Clinical performance of SARS-CoV-2 antigen-detection rapid diagnostic test using SERS-based lateral flow immunoassay.

Background: 'ACROSIS COVID-19 Ag (NPS)' kit (SG Medical, Seoul, Korea) is a newly developed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen-detection rapid diagnostic test (Ag-RDT) using surface-enhanced Raman scattering (SERS)-based lateral flow immunoassay (LFIA). We evaluated its clinical performance compared with STANDARD Q COVID-19 Ag (SD Biosensor, Suwon, Korea), a previously approved Ag-RDT. Methods: A total of 286 nasopharyngeal swab specimens were collected: 104 positive and 182 negative specimens in SARS-CoV-2 real-time reverse-transcription polymerase-chain-reaction (rRT-PCR). SARS-CoV-2-positive specimens were divided according to the cycle threshold (Ct) value in rRT-PCR. The clinical performance of ACROSIS was compared with that of STANDARD Q. Results: ACROSIS showed significantly higher sensitivity than STANDARD Q (92.3% vs. 85.6%, P = 0.02), especially in specimens with 25 ≤ Ct < 30 (78.6% vs. 42.9%). The Ct values of RdRp/S genes for 95% detection rates by ACROSIS and STANDARD Q were 25.8 and 23.0, respectively. Conclusions: This is the first study that evaluated the performance of ACROSIS compared with STANDARD Q. The overall clinical performance of ACROSIS was superior to that of STANDARD Q, especially in specimens with 25 ≤ Ct < 30. ACROSIS could be useful for SARS-CoV-2 Ag detection even in relatively low viral load specimens.

Patterns of inflammatory immune responses in patients with septic shock receiving vitamin C, hydrocortisone, and thiamine: clustering analysis in Korea.

BACKGROUND: Sepsis is characterized by heterogeneous immune responses that may evolve during the course of illness. This study identified inflammatory immune responses in septic patients receiving vitamin C, hydrocortisone, and thiamine. METHODS: This was a single-center, post-hoc analysis of 95 patients with septic shock who received the vitamin C protocol. Blood samples were drawn on days 1-2, 3-4, and 6-8 after shock onset. Group-based multi-trajectory modeling was used to identify immune trajectory groups. RESULTS: The median age was 78 years (interquartile range, 70-84 years), and 56% were male. Clustering analysis identified group 1 (n=41), which was characterized by lower interleukin (IL)-6, tumor necrosis factor (TNF)-α, and IL-10 levels, and these levels remained stationary or mildly increased until day 7. Conversely, group 2 (n=54) expressed initially higher IL-6, TNF-α, and IL-10 levels that decreased rapidly by day 4. There was a nonsignificant increase in lymphocyte count and a decrease in C-reactive protein level until day 7 in group 2. The intensive care unit mortality rate was significantly lower in group 2 (39.0% vs. 18.5%, P=0.03). Group 2 also had a significantly higher decrease in the mean (standard deviation) vasopressor dose (norepinephrine equivalent: -0.09±0.16 µg/kg/min vs. -0.23±0.31 µg/kg/min, P<0.001) and Sequential Organ Failure Assessment score (0±5 vs. -4±3, P=0.002) between days 1 and 4. CONCLUSIONS: There may be different subphenotypes in septic patients receiving the vitamin C protocol.

Immune Responses After Vaccination With Primary 2-Dose ChAdOx1 Plus a Booster of BNT162b2 or Vaccination With Primary 2-Dose BNT162b2 Plus a Booster of BNT162b2 and the Occurrence of Omicron Breakthrough Infection.

