Quantitative Analysis and Enantiomeric Separation of Ephedra Alkaloids in Ma Huang Related Products by HPLC-DAD and UPLC-MS/MS

Ephedra is a genus of the Ephedraceae family and is found in temperate regions, such as Central Asia and Europe. Among the various ephedra species, Ma Huang (Ephedra herb) is derived from the aerial parts of Ephedra sinica Stapf, Ephedra equisetina Bunge, and Ephedra intermedia Schrenk & C.A. Mey. Ma Huang contains various ephedra alkaloids, including (-)-ephedrine, (+)-pseudoephedrine, (-)-norephedrine, (+)-norpseudoephedrine, (-)-methylephedrine, and (+)-methylpseudoephedrine, which are found naturally as single enantiomers, although they can be prepared as racemates. Although the use of Ma Huang in foods is prohibited in Korea, products containing Ma Huang can be imported, and so it is necessary to develop a suitable analytical technique for the detection of Ma Huang in foods. Herein, we report the development of analytical methods for the detection of ephedra alkaloids in products containing Ma Huang. Following sample purification by solid phase extraction, quantitative analysis was performed using ultra-performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-MS/MS). Additionally, the enantiomers were successfully separated using HPLC-DAD. We successfully analyzed various food samples, where the ephedra alkaloids were qualitatively and quantitatively determined, and the enantiomers were separated. It is expected that these methods may contribute toward preventing the distribution of illegal products containing Ma Huang.

A Case Series of Trauma Resuscitation in the Intensive Care Unit Bypassing the Emergency Room During the Conversion to a COVID-19 Only Hospital

When a patient with severe trauma is admitted to the emergency room (ER), they are evaluated before transfer to either the intensive care unit (ICU) or operating room. To minimize the time until a definitive treatment can be provided, direct operating room resuscitation can be performed. In this hospital the ER was closed during the hospital’s transition to a coronavirus disease 2019-dedicated hospital, and direct ICU resuscitation for patients with trauma was performed for a short period. To perform effective trauma resuscitation, all ICU beds were reorganized to achieve a modified, experienced nurse: patient ratio (1:2-3) and 2 beds were assigned for trauma ICU resuscitation alone. The equipment for initial resuscitation was installed and ICU nurses received training. Consultations with the hospital administration, nursing, and pharmaceutical departments were completed in advance to avoid formal problems. Conversion of the ICU for direct resuscitation procedures was performed in 4 patients.

COVID-19 in a Patient Previously Exposed to Toxic Disinfectant from a Humidifier

In August, 2011, the Korean Public Health Surveillance declared an outbreak of pulmonary disease due to the inhalation of humidifier disinfectants (HDs), which led to approximately 20,000 deaths. In March, 2020, the World Health Organization declared coronavirus disease-2019 (COVID-19) a pandemic. In this Case Report, we present a rare case of a patient who inhaled toxic HDs and developed COVID-19. He was young and had a low risk of severe COVID-19, however, he had a critical course to recovery. He was admitted to the intensive care unit and administered high-flow oxygen via a nasal cannula. He received dexamethasone injections each day for 10 days and his condition began to improve on hospital Day 6, although radiographical findings revealed no improvement. He was discharged on hospital Day 26. Despite the patient’s chronic lung disease becoming asymptomatic, HDs could be an important risk factor affecting the clinical course of COVID-19.

A Case Report of Tracheostomy for a Patient with COVID-19: How to Minimize Medical Staff and Patient Risks

Coronavirus disease was first reported in December 2019, and the World Health Organization declared it as a pandemic on March 11, 2020. The virus is known to attack various vital organs, including the respiratory system. Patients sometimes require positive pressure ventilation and tracheostomy. Because tracheostomy is a droplet-spreading procedure, medical staff should protect themselves against the risk of transmission of this contagious viral disease. In our case, we performed tracheostomy for a 70-year-old man with coronavirus disease 2019 (COVID-19) who had required more oxygen with gradual weakness of respiratory muscle to maintain his arterial oxygen saturation. We focused on the risks of the medical staffs and patients, and minimized them at the same time using temporary balloon over-inflation, pre-operative adjustment of endotracheal tube position, and attachment of a transparent film dressing to the surgical field without stopping the ventilator while following routine safety measures. Fourteen days after the tracheostomy, all participating medical staff members were healthy and asymptomatic. The patient was discharged 105 days after the COVID-19 diagnosis.

