Restoring susceptibility to ß-lactam antibiotics in methicillin-resistant Staphylococcus aureus.

Infections by Staphylococcus aureus have been treated historically with ß-lactam antibiotics. However, these antibiotics have become obsolete in methicillin-resistant S. aureus by acquisition of the bla and mec operons. The presence of the ß-lactam antibiotic is detected by the sensor domains of BlaR and/or MecR, and the information is transmitted to the cytoplasm, resulting in derepression of the antibiotic-resistance genes. We hypothesized that inhibition of the sensor domain would shut down this response system, and ß-lactam susceptibility would be restored. An in silico search of 11 million compounds led to a benzimidazole-based hit and, ultimately, to the boronate 4. The X-ray structure of 4 is covalently engaged with the active-site serine of BlaR. Compound 4 potentiates by 16- to 4,096-fold the activities of oxacillin and of meropenem against methicillin-resistant S. aureus strains. The combination of 4 with oxacillin or meropenem shows efficacy in infected mice, validating the strategy.

A dual-action antibiotic that kills Clostridioides difficile vegetative cells and inhibits spore germination.

Clostridioides difficile infection (CDI) is the most lethal of the five CDC urgent public health treats, resulting in 12,800 annual deaths in the United States alone [Antibiotic Resistance Threats in the United States, 2019 (2019), www.cdc.gov/DrugResistance/Biggest-Threats.html]. The high recurrence rate and the inability of antibiotics to treat such infections mandate discovery of new therapeutics. A major challenge with CDI is the production of spores, leading to multiple recurrences of infection in 25% of patients [C. P. Kelly, J. T. LaMont, N. Engl. J. Med. 359, 1932-1940 (2008)], with potentially lethal consequence. Herein, we describe the discovery of an oxadiazole as a bactericidal anti-C. difficile agent that inhibits both cell-wall peptidoglycan biosynthesis and spore germination. We document that the oxadiazole binds to the lytic transglycosylase SleC and the pseudoprotease CspC for prevention of spore germination. SleC degrades the cortex peptidoglycan, a critical step in the initiation of spore germination. CspC senses germinants and cogerminants. Binding to SleC is with higher affinity than that to CspC. Prevention of spore germination breaks the nefarious cycles of CDI recurrence in the face of the antibiotic challenge, which is a primary cause of therapeutic failure. The oxadiazole exhibits efficacy in a mouse model of recurrent CDI and holds promise in clinical treatment of CDI.

Catalytic process of anhydro-N-acetylmuramic acid kinase from Pseudomonas aeruginosa.

The bacterial cell envelope is the structure with which the bacterium engages with, and is protected from, its environment. Within this envelop is a conserved peptidoglycan polymer which confers shape and strength to the cell envelop. The enzymatic processes that build, remodel, and recycle the chemical components of this cross-linked polymer are preeminent targets of antibiotics and exploratory targets for emerging antibiotic structures. We report a comprehensive kinetic and structural analysis for one such enzyme, the Pseudomonas aeruginosa anhydro-N-acetylmuramic acid (anhNAM) kinase (AnmK). AnmK is an enzyme in the peptidoglycan-recycling pathway of this pathogen. It catalyzes the pairing of hydrolytic ring opening of anhNAM with concomitant ATP-dependent phosphoryl transfer. AnmK follows a random-sequential kinetic mechanism with respect to its anhNAM and ATP substrates. Crystallographic analyses of four distinct structures (apo AnmK, AnmK:AMPPNP, AnmK:AMPPNP:anhNAM, and AnmK:ATP:anhNAM) demonstrate that both substrates enter the active site independently in an ungated conformation of the substrate subsites, with protein loops acting as gates for anhNAM binding. Catalysis occurs within a closed conformational state for the enzyme. We observe this state crystallographically using ATP-mimetic molecules. A remarkable X-ray structure for dimeric AnmK sheds light on the precatalytic and postcatalytic ternary complexes. Computational simulations in conjunction with the high-resolution X-ray structures reveal the full catalytic cycle. We further report that a P. aeruginosa strain with disrupted anmK gene is more susceptible to the ß-lactam imipenem compared to the WT strain. These observations position AnmK for understanding the nexus among peptidoglycan recycling, susceptibility to antibiotics, and bacterial virulence.

Antioxidant Efficacy of Hwangryunhaedok-tang through Nrf2 and AMPK Signaling Pathway against Neurological Disorders In Vivo and In Vitro.

