Developing a Loop-Mediated Isothermal Amplification Assay for the Rapid Detection of Seven Respiratory Viruses including SARS-CoV-2.

Background and Objectives: The coronavirus disease (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), continues to be a pandemic even in 2022. As the initial symptoms of COVID-19 overlap with those of infections from other respiratory viruses, an accurate and rapid diagnosis of COVID-19 is essential for administering appropriate treatment to patients. Currently, the most widely used method for detecting respiratory viruses is based on real-time polymerase chain reaction (PCR) and includes reverse-transcription real-time quantitative PCR (RT-qPCR). However, RT-qPCR assays require sophisticated facilities and are time-consuming. This study aimed to develop a real-time quantitative loop-mediated isothermal amplification (RT-qLAMP) assay and compare its analytical performance with RT-qPCR. Materials and Methods: A total of 315 nasopharyngeal swabs from patients with symptoms of respiratory infections were included in this study. A primary screening of the specimens was performed using RT-qPCR. RNA/DNA from standard strains for respiratory viruses and heat-inactivated preparations of standard strains for SARS-CoV-2 were used to evaluate the accuracy and target specificity of the RT-qLAMP assay. Results: We successfully developed an RT-qLAMP assay for seven respiratory viruses: respiratory syncytial virus (RSV) A, RSV B, adenovirus, influenza (Flu) A (H1N1 and H3N2), Flu B, and SARS-CoV-2. RT-qLAMP was performed in a final reaction volume of 9.6 µL. No cross-reactivity was observed. Compared with the RT-PCR results, the sensitivity and specificity of the RT-qLAMP assay were 95.1% and 100%, respectively. The agreement between the two methods was 97.1%. The median amplification time to RT-qLAMP positivity was 22:34 min (range: 6:80-47:98 min). Conclusions: The RT-qLAMP assay requires a small number of reagents and samples and is performed with an isothermal reaction. This study established a fast, simple, and sensitive test that can be applied to point-of-care testing devices to facilitate the detection of respiratory viruses, including SARS-CoV-2.

Successful Treatment of Thrombotic Thrombocytopenic Purpura in a Patient with Mixed Connective Tissue Disease.

BACKGROUND: Although the survival rate of thrombocytopenic purpura (TTP) has increased significantly due to the introduction of therapeutic plasma exchange (TPE). TTP in patients with mixed connective tissue disease (MCTD) has a very high mortality rate and a very small number of reported cases. In TTP, daily TPE is administered until a treatment response is achieved; however, in practice, TPE is often not performed for such long durations. METHODS: We report a case of TTP with MCTD in a female patient. She had developed thrombocytopenia and hemolytic anemia 9 months after delivery. She had status epilepticus and lapsed into a coma. RESULTS: The patient was successfully treated with extended sessions of TPE with corticosteroids and rituximab. CONCLUSIONS: Although the TTP regimen has not yet been established and remains controversial, this report demonstrates the importance of continuing daily TPE until achieving a treatment response.

Practical Considerations for Clinical Laboratories in Top-down Approach for Assessing the Measurement Uncertainty of Clinical Chemistry Analytes.

Background: The top-down (TD) approach using internal quality control (IQC) data is regarded a practical method for estimating measurement uncertainty (MU) in clinical laboratories. We estimated the MU of 14 clinical chemistry analytes using the TD approach and evaluated the effect of lot changes on the MU. Methods: MU values were estimated using subgrouping by reagent lot changes or using the data as a whole, and both methods were compared. Reagent lot change was simulated using randomly generated data, and the mean values and MU for two IQC datasets (different QC material lots) were compared using statistical methods. Results: All MU values calculated using subgrouping were lower than the total values; however, the average differences were minimal. The simulation showed that the greater the increase in the extent of the average shift, the larger the difference in MU. In IQC data comparison, the mean values and MU exhibited statistically significant differences for most analytes. The MU calculation methods gave rise to minimal differences, suggesting that IQC data in clinical laboratories show no significant shift. However, the simulation results demonstrated that notable differences in the MU can arise from significant variations in IQC results before and after a reagent lot change. Additionally, IQC material lots should be treated separately when IQC data are collected for MU estimation. Conclusions: Lot changes in IQC data are a key factor affecting MU estimation and should not be overlooked during MU estimation.

Verification and Establishment of Reference Intervals for Prothrombin Time and Activated Partial Thromboplastin Time for Children in a Clinical Laboratory.