Development and Validation of a Novel Warfarin Dosing Algorithm for Korean Patients With VKORC1 1173C

BACKGROUND: Differences in the performance of suggested warfarin dosing algorithms among different ethnicities and genotypes have been reported; this necessitates the development of an algorithm with enhanced performance for specific population groups. Previous warfarin dosing algorithms underestimated warfarin doses in VKORC1 1173C carriers. We aimed to develop and validate a new warfarin dosing algorithm for Korean patients with VKORC1 1173C.METHODS: A total of 109 patients carrying VKORC1 1173CT (N=105) or 1173CC (N=4) were included in this study. Multiple regression analysis was performed to deduce a new dosing algorithm. Following literature searches for genotype-guided warfarin dosing algorithms, 21 algorithms were selected and evaluated using the correlation coefficient (ρ) of actual dose and estimated dose, mean error, and root mean square error.RESULTS: The developed algorithm is as follows: maintenance dose (mg/week)=exp [3.223−0.009×(age)+0.577×(body surface area [BSA])+0.178×(sex)−0.481×(CYP2C9 genotype)+0.227×(VKORC1 genotype)]. Integrated variables explained 44% of the variance in the maintenance dose. The predicted and actual doses showed moderate correlation (ρ=0.641) with the best performance with a mean error of −1.30 mg/week. The proportion of underestimated groups was 17%, which was lower than with the other algorithms.CONCLUSIONS: This is the first study to develop and validate a warfarin dosing algorithm based on data from VKORC1 1173C carriers; it showed superior predictive performance compared with previously published algorithms.

Impact of Sarcopenia on the Risk of Erosive Esophagitis / 대한소화기학회지

BACKGROUND/AIMS: An association between obesity and erosive esophagitis has been reported, but the effects of sarcopenia and obesity on erosive esophagitis are unknown. This study examined the relationship between obesity, sarcopenia, sarcopenic obesity, and erosive esophagitis in a large population of asymptomatic men and women.METHODS: This study analyzed 32,762 subjects who underwent a comprehensive health check-up, which included upper gastrointestinal endoscopy, from August 2006 to December 2011 by a cross-sectional study. Sarcopenia was defined as a decrease in the appendicular skeletal muscle mass (ASM)/body weight value of two SD or more below the normal means for a younger reference group.RESULTS: The study was carried out on four groups according to obesity and sarcopenic status: normal, obesity, sarcopenic, and sarcopenic obese group. In a multivariable model, the risk of erosive esophagitis was higher in the obese (adjusted OR [aOR] 1.35, 95% CI 1.22–1.49), sarcopenic (aOR 2.12, 95% CI 1.40–3.19), and sarcopenic obese groups (aOR 1.54, 95% CI 1.27–1.87) than in the normal group. The risk of erosive esophagitis was higher in the sarcopenic and sarcopenic obese groups than the obese group; the ORs were 1.63 (95% CI 1.08–2.47) and 1.22 (95% CI 1.01–1.46), respectively. In dose-response analysis, increasing sarcopenia severity showed a positive and graded relationship with the overall, Los Angeles (LA)-B or higher grade, and LA-C erosive esophagitis.CONCLUSIONS: This study suggests that sarcopenia is strongly and progressively associated with erosive esophagitis.

Bevacizumab induced intestinal perforation in patients with colorectal cancer

PURPOSE: Bevacizumab has been used as a promising drug for metastatic colorectal cancer in combination with chemotherapeutic agents. However, it has a few serious adverse effects, such as intestinal bleeding or perforation. The purpose of this study is to identify the clinical characteristics of intestinal perforation induced by bevacizumab in colorectal cancer patients.METHODS: From January 2007 to June 2018, a total of 488 patients underwent chemotherapy with bevacizumab for metastatic colorectal cancer. Medical records were reviewed retrospectively.RESULTS: Nine patients (1.8%) were identified with intestinal perforation induced with bevacizumab. The median age was 59 years (range, 36–68 years). The primary tumor site was the sigmoid colon in six patients, the rectum in three patients. The liver was the most common metastatic organ (7 patients). Perforation sites were primary tumor site of the colorectum in four patients and the small bowel in five patients. Intestinal perforation was developed after a median of 3 chemotherapy cycles (range, 1–15 cycles), and a median of 7 days (range, 3–32 days) after chemotherapy. One patient expired due to sepsis.CONCLUSION: Bevacizumab induced intestinal perforation is a lethal adverse effect in patients with colorectal cancers. The characteristics of intestinal perforation varied according to perforation site, previous chemotherapy cycles, and clinical course. Careful monitoring is necessary with the use of bevacizumab in conjunction with chemotherapeutic agents.