BACKGROUND: Before the omicron era, health care workers were usually vaccinated with either the primary 2-dose ChAdOx1 nCoV-19 (Oxford-AstraZeneca) series plus a booster dose of BNT162b2 (Pfizer-BioNTech) (CCB group) or the primary 2-dose BNT162b2 series plus a booster dose of BNT162b2 (BBB group) in Korea. METHODS: The two groups were compared using quantification of the surrogate virus neutralization test for wild type severe acute respiratory syndrome coronavirus 2 (SVNT-WT), the omicron variant (SVNT-O), spike-specific IgG, and interferon-gamma (IFN-γ), as well as the omicron breakthrough infection cases. RESULTS: There were 113 participants enrolled in the CCB group and 51 enrolled in the BBB group. Before and after booster vaccination, the median SVNT-WT and SVNT-O values were lower in the CCB (SVNT-WT [before-after]: 72.02-97.61%, SVNT-O: 15.18-42.29%) group than in the BBB group (SVNT-WT: 89.19-98.11%, SVNT-O: 23.58-68.56%; all P < 0.001). Although the median IgG concentrations were different between the CCB and BBB groups after the primary series (2.677 vs. 4.700 AU/mL, respectively, P < 0.001), they were not different between the two groups after the booster vaccination (7.246 vs. 7.979 AU/mL, respectively, P = 0.108). In addition, the median IFN-γ concentration was higher in the BBB group than in the CCB group (550.5 and 387.5 mIU/mL, respectively, P = 0.014). There was also a difference in the cumulative incidence curves over time (CCB group 50.0% vs. BBB group 41.8%; P = 0.045), indicating that breakthrough infection occurred faster in the CCB group. CONCLUSION: The cellular and humoral immune responses were low in the CCB group so that the breakthrough infection occurred faster in the CCB group than in the BBB group.

Exploring the vaccine-induced immunity against severe acute respiratory syndrome coronavirus 2 in healthcare workers.

We aimed to analyze the kinetics of T-cell-mediated and B-cell-mediated humoral immune responses against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) before and after booster vaccination, as well as the impacts of the in vitro test results the type of vaccination on the prediction of SARS-CoV-2 infection. A total of 240 healthcare workers vaccinated twice were serially tested using an interferon gamma release assay (IGRA) and a neutralizing antibody (nAb). At the end of the study, we investigated the history of SARS-CoV-2 infection of all the enrolled participants to analyze the effects of the test results and the type of vaccination on SARS-CoV-2 infection. Overall, the positive rates were 52.3% and 80.0% for IGRA and 84.6% and 100% for the nAb test before and after booster vaccination, respectively. However, the positive rates were 52.8% for IGRA and 100% for nAb 3 months after booster vaccination. The in vitro test results and the type of vaccination were not associated with SARS-CoV-2 infection. The antibody response caused by the SARS-CoV-2 vaccination lasted more than 6 months, although the response of the T-cells disappeared rapidly after 3 months. However, these in vitro results and the type of vaccination cannot be used for predicting the risk of SARS-CoV-2 infection.

Performance evaluation of newly developed surrogate virus neutralization tests for detecting neutralizing antibodies against SARS-CoV-2.

We evaluated newly developed surrogate virus neutralization tests (sVNT) for detecting neutralizing antibodies (NAbs) against the receptor binding domain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). VERI-Q SARS-CoV-2 Neutralizing Antibody Detection ELISA Kit (MiCo BioMed, Gyeonggi-do, Republic of Korea, hereafter, "eCoV-CN") is an enzyme-linked immunosorbent assay-based sVNT, and VERI-Q SARS-CoV-2 Neutralizing Antibody Rapid Test Kit (MiCo BioMed, hereafter, "rCoV-RN") is a point-of-care lateral-flow immunochromatography test with auto-scanner. A total of 411 serum samples were evaluated. Both evaluations used a 50% plaque reduction neutralization test (PRNT50) as the gold standard. Compared with PRNT50, the eCoV-CN showed 98.7% positive percent agreement (PPA), 96.8% negative percent agreement (NPA), 97.4% total percent agreement (TPA), with kappa values of 0.942. The rCoV-RN showed 98.7% PPA, 97.4% NPA, 97.8% TPA, and kappa values of 0.951, comparing to PRNT50. Neither assay indicated cross-reactivity for other pathogens, and the signal indexes were statistically significantly correlated to the PRNT50 titer. The two evaluated sVNTs show comparable performances to the PRNT50 with the advantages of technical simplicity, speed, and do not require cell culture facilities.

Estimating the measurement uncertainties of the international sensitivity index of 12 thromboplastins through Monte Carlo simulation.

