Feasibility of Loop-Mediated Isothermal Amplification for Rapid Detection of Methicillin-Susceptible and Methicillin-Resistant Staphylococcus aureus in Tissue Samples

Background@#To date, few studies have investigated the feasibility of the loop-mediated isothermal amplification (LAMP) assay for identifying pathogens in tissue samples. This study aimed to investigate the feasibility of LAMP for the rapid detection of methicillin-susceptible or methicillin-resistant Staphylococcus aureus (MSSA or MRSA) in tissue samples, using a bead-beating DNA extraction method. @*Methods@#Twenty tissue samples infected with either MSSA (n = 10) or MRSA (n = 10) were obtained from patients who underwent orthopedic surgery for suspected musculoskeletal infection between December 2019 and September 2020. DNA was extracted from the infected tissue samples using the bead-beating method. A multiplex LAMP assay was conducted to identify MSSA and MRSA infections. To recognize the Staphylococcus genus, S. aureus, and methicillin resistance, 3 sets of 6 primers for the 16S ribosomal ribonucleic acid (rRNA) and the femA and mecA genes were used, respectively. The limit of detection and sensitivity (detection rate) of the LAMP assay for diagnosing MSSA and MRSA infection were analyzed. @*Results@#The LAMP result was positive for samples containing 10 3 colony-forming unit (CFU)/mL for 16S rRNA, 10 4 CFU/mL for femA, and 10 5 CFU/mL formecA. The limits of detection for 16S rRNA and femA were not different between MSSA and MRSA. For the 10 MSSA-positive samples, the LAMP assay showed 100% positive reactions for 16S rRNA and femA and a 100% negative reaction for mecA. For the 10 MRSA-positive samples, the LAMP assay showed 100% positive reactions for 16S rRNA and mecA but only 90% positive reactions for femA. The sensitivity (detection rate) of the LAMP assay for identifying MSSA and MRSA in infected tissue samples was 100% and 90%, respectively. @*Conclusions@#The results of this study suggest that the LAMP assay performed with tissue DNA samples can be a useful diagnostic method for the rapid detection of musculoskeletal infections caused by MSSA and MRSA.

Performance Evaluation of Biozentech Malaria Scanner in Plasmodium knowlesi and P. falciparum as a New Diagnostic Tool

The computer vision diagnostic approach currently generates several malaria diagnostic tools. It enhances the accessible and straightforward diagnostics that necessary for clinics and health centers in malaria-endemic areas. A new computer malaria diagnostics tool called the malaria scanner was used to investigate living malaria parasites with easy sample preparation, fast and user-friendly. The cultured Plasmodium parasites were used to confirm the sensitivity of this technique then compared to fluorescence-activated cell sorting (FACS) analysis and light microscopic examination. The measured percentage of parasitemia by the malaria scanner revealed higher precision than microscopy and was similar to FACS. The coefficients of variation of this technique were 1.2-6.7% for Plasmodium knowlesi and 0.3-4.8% for P. falciparum. It allowed determining parasitemia levels of 0.1% or higher, with coefficient of variation smaller than 10%. In terms of the precision range of parasitemia, both high and low ranges showed similar precision results. Pearson’s correlation test was used to evaluate the correlation data coming from all methods. A strong correlation of measured parasitemia (r2=0.99, P<0.05) was observed between each method. The parasitemia analysis using this new diagnostic tool needs technical improvement, particularly in the differentiation of malaria species.