Lack of Association between Helicobacter pylori Infection and Various Markers of Systemic Inflammation in Asymptomatic Adults / 대한소화기학회지

BACKGROUND/AIMS: Helicobacter pylori (H. pylori) infection has been known to cause various extra-gastric diseases, which may be mediated by an increase in systemic inflammation. Thus, we examined the association between H. pylori infection and various markers of systemic inflammation in a large sample of asymptomatic adults. METHODS: Cross-sectional data were obtained from 17,028 adults who completed routine health check-ups. H. pylori infection status was determined using a serum immunoglobulin G test, and systemic inflammation was assessed using the C-reactive protein (CRP) levels, neutrophil/lymphocyte ratio (NLR), and platelet/lymphocyte ratio (PLR). RESULTS: Multiple linear regression model-adjusted for potential confounders-revealed that H. pylori infection was not associated with CRP levels (coefficient: −0.012, 95% confidence interval [CI]: −0.037, 0.012, p=0.319), NLR (coefficient: 0.055, 95% CI: −0.027, 0.138, p=0.192), or PLR (coefficient: 1.798, 95% CI: −1.979, 5.574, p=0.351). In a multivariable logistic regression model, H. pylori infection was not associated with the risk of CRP levels being elevated to ≥0.1 mg/dL (odds ratio: 0.96, 95% CI: 0.81, 1.08) or ≥0.3 mg/dL (odds ratio: 1.02, 95% CI: 0.84, 1.19). In the multivariable model, CRP levels elevated to ≥0.1 mg/dL were significantly associated with body mass index, current smoking status, hypertension, and diabetes mellitus. Regular exercise and high-density lipoprotein cholesterol were factors that minimized the elevation of CRP levels. CONCLUSIONS: Chronic infection with H. pylori was not associated with various inflammatory markers. Further investigation is needed to clarify the interaction between H. pylori infection, systemic inflammation, and extra-gastric disease.

Associations between Atopic Dermatitis and Risk of Gastric Cancer: A Nationwide Population-based Study / 대한소화기학회지

BACKGROUND/AIMS: Epidemiologic and clinical data indicate that allergies may be associated with reduced risks for several cancers; however, to date, only a few studies have examined the associations between allergies and gastric cancer. This study aimed to examine the associations between allergies and gastric cancer using a large population-based dataset. METHODS: This cross-sectional study obtained data from the Korea National Health and Nutrition Examination Survey between 2010 and 2014, involving a total of 24,089 participants. The associations between allergies and gastric cancer were analyzed using univariable and multivariable logistic regression analyses with complex sampling, while adjusting for confounding factors that included age, sex, body mass index, smoking status, alcohol intake, and level of education. RESULTS: Multivariable logistic regression analyses that were adjusted for the potential confounders determined that a history of allergic diseases tended to be associated with reduced risk of gastric cancer; however, this relationship was not statistically significant (any allergy: odds ratio [OR], 0.62; 95% confidence interval [CI], 0.34-1.12; atopic dermatitis: OR, 0.34; 95% CI, 0.50-1.72; allergic rhinitis: OR, 0.71; 95% CI, 0.34-1.46; asthma: OR, 0.44; 95% CI, 0.15-1.29). Multivariable analysis showed that a history of atopic dermatitis was associated with reduced risk of gastric cancer in men (OR, 0.16; 95% CI, 0.03-0.75). CONCLUSIONS: This findings of this study suggest that individuals with allergies tend to have a reduced risk of gastric cancer, without a statistically significant association. Furthermore, atopic dermatitis was associated with reduced risk of gastric cancer, particularly in men.

