Rapid Direct Identification of Microbial Pathogens and Antimicrobial Resistance Genes in Positive Blood Cultures Using a Fully Automated Multiplex PCR Assay.

This study assessed the performance of the BioFire Blood Culture Identification 2 (BCID2) panel in identifying microorganisms and antimicrobial resistance (AMR) profiles in positive blood cultures (BCs) and its influence on turnaround time (TAT) compared with conventional culture methods. We obtained 117 positive BCs, of these, 102 (87.2%) were correctly identified using BCID2. The discordance was due to off-panel pathogens detected by culture (n = 13), and additional pathogens identified by BCID2 (n = 2). On-panel pathogen concordance between the conventional culture and BCID2 methods was 98.1% (102/104). The conventional method detected 19 carbapenemase-producing organisms, 14 extended-spectrum beta-lactamase-producing Enterobacterales, 18 methicillin-resistant Staphylococcus spp., and four vancomycin-resistant Enterococcus faecium. BCID2 correctly predicted 53 (96.4%) of 55 phenotypic resistance patterns by detecting AMR genes. The TAT for BCID2 was significantly lower than that for the conventional method. BCID2 rapidly identifies pathogens and AMR genes in positive BCs.

Evaluation of a sterile, filter-based, in-house method for rapid direct bacterial identification and antimicrobial susceptibility testing using positive blood culture.

This study aimed to assess the performance of our in-house method for rapid direct bacterial identification (ID) and antimicrobial susceptibility testing (AST) using a positive blood culture (BC) broth. For Gram-negative bacteria, 4 mL of BC broth was aspirated and passed through a Sartorius Minisart syringe filter with a pore size of 5 µm. The filtrate was then centrifuged and washed. A small volume of the pellet was used for ID, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and for AST, using automated broth microdilution. For Gram-positive cocci, 4 mL of BC broth was passed through the Minisart syringe filter. Then, 4 mL of sterile distilled water was injected in the direction opposite to that of the filtration to collect the bacterial residue trapped in the filter. Compared with the conventional method performed with pure colonies on agar plates, 94.0% (234/249) were correctly identified using the in-house method, with rates of 91.4% (127/139) and 97.3% (107/110) for Gram-positive and Gram-negative isolates, respectively. Of 234 correctly identified isolates, 230 were assessed by AST. Categorical agreement and essential agreement were 93.3% and 94.5%, respectively, with a minor error rate of 3.8%, a major error rate of 3.4%, and a very major error rate of 1.6%. Our in-house preparation method showed good performance in rapid direct ID and AST using positive BC broths compared to the conventional method. This simple method can shorten the conventional turnaround time for ID and AST by at least 1 day, potentially contributing to better patient management.

Development and Validation of a U-HPLC-MS/MS Method for the Concurrent Measurement of four Immunosuppressants in Whole Blood.

BACKGROUND: This study aimed to develop and validate a U-HPLC-MS/MS method for simultaneous determination of four immunosuppressants in human whole blood. METHODS: The method was based on the injection of 20 µL of calibrators and controls pretreated with the liquid phase extraction method for chromatography separation and mass spectrometry determination. LPE offline was performed by adding 0.1 mol/L ZnSO4 and acetonitrile, while separation of target compounds was achieved within 2.5 minutes by a Zorbax Eclipse XDB-C8 column using ammonium acetate and ACN mixed with formic acid as solvents. RESULTS: The assay offers ng/mL detection limits (from 1.1 to 12.4 ng/mL), accuracy (% deviation from -4.4% to 5.6%), precision (CV less than 15% at all QC levels), and linearity (from 23.4 to 948 ng/mL for CsA, from 2.11 to 45.5 ng/mL for TAC, SIR and EVR). The recovery and matrix results were acceptable, and the carryover was less than 1%. The results of method comparison show that IA-based methods overestimated the concentration of drugs compared with the MS-based method. Comparing our MS-based method with external LC-MS/MS showed that the results were within 2 SDs. CONCLUSIONS: We have developed a reliable assay for the analysis of CsA, TAC, SIR and EVR in whole blood using U-HPLC-MS/MS.

Analysis of bone marrow supernatant neutrophil gelatinase-associated lipocalin and hematological parameters in hematological malignancy.