BACKGROUND: Measurement uncertainty (MU) estimation has become an important process in clinical laboratories; however, calculating the MUs of the international sensitivity index (ISI) of thromboplastins is difficult because of the complex mathematical calculations required in calibration. Therefore, this study quantifies the MUs of ISIs through the Monte Carlo simulation (MCS), which involves random sampling of numerical values to solve a complex mathematical calculation. METHODS: Eighty blood plasmas and commercially available certified plasmas (ISI Calibrate) were used to assign the ISIs of each thromboplastin. Prothrombin times were measured using reference thromboplastin and 12 commercially available thromboplastins (Coagpia PT-N, PT Rec, ReadiPlasTin, RecombiPlasTin 2G, PT-Fibrinogen, PT-Fibrinogen HS PLUS, Prothrombin Time Assay, Thromboplastin D, Thromborel S, STA-Neoplastine CI Plus, STA-Neoplastine R 15, and STA-NeoPTimal) with two automated coagulation instruments: ACL TOP 750 CTS (ACL TOP; Instrumentation Laboratory, Bedford, MA, USA) and STA Compact (Diagnostica Stago, Asnières-sur-Seine, France). Then, the MUs of each ISI were simulated through MCS. RESULTS: The MUs of ISIs ranged from 9.7 % to 12.1 % and 11.6 % to 12.0 % when blood plasma and ISI Calibrate were used, respectively. For some thromboplastins, the ISI claimed by manufacturers significantly differed from the estimated results. CONCLUSIONS: MCS is adequate to estimate the MUs of ISI. These results would be clinically useful for estimating the MUs of the international normalized ratio in clinical laboratories. However, the claimed ISI significantly differed from the estimated ISI of some thromboplastins. Therefore, manufacturers should provide more accurate information about the ISI value of thromboplastins.

Evaluation of ichroma™ COVID-19 interferon gamma release assay for detection of vaccine-induced immunity in healthcare workers.

OBJECTIVES: We compared the performance of a new interferon gamma release assay (IGRA) format assay, the ichroma™ COVID-19 IGRA (IGRA-SARS), with that of the widely used QuantiFERON SARS-CoV-2 ELISA kit (QFN-SARS) in vaccinated healthcare workers (HCWs). Additionally, we analyzed the long-term changes in IGRA results after the final vaccine dose. METHODS: A total of 383 specimens from 281 HCWs were enrolled in this study, and the results of SARS-IGRA and QFN-SARS assays were compared. In addition, we performed the receive operator curve analysis to estimate the optimal cut-off value for IGRA-SARS. RESULTS: For all specimens, IGRA-SARS and QFN-SARS showed 75.7% and 64.2% of the positive results, respectively. The absolute agreement between IGRA-SARS and QFN-SARS was 80.0%, and the Fleiss' κ value was 0.525, indicating moderate agreement. ROC curve analysis of the IGRA-SARS results showed a cut-off value of >0.254 IU/mL, which was consistent with the manufacturer's specifications. The positive rates of both IGRA assays decreased significantly after a postvaccination period of 6 months. CONCLUSIONS: IGRA-SARS showed acceptable performance in the detection of vaccine-induced immunity against COVID-19; however, harmonization of IGRA assays has not yet been achieved. Additionally, the significant decline of positive rates of IGRA after the last vaccination would support the necessity of booster vaccination after a postvaccination period of 6 months.

Establishment of Reference Intervals of Cytokeratin 19 Fragment Antigen 21-1 in Korean Adults.

Cytokeratin 19 fragment antigen 21-1 (CYFRA 21-1) is useful for predicting and monitoring non-small cell lung cancer prognosis. We established reference intervals (RIs) of CYFRA 21-1 in Korean adults, including those older than 60 years. Data of 4,098 apparently healthy subjects (age range, 20-87 years) were analyzed after excluding those with a history of malignancy, high tumor marker concentrations (except CYFRA 21-1), and/or abnormal findings on a chest computed tomography scan through medical chart review. After removing two outliers, RIs of CYFRA 21-1 were determined using data of 4,096 subjects based on the non-parametric method (2.5th and 97.5th percentiles) according to CLSI guidelines EP28-A3c. The subjects were divided into two and four groups according to sex and age (20-40, 41-50, 51-60, and >60 years), respectively, and the median CYFRA 21-1 concentration was compared between the groups. The RI of CYFRA 21-1 was 0.66-3.84 ng/mL, applicable to both men and women. Regardless of sex, the CYFRA 21-1 concentration increased with age, suggesting that age-dependent RIs of CYFRA 21-1 should be applied. Rather than using a single RI provided by the manufacturer, the RI of CYFRA 21-1 should be continually verified and established in each clinical laboratory.

Prevalence and detection of Tropheryma whipplei in the stools of Korean patients with diarrhea using real-time PCRs.