Performance Evaluation of Biozentech Malaria Scanner in Plasmodium knowlesi and P. falciparum as a New Diagnostic Tool

The computer vision diagnostic approach currently generates several malaria diagnostic tools. It enhances the accessible and straightforward diagnostics that necessary for clinics and health centers in malaria-endemic areas. A new computer malaria diagnostics tool called the malaria scanner was used to investigate living malaria parasites with easy sample preparation, fast and user-friendly. The cultured Plasmodium parasites were used to confirm the sensitivity of this technique then compared to fluorescence-activated cell sorting (FACS) analysis and light microscopic examination. The measured percentage of parasitemia by the malaria scanner revealed higher precision than microscopy and was similar to FACS. The coefficients of variation of this technique were 1.2-6.7% for Plasmodium knowlesi and 0.3-4.8% for P. falciparum. It allowed determining parasitemia levels of 0.1% or higher, with coefficient of variation smaller than 10%. In terms of the precision range of parasitemia, both high and low ranges showed similar precision results. Pearson’s correlation test was used to evaluate the correlation data coming from all methods. A strong correlation of measured parasitemia (r2=0.99, P<0.05) was observed between each method. The parasitemia analysis using this new diagnostic tool needs technical improvement, particularly in the differentiation of malaria species.

Clinical Usefulness of LabChip Real-time PCR using Lab-On-a-Chip Technology for Diagnosing Malaria

As malaria remains a major health problem worldwide, various diagnostic tests have been developed, including microscopy-based and rapid diagnostic tests. LabChip real-time PCR (LRP) is a small and portable device used to diagnose malaria using lab-on-a-chip technology. This study aimed to evaluate the diagnostic performance of LRP for detecting malaria parasites. Two hundred thirteen patients and 150 healthy individuals were enrolled from May 2009 to October 2015. A diagnostic detectability of LRP for malaria parasites was compared to that of conventional RT-PCR. Sensitivity of LRP for Plasmodium vivax, P. falciparum, P. malariae, and P. ovale was 95.5%, 96.0%, 100%, and 100%, respectively. Specificity of LRP for P. vivax, P. falciparum, P. malariae, and P. ovale was 100%, 99.3%, 100%, and 100%, respectively. Cohen’s Kappa coefficients between LRP and CFX96 for detecting P. vivax, P. falciparum, P. malariae, and P. ovale were 0.96, 0.98, 1.00, and 1.00, respectively. Significant difference was not observed between the results of LRP and conventional RT-PCR and microscopic examination. A time required to amplify DNAs using LRP and conventional RT-PCR was 27 min and 86 min, respectively. LRP amplified DNAs 2 times more fast than conventional RT-PCR due to the faster heat transfer. Therefore, LRP could be employed as a useful tool for detecting malaria parasites in clinical laboratories.

Clinical Significance of a High SARS-CoV-2 Viral Load in the Saliva

Background@#Patients with coronavirus disease 2019 (COVID-19) can unknowingly spread the virus to several people during the early subclinical period. @*Methods@#We evaluated the viral dynamics in various body fluid specimens, such as nasopharyngeal swab, oropharyngeal swab, saliva, sputum, and urine specimens, of two patients with COVID-19 from hospital day 1 to 9. Additional samples of the saliva were taken at 1 hour, 2 hours, and 4 hours after using a chlorhexidine mouthwash. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral load was determined by real-time reverse transcriptase polymerase chain reaction (rRT-PCR). @*Results@#SARS-CoV-2 was detected from all the five specimens of both patients by rRT-PCR. The viral load was the highest in the nasopharynx (patient 1 = 8.41 log10 copies/mL; patient 2 = 7.49 log10 copies/mL), but it was also remarkably high in the saliva (patient 1 = 6.63 log10 copies/mL; patient 2 = 7.10 log10 copies/mL). SARS-CoV-2 was detected up to hospital day 6 (illness day 9 for patient 2) from the saliva of both patients. The viral load in the saliva decreased transiently for 2 hours after using the chlorhexidine mouthwash. @*Conclusion@#SARS-CoV-2 viral load was consistently high in the saliva; it was relatively higher than that in the oropharynx during the early stage of COVID-19. Chlorhexidine mouthwash was effective in reducing the SARS-CoV-2 viral load in the saliva for a short-term period.