BACKGROUND: Prothrombin time (PT) and activated partial thromboplastin time (aPTT) are routine coagulation tests. The absence of age-dependent reference interval (RI) may lead to abnormal results in children. In this study, we aimed to verify adult-based RIs in children and establish pediatric RIs for PT and aPTT. METHODS: We analyzed PT and aPTT results. Samples from inpatients and outpatients aged 1 - 18 years, considered healthy subjects, were divided into six groups by age. Verification and establishment of RIs were conducted. RESULTS: Applying adult-based RIs to pediatrics was statistically invalid for individuals aged 10 - 18 years for PT and invalid throughout childhood for aPTT. The new RI of PT for individuals aged 10 - 18 years was 11.1 - 14.1 seconds and that of aPTT for individuals aged 1 - 9 years was 28.2 - 46.0 seconds. CONCLUSIONS: Pediatric RIs were higher than adult-based RIs. Using pediatric RI can save time, labor, and costs to make clinical decision.

Anti-LW Confused with Anti-D: a Case Transfused RhD-positive RBCs and Literature Review.

BACKGROUND: Anti-LW is rarely identified, and it is known to have little clinical importance. However, it is often difficult to differentiate anti-LW from anti-D. METHODS: Here, we report a case where anti-LW was identified for pretransfusion testing in a patient suspected of having lymphoma. RESULTS: His blood group was O RhD-positive. Anti-D specificity, weak panreactivity and 2+ reactivity in auto control were shown as a result of antibody identification. The reactions became weaker with DTT-treated RBCs, which confirmed the specificity of an anti-LW. The patient received 8 units of O RhD-positive pRBC before discharge without any transfusion reaction. CONCLUSIONS: In conclusion, anti-LWs may show mimicking specificity of anti-D for antibody identification testing, but their clinical significance is completely different. Therefore, their prompt identification is important.

Septic shock caused by Slackia exigua in a patient with diabetes.

We report a case of septic shock caused by Slackia exigua, an obligatory anaerobic gram-positive rod, in an 82-year-old woman with diabetes. Dental assessment revealed a palatal lesion and untreated periodontitis. Although a resident species in the oral cavity and associated with localized disorders, S. exigua can cause extra-oral diseases, which can be fatal in individuals with risk factors, such as diabetes. Thus, control of oral lesions caused by S. exigua is important to prevent systemic infection.

Relationship between Brain Tissue Changes and Blood Biomarkers of Cyclophilin A, Heme Oxygenase-1, and Inositol-Requiring Enzyme 1 in Patients with Alzheimer's Disease.

Cyclophilin A (CypA), heme oxygenase-1 (HO-1), and inositol-requiring enzyme 1 (IRE1) are believed to be associated with Alzheimer's disease (AD). In this study, we investigated the association between gray matter volume (GMV) changes and blood levels of CypA, HO-1, and IRE1 in cognitively normal (CN) subjects and those with amnestic mild cognitive impairment (aMCI) and AD. Forty-five elderly CN, 34 aMCI, and 39 AD subjects were enrolled in this study. The results of voxel-based multiple regression analysis showed that blood levels of CypA, HO-1, and IRE1 were correlated with GMV on brain magnetic resonance imaging (MRI) in the entire population (p = 0.0005). The three serum protein levels were correlated with GMV of signature AD regions in the population as a whole. CypA values increased with increasing GMV in the occipital gyrus (r = 0.387, p < 0.0001) and posterior cingulate (r = 0.196, p = 0.034). HO-1 values increased with increasing GMV at the uncus (r = 0.307, p = 0.0008), lateral globus pallidus and putamen (r = 0.287, p = 0.002), and hippocampus (r = 0.197, p = 0.034). IRE1 values decreased with increasing GMV at the uncus (r = -0.239, p = 0.010) and lateral globus pallidus and putamen (r = -0.335, p = 0.0002). Associations between the three serum protein levels and regional GMV indicate that the blood levels of these biomarkers may reflect the pathological mechanism of AD in the brain.

Need for Confirmatory Neutralization Tests for Hepatitis B Surface Antigen Tests in Populations with Intermediate Prevalence.