Alzheimer's disease (AD) is a representative cause of dementia and is caused by neuronal loss, leading to the accumulation of aberrant neuritic plaques and the formation of neurofibrillary tangles. Oxidative stress is involved in the impaired clearance of amyloid beta (Aß), and Aß-induced oxidative stress causes AD by inducing the formation of neurofibrillary tangles. Hwangryunhaedok-tang (HHT, Kracie K-09®), a traditional herbal medicine prescription, has shown therapeutic effects on various diseases. However, the studies of HHT as a potential treatment for AD are insufficient. Therefore, our study identified the neurological effects and mechanisms of HHT and its key bioactive compounds against Alzheimer's disease in vivo and in vitro. In a 5xFAD mouse model, our study confirmed that HHT attenuated cognitive impairments in the Morris water maze (MWM) test and passive avoidance (PA) test. In addition, the prevention of neuron impairment, reduction in the protein levels of Aß, and inhibition of cell apoptosis were confirmed with brain tissue staining. In HT-22 cells, HHT attenuates tBHP-induced cytotoxicity, ROS generation, and mitochondrial dysfunction. It was verified that HHT exerts a neuroprotective effect by activating signaling pathways interacting with Nrf2, such as MAPK/ERK, PI3K/Akt, and LKB1/AMPK. Among the components, baicalein, a bioavailable compound of HHT, exhibited neuroprotective properties and activated the Akt, AMPK, and Nrf2/HO-1 pathways. Our findings indicate a mechanism for HHT and its major bioavailable compounds to treat and prevent AD and suggest its potential.

Application of 2D-ITC to the Elucidation of the Enzymatic Mechanism of N-Acetylmuramic Acid/N-Acetylglucosamine Kinase (AmgK) from Pseudomonas aeruginosa.

Characterization of the turnover mechanism of bisubstrate enzymes is a tedious task. Molecular tools for studying the enzymatic mechanism are not readily available for all enzymes (e.g., radioactive substrates, substrate-competitive inhibitors, etc.). Wang and Mittermaier recently introduced two-dimensional isothermal titration calorimetry (2D-ITC) for determining the bisubstrate mechanism at high resolution while simultaneously quantifying the kinetic parameters for substrate turnover in a single reporter-free experiment. We demonstrate the utility of 2D-ITC in studying N-acetylmuramic acid/N-acetylglucosamine kinase (AmgK) from Pseudomonas aeruginosa. This enzyme is involved in cytoplasmic cell-wall-recycling events as a step in the peptidoglycan salvage pathway. Furthermore, AmgK phosphorylates N-acetylglucosamine and N-acetylmuramic acid, linking the recycling events to de novo cell-wall synthesis. We document in a 2D-ITC experiment that AmgK follows an ordered-sequential mechanism, where ATP binds first and ADP is released last. We also show that classical enzyme kinetic methods support the results of 2D-ITC and that 2D-ITC could overcome the shortcomings of these classical methodologies. We provide evidence for inhibition of AmgK by the catalytic product ADP, but not by the phosphorylated sugar product. These results provide a full kinetic characterization of the bacterial kinase AmgK. This work highlights 2D-ITC as a versatile tool for the mechanistic evaluation of bisubstrate enzymes, as an alternative for classical methods.

A Double-Blind, Randomized, Placebo-Controlled, Phase II Clinical Study To Evaluate the Efficacy and Safety of Camostat Mesylate (DWJ1248) in Adult Patients with Mild to Moderate COVID-19.

Although several antiviral agents have become available for coronavirus disease 2019 (COVID-19) treatment, oral drugs are still limited. Camostat mesylate, an orally bioavailable serine protease inhibitor, has been used to treat chronic pancreatitis in South Korea, and it has an in vitro inhibitory potential against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This study was a double-blind, randomized, placebo-controlled, multicenter, phase 2 clinical trial in mild to moderate COVID-19 patients. We randomly assigned patients to receive either camostat mesylate (DWJ1248) or placebo orally for 14 days. The primary endpoint was time to clinical improvement of subject symptoms within 14 days, measured using a subjective 4-point Likert scale. Three hundred forty-two patients were randomized. The primary endpoint was nonsignificant, where the median times to clinical improvement were 7 and 8 days in the camostat mesylate group and the placebo group, respectively (hazard ratio [HR] = 1.09; 95% confidence interval [CI], 0.84 to 1.43; P = 0.50). A post hoc analysis showed that the difference was greatest at day 7, without reaching significance. In the high-risk group, the proportions of patients with clinical improvement up to 7 days were 45.8% (50/109) in the camostat group and 38.4% (40/104) in the placebo group (odds ratio [OR] = 1.33; 95% CI, 0.77 to 2.31; P = 0.31); the ordinal scale score at day 7 improved in 20.0% (18/90) of the camostat group and 13.3% (12/90) of the placebo group (OR = 1.68; 95% CI, 0.75 to 3.78; P = 0.21). Adverse events were similar in the two groups. Camostat mesylate was safe in the treatment of COVID-19. Although this study did not show clinical benefit in patients with mild to moderate COVID-19, further clinical studies for high-risk patients are needed. (This trial was registered with ClinicalTrials.gov under registration no. NCT04521296).