BACKGROUND: Neutrophil gelatinase-associated lipocalin (NGAL) is a urine biomarker related to acute renal injury. Whereas several studies have evaluated NGAL levels in hematological malignancy, using peripheral blood (PB). Recently, bone marrow (BM) NGAL level was reported to be higher than PB NGAL level in individuals with hematological malignancy, suggesting that BM NGAL would reflect BM microenvironment better than PB NGAL. We measured BM NGAL levels in patients with hematological malignancy, comparing those with NGAL levels in normal BM. We evaluated the association of BM NGAL with hematological parameters including neutrophil counts. METHODS: BM samples were collected from 107 patients who underwent BM examination. Immunoassays were used to assess NGAL levels. Data on hematological parameters were collected from medical records. Intergroup comparisons were performed using the Kruskal-Wallis H test and Pearson chi-square test. Single and multiple regression analyses were performed to analyze the relationships. RESULTS: The independent factors that affected the BM NGAL level were neutrophil counts and BM band neutrophil%, while neutrophil count was the main influencing factor. The acute myeloid leukemia (n = 18) and myelodysplastic syndrome (n = 25) groups showed statistically lower BM NGAL levels than patients with normal BM. The myeloproliferative neoplasm group (n = 34) showed higher BM NGAL levels than patients with normal BM, but this difference was not statistically significant. Neutrophil counts and BM band neutrophil% showed intergroup patterns similar to those of BM NGAL levels. CONCLUSION: BM NGAL was related to neutrophil count and BM band neutrophil%, showing different levels according to hematological malignant disease entities.

Single-dose etoposide is an effective and safe protocol for stem cell mobilization in patients with multiple myeloma.

BACKGROUND: Single-dose etoposide was used an outpatient-based protocol for mobilization in patients with multiple myeloma (MM) for autologous stem cell transplantation (ASCT). Thus, we retrospectively analyzed the efficacy and safety of our one-day protocol in comparison with that of previous methods. METHODS: We retrospectively analyzed 168 patients with MM who underwent peripheral blood stem cell collection for upfront ASCT between 2008 and 2018. The mobilization protocols included G-CSF alone (G-mobilization), one-day 375 mg/m2 of etoposide (E375), two-days of 375 mg/m2 of etoposide (E750), or one-day high-dose (3.5 g/m2 ) cyclophosphamide (HD CY). For comparison of efficacy of each protocol, collected CD34+ cells over 4 × 106 /kg and under 2 × 106 /kg were defined as "adequate harvest" and "harvest failure," respectively. RESULTS: The median number of collected CD34+ cells was 5.58 × 106 /kg in patients receiving single-dose etoposide, and the percentage of uncomplicated optimal harvest of E375 (65.6%, 21/32) was significantly higher than that of E750 (41.9%, 13/31) and HD CY (31.3%, 15/48). The E375 showed the highest rate of adequate harvest (96.9%, 31/32) compared to that of E750 (87.1%), HD CY (75.0%), and G-mobilization (59.6%). Most E375 patients achieved adequate harvest without complication (29/32, 90.6%), the CD34+ cell collection yield on apheresis days one and two of E375 was not significantly different from that of E750, and no harvest failures occurred for E375. Neutrophil and platelet engraftments were significantly faster in E375 than other groups (P < .001). CONCLUSIONS: The use of single-dose etoposide could be an effective and safe method for mobilization in patients with MM.

Posterior circulation involvement and collateral flow pattern in moyamoya disease with the RNF213 polymorphism.

PURPOSE: Moyamoya disease is a chronic cerebrovascular disorder characterized by progressive stenosis of the circle of Willis with a compensatory collateral vessel network. Recent studies have identified the ring finger protein 213 gene (RNF213) as the unique susceptibility gene for moyamoya disease. The purpose of this study was to compare clinical features of moyamoya disease, especially angiographic findings, between patients with and without the RNF213 mutation. METHODS: Blood samples from 35 patients with moyamoya disease were obtained between May 2016 and May 2017. Information on age at the time of diagnosis, sex, and initial symptom were obtained via retrospective chart review. Angiographic records were evaluated. RESULTS: RNF213 variants were detected in the 28 of 35 patients (80%), including all pediatric patients (100%) and 18 of 25 adult patients (72%) in our cohort. Leptomeningeal collateral flow from posterior to anterior circulation was more frequent in the RNF213-negative group than in the RNF213-positive group (100% versus 38.9%; p = 0.020). Posterior cerebral arterial territorial involvement was more frequently observed in RNF213-positive patients than in RNF213-negative patients (50% versus 0%; p = 0.027). CONCLUSIONS: RNF213 may play a significant role in the development of collateral anastomoses.