OBJECTIVE: Hepatitis B surface antigen (HBsAg) is known as the hallmark of hepatitis B virus (HBV) infection. This study aimed to determine whether an HBsAg neutralization test is necessary to accurately interpret HBsAg test results. METHODS: Initially reactive HBsAg specimens from a 5-year period, with cutoff index values between 1.0 and 2.0, were subjected to neutralization confirmatory testing using an Elecsys HBsAg Confirmatory test kit (Roche Diagnostics GmbH. Mannheim, Germany). RESULTS: The neutralization test showed 46.1% positive (confirmed positive group) and 53.9% negative (confirmed negative group) results from the total specimens. Among the confirmed negative group, 79.5% of patients were confirmed to be negative for the current infection, whereas 4 patients in the chronic hepatitis B subgroup showed a neutralization percentage close to 40%. More than half of patients in the confirmed positive group were considered to be in the hepatitis B e antigen-negative inactive HBsAg carrier phase. CONCLUSION: In populations with intermediate HBV prevalence, a neutralization test is necessary to confirm an HBsAg result and reduce the false positive and false negative rates of initial HBsAg tests.

Bacteremia Caused by Moraxella Osloensis: a Fatal Case of an Immunocompromised Patient and Literature Review.

BACKGROUND: Moraxella osloensis rarely causes infection in humans, and most of the reported cases are not fatal. It is often difficult to identify M. osloensis using conventional biochemical methods. METHODS: Here, we report a bacteremia case caused by M. osloensis in a patient with advanced lung cancer who initially presented symptoms of fever. RESULTS: Blood culture revealed growth of a gram-negative bacterium, which was identified as M. osloensis through 16S rRNA gene sequencing and MALDI-TOF analyses. The patient could not recover from sepsis with empirical treatment. CONCLUSIONS: As M. osloensis can cause serious infections in immunocompromised patients, its prompt identification is important.

Evaluation of HCV RNA by PCR and Signal-to-Cutoff Ratios of HCV Antibody Assays for Diagnosis of HCV Infection.

OBJECTIVE: In this study, we assessed whether a hepatitis C virus (HCV) RNA test could replace recombinant immunoblot assay (RIBA) and reduce unnecessary supplemental tests as the signal-to-cutoff (S/Co) ratio from anti-HCV antibody (Ab) tests. METHODS: Anti-HCV Ab tests were performed to screen for HCV infections, and RIBA and real-time polymerase chain reaction were performed for HCV RNA to confirm HCV infection. Receiver operating characteristic curves were evaluated to determine the optimal S/Co ratios for predicting HCV infection. RESULTS: The cutoff value for the S/Co ratio was 3.63 for predicting RIBA results and 10.6 for predicting HCV RNA results. Our data suggested that an S/Co ratio ≥10.6 indicated a high risk of active HCV infection. An S/Co ratio of 3.63 to 10.6 needed further evaluation and repeat HCV RNA testing. No further testing was required for S/Co ratios <3.63 or ≥10.6. CONCLUSION: We determined that the S/Co ratio of the anti-HCV Ab test provides useful information to confirm HCV infections, including the need for further laboratory testing or clinical follow-up.

Introduction of a Borderline Range for the Interpretation of QuantiFERON-TB Gold In-Tube Results in Korean Healthcare Workers.

BACKGROUND: South Korea is the only Organisation for Economic Co-operation and Development (OECD) country with a high incidence of tuberculosis (TB). Healthcare workers (HCWs) have an increased risk of TB infection. QuantiFERON-TB Gold In-Tube (QFT-GIT) testing is performed for occupational health screening to detect latent TB infection (LTBI). METHODS: We evaluated the introduction of new criteria for borderline ranges for interferon-gamma release assay (IGRA) results in HCWs. The results of QFT-GIT tests in HCWs in 2017 and 2018 were collected, with high-risk HCWs having two serial test results. Existing dichotomous criteria and new criteria with borderline ranges (bor-derline negative [BN], 0.20 - 0.34 IU/mL; borderline positive [BP], 0.35 - 0.99 IU/mL) were applied to each test re-sult. RESULTS: After applying the borderline range, 26.4% of the positive results were classified as BP (4% of total results), while 4.2% of the negative results were classified as BN (3.6% of total results). Among seven HCWs with initial results in the borderline range, two had repeated borderline results while 71.4% had low negative results. CONCLUSIONS: We recommend the introduction of borderline ranges in the interpretation of QFT-GIT results to reduce unnecessary TB therapy in HCWs.

Etest Methods for Screening Heterogeneous Vancomycin-Intermediate Staphylococcus aureus (hVISA) strains.