BACKGROUND: The prevalence of Tropheryma whipplei varies depending on age, region, and underlying disease. We estimated the prevalence of T. whipplei in the stools of Korean patients with diarrhea using real-time PCR (RT-PCR) and compared three RT-PCR targets, rpoB, hsp65, and Dig15. METHODS: A total of 1404 nucleic acid samples extracted from the stools of Korean patients with diarrhea were tested using an initial RT-PCR targeting T. whipplei-specific regions of 16S-23S rRNA intergenic spacer. Subsequently, the samples positive for the initial RT-PCR were tested using the follow-up RT-PCRs targeting rpoB, hsp65, and Dig15 and analyzed by sequencing to confirm the presence of T. whipplei. We estimated the prevalence of T. whipplei and compared them according to gender and age. We also compared the performance of three targets in the follow-up RT-PCRs. RESULTS: T. whipplei was detected in 1.4% of all samples (20 of 1404), and there were no differences according to gender and age. In pediatric samples (≤ 19 years), T. whipplei was detected higher in children aged 6-19 than in those aged 1-5 (2.7% vs. 0.7%, P = 0.01). Sensitivities of the rpoB, hsp65, and Dig15 RT-PCR were 50.0%, 85.0%, and 95.0%, respectively; specificities were 100.0%, 100.0%, and 84.6%, respectively. CONCLUSIONS: This is the first study that estimated the prevalence of T. whipplei in the stools of Korean patients with diarrhea. This study demonstrated the presence of T. whipplei in stools of Koreans, even though the bacterium was detected low. The RT-PCRs targeting hsp65 and Dig15 showed reliable performance, and a multiplex PCR including these targets is expected to be useful for T. whipplei detection.

Real-World Accuracy of a SARS-CoV-2 Rapid Diagnostic Tests in the Republic of Korea.

Antigen rapid diagnostic tests (RDTs) became the most important tool for the diagnosis of the coronavirus disease 2019 (COVID-19), however there have been very few evaluations of the accuracy of the RDTs in actual use. In this study, we investigated the performance accuracy of the RDT, the STANDARD Q COVID-19 Ag (STANDARD Q), in the Republic of Korea. We collected a total of 5,792 results that underwent both RDT and reverse transcription polymerase chain reaction simultaneously, and overall sensitivity and specificity of the STANDARD Q were 57.6% and 99.9%, respectively. With binomial logistic regression analysis, we estimated that about half of the COVID-19 patients with a cycle threshold value of 25 for E and RdRP were RDT-negative. These results suggest that the clinical sensitivity of RDTs against severe acute respiratory syndrome coronavirus 2 is considerably low in a real-world setting, and we recommend that limitations of RDTs should be considered when setting up COVID-19 test strategies.

Comparison of simulated candidemia detection during prophylactic antifungal therapy.

BACKGROUND: We investigated the neutralization performance of various automated blood culture systems for antifungal agents with regard to the most commonly isolated Candida species. METHODS: In this study, we evaluated the time to detection (TTD) of simulated candidemia for 6 Candida spp. (C. albicans, C. auris, C. glabrata, C. krusei, C. parapsilosis, and C. tropicalis) in 3 automated blood culture systems (BACTEC™ FX, BACT/ALERT® 3D, and BACT/ALERT® VIRTUO®), with or without trough and peak levels of eight antifungal agents (amphotericin B, anidulafungin, caspofungin, fluconazole, itraconazole, micafungin, posaconazole, and voriconazole). RESULTS: Caspofungin and micafungin significantly prolonged the TTDs for most of the tested strains in the 3 blood culture instruments, especially at peak concentrations. CONCLUSION: Peak concentrations of caspofungin and micafungin influence the performance of blood culture detection systems. Therefore, one should be careful about the possibility of prolonged TTDs for candidemia when using the abovementioned antifungal agents.

Arsenicicoccus cauae sp. nov., isolated from the blood of a pediatric gastroenteritis patient.