The first case of congenital blood chimerism in two of the triplets in Korea.

BACKGROUND: Chimeras are composed of two or more different populations that originated from different zygotes. Blood chimerism arising from twins have been reported in the literature. Herein, we report the first blood group chimerism in triplets. METHODS: ABO blood grouping was carried out by manual tile methods (Merck Millipore, UK) and micro-column agglutination method (Bio-Rad, Cressier sur Morat, Switzerland). Flow cytometric analysis was performed with Anti-A-PE conjugated monoclonal antibodies (BD Biosciences, San Jose, CA, USA) and FACS Canto II (BD Biosciences). Molecular analysis was performed with allele-specific polymerase chain reaction (AS-PCR) and direct sequencing of the exons 6 and 7. RESULTS: Mixed-field agglutination and weak agglutination against anti-A were revealed by ABO blood grouping. Flow cytometric analysis revealed the presence of both A cells and O cells. AS-PCR and sequencing showed two neonates with chimerism, with each neonate`s genotype being A102/O01/O02. CONCLUSION: This is the first recorded case of blood chimera from a triplet in Korea. We recommend full investigation of blood group chimerism in neonates with ABO discrepancy, as blood chimerism is subject to certain caution in the clinical environment.

Clinical performance evaluation of the Sofia RSV FIA rapid antigen test for diagnosis of respiratory syncytial virus infection.

A recently introduced Sofia respiratory syncytial virus (RSV) fluorescent immunoassay (FIA) was evaluated against the BinaxNOW RSV card and the SD Bioline RSV test using 348 respiratory samples. The Sofia, BinaxNOW, and SD Bioline kits showed sensitivities of 66%, 65%, and 64%, respectively, for detecting RSV-A, and 71%, 63%, and 65% for detecting RSV-B, respectively.

Comparative Assessment of Risk and Turn-Around Time between Sequence-Based Typing and Next-Generation Sequencing for HLA Typing.

This study compared laboratory risk and turn-around time (TAT) between sequence-based typing (SBT) and next-generation sequencing (NGS) for human leukocyte antigen (HLA) typing. For risk assessment, we utilized the risk priority number (RPN) score based on failure mode and effect analysis (FMEA) and a risk acceptability matrix (RAM) according to the Clinical Laboratory Standards Institute (CLSI) guidelines (EP23-A). Total TAT was documented for the analytical phase, and hands-on time was defined as manual processes conducted by medical technicians. NGS showed a significantly higher total RPN score than SBT (1169 vs. 465). NGS indicated a higher mean RPN score, indicating elevated severity and detectability scores in comparison to SBT (RPN 23 vs. 12, p = 0.001; severity 5 vs. 3, p = 0.005; detectability 5 vs. 4, p < 0.001, respectively). NGS required a greater number of steps than SBT (44 vs. 25 steps), all of which were acceptable for the RAM. NGS showed a longer total TAT, total hands-on time, and hands-on time per step than SBT (26:47:20 vs. 12:32:06, 03:59:35 vs. 00:47:39, 00:05:13 vs. 00:01:54 hh:mm:ss, respectively). Transitioning from SBT to NGS for HLA typing involves increased risk and an extended TAT. This study underscored the importance of evaluating these factors to optimize laboratory efficiency in HLA typing.

Comparison of Neutralizing Activity between Vaccinated and Unvaccinated Hospitalized COVID-19 Patients Infected with Delta, Omicron BA.1, or Omicron BA.2 Variant.