The importance of heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) as a preceding stage for the development of vancomycin-resistant S. aureus is growing. We analyzed the prevalence of hVISA among bacteremia with methicillin-resistant S. aureus (MRSA) using two Etests and evaluated the clinical characteristics and outcomes. Ninety-eight MRSA isolates from blood were collected at two University hospitals in Korea. Macrodilution Etest and glycopeptide resistance detection Etests were used for detection of hVISA. Staphylococcal cassette chromosome mec (SCCmec) typing was performed by multiplex PCR. Clinical data were collected retrospectively from patient medical records. About 30% of MRSA strains were identified as hVISA. Diabetes mellitus was associated (P = 0.047) with hVISA infections. The hVISA isolates were associated with high teicoplanin MIC and multidrug resistance (P = 0.001). SCCmec type II accounted for the majority (79.3%) of hVISA strains. The prevalence of hVISA strains was increased and can lead to the development of multidrug-resistant strains. Patients with diabetes were found to have a greater risk for infection with hVISA strains. As the impact of hVISA on clinical outcome is not yet clear, large-scale studies about clinical outcomes and optimal detection methods of hVISA are needed. In conclusion, hVISA strains have a high prevalence in bloodstream MRSA infections. Awareness of the increase in hVISA strains should motivate laboratories to establish a system to detect and monitor hVISA.

Clinical Aspects of the Dense Fine Speckled Pattern in Indirect Immunofluorescence-Antinuclear Antibody Screening and Its Association with DFS70 Autoantibodies.

GOALS: The existence of anti-dense fine speckled (DFS) 70 autoantibodies are considered an exclusionary biomarker for systemic autoimmune rheumatic diseases (SARDs). Several tests to confirm the presence of anti-DFS70 autoantibodies have been introduced, and the use of them in specimens with a DFS pattern in indirect immunofluorescence-antinuclear antibody (IIF-ANA) testing has been suggested. However, these additional tests have only been evaluated in a small number of samples; their clinical usefulness therefore requires further evaluation. METHODS: A total of 213 serum specimens showing DFS (n=155) or homogeneous (H; n=58) patterns were included. All specimens were tested by western blotting (WB) and enzyme immunoassay (EIA). Clinical information regarding SARDs was analyzed. RESULTS: The detection rates for WB and EIA for anti-DFS70 autoantibodies in specimens with a DFS pattern were 86.5% and 73.5%, respectively. Detection rates in specimens with a low IIF-ANA titer were significantly lower than those in specimens with a high titer. The detection rate of anti-DFS70 autoantibodies in 58 specimens with an H pattern was 10.3% (6/58). Among 155 subjects with a DFS pattern in IIF-ANA staining, only five were diagnosed with SARD. CONCLUSIONS: There is little need to confirm the presence of anti-DFS70 autoantibodies using other methods. When a DFS pattern is observed in IIF-ANA staining, it is more important to confirm the presence of other autoantibodies related to SARDs than to identify anti-DFS70 autoantibodies. Finally, more careful interpretation of IIF-ANA to specimens with a low IIF-ANA titer is needed.

Clinical use of anti-DFS70 autoantibodies.

The dense fine speckled (DFS) nuclear pattern is one of the most common indirect immunofluorescence (IIF) patterns detected during routine anti-nuclear antibody (ANA) screening. There is a negative association between anti-DFS70 status and systemic autoimmune rheumatic disease (SARD), especially in the absence of concomitant SARD-specific autoantibodies. The purpose of this study was to determine the need for confirming anti-DFS70 status when a DFS pattern is observed in IIF-ANA. The frequency of anti-DFS70 detection on Western blot and the positive rate of connective tissue disease (CTD)-related autoantibody screening with a fluorescence-based enzyme immunoassay was evaluated in DFS (n = 182) and non-DFS (n = 359) groups. Specific autoantibodies against 15 autoantigens were identified by line immunoassay. We evaluated the frequency of cases of DFS mistaken for non-DFS and non-DFS cases mistaken for DFS, as well as the clinical impacts of these misinterpretations. Among cases of IIF-ANA with an observable DFS pattern, 68.1% had only anti-DFS70 without CTD-related autoantibodies, 20.3% were false positive for IIF-ANA, and the remaining 11.5% had CTD-related autoantibodies independent of anti-DFS70 status. These results indicated that CTD-related autoantibodies may be present with or without anti-DFS70 even if a DFS pattern is observed in IIF-ANA. Among patients who are ANA negative or have a low probability of SARD, an anti-DFS70 confirmation test has no clinical benefit and cannot replace specific tests for detecting CTD-related autoantibodies. Specific tests to detect CTD-related autoantibodies should be performed instead of anti-DFS70 confirmation tests when a DFS pattern is observed in IIF-ANA.

Neutrophil-to-Monocyte-Plus-Lymphocyte Ratio as a Potential Marker for Discriminating Pulmonary Tuberculosis from Nontuberculosis Infectious Lung Diseases.