Nomograms Predicting Platinum Sensitivity, Progression-Free Survival, and Overall Survival Using Pretreatment Complete Blood Cell Counts in Epithelial Ovarian Cancer / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: This study was conducted to evaluate the prognostic significance of pre-treatment complete blood cell count (CBC), including white blood cell (WBC) differential, in epithelial ovarian cancer (EOC) patients with primary debulking surgery (PDS) and to develop nomograms for platinum sensitivity, progression-free survival (PFS), and overall survival (OS). MATERIALS AND METHODS: We retrospectively reviewed the records of 757 patients with EOC whose primary treatment consisted of surgical debulking and chemotherapy at Samsung Medical Center from 2002 to 2012. We subsequently created nomograms for platinum sensitivity, 3-year PFS, and 5-year OS as prediction models for prognostic variables including age, stage, grade, cancer antigen 125 level, residual disease after PDS, and pre-treatment WBC differential counts. The models were then validated by 10-fold cross-validation (CV). RESULTS: In addition to stage and residual disease after PDS, which are known predictors, lymphocyte and monocyte count were found to be significant prognostic factors for platinum-sensitivity, platelet count for PFS, and neutrophil count for OS on multivariate analysis. The area under the curves of platinum sensitivity, 3-year PFS, and 5-year OS calculated by the 10-fold CV procedure were 0.7405, 0.8159, and 0.815, respectively. CONCLUSION: Prognostic factors including pre-treatment CBC were used to develop nomograms for platinum sensitivity, 3-year PFS, and 5-year OS of patients with EOC. These nomograms can be used to better estimate individual outcomes.

Normative Hearing Threshold Levels in Koreans with Normal Tympanic Membranes and Estimated Prevalence of Hearing Loss

OBJECTIVES: We investigated the normative data on the hearing threshold levels of Koreans with normal tympanic membranes and the prevalence of hearing loss (HL) and nonserviceable hearing using the data from the Korea National Health and Nutrition Examination Surveys (KNHANES) during 2010–2012. METHODS: Data obtained from 16,673 participants ≥12-year-of-age with normal tympanic membranes who completed audiometric testing. We defined HL as the pure tone average (PTA) >25 dB hearing level at 500, 1,000, 2,000, and 3,000 Hz and non-serviceable hearing as PTA >40 dB hearing level. RESULTS: The hearing levels at some frequencies (0.5, 3, and 6 kHz) did not differ in between the 10's and 20's, but the hearing thresholds at all frequencies increased gradually from the 30's. The hearing thresholds were higher in men than in women at high frequencies (3, 4, and 6 kHz) in the 30's and older. The prevalence of HL in either ear was 16.5% (estimates of 5.9 million), from 2.4% in the 10's up to 75.4% in the 70's and older. The prevalence of nonserviceable hearing in either ear was 6.8% (estimates of 2.5 million) and that of bilateral nonserviceable hearing was 2.5% (estimates of 0.9 million). CONCLUSION: Hearing loss aggravated from the 30's at all frequencies and men showed poorer hearing levels than women at high frequencies. Hearing loss was a common condition and the prevalence of non-serviceable hearing in either ear, which needs hearing rehabilitation to help social communication, was 6.8%. Normative pure tone thresholds at each frequency can be used as referent values when counseling patients complaining of hearing loss.

T2-Weighted Liver MRI Using the MultiVane Technique at 3T: Comparison with Conventional T2-Weighted MRI