BACKGROUND: Understanding the immune response to evolving viral strains is crucial for evidence-informed public health strategies. The main objective of this study is to assess the influence of vaccination on the neutralizing activity of SARS-CoV-2 delta and omicron infection against various SARS-CoV-2 variants. METHODS: A total of 97 laboratory-confirmed COVID-19 cases were included. To assess the influence of vaccination on neutralizing activity, we measured the neutralizing activity of SARS-CoV-2 delta or omicron (BA.1 or BA.2) infection against wild-type (WT), delta, BA.1, and BA.2, with the results stratified based on vaccination status. RESULTS: The neutralizing activity against the WT, delta, and omicron variants (BA.1 and BA.2) was significantly higher in the vaccinated patients than those in the unvaccinated patients. In the unvaccinated individuals infected with the delta variant, the decrease in binding to BA.1 and BA.2 was statistically significant (3.9- and 2.7-fold, respectively) compared to the binding to delta. In contrast, vaccination followed by delta breakthrough infection improved the cross-neutralizing activity against omicron variants, with only 1.3- and 1.2-fold decreases in BA.1 and BA.2, respectively. Vaccination followed by infection improved cross-neutralizing activity against WT, delta, and BA.2 variants in patients infected with the BA.1 variant, compared to that in unvaccinated patients. CONCLUSIONS: Vaccination followed by delta or BA.1 infection is associated with improved cross-neutralizing activity against different SARS-CoV-2 variants. The enhanced protection provided by breakthrough infections could have practical implications for optimizing vaccination strategies.

Use of a platform with lens-free shadow imaging technology to monitor natural killer cell activity.

Natural killer (NK) cells are a crucial component of the innate immune system. This study introduces Cellytics NK, a novel platform for rapid and precise measurement of NK cell activity. This platform combines an NK-specific activation stimulator cocktail (ASC) and lens-free shadow imaging technology (LSIT), using optoelectronic components. LSIT captures digital hologram images of resting and ASC-activated NK cells, while an algorithm evaluates cell size and cytoplasmic complexity using shadow parameters. The combined shadow parameter derived from the peak-to-peak distance and width standard deviation rapidly distinguishes active NK cells from inactive NK cells at the single-cell level within 30 s. Here, the feasibility of the system was demonstrated by assessing NK cells from healthy donors and immunocompromised cancer patients, demonstrating a significant difference in the innate immunity index (I3). Cancer patients showed a lower I3 value (161%) than healthy donors (326%). I3 was strongly correlated with NK cell activity measured using various markers such as interferon-gamma, tumor necrosis factor-alpha, perforin, granzyme B, and CD107a. This technology holds promise for advancing immune functional assays, offering rapid and accurate on-site analysis of NK cells, a crucial innate immune cell, with its compact and cost-effective optoelectronic setup, especially in the post-COVID-19 era.

Molecular characterization of human respiratory syncytial virus in Seoul, South Korea, during 10 consecutive years, 2010-2019.

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections and hospitalization in infants and young children. Here, we analyzed the genetic diversity of RSV using partial G gene sequences in 84 RSV-A and 78 RSV- B positive samples collected in Seoul, South Korea, for 10 consecutive years, from 2010 to 2019. Our phylogenetic analysis revealed that RSV-A strains were classified into either the ON1 (80.9%) or NA1 (19.0%) genotypes. On the other hand, RSV-B strains demonstrated diversified clusters within the BA genotype. Notably, some sequences designated as BA-SE, BA-SE1, and BA-DIS did not cluster with previously identified BA genotypes in the phylogenetic trees. Despite this, they did not meet the criteria for the assignment of a new genotype based on recent classification methods. Selection pressure analysis identified three positive selection sites (amino acid positions 273, 274, and 298) in RSV-A, and one possible positive selection site (amino acid position 296) in RSV-B, respectively. The mean evolutionary rates of Korean RSV-A from 1999 to 2019 and RSV-B strains from 1991 and 2019 were estimated at 3.51 × 10-3 nucleotides (nt) substitutions/site/year and 3.32 × 10-3 nt substitutions/site/year, respectively. The population dynamics in the Bayesian skyline plot revealed fluctuations corresponding to the emergence of dominant strains, including a switch of the dominant genotype from NA1 to ON1. Our study on time-scaled cumulative evolutionary analysis contributes to a better understanding of RSV epidemiology at the local level in South Korea.

Label-Free CD34+ Cell Identification Using Deep Learning and Lens-Free Shadow Imaging Technology.