Seroprevalence of Anti-SARS-CoV-2 Antibodies among Outpatients in Southwestern Seoul, Korea

Serosurveillance studies reveal the actual disease burden and herd immunity level in the population. In Seoul, Korea, a cross-sectional investigation showed 0.07% anti-severe acute respiratory syndrome coronavirus-2 antibody seropositivity among 1,500 outpatients of the university hospitals. Low seroprevalence reflects well-implemented social distancing.Serosurveillance should be repeated as the pandemic progresses.

Evaluation of Diagnostic Performance of Three Real-Time PCR Assays for the Detection of Mycobacteria Species / 대한임상미생물학회지

Background@#The disease burden caused by Mycobacterium tuberculosis (MTB) complex continues to decrease in most countries. However, the diseases caused by the nontuberculous mycobacteria (NTM) become a public health problem. This study aimed to compare the diagnostic accuracy of three real-time PCR assays: AdvanSure TB/NTM real-time PCR kit (AdvanSure; LG Chem., Korea), Genedia MTB/NTM detection kit (Genedia; Green Cross MS, Korea), and PowerChek MTB/NTM Real-time PCR kit (PowerChek; Kogenebiotech, Korea) for the detection of MTB complex and NTM. @*Methods@#Total 102 acid-fast bacilli (AFB) smear-positive and 177 smear-negative specimens from Korea University Medical Center, Guro Hospital, were enrolled. The AFB smear-positive and negative specimens were collected from November 2016 to October 2017 and November to December 2018, respectively. DNA extraction was performed using Genedia Mycobacteria DNA prep Kit (Green Cross MS, Korea). The statistical analysis was performed using MedCalc 18.11.6 (MedCalc Software, Belgium). @*Results@#Among 261 specimens, 64 showed MTB complex growth and 28 exhibited NTM growth. The sensitivity, specificity, positive predictive value, and negative predictive value of AdvanSure/Genedia/PowerChek kits for MTB were 96.9%/95.3%/96.9%, 98.5%/99.5%/98.5%, 58.9%/80.9%/58.9%, and 99.9%/99.9%/99.9%. Whereas those for NTM detection were 81.5%/44.4%/88.9%, 99.6%/100.0%/98.7%, 57.3%/100.0%/32.8% and 99.9%/99.6%/99.9%, respectively. The area under the receiver operating characteristic curve of AdvanSure and PowerChek for NTM detection was statistically different from that of Genedia (P<0.0001). @*Conclusion@#Three real-time PCR assays were reliable for MTB complex in AFB-positive and -negative specimens. There was a difference between these three reagents for the accuracy of NTM detection.

Hematopoietic Stem Cell Registry Report: 2018 / 대한수혈학회지

Background@#Hematopoietic stem cell (HSC) transplantation is an important therapeutic option for many malignant and non-malignant diseases. The whole transplant process involves multiple areas and complex steps. The laboratory procedures include the collection, processing, and storage of HSC. The HSC registry aims to identify the current situation and draw improvement points by voluntarily registering the information of an HSC graft collected by each institute sharing the analyzed data. This study analyzed and shared the data for 2018. @*Methods@#Data for 2018 registered at the HSC registry website (www.ksfa-registry.org) was downloaded and analyzed. The data were to enter the information of each collection and include the demography of the donors, transplant type, instrument, vascular access, mobilization modality, and the number of CD34＋ cells. @*Results@#Two thousand eight hundred eighty-eight collection datasets from 1,373 donors were registered from 19 institutes, which was slightly higher than that reported in 2017. The number of collections in one patient was in the range of 1∼17 times, and the average was two times. In allogeneic HSCT, the number of related donors was higher than that of unrelated donors. The frequency of collecting more than four times per donor was 25.2% for autologous donors, compared to 95.4% for allogeneic donors less than twice. @*Conclusion@#The HSC registry is not limited to identifying the current situation and sharing the analyzed data, but is expected to contribute to the development of guidelines, education of human resources, and the standardization of laboratory procedures involved in hematopoietic stem cell transplantation.