Synthesis of Muramyl-δ-Lactam in Spore Peptidoglycan of Clostridioides difficile.

Clostridioides difficile is a spore-forming human pathogen responsible for significant morbidity and mortality. Infections by this pathogen ensue dysbiosis of the intestinal tract, which leads to germination of the spores. The process of spore formation requires a transition for the cell-wall peptidoglycan of the vegetative C. difficile to that of spores, which entails the formation of muramyl-δ-lactam. We describe a set of reactions for three recombinant C. difficile proteins, GerS, CwlD, and PdaA1, with the use of four synthetic peptidoglycan analogs. CwlD and PdaA1 excise the peptidoglycan stem peptide and the acetyl moiety of N-acetyl muramate, respectively. The reaction of CwlD is accelerated in the presence of GerS. With the use of a suitable substrate, we document that PdaA1 catalyzes a novel zinc-dependent transamidation/transpeptidation reaction, an unusual reaction that requires excision of the stem peptide as a pre-requisite.

Protective Role of Ethanol Extract of Cibotium barometz (Cibotium Rhizome) against Dexamethasone-Induced Muscle Atrophy in C2C12 Myotubes.

Sarcopenia is a progressive muscle disease characterized by the loss of skeletal muscle mass, strength, function, and physical performance. Since the disease code was assigned, attention has been focused on natural products that can protect against muscle atrophy. Cibotium barometz (Cibotium Rhizome) has been used as an herbal medicine for the treatment of bone or joint diseases in Asian countries. However, no studies have identified the mechanism of action of Cibotium Rhizome on muscle atrophy related to sarcopenia at the site of myotubes. The aim of this study was to investigate the improvement effect of the ethanol extract of Cibotium Rhizome (ECR) on dexamethasone-induced muscle atrophy in an in vitro cell model, i.e., the C2C12 myotubes. High-performance liquid chromatography was performed to examine the phytochemicals in ECR. Seven peaks in the ECR were identified, corresponding to the following compounds: protocatechuic acid, (+)-catechin hydrate, p-coumaric acid, ellagic acid, chlorogenic acid, caffeic acid, and ferulic acid. In atrophy-like conditions induced by 100 µM dexamethasone for 24 h in C2C12, ECR increased the expression of the myosin heavy chain, p-Akt, the p-mammalian target of rapamycin (mTOR), p-p70S6K, and repressed the expression of regulated in development and DNA damage responses 1 (REDD1), kruppel-like factor 15 (KLF 15), muscle atrophy F-box, and muscle-specific RING finger protein-1 in C2C12. In addition, ECR alleviated dexamethasone-induced muscle atrophy by repressing REDD1 and KLF15 transcription in C2C12 myotubes, indicating the need for further studies to provide a scientific basis for the development of useful therapeutic agents using ECR to alleviate the effects of skeletal muscle atrophy or sarcopenia.

Evaluation of the Diagnostic Accuracy of Antibody Assays for Patients with Scrub Typhus.