Accurate and efficient classification and quantification of CD34+ cells are essential for the diagnosis and monitoring of leukemia. Current methods, such as flow cytometry, are complex, time-consuming, and require specialized expertise and equipment. This study proposes a novel approach for the label-free identification of CD34+ cells using a deep learning model and lens-free shadow imaging technology (LSIT). LSIT is a portable and user-friendly technique that eliminates the need for cell staining, enhances accessibility to nonexperts, and reduces the risk of sample degradation. The study involved three phases: sample preparation, dataset generation, and data analysis. Bone marrow and peripheral blood samples were collected from leukemia patients, and mononuclear cells were isolated using Ficoll density gradient centrifugation. The samples were then injected into a cell chip and analyzed using a proprietary LSIT-based device (Cellytics). A robust dataset was generated, and a custom AlexNet deep learning model was meticulously trained to distinguish CD34+ from non-CD34+ cells using the dataset. The model achieved a high accuracy in identifying CD34+ cells from 1929 bone marrow cell images, with training and validation accuracies of 97.3% and 96.2%, respectively. The customized AlexNet model outperformed the Vgg16 and ResNet50 models. It also demonstrated a strong correlation with the standard fluorescence-activated cell sorting (FACS) technique for quantifying CD34+ cells across 13 patient samples, yielding a coefficient of determination of 0.81. Bland-Altman analysis confirmed the model's reliability, with a mean bias of -2.29 and 95% limits of agreement between 18.49 and -23.07. This deep-learning-powered LSIT offers a groundbreaking approach to detecting CD34+ cells without the need for cell staining, facilitating rapid CD34+ cell classification, even by individuals without prior expertise.

Proof-of-Concept: Smartphone- and Cloud-Based Artificial Intelligence Quantitative Analysis System (SCAISY) for SARS-CoV-2-Specific IgG Antibody Lateral Flow Assays.

Smartphone-based point-of-care testing (POCT) is rapidly emerging as an alternative to traditional screening and laboratory testing, particularly in resource-limited settings. In this proof-of-concept study, we present a smartphone- and cloud-based artificial intelligence quantitative analysis system (SCAISY) for relative quantification of SARS-CoV-2-specific IgG antibody lateral flow assays that enables rapid evaluation (<60 s) of test strips. By capturing an image with a smartphone camera, SCAISY quantitatively analyzes antibody levels and provides results to the user. We analyzed changes in antibody levels over time in more than 248 individuals, including vaccine type, number of doses, and infection status, with a standard deviation of less than 10%. We also tracked antibody levels in six participants before and after SARS-CoV-2 infection. Finally, we examined the effects of lighting conditions, camera angle, and smartphone type to ensure consistency and reproducibility. We found that images acquired between 45° and 90° provided accurate results with a small standard deviation and that all illumination conditions provided essentially identical results within the standard deviation. A statistically significant correlation was observed (Spearman correlation coefficient: 0.59, p = 0.008; Pearson correlation coefficient: 0.56, p = 0.012) between the OD450 values of the enzyme-linked immunosorbent assay and the antibody levels obtained by SCAISY. This study suggests that SCAISY is a simple and powerful tool for real-time public health surveillance, enabling the acceleration of quantifying SARS-CoV-2-specific antibodies generated by either vaccination or infection and tracking of personal immunity levels.

Performance Evaluation of Three Antibody Binding Assays, a Neutralizing Antibody Assay, and an Interferon-Gamma Release Assay for SARS-CoV-2 According to Vaccine Type in Vaccinated Group.

We evaluated the performance of SARS-CoV-2 assays in the vaccinated group using receptor-binding domain antibody assays (RBD Ab assay), neutralizing antibody assay (nAb assay), and interferon-gamma release assay (IGR assay). We also compared the performance of the SARS-CoV-2 assays based on vaccine type in a large population. We collected 1851 samples from vaccinated individuals with vector, mix-and-match (MM), and mRNA vaccines. The performance of the RBD Ab assays was assessed by SARS-CoV-2 IgG II Quant (Abbott Laboratories, Sligo, Ireland), SARS-CoV-2 IgG (Beckman Coulter, CA, USA), and anti-SARS-CoV-2 S (Roche Diagnostics GmbH, Mannheim, Germany). The nAb assay was assessed by cPass SARS-CoV-2 neutralization antibody detection kits (GenScript, NJ, USA). The IGR assay was assessed by QuantiFERON (Qiagen, Venlo, The Netherlands). Median values of the RBD Ab assays and nAb assay sequentially increased after the first and second vaccinations. RBD Ab assays and nAb assay showed very strong correlations. The median values of the RBD Ab, nAb, and IGR were higher in the mRNA vaccine group than in the vector and MM vaccine groups. The agreement and correlation among the RBD Ab assays, nAb assay, and IGR assay were higher in the mRNA vaccine group than in the vector and MM vaccine groups. We compared the performance of the RBD Ab assay, nAb assay, and IGR assay based on the vaccine types using the RBD Ab, nAb, and IGR assays. This study provides a better understanding of the assessment of humoral and cellular immune responses after vaccination.