Cellulitis Caused by a Novel Cupriavidus Species Strain J1218 Identified by Whole Genome Sequencing / 대한임상미생물학회지

We report a case of cellulitis caused by a novel Cupriavidus species identified using whole-genome sequence analysis. Subcutaneous tissue biopsies from the left lower leg of a 67-year-old man who suffered from cellulitis were cultured. Round, convex, gray and non-hemolytic colonies were recovered after 72-h incubation. 16S rRNA sequence analysis showed 98.6% similarity with Cupriavidus basilensis DSM 11853(T) in the NCBI database and 99.9% similarity with C. basilensis KF708 in the EzBioCloud database. Genomic analysis using the MiSeq platform (Illumina, USA) and the TrueBac ID database (ChunLab, Korea) revealed that the average nucleotide identity (ANI) of this strain with C. basilensis DSM 11853(T) was 87.6%. The patient was treated with oral cefditoren pivoxil for 9 weeks. This study is the first to report cellulitis caused by Cupriavidus species strain J1218.

An Automated Draft Report Generator for Peripheral Blood Smear Examinations Based on Complete Blood Count Parameters

BACKGROUND: Complete blood count (CBC) results play an important role in peripheral blood smear (PBS) examinations. Many descriptions in PBS reports may simply be translated from CBC parameters. We developed a computer program that automatically generates a PBS draft report based on CBC parameters and age- and sex-matched reference ranges. METHODS: The Java programming language was used to develop a computer program that supports a graphical user interface. Four hematology analyzers from three different laboratories were tested: Sysmex XE-5000 (Sysmex, Kobe, Japan), Sysmex XN-9000 (Sysmex), DxH800 (Beckman Coulter, Brea, CA, USA), and ADVIA 2120i (Siemens Healthcare Diagnostics, Eschborn, Germany). Input data files containing 862 CBC results were generated from hematology analyzers, middlewares, or laboratory information systems. The draft reports were compared with the content of input data files. RESULTS: We developed a computer program that reads CBC results from a data file and automatically writes a draft PBS report. Age- and sex-matched reference ranges can be automatically applied. After examining PBS, users can modify the draft report based on microscopic findings. Recommendations such as suggestions for further evaluations are also provided based on morphological findings, and they can be modified by users. The program was compatible with all four hematology analyzers tested. CONCLUSIONS: Our program is expected to reduce the time required to manually incorporate CBC results into PBS reports. Systematic inclusion of CBC results could help improve the reliability and sensitivity of PBS examinations.

Status of ABO Incompatible Blood Transfusion in Korea and Preventon / 대한수혈학회지

No abstract available.

The inflammatory response of neutrophils in an in vitro model that approximates the postcardiac arrest state

PURPOSE: Postcardiac arrest syndrome (PCAS) shares many features with sepsis including plasma cytokine elevation with dysregulation of cytokine production, and the presence of endotoxin in plasma. PCAS is closely related to ischemia-reperfusion injury. During ischemia-reperfusion injury, neutrophil, which is the first line of innate immunity, plays a major role. In this study, we investigated the inflammatory response of human neutrophils in an in vitro model which we simulated with hypoxia-normoxia and hypoxia-hyperoxia environments. METHODS: After separation of neutrophils from the whole blood, they were divided into 3 experimental groups: normoxia-normoxia, hypoxia-normoxia, and hypoxia-hyperoxia groups. The production of H₂O₂, the expression of Toll-like receptor 4 (TLR₄) receptor, and the extent of apoptosis of the neutrophils were checked. RESULTS: The in vitro hypoxia-normoxia and -hyperoxia models, which simulated the PCAS, showed initiation of the neutrophils' inflammatory reaction by hypoxia insult. Lipopolysaccharide amplifies such inflammation; therefore, prevention of secondary infection may be critical in postresuscitation patients. Temporary hyperoxia following hypoxic insult showed no difference in inflammatory reaction compared with hypoxia-normoxia. Rather, temporary hyperoxia may suppress or minimize inflammation by attenuation of TLR4 receptor. CONCLUSION: It is well known that continuous hyperoxygenation after successful cardiac arrest harms patients, but temporary hyperoxygenation with 100% O₂ in a clinical situation may be helpful.