A Gram-stain-positive coccus was isolated from the blood of a paediatric patient suffering from gastroenteritis. The taxonomic position of this catalase-positive, non-motile, non-spore-forming facultative anaerobe designated as strain MKL-02T was investigated using a polyphasic approach. Colonies grown on tryptic soy agar with 10 % sheep blood were circular, creamy yellow, and convex. Phylogenetic analysis based on 16S rRNA gene and whole-genome sequences revealed that this strain was most closely related to Arsenicicoccus bolidensis CCUG 47306T within the cluster of the genus Arsenicicoccus. Average nucleotide identity and digital DNA-DNA hybridization values between strain MKL-02T and A. bolidensis DSM 15745T, A. dermatophillus DSM 25571T and A. piscis DSM 22760T were 89.5 and 37.0 %, 79.6 and 22.4 %, and 75.9 and 21.0 %, respectively. The genomic size of strain MKL-02T was 3 423 857 bp with a 72.7 mol% G+C content. Growth was observed at 10-45 °C (optimum, 37-40 °C) and pH 6.0-10.0 (optimum, pH 7.0), in the presence of 0-10 % (w/v) NaCl (optimum, 0.5 %). Cells of strain MKL-02T were non-motile cocci and 0.50-0.60 µm long, as determined by transmission electron microscopy. The strain was catalase-positive and oxidase-negative. The major fatty acid type (>10 % of total) was C15 : 0. The polar lipid profile consisted of two unidentified phospholipids, three unidentified lipids and an unidentified aminophospholipid. The strain contained MK-8 (H4) as the predominant menaquinone. Based on phylogenetic and phenotypic considerations, it is proposed that strain MKL-02T be classified as a new species, named Arsenicicoccus cauae sp. nov. The type strain is MKL-02T (=NCCP 16967T=JCM 34624T).

Positivity of Rapid Antigen Testing for SARS-CoV-2 With Serial Followed-up Nasopharyngeal Swabs in Hospitalized Patients due to COVID-19.

Despite the accuracy of nucleic acid amplification tests (NAATs), rapid antigen tests (RATs) for severe acute respiratory syndrome coronavirus-2 are widely used as point-of-care tests. A total of 282 pairs of reverse transcription-polymerase chain reaction and Standard Q COVID-19 Ag tests were serially conducted for 68 patients every 3-4 days until their discharge. Through a field evaluation of RATs using direct nasopharyngeal swabs, the sensitivities were 84.6% and 87.3% for E and RNA-dependent RNA polymerase (RdRp) genes, respectively, for specimens with cycle thresholds (Cts) < 25. The Ct values of E and RdRp genes for 95% detection rates by RATs were 16.9 and 18.1, respectively. The sensitivity of RAT was 48.4% after the onset of symptoms, which was not sufficient. RAT positivity gradually decreased with increased time after symptom onset and had continuously lower sensitivity than NAATs.

Red and white blood cell morphology characterization and hands-on time analysis by the digital cell imaging analyzer DI-60.

BACKGROUND: The Sysmex DI-60 digital morphology analyzer is a fully automated, cell-locating image analysis system. This study aimed to evaluate the analytical performance of DI-60. METHODS: A total of 822 peripheral blood smears were used. The diagnostic performance of DI-60 in terms of red blood cell (RBC) morphology characterization, white blood cell (WBC) differentials, and the total assay time including hands-on time was evaluated. RESULTS: In comparison with manual slide review, DI-60 demonstrated acceptable accuracy in recognizing polychromasia, target cells, and ovalocytes. However, for schistocytes, DI-60 demonstrated low specificity (10.4%) despite the high sensitivity (97.2%). In the precision analysis of RBC morphology characterization, borderline samples harboring specific RBCs showed inconsistencies in the positive results among 20 replicates. Particularly, 6 of 10 samples showed inconsistencies in the precision for schistocytes. For WBC differentials, the overall agreement between pre-classification results and user-verified results was 89.4%. Except for basophils, normal WBCs showed a good correlation between DI-60 (after user verification) and manual counts. The sensitivities in detecting immature granulocytes, blasts, atypical lymphocytes, and normoblasts were 85.9%, 92.0%, 37.5%, and 77.6%, respectively. Although the total assay time of DI-60 was longer than that of manual review, the hands-on time was considerably shorter with a difference of 144.1 s/slide for abnormal samples. CONCLUSION: DI-60 demonstrated acceptable performance for normal samples. However, for abnormal WBC differentials and RBC morphology characterization, it should be utilized carefully. DI-60 may contribute to an improvement in laboratory efficiency with increased feasibility.

Field evaluation of seven SARS-COV-2 antigen assays.