Field-Portable Leukocyte Classification Device Based on Lens-Free Shadow Imaging Technique.

The complete blood count (CBC) is one of the most important clinical steps in clinical diagnosis. The instruments used for CBC are usually expensive and bulky and require well-trained operators. Therefore, it is difficult for medical institutions below the tertiary level to provide these instruments, especially in underprivileged countries. Several reported on-chip blood cell tests are still in their infancy and do not deviate from conventional microscopic or impedance measurement methods. In this study, we (i) combined magnetically activated cell sorting and the differential density method to develop a method to selectively isolate three types of leukocytes from blood and obtain samples with high purity and concentration for portable leukocyte classification using the lens-free shadow imaging technique (LSIT), and (ii) established several shadow parameters to identify the type of leukocytes in a complete leukocyte shadow image by shadow image analysis. The purity of the separated leukocytes was confirmed by flow cytometry. Several shadow parameters such as the "order ratio" and "minimum ratio" were developed to classify the three types of leukocytes. A shadow image library corresponding to each type of leukocyte was created from the tested samples. Compared with clinical reference data, a correlation index of 0.98 was obtained with an average error of 6% and a confidence level of 95%. This technique offers great potential for biological, pharmaceutical, environmental, and clinical applications, especially where point-of-care detection of rare cells is required.

Usefulness of Combining Sputum and Nasopharyngeal Samples for Viral Detection by Reverse Transcriptase PCR in Adults Hospitalized with Acute Respiratory Illness.

Nasopharyngeal swabs (NPS) or washings have traditionally been used to diagnose respiratory tract infections. Reverse transcriptase PCR (RT-PCR) is widely used for rapid viral detection using samples from the upper respiratory tract. However, RT-PCR is rarely applied to sputum samples, mainly due to the viscosity of sputum. Thus, we assessed the detection rates of respiratory viruses from NPS, sputum samples, and combined NPS and sputum samples using multiplex RT-PCR (Allplex respiratory panels I, II, and III; Seegene, Seoul, South Korea). Paired NPS and sputum samples were collected from 219 patients admitted to the hospital with acute respiratory illnesses from October to December 2019. RT-PCR was performed on each sample for virus detection. Combined samples for virus detection were produced using remnant NPS and sputum samples with a positive virus signal. Respiratory viral nucleic acid was identified in 92 (42%) of 219 patients. Among the 92 viral detections, 61 (28%) were detected by both NPS and sputum samples. Twenty-four (11%) were sputum positive/NPS negative, and seven (3%) were sputum negative/NPS positive. For the combined NPS-sputum samples (n = 92), all paired samples positive in both specimens (n = 61) were also positive in the combined NPS-sputum sample. Twenty-seven (87%) of the 31 discordant paired samples were positive in the combined samples. Out of the total of 103 viruses identified before combining the samples, the detection rate of the combined samples was 94% (97/103), which was higher than the detection rates of sputum (88%; 91/103) and NPS (71%; 73/103). Because additional tests incur additional costs, our findings suggest that combining samples instead of testing separate samples using RT-PCR is likely the most cost-effective method of viral testing for patients with acute respiratory illnesses. IMPORTANCE This study reveals that RT-PCR utilizing sputum significantly increased the detection rate for respiratory viral nucleic acids among adult patients admitted to the hospital, compared to nasopharyngeal swabs (NPS). Notably, combined samples of sputum and NPS maintained the majority of the improved sputum detection rate with only a few positive signal losses from NPS samples. In order to detect respiratory viruses in adult patients with acute respiratory illness, it is important to choose the optimal respiratory samples. This study helped to improve our understanding of this process.

Guidelines for Mobile Laboratories for Molecular Diagnostic Testing of COVID-19.