Identification of the ABO*cis-AB04 Allele With a Unique Substitution C796A: The First Case in Korea

No abstract available.

Anti-H Antibody Showing Agglutination in the Anti-Human Globulin Phase in a Korean Patient

No abstract available.

Actual Incidence of Transfusion-Related Adverse Reactions Compared with Transfusion-Related Signs or Symptoms and by Each Blood Product / 대한수혈학회지

BACKGROUND: Transfusion-related adverse reaction is detected based on patients' adverse signs or symptoms during or after transfusion. We analyzed the actual incidence of transfusion-related adverse reactions by investigating diagnosed cases among reported signs or symptoms, and reexamined our transfusion-related adverse reaction reporting system. METHODS: From January to June, 2015, there were 4,234 cases of transfusion and 18,191 units of blood product were used. During transfusion, patients' signs or symptoms were checked and reported by the medical team at least three times, 5 minutes after transfusion started, during transfusion, and after transfusion, using the electronic reporting system in the blood bank. A laboratory medicine doctor investigated reported signs or symptoms by reviewing patients' electronic medical records, diagnosed transfusion-related adverse reaction by textbook definition, and surveyed actual incidence. In addition, incidence of transfusion-related signs or symptoms and transfusionrelated adverse reaction by each blood product was determined. RESULTS: Out of 1,091 transfusion-related signs or symptoms, only 226 cases (20.71%) were diagnosed with transfusion-related adverse reaction. Among these, most common cases were febrile nonhemolytic reaction with incidence of 0.91%, followed by allergic reaction with 0.32%. The incidence of transfusion-related adverse reaction by each blood product was highest for leukocyte-reduced red blood cells 3.41% and apheresis platelets 2.59%. Febrile nonhemolytic reaction was mainly related to red blood cells and allergic reaction was mainly related to platelets. CONCLUSION: The actual incidence of transfusion-related adverse reaction was only 20% of transfusion-related signs or symptoms. Therefore, reforming the reporting system and transfusion-related clinical inspection and education are required.

Consideration of Discontinuance of Malaria Antibody Test for Blood Donor Screening / 대한수혈학회지

BACKGROUND: Since 2001, the Korean Red Cross has performed malaria antibody test for blood donors in malaria-risk areas to prevent transfusion-transmitted malaria. However, due to insufficient sensitivity and specificity the malaria antibody assay is not considered an efficient screening method. Therefore, we have considered discontinuing malaria antibody testing for blood donors. METHODS: We analyzed the results of malaria antibody test from 2001 to 2014 utilizing data from the Blood Information Management System of the Korean Red Cross. RESULTS: Among 16,650,812 donations tested from 2001 to 2014, 50,143 donations (0.30%) showed positive results. However, there was no truly infected case at the time of donation. The positive rate among blood donations was between 34 and 39 per 10,000 in 2001, but between 9 and 10 per 10,000 in 2014. There was no interregional disparity in the positive rate of blood donations. CONCLUSION: Korea is in a malaria elimination phase and malaria antibody testing in limited areas is not effective, therefore we propose discontinuing the malaria antibody test.

Current Status of Pretransfusion Tests and Equipments in Small and Medium Sized Hospitals in Korea / 대한수혈학회지