OBJECTIVE: To assess the value of applying MultiVane to liver T2-weighted imaging (T2WI) compared with conventional T2WIs with emphasis on detection of focal liver lesions. MATERIALS AND METHODS: Seventy-eight patients (43 men and 35 women) with 86 hepatic lesions and 20 pancreatico-biliary diseases underwent MRI including T2WIs acquired using breath-hold (BH), respiratory-triggered (RT), and MultiVane technique at 3T. Two reviewers evaluated each T2WI with respect to artefacts, organ sharpness, and conspicuity of intrahepatic vessels, hilar duct, and main lesion using five-point scales, and made pairwise comparisons between T2WI sequences for these categories. Diagnostic accuracy (Az) and sensitivity for hepatic lesion detection were evaluated using alternative free-response receiver operating characteristic analysis. RESULTS: MultiVane T2WI was significantly better than BH-T2WI or RT-T2WI for organ sharpness and conspicuity of intrahepatic vessels and main lesion in both separate reviews and pairwise comparisons (p < 0.001). With regard to motion artefacts, MultiVane T2WI or BH-T2WI was better than RT-T2WI (p < 0.001). Conspicuity of hilar duct was better with BH-T2WI than with MultiVane T2WI (p = 0.030) or RT-T2WI (p < 0.001). For detection of 86 hepatic lesions, sensitivity (mean, 97.7%) of MultiVane T2WI was significantly higher than that of BH-T2WI (mean, 89.5%) (p = 0.008) or RT-T2WI (mean, 84.9%) (p = 0.001). CONCLUSION: Applying the MultiVane technique to T2WI of the liver is a promising approach to improving image quality that results in increased detection of focal liver lesions compared with conventional T2WI.

Analysis of Training Needs with Roles in College & University Foodservice Dietitians

College and university foodservice dietitians have to be competent for playing a role as the future food service executive as well as the present foodservice administrator in the promising foodservice industry field. The study conducted a survey to examine training needs corresponding to educational contents for the purpose of helping them acquire a new knowledge related to self-development and duty through educational training. The results of this study suggested that training needs were differed by general characteristics of dietitians, operational characteristics of food services, and training subjects. A future study should develop systematic training strategies for dietitians.

Construction of A Stable Full-Length cDNA Clone of Japanese Encephalitis Virus Strain SA14-14-2 Using Low Copy Number Plasmid

Recently the reverse genetics system contributed to the progresses in the investigation of positive-stranded RNA viruses. Here, we report the successful construction of a stable full-length infectious cDNA clone of the live attenuated JEV vaccine strain SA14-14-2. The eleven kilobase viral RNA genome was reverse transcribed, amplified as four overlapping DNA fragments and successively ligated into the low copy number plasmid pACYC184, which contains the p15A origin of replication. In vitro-transcribed RNAs had a specific infectivity of approximately 104 PFU/microgram RNA, and the resulting virus exhibited growth kinetics and plaque morphology similar to the parental virus in cell culture. The structural and functional integrity of the cDNA clone was stably maintained even after at least 150 generations in Escherichia coli strain TOP10. The cDNA clone was engineered to contain single nucleotide change to create a XhoI site and knock out a XbaI site (A to C at nt 9134) acting as a genetic marker. This genetic marker was retained in the recovered progeny virus. Our results suggest that the instability of the full-length infectious JEV cDNA clone can be overcome by employing low copy number plasmid pACYC184. This infectious JEV cDNA clone will aid future studies of pathogenesis, virulence, and replication. Furthermore, it will facilitate the development of SA14-14-2 based recombinant vaccines.

Construction of A Stable Full-Length cDNA Clone of Japanese Encephalitis Virus Strain SA14-14-2 Using Low Copy Number Plasmid

Recently the reverse genetics system contributed to the progresses in the investigation of positive-stranded RNA viruses. Here, we report the successful construction of a stable full-length infectious cDNA clone of the live attenuated JEV vaccine strain SA14-14-2. The eleven kilobase viral RNA genome was reverse transcribed, amplified as four overlapping DNA fragments and successively ligated into the low copy number plasmid pACYC184, which contains the p15A origin of replication. In vitro-transcribed RNAs had a specific infectivity of approximately 104 PFU/microgram RNA, and the resulting virus exhibited growth kinetics and plaque morphology similar to the parental virus in cell culture. The structural and functional integrity of the cDNA clone was stably maintained even after at least 150 generations in Escherichia coli strain TOP10. The cDNA clone was engineered to contain single nucleotide change to create a XhoI site and knock out a XbaI site (A to C at nt 9134) acting as a genetic marker. This genetic marker was retained in the recovered progeny virus. Our results suggest that the instability of the full-length infectious JEV cDNA clone can be overcome by employing low copy number plasmid pACYC184. This infectious JEV cDNA clone will aid future studies of pathogenesis, virulence, and replication. Furthermore, it will facilitate the development of SA14-14-2 based recombinant vaccines.