There is a global demand for rapid diagnostic tests (RDTs) for Coronavirus disease 2019 (COVID-19), and the interest in their clinical compliance is growing. In this study, we evaluated the clinical compliance of seven different severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen RDTs. Nasopharyngeal/oropharyngeal swab specimens from COVID-19-confirmed cases and reverse-transcription PCR (RT-PCR) screening were used to evaluate the performance of seven RDTs. Using the RT-PCR and RDT results, we predicted the cycle threshold (Ct) of each target gene (E, RdRP, and N genes) which 50% (Ct50) and 95% (Ct95) detection rates were achieved in the RDTs. A total of 482 specimens were enrolled in our study: 316 specimens from COVID-19-confirmed cases and 166 RT-PCR-negative specimens. The median values of Ct50 and Ct95 for the seven RDTs were in the ranges of ranged 24.3-30.9 and 19.3-22.6 for E, 25.5-31.5 and 20.9-24.0 for RdRP, and 26.8-32.3 and 22.7-25.7 for N, respectively. The RDTs showed acceptable compliance only for specimens with high viral burdens (Ct < 20). However, the false-negative rate increased by more than 50% for most of the RDTs in low-viral burden specimens (Ct> 30). These results suggest that RDTs should not be used without molecular assays for COVID-19 screening for asymptomatic patients because of their high false-negative rates.

Performance evaluation of newly developed fluorescence immunoassay-based interferon-gamma release assay for the diagnosis of latent tuberculosis infection in healthcare workers.

The ichroma™ IGRA-TB (Boditech Med Inc., Chuncheon, Republic of Korea) is an automated fluorescent immunoassay-based point-of-care interferon-gamma release assay for detecting latent tuberculosis infection. We evaluated this assay with 408 health care workers, and demonstrated its acceptable performances comparing to QuantiFERON-TB Gold-Plus (QFT-Plus; Qiagen, Germantown, MD).

Biomarkers Associated with Failure of Liberation from Oxygen Therapy in Severe COVID-19: A Pilot Study.

This study aimed to investigate whether clinical and laboratory biomarkers can identify patients with COVID-19 who are less likely to be liberated from oxygen therapy. This was a retrospective study comparing 18 patients in the weaning failure group with 38 patients in the weaning success group. Weaning failure was defined as death or discharge with an oxygen device before day 28 after hospital admission or requiring oxygen support as of day 28. The median quick Sequential Organ Failure Assessment (qSOFA) score was significantly higher and the median SpO2/FiO2 was significantly lower in the weaning failure group. The laboratory biomarkers, procalcitonin (PCT) and D-dimer, were significantly higher in the weaning failure group, as were the biomarkers of endothelial injury, such as angiopoietin-2 (Ang-2) and Ang-2/Ang-1, and tumor necrosis factor-α (TNF-α). Patients' qSOFA scores, SpO2/FiO2, and PCT, D-dimer, Ang-2, Ang-2/Ang-1, endocan (4-day and 7-day increases), and TNF-α levels predicted weaning failure; 7-day endocan levels were the best predictor of weaning failure with an AUC of 0.81 (95% CI, 0.67-0.94). We identified clinical and laboratory parameters, including plasma biomarkers of endothelial injury, that may be considered as biomarkers for predicting failure of liberation from oxygen therapy in patients with severe COVID-19.

Clinical Utility of Serum Holotranscobalamin Measurements in Patients with First-Ever Ischemic Stroke.

BACKGROUND: Whether holotranscobalamin (holoTC) indicates B12 deficiency more sensitively than total vitamin B12 (B12) is unclear. This study is aimed at determining the impact of serum holoTC level as a risk factor for ischemic stroke and investigating its association with disease severity and short-term outcomes. METHODS: Serum holoTC, total B12, and homocysteine levels were compared between 130 stroke patients and 138 healthy controls. Biomarker level correlations with disease severity and stroke functional outcomes were investigated. RESULTS: holoTC levels were lower and homocysteine levels were higher in stroke patients than in healthy controls (P < 0.05). The holoTC/total B12 ratio and homocysteine level significantly predicted ischemic stroke in the multivariable regression analysis (P < 0.05). Along with hyperhomocysteinemia, patients more often had holoTC than total B12 deficiency (6.2% vs. 3.1%). holoTC levels negatively correlated with homocysteine levels (partial R -0.165, P < 0.05) in stroke patients in multiple linear regression analyses, but not total B12 levels. The holoTC level and holoTC/total B12 ratio, but not homocysteine and total B12 levels, negatively correlated with the National Institute of Health Stroke Scale (partial R, -0.405 and -0.207, respectively, P < 0.01). CONCLUSIONS: Measurements of serum holoTC levels combined with total B12 and homocysteine levels may provide valuable information for predicting ischemic stroke and its severity and short-term outcomes of ischemic stroke patients.