BACKGROUND: The appropriate procedures and equipment for the pretransfusion test are fundamental to a safe blood transfusion. The present study aimed to assess the current status of procedures and equipment for pretransfusion tests at small- and medium-sized medical institutions, as well as to use this basic raw data to better manage blood transfusions at these institutions. METHODS: Offline and online questionnaire surveys were performed at institutions that used between 24 and 1,000 units of blood products in 2014. A total of 338 institutions participated, and the survey results were subsequently analyzed. RESULTS: Among 307 institutions where on-site ABO blood typing was performed, 15.0%, 2.1%, and 43.5% did not conduct ABO serum typing, RhD typing, and irregular antibody screening tests, respectively, and 12.8% only conducted the saline phase for crossmatching. Moreover, among 338 institutions, only 66.7% of blood banks had centrifuges, 84.5% had 37℃ incubators, 41.1% had slide view boxes; in addition, 66.1% and 18.6% had refrigerators and deep freezers, respectively, for blood storage. CONCLUSION: Certain small- and medium-sized institutions did not have the essential equipment required to operate as blood banks. Moreover, they also needed to improve their testing procedures. To address these issues, the initiation of systematic training programs and the employment of institutional strategies are necessary to enhance testing procedures and equipment, respectively.

A Case of Primary Anti-D Alloimmunization by RHD (c.1227G>A) DEL Red Blood Cell Transfusion / 대한수혈학회지

The Rh blood group D antigen is the most immunogenic of all antigens, next to ABO antigens. Anti-D immunization is clinically important since it may cause clinical problems, such as severe hemolytic transfusion reactions and hemolytic disease of the newborn. DEL is an extremely weak D variant that cannot be detected by basic serologic typing and is typed as D-negative without the absorption-elution techniques and RHD genotyping. Of the DEL phenotype, RHD (c.1227G>A) allelic variant is the most common in Korea. The DEL phenotype has been considered to carry only a few D antigens to induce anti-D immunization, but a few cases have reported that this allelic variant is capable of inducing anti-D immunization in a D-negative recipient, for which it is clinical significant. Herein, we present a case of primary anti-D alloimmunization in a RhD negative patient after receiving RHD (c.1227G>A) DEL red cell transfusion identified by serological and molecular tests, including RHD genotyping.

Effect of Irradiation on Microparticles in Red Blood Cell Concentrates

Changes in microparticles (MP) from red blood cell (RBC) concentrates in the context of irradiation have not been investigated. The aim of this study was to evaluate how irradiation affects the number of MPs within transfusion components. Twenty RBC concentrates, within 14 days after donation, were exposed to gamma rays (dose rate: 25 cGy) from a cesium-137 irradiator. Flow cytometry was used to determine the numbers of MPs derived from RBC concentrates before and 24 hr after irradiation. The mean number of MPs (±standard deviation) in RBC concentrates was 21.9×10(9)/L (±22.7×10(9)/L), and the total number of MPs ranged from 2.6×10(9)/L to 96.9×10(9)/L. The mean number of MPs increased to 22.6×10(9)/L (±31.6×10(9)/L) after irradiation. Before irradiation, the CD41-positive and CD235a-positive MPs constituted 9.5% (1.0×10(9)/L) and 2.2% (263×10(6)/L) of total MPs, respectively. After irradiation, CD41-positive MPs increased to 12.1% (1.5×10(9)/L) (P=0.014), but the CD235a-positive MPs decreased to 2.0% (214×10(6)/L) of the total MPs (P=0.369). Irradiation increases the number of CD41-positive MPs within RBC concentrates, suggesting the irradiation of RBC concentrates could be associated with thrombotic risk of circulating blood through the numerical change.

Emerging Infectious Disease and Safety of Blood Components / 대한수혈학회지

Recently, the emergency of Middle East Respiratory Syndrome in Korea raised public concern regarding emerging infectious disease (EID) and affected the strategy for blood safety. Although some cases of EID such as West Nile virus in the United States and hepatitis E virus in Japan emerged in a restricted area, the emergence can rapidly affect the situation in other countries in a globalized society with advancements in transportation and international exchange. Because the risk of transfusion-mediated infection may occur by the agent of EID which can be transmitted through blood, the strategy for safety must be considered for agents that were not tested in the stage of blood donor screening. We reviewed the characteristics of transfusion-transmissible EIDs raising concerns worldwide and the strategies which had been adopted.