Evaluation of JEV and BVDV Clearance During the Purification of Recombinant HPV-16 L1 Virus-Like Particles

Insect cell-derived biotechnological products have a potential for viral contamination from cell line sources themselves or from adventitious introduction of virus during production. The objective of this study was to establish techniques for viral clearance validation of insect cell-derived recombinant human papillomavirus (HPV)-16 type L1 virus-like particles (VLPs) using Japanese encephalitis virus (JEV) and bovine viral diarrhea virus (BVDV) as relevant viruses. The downstream process for the production of recombinant HPV-16 L1 VLPs was sequentially carried out employing detergent lysis (NP-40/PBS), sonication, sucrose cushion centrifugation, and cesium chloride (CsCl) equilibrium density centrifugation. Recombinant HPV-16 L1 capsid protein (56 kD) expressed in Sf9 cell culture was clearly detected by SDS-PAGE and Western blotting analysis. Each purification step was evaluated to determine reduction factor for viral clearance by infectivity assay. In individual purification steps, detergent treatment (0.50% v/v, NP-40/PBS) and CsCl equilibrium density centrifugation were found to be effective in JEV and BVDV clearance. Overall cumulative reduction factors of JEV and BVDV infectivity titer for the purification procedure implemented in this study were 12.53 and 10.05 log TCID(50)/pool, respectively. The results suggest that the purification procedure employed in this study for the HPV-16 L1 VLPs produced from recombinant baculovirus-infected Sf9 cells will be effective over 10 log TCID(50)/pool reduction factor in the clearance of enveloped, adventitious viruses with a buoyant density lower than approximately 1.23 g/ml.

Development of One-step Real-time Reverse Transcription-polymerase Chain Reaction in Combination with Automated RNA Extraction for Detection and Quantitation of Hepatitis A Virus

One-step real-time reverse transcription-polymerase chain reaction (RT-PCR) assay using the MagNA Pure LC and LightCycler(TM) system was developed and validated for the detection and quantitation of hepatitis A virus (HAV) RNA. The assay was evaluated using in-house synthetic HAV RNA standard. The real-time RT-PCR assay could quantitate a dynamic range of HAV RNA standard between 10(2) and 10(8) copies per reaction. The regression coefficient of the standard curve was an 0.99. The detection limit of the assay was 31.3 RNA copies per reaction. The coefficient variations (CVs) of the assay in combination with automated RNA extraction were less than 1.91% in both intra- and inter-assay. The real-time RT-PCR assay for quantitative detection of HAV would serve a useful method for improving the safety of biological products.

Evaluation of Limit of Detection and Range of Quantitation for RT-PCR, Real-Time RT-PCR and RT-PCR-ELISA Detection of Bovine Viral Diarrhoea Virus Contamination in Biologics Derived from Cell Cultures

Risk of viral contamination is one of major concerns common to all biologics derived from cultivated cells. Bovine viral diarrhoea virus (BVDV) has widely been known as a contaminant of cell culture-derived vaccines. The objective of the study was to assess the limit of detection and range of quantitation of the detection methods for BVDV using a reverse transcription-polymerase chain reaction (RT-PCR) assay, real-time RT-PCR assay, and RT-PCR-ELISA. One milliliter of cell culture supernatant containing 106.5+/-0.2 median tissue culture infectious dose (TCID50)/ml of BVDV NADL strain was subjected to RNA isolation. The isolated RNA was 10-fold serially diluted and each diluted sample (10-1 to 10-6) was subjected to RT-PCR on a GeneAmpR PCR System 9700 and/or LightCycler(TM). The amplified products were analyzedly (1) agarose gel electrophoresis for RT-PCR assay, (2) melting curve analysis for real-time RT-PCR assay (in this case a program is automatically linked to amplification step), and (3) ELISA using capture and detection probes for RT-PCR-ELISA. The limit of detection of the 3 assay methods was equally estimated to be 316 TCID50/ml of starting virus culture supernatant subjected to the assay. The quantitation range of real-time RT-PCR assay and RT-PCR-ELISA was estimated to be from 3.16x105 to 3.16x102 TCID50/ml of starting virus culture supernatant. The overall results suggested that the 3 assay methods for BVDV detection can be reliably applied to evaluate BVDV contamination in biologics derived from cell cultures.