With the rapid spread of the coronavirus disease (COVID-19), the need for rapid testing and diagnosis and consequently, the demand for mobile laboratories have increased. Despite this need, there are no clear guidelines for the operation, maintenance, or quality control of mobile laboratories. We provide guidelines for the operation, management, and quality control of mobile laboratories, and specifically for the implementation and execution of COVID-19 molecular diagnostic testing. These practical guidelines are primarily based on expert opinions and a laboratory accreditation inspection checklist. The scope of these guidelines includes the facility, preoperative evaluation, PCR testing, internal and external quality control, sample handling, reporting, laboratory personnel, biosafety level, and laboratory safety management. These guidelines are useful for the maintenance and operation of mobile laboratories not only in normal circumstances but also during public health crises and emergencies.

Humoral and Cellular Immune Responses to Vector, Mix-and-Match, or mRNA Vaccines against SARS-CoV-2 and the Relationship between the Two Immune Responses.

We investigated how differences in age, sex, or vaccine type can affect humoral and cellular immune responses after vaccination with vector (ChAdOx1 nCoV-19), mix-and-match (first, ChAdOx1 nCoV-19, and second, BNT162b2), or mRNA (BNT162b2 or mRNA-1273) vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Venous blood was collected from 573 subjects (vector, 396; mix-and-match, 96; and mRNA, 81) before the first vaccination (T0), 7 to 8 weeks (vector) or 3 to 4 weeks (mRNA) after the first vaccination (T1), and 3 to 4 weeks after the second vaccination (T2). The humoral and cellular immune responses were evaluated using Elecsys anti-SARS-CoV-2 (Roche), Alinity SARS-CoV-2 IgG II Quant (Abbott), cPass SARS-CoV-2 neutralization antibody detection (GenScript), and QuantiFERON SARS-CoV-2 (Qiagen) kits. At T1, the levels of the receptor-binding domain antibodies (RBD Ab) and neutralizing antibodies (NAb) decreased with aging, but interferon gamma release (IGR) levels increased. The RBD Ab, NAb, and IGR levels were higher in females than in males at T1 and T2. The NAb levels were higher in the mix-and-match and mRNA vaccine groups than in the vector vaccine group at T2. The RBD Ab and IGR levels were higher in the mRNA vaccine group than in the vector or mix-and-match vaccine groups at T2. The optimal cutoffs for RBD Ab and NAb, which were used to determine the presence of T cell responses, were 5.7 binding antibody units per milliliter (BAU mL-1) and 12.0 IU mL-1, respectively. Age, sex, and vaccine type affected the humoral and cellular immune responses, and T cell responses could be estimated from RBD Ab and NAb levels. IMPORTANCE There have been few studies that comprehensively evaluated factors affecting immune responses and the correlation between humoral and cellular immune responses after vector, mix-and-match, and mRNA vaccines against SARS-CoV-2. Therefore, we analyzed the effects of age, sex, and the different vaccine regimens on the immune responses to vaccination against SARS-CoV-2. The correlation between humoral and cellular immune responses and the cutoffs were derived for RBD antibodies and neutralizing antibodies to predict the presence of the cellular immune responses. In this comprehensive study, we demonstrated that there were differences in the immune responses induced after vaccination depending on the age and sex of an individual. Among the three vaccine regimens, the mix-and-match and mRNA vaccines induced the most robust immune responses. Finally, the proposed optimal cutoffs for RBD and neutralizing antibodies may be useful for predicting cellular immune responses when assays for cellular immune responses are not available.

Evaluation of Plasmodium vivax ELISA for the blood screen.

Plasmodium vivax malaria is the indigenous strain in the Republic of Korea (ROK). Plasmodium vivax can be transmitted through the transfusions of various blood components, which became a severe problem with the safety of blood transfusions and blood-related products in ROK. We evaluated a P. vivax-specific enzyme-linked immunosorbent assay (Genedia Malaria Ab ELISA 2.0, Green Cross, ROK) with blood samples from four groups: 251 samples from P. vivax-infected patients, 39 samples from post-treatment patients upon follow-up, 200 samples from healthy volunteers and 421 samples from domestic travellers to and from high endemic areas of ROK. The positive cases from the ELISA test were confirmed by both Giemsa microscopic and polymerase chain reaction methods. The clinical sensitivity and specificity of detecting P. vivax with ELISA test were 94.4% and 99.0%, respectively. Thirteen of 421 domestic travellers (3.0%) to endemic areas tested positive. The results indicate the effectiveness of detecting antibodies against P. vivax in blood with Genedia Malaria Ab ELISA 2.0 test in a large blood screen setting.