OBJECTIVE: To determine whether NMLR has more statistical strength than NLR in discriminating TB from non-TB infectious lung diseases. METHODS: Among patients who underwent 3 or more TB culture tests with molecular study between January 2016 and December 2017, 110 patients with TB, and 159 patients diagnosed with non-TB infectious lung diseases were enrolled. The original complete blood count (CBC) parameters and modified CBC indices, including NLR and NMLR, were analyzed. RESULTS: The NLR and NMLR were significantly lower in TB patients than in patients with other infectious lung diseases. However, the area under the curve (AUC) for NMLR (0.90; 95% confidence interval [CI], 0.86-0.93) was significantly greater than that for NLR (0.88 [0.84-0.92]). CONCLUSIONS: The neutrophil-to-monocyte-plus-lymphocyte ratio (NMLR) can be used as a new index that is more powerful than neutrophil-to-lymphocyte ratio (NLR) in discriminating tuberculosis (TB) from non-TB infectious lung diseases.NMLR had more statistical strength than NLR in discriminating TB from non-TB infectious lung diseases.

Performance evaluation of cobas HBV real-time PCR assay on Roche cobas 4800 System in comparison with COBAS AmpliPrep/COBAS TaqMan HBV Test.

BACKGROUND: Hepatitis B virus (HBV) nucleic acid amplification testing (NAAT) is important for the diagnosis and management of HBV infection. We evaluated the analytical performance of the cobas HBV NAAT (Roche Diagnostics GmbH, Mannheim, Germany) on the cobas 4800 System in comparison with COBAS AmpliPrep/COBAS TaqMan HBV Test (CAP/CTM HBV). METHODS: Precision was evaluated using three levels of cobas HBV/HCV/HIV-1 Control Kit, and linearity was evaluated across the anticipated measuring range (10.0-1.0×109 IU/mL) at seven levels using clinical samples. Detection capability, including limit of blank (LOB), limit of detection (LOD) and limit of quantitation (LOQ), was verified using the 4th WHO International Standard for HBV DNA for NAT (NIBSC code: 10/266). Correlation between the two systems was compared using 205 clinical samples (102 sera and 103 EDTA plasma). RESULTS: Repeatability and total imprecision (coefficient of variation) ranged from 0.5% to 3.8% and from 0.5% to 3.5%, respectively. Linearity (coefficient of determination, R2) was 0.999. LOB, LOD and LOQ were all acceptable within the observed proportion rate (85%). Correlation was very high between the two systems in both serum and plasma samples (correlation coefficient [r]=0.995). CONCLUSIONS: The new cobas HBV real-time PCR assay on the cobas 4800 System showed reliable analytical performances.

Misidentification of Acinetobacter baumannii as Alcaligenes faecalis by VITEK 2 System; Case Report.

Acinetobacter baumannii (A. baumannii) is widely known as an opportunistic bacterial pathogen that causes infection more frequently among immunocompromised individuals. Our case demonstrated the limitation of the current VITEK 2 system for the idendification of A. baumannii. Four clinical isolates of A. baumannii were identified as Alcaligenes faecalis by the VITEK 2 system. Misidentification might lead to unnecessary tests and inappropriate treatments. Additional methods appear to be helpful for the accurate identification of A. baumannii for clinical microbiology laboratories.

Modified Complete Blood Count Indices as Predicting Markers of Preeclampsia from Gestational Hypertension: Neutrophil to Lymphocyte Ratio, Platelet to Lymphocyte Ratio, and Platelet to Neutrophil Ratio.

BACKGROUND: To evaluate modified complete blood count (CBC) indices as a predicting marker of preeclampsia (PE) from gestational hypertension (GH), we analyzed the neutrophil to lymphocyte ratio (NLR), platelet to lymphocyte ratio (PLR), and platelet to neutrophil ratio (PNR). PNR was a newly designed index in this study based on results of PE patients having a tendency toward higher neutrophil count and lower platelet count compared to normal pregnant women in previous studies. METHODS: We recruited 86 normal pregnant women, 33 patients with GH and 68 patients with PE. Subjects with any history of membrane rupture, infection, or multiple pregnancies were excluded. PNR, NLR, and PLR values including other CBC indices were statistically analyzed. RESULTS: NLR value of PE patients was significantly higher than GH patients (p = 0.011). PNR value was the most statistically significant index separating patients with PE and GH (p < 0.001). PLR value was lower in patients with PE compared to GH; however, statistical significance was low. CONCLUSIONS: NLR as well as PNR is a useful index to help predicting progression from GH to PE. Further studies are required to evaluate the full extent of utility of PNR as a predictive index in PE patients.