This study was carried out to evaluate the accuracy of various antibody tests for scrub typhus, namely, the indirect immunofluorescence assay (IFA) from the Korea Centers for Disease Control and Prevention (KCDC) and four commercial kits (companies A to D). The test accuracy was based on the diagnosis of scrub typhus, as defined by a positive PCR or culture. In total, serum samples from 97 patients with scrub typhus and 200 non-scrub typhus patients were tested. The respective sensitivity and specificity of each test were as follows. For the KCDC IFA, sensitivity and specificity were 55.7% (95% confidence interval [CI], 45.2 to 65.8%) and 94.8% (95% CI, 90.4 to 97.3%) for IgM and 42.3% (95% CI, 32.3 to 52.7%) and 96.3% (95% CI, 92.6 to 98.5%) for IgG, with diagnostic cutoffs of ≥1:16 for IgM and ≥1:256 for IgG. For kit A, the sensitivity and specificity were 70.1% (95% CI, 59.8 to 78.8%) and 74.6% (95% CI, 67.6 to 80.6%) for total immunoglobulins, with a cutoff of ≥1:40. For kit B, the sensitivity and specificity were 64.3% (95% CI, 51.9 to 75.1%) and 94.9% (95% CI, 81.4 to 99.1%) for IgM and 67.1% (95% CI, 54.8 to 77.6%) and 74.4% (95% CI, 57.6 to 86.4%) for IgG. For kit C, the sensitivity and specificity were 53.6% (95% CI, 43.2 to 63.7%) and 99.5% (95% CI, 96.8 to 100%) for IgM and 36.1% (95% CI, 26.8 to 46.5%) and 100% (95% CI, 97.6 to 100%) for IgG. For kit D, the sensitivity and specificity were 73.2% (95% CI, 63.1 to 81.4%) and 89.5% (95% CI, 84.2 to 93.2%) for total immunoglobulins. These results are all unsatisfactory, highlighting an urgent need for the development of more highly sensitive and specific tests.

Molecular detection and identification of Culex flavivirus in mosquito species from Jeju, Republic of Korea.

BACKGROUND: Mosquito-borne flaviviruses are prime pathogens and have been a major hazard to humans and animals. They comprise several arthropod-borne viruses, including dengue virus, yellow fever virus, Japanese encephalitis virus, and West Nile virus. Culex flavivirus (CxFV) is a member of the insect-specific flavivirus (ISF) group belonging to the genus Flavivirus, which is widely distributed in a variety of mosquito populations. METHODS: Viral nucleic acid was extracted from adult mosquito pools and subjected to reverse transcriptase nested polymerase chain reaction (PCR) using target-specific primers for detecting CxFV nonstructural protein 5 (NS5). The PCR-positive samples were then sequenced, and a phylogenetic tree was constructed, including reference sequences obtained from GenBank. RESULTS: 21 pools, belonging to Culex pipiens pallens (Cx. p. pallens) were found to be positive for the CxFV RNA sequence, with a minimum infection rate of 14.5/1000 mosquitoes. The phylogenetic analysis of the NS5 protein sequences indicated that the detected sequences were closely related to strains identified in China, with 95-98% sequence similarities. CONCLUSION: Our findings highlight the presence of CxFV in Cx. p. pallens mosquito species in Jeju province, Republic of Korea. This is the first study reporting the prevalence of CxFV in Culex Pipiens (Cx. pipiens) host in the Jeju province, which can create possible interaction with other flaviviruses causing human and animal diseases. Although, mosquito-borne disease causing viruses were not identified properly, more detailed surveillance and investigation of both the host and viruses are essential to understand the prevalence, evolutionary relationship and genetic characteristic with other species.

Pharmacokinetics and safety of evogliptin in hepatically impaired patients.

AIMS: Evogliptin is a potent and selective dipeptidyl peptidase-4 inhibitor for glycaemic control in patients with type 2 diabetes mellitus. Since evogliptin is mainly eliminated through hepatic metabolism, we investigated the pharmacokinetics (PKs) and safety characteristics of evogliptin in Korean patients with mild or moderate hepatic impairment. METHODS: An open-label, parallel-group study was conducted in patients with mild or moderate hepatic impairment and healthy control subjects matched to each patient for sex, age and body mass index. A single dose (5 mg) of evogliptin was administered orally, and serial blood samples were collected over 120 h to assess the PK profile of evogliptin and its main metabolites (M7 and M8). RESULTS: Patients with mild hepatic impairment and their matched healthy controls showed similar maximum concentration (Cmax ) and area under the concentration-time curve values from 0 to 120 h (AUClast ); the geometric mean ratio (GMR) and 90% confidence interval (CI) were 1.04 (0.80, 1.35) and 1.01 (0.90, 1.14), respectively. Exposure to evogliptin (Cmax and AUClast ) was increased by about 40% in patients with moderate hepatic impairment-the GMR and 90% CI were 1.37 (1.09, 1.72) and 1.44 (1.18, 1.75), respectively. The metabolic ratios of M7 and M8 were lower in patients with moderate hepatic impairment than in matched healthy controls. Evogliptin was well tolerated by both patients and healthy subjects. CONCLUSION: Although evogliptin exposure was increased in patients with moderate hepatic impairment, the increase is unlikely to affect safety and efficacy adversely, and no dose adjustment is warranted.

