration serum panel (Ewha QC sera panel B) for 11 serotypes.

Pneumococcal conjugate vaccines (PCVs) have been effective in reducing the disease burden caused by Streptococcus pneumoniae. The first licensed PCV (PCV7) was composed of capsular polysaccharides from seven serotypes. This was followed by PCV10, then PCV13, and currently there are a number of higher valency vaccines in development. As part of licensure, new vaccine iterations require assessment of immunogenicity. Since some antibodies can be non-functional, measuring functional antibodies is desirable. To meet this need, opsonophagocytic assays (OPAs) have been developed. Previous studies have shown there can be significant variations in OPA results from different laboratories. We have previously shown that standardizing OPA data using reference serum 007sp can decrease this variation. To extend this approach to additional serotypes, a panel of sera was tested by five laboratories using a multiplexed OPA for serotypes 2, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20B, 22F, and 33F. Each sample was tested in five runs with 007sp tested three times in each run. Results were analyzed using a mixed effects ANOVA model. Standardization of the results significantly decreased the inter-laboratory variation for some serotypes. For serotypes 2, 8, and 11A, the variability was reduced by 40%, 45%, and 40%, respectively. For serotypes 12F, 17F, and 20B, the reductions were more modest (14%, 19%, and 24%, respectively). Standardization had little effect for the remaining serotypes. In many cases, the impact of normalization was blunted by the results from five sera that were collected after an extended post-vaccination interval. We have previously reported consensus values for 007sp for 13 serotypes, as well as the creation of a calibration serum panel ("Ewha Panel A"). Here, we report consensus values for 11 additional serotypes for 007sp and the creation of a second serum panel ("Ewha Panel B"). These consensus values will facilitate the development of next-generation PCVs.

Hyaluronic acid binding to CD44S is indiscriminate of molecular weight.

The ubiquitous presence of hyaluronic acid (HA) in the extracellular matrix (ECM) of both healthy and diseased tissues underscores its importance in human physiology. Previous studies suggest that HA can be used as a probe to qualitatively monitor cell surface levels of CD44 and other important HA receptors; however, these studies use mixtures of HA at various molecular weights. Using fluorescently labeled HA, we evaluated the apparent differences of low (25 kilodalton) and high (700 kilodalton) molecular weight HA interacting with breast cancer cell lines of varying levels of CD44. Our results confirm that CD44 expression and the apparent level of HA interaction correlates with molecular weight. Importantly, we show that HA only binds a small fraction of the major CD44 isoform, CD44S, on cell surfaces and that CD44S interactions account for <50% of the total HA bound to cell surfaces. Although increased fluorescence level correlates with higher molecular weight of HA, this appears to be an artifact of chain length and not a result of multivalent binding between HA and CD44S. Accordingly, we verify that HA binding characteristics of cell surfaces is similar to previous artificial membrane models which proposed that HA anchors to CD44S and forms a non-binding corona of HA that extends beyond the surface.

Early Experience Managing a High-volume Academic Orthopaedic Department During the Coronavirus Pandemic in New York City.

Our orthopaedic surgery department at Montefiore Medical Center and Albert Einstein College of Medicine is located within the Bronx, a borough of New York City, and serves a densely populated urban community. Since the beginning of the novel coronavirus outbreak in New York City, the medical center was forced to rapidly adapt to the projected influx of critically ill patients. The aim of this report is to outline how our large academic orthopaedic surgery department adopted changes and alternative practices in response to the most daunting challenge to public health in our region in over a century. We hope that this report provides insight for others facing similar challenges.

Graphene surface contacts of tin disulfide transistors for switching performance improvement and contact resistance reduction.

We investigated the performance improvement of tin disulfide channel transistors by graphene contact configurations. From its two-dimensional nature, graphene can make electric contacts only at the outermost layers of the channel. For intralayer current flow, two graphene flakes are contacted at the channel's top or bottom layer. For interlayer current flow, one flake is contacted at the top and bottom of each layer. We compared the transistor performance in terms of current magnitude, mobility, and subthreshold swing between the configurations. From such observations, we deduced that device characteristics depend on resistivity or doping level of individual graphene flakes. We also found that interlayer flow excels in the on-current magnitude and the mobility, and that top-contact configuration excels in the subthreshold swing.

Development for Clinical Use of a Multiplexed Immunoassay Using Sputum Samples for Streptococcus pneumoniae: a Non-Culture-Based Approach for Serotype-Specific Detection.

The multiplexed immunoassay (MIA) is an automated, monoclonal antibody-based serotyping assay that uses culture lysates of Streptococcus pneumoniae This study describes the development and validation of applying MIA directly to sputum samples for the serotype-specific detection of S. pneumoniae Sputum optimization involved liquefaction and fractionation. The subjects included 173 adult patients from whom both pneumococcal isolates cultured from sputum samples and the corresponding sputum samples were available at the Korea University Hospital from March 2012 to June 2015. Pneumococcal lysates and the sputum fraction were separately evaluated by MIA with a set A reaction to identify 27 serotypes (24 vaccine serotypes and serotypes 6C, 6D, and 11E). MIA results were validated by multiplex PCR (mPCR). Among the 173 patients analyzed, the pneumococcal isolate MIA detected a single set A serotype in 104 patients, and the corresponding sputum MIA showed concordant results with additional multiple serotypes in 21 patients. For the remaining 69 patients whose pneumococcal isolates were not determined to be set A serotypes by the pneumococcal isolate MIA, the corresponding sputum MIA identified additional set A serotypes (single serotypes, n = 17; multiple serotypes, n = 4). Serotypes 3 and 11A/D/F were the most commonly detected serotypes in both the pneumococcal isolate and sputum MIA analyses. However, serotype 8 was the most prevalent serotype detected only by the sputum MIA. The results of mPCR, performed for validation, showed a high concordance with the results of the sputum MIA. In conclusion, MIA using sputum samples enables the accurate, rapid, direct, and serotype-specific detection of S. pneumoniae, which may improve postvaccination serotype surveillance.