A Quantitative Assay of Japanese Encephalitis Virus as a Model Virus for Viral Clearance Validation in Insect Cell-Derived Biotechnology Products

Commercial interests towards insect cell cultures have greatly been increased recently, in part due to the widespread use of insect virus-based vectors for efficient expressions of foreign proteins. Insect cell-derived biotechnology products should be free of adventitious agents such as arboviruses and mycoplasmas. The objective of this study was to establish techniques for the viral safety evaluation of insect cell-derived biotechnology products using Japanese encephalitis virus (JEV) as a model, since JEV is a member of arthropod-borne flaviviruses which has been known to be infectious to insect cells such as Sf9 cells. Quantitative assays for viral infectivity, concentrations of an antigen or a genome are a prerequisite for the studies of viral clearance validation. Here, we report the development of a quantitative assay for JEV RNA using real-time reverse transcription-polymerase chain reaction (RT-PCR). The assay was performed using LightCycler and RNA amplification kit SYBR Green I. Viral RNA extracted from stock suspension of JEV with known infectivity titer was made to 10-fold serial dilution, and each dilution was subjected to the assay for the generation of a standard curve. A JEV specific primer was selected from 3' untranslated region with the expected band size of 323 base pairs. The real-time RT-PCR assay resulted in a successful amplification within 5 log dilution ranges of JEV RNA samples, and the sensitivity of the assay was calculated to be approximately 15 TCID50 per reaction. Highly reproducible standard curves were obtained from experiments performed on three different days. The real-time RT-PCR assay for the quantification of JEV will be an efficient alternative tool for viral clearance validation studies of insect cell-derived biotechnology products.

Age Related Seroepidemiological Study of Diphtheria among Koreans / 감염

BACKGROUND: The incidence of diphtheria has been markedly reduced and almostly eradicated by widespread use of DTP vaccines in developed countries. However, outbreaks of this disease may be occurred under some circumstances of ineffective immunization. In recent time, some studies reported persistent outbreaks of diphtheria in developed countries and indicated the existence of a large pool of susceptible individuals with potential for epidemic infection. In Korea, diphtheria vaccination has been well maintained since 1956 with high acceptant vaccination rates. So, there has been no reported diphtheria patient since 1987. But, there has been few study to diphtheria serosuvey, and no assessment of diphtheria immunization. Also, we do not use Td vaccine in adults for maintenance of diphtheria immunity. In this aspect, we conducted age related seroepidemiology of diphtheria and indirectly assessed the immunity of diphtheria vaccines, used in Korea. METHODS: For the evaluation of age related serosurvey of diphtheria immunity in Korean populations, study subjects below 10 years old aged children were classified into 10 groups (A~J) with one year interval, and beyond this age to 60 years old aged adults were classified into 5 group (K~O) with 10 years interval. And the adults over 60 years old age was classified into the last group (P). The numbers of each group were 100, and sex distributions of each group were almostly equal. And for the indirect assessment of diphtheria immunization in Korean children, children under 15 years old were classified into 6 groups (I~ VI) according to the status of DTaP vaccination. The numbers of this each group were 30, and sex ratio was almostly equal. Detection of specific IgG antibody to diphtheria toxin were determined by ELISA (contained fragment A & B toxin; IBL, Germany). RESULTS: In age related groups, the antibody titers to diphtheria toxin were well maintained until 10 years old age group, thereafter the titers abruptly decreased below 0.1 IU/mL and then slightly elevated after 30 years old age group and then maintained with low levels. In the groups related DTaP vaccine status, the antibody titers were very low (below 0.07 IU/mL) in prevaccination status, but the titers after primary vaccinations were markedly increased and maintained (above 0.6 IU/mL) until 15 years. And diphtheria antitoxin levels in the groups (L, M, N) showed no significant differences between males and females. CONCLUSION: The results of our study showed that the antibody titers to diphtheria toxin in the 20~50 years old aged groups dramatically decreased. This result indicated that vaccine induced diphtheria immunity did not last throughout life, and Td vaccination program in adults should be considered for maintenance of diphtheria immunity. And the immunity to diphtheria in Korean children indicated that 3 timesprimary and 2 times booster diphtheria immunizations were optimal.