Performance assessment of ASTA MicroIDSys, a new matrix assisted laser desorption ionization-time of flight mass spectrometry system, for identification of viridans group streptococci.

The performance of the ASTA MicroIDSys system (ASTA), a new matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) system, was evaluated for the identification of viridans group streptococci (VGS) and compared with the results obtained with the Bruker Biotyper system (Bruker Daltonics). A total of 106 Streptococcus reference strains belonging to 24 species from the bacterial strain bank was analyzed using the two MALDI-TOF MS systems. Of the 106 reference strains tested, ASTA MicroIDSys and Bruker Biotyper correctly identified 84.9% and 81.1% at the species level, 100% and 97.2% at the group level and 100% and 98.1% at the genus level, respectively. The difference between the two systems was not statistically significant (P = 0.289). Out of 24 species, 13 species were accurately identified to the species level with 100% accurate identification rates with both systems. The accurate identification rates at the species level of ASTA MicroIDSys and Bruker Biotyper were 100% and 87.5% for the S. anginosus group; 78.4% and 73.5% for the S. mitis group; 91.7% and 91.7% for the S. mutans group; and 100% and 100% for the S. salivarius group, respectively. The ASTA MicroIDSys showed an identification performance equivalent to that of the Bruker Biotyper for VGS. Therefore, it would be useful for the identification of VGS strains in clinical microbiology laboratories.

Follow-up investigation of antibody titers and diagnostic antibody cutoff values in patients with scrub typhus in South Korea.

BACKGROUND: Scrub typhus is a mite-borne infectious disease caused by Orientia tsutsugamushi. Few follow-up studies have assessed antibody titers using serologic tests from various commercial laboratories and the Korea Centers for Disease Control and Prevention (KCDC). METHODS: A prospective study to assess the antibody titers in patients with scrub typhus and seroprevalence in individuals undergoing health checkups was conducted using results of immunofluorescence antibody assays (IFAs) and serologic tests, used by the KCDC and commercial laboratories, respectively. The following tests were performed simultaneously: (i) indirect IFA used by the KCDC to detect immunoglobulin (Ig) M and IgG, (ii) IFA used by a commercial laboratory to detect total Ig, and (iii) antibody tests using two commercially available kits. RESULTS: When the IgM and IgG cutoff values (≥1:16 and ≥1:256, respectively) used in the IFA and the total IgG cutoff values (≥1:40) were used in prospective follow-up investigations, the antibody positivity rates of 102 patients with scrub typhus were 44.1, 35.3, and 57.6%, respectively, within 5 days of symptom onset. Among 91 individuals who recovered from scrub typhus, the follow-up IgM, IgG, and total Ig positivity rates for 13 years were 37.4% (34/91), 22.0% (20/91), and 76.9% (70/91), respectively. Among 216 individuals undergoing health checkups, the seroprevalence of IgM was 4.2% (9/216); no seroprevalence of IgG was observed. CONCLUSIONS: IFAs used by the KCDC and the commercial laboratory and rapid commercial kits could not distinguish between patients who had recovered from scrub typhus and those who are currently infected with O. tsutsugamushi. In South Korea and other countries, where low antibody cutoff values are used, upward adjustments of cutoff values may be necessary.

A case of SFTS coinfected with E. coli bacteremia.

BACKGROUND: Severe fever thrombocytopenia syndrome virus (SFTSV) is the causative agent of severe fever thrombocytopenia syndrome (SFTS). SFTS is an emerging infectious disease, characterized by high fever, gastrointestinal symptoms, leukopenia, thrombocytopenia, and a high mortality rate. Until now, little importance has been given to the association of SFTS with leukocytosis and bacterial co-infection. CASE PRESENTATION: A 51-year old man visited our hospital with fever and low blood pressure. He was a farmer by occupation and often worked outdoors. He had a Foley catheter inserted due to severe BPH. Laboratory tests revealed thrombocytopenia, elevated liver function, and elevated CRP levels. He had marked leukocytosis, proteinuria, hematuria, and conjunctival hemorrhage. Initially, we thought that the patient was suffering from hemorrhagic fever with renal syndrome (HFRS). However, we confirmed SFTS through PCR and increasing antibody titer. However, his blood culture also indicated E. coli infection. CONCLUSION: SFTS displays characteristics of fever, thrombocytopenia, elevated liver function, and leukocytopenia. We described a case of SFTS with leukocytosis due to coinfection with E. coli. Since patients with SFTS usually have leukocytopenia, SFTS patients with leukocytosis are necessarily evaluated for other causes of leukocytosis. Here, we report the first case of an SFTS with concurrent E. coli bacteremia.

The Effect of Long-lasting Permethrin Impregnated Socks on Tick Bite in Korea.

BACKGROUND: The risk of tick-borne diseases is decreased by increasing awareness and knowledge through prevention education. The aim of the present study was to evaluate the effect of long-lasting permethrin impregnated (LLPI) socks for tick bites. METHODS: A randomized open label study was conducted to determine the effectiveness of LLPI socks for prevention of tick bites among 367 adults living in a rural area. Participants completed questionnaires at the start of follow-up (July 2014) and at the end of follow-up (December 2014), and tick bites were reported. RESULTS: A total of 332 subjects completed the follow-up survey. The tick bite rate of the two groups was not significantly different (3.6% vs. 3.1%). But the tick bite rate of lower extremities of subjects wearing LLPI socks was significantly lower compared to that of subjects wearing general socks. CONCLUSION: The tick bite rate was not different between the two groups, but the tick bite rate of lower extremities of LLPI was significantly lower than general groups. Further study is needed to investigate the effect of LLPI clothes with larger populations.

Asymptomatic-anaplasmosis confirmation using genetic and serological tests and possible coinfection with spotted fever group Rickettsia: a case report.

BACKGROUND: Anaplasmosis is an emerging acute febrile disease that is caused by a bite of an Anaplasma phagocytophilum-infected hard tick. As for healthy patients, reports on asymptomatic anaplasmosis resulting from such tick bites are rare. CASE PRESENTATION: A 55-year-old female patient visited the hospital with a tick bite in the right infraclavicular region. The tick was suspected to have been on the patient for more than 10 days. PCR and an indirect immunofluorescence assay (IFA) were performed to identify tick-borne infectious diseases. The blood sample collected at admission yielded a positive result in nested PCR targeting Ehrlichia- or Anaplasma-specific genes groEL and ankA. Subsequent sequencing confirmed the presence of A. phagocytophilum, and seroconversion was confirmed by the IFA involving an A. phagocytophilum antigen slide. PCR detected no Rickettsia-specific genes [outer membrane protein A (ompA) or surface cell antigen 1 (sca1)], but seroconversion of spotted fever group (SFG) rickettsiosis was confirmed by an IFA. CONCLUSIONS: This study genetically and serologically confirmed an asymptomatic A. phagocytophilum infection. Although SFG rickettsiosis was not detected genetically, it was detected serologically. These findings indicate the possibility of an asymptomatic coinfection: anaplasmosis plus SFG rickettsiosis. It is, therefore, crucial for clinicians to be aware of potential asymptomatic anaplasmosis following a tick bite.

First identification of Anaplasma phagocytophilum in both a biting tick Ixodes nipponensis and a patient in Korea: a case report.

BACKGROUND: Human granulocytic anaplasmosis (HGA) is a tick-borne infectious disease caused by Anaplasma phagocytophilum. To date, there have been no reported cases of A. phagocytophilum infection found in both the biting tick and the patient following a tick bite. CASE PRESENTATION: An 81-year-old woman presented with fever following a tick bite, with the tick still intact on her body. The patient was diagnosed with HGA. The tick was identified as Ixodes nipponensis by morphological and molecular biological detection methods targeting the 16S rRNA gene. The patient's blood was cultured after inoculation into the human promyelocytic leukemia cell line HL-60. A. phagocytophilum growth was confirmed via culture and isolation. A. phagocytophilum was identified in both the tick and the patient's blood by Anaplasma-specific groEL- and ankA-based nested polymerase chain reaction followed by sequencing. Moreover, a four-fold elevation in antibodies was observed in the patient's blood. CONCLUSION: We report a case of a patient diagnosed with HGA following admission for fever due to a tick bite. A. phagocytophilum was identified in both the tick and the patient, and A. phagocytophilum was successfully cultured. The present study suggests the need to investigate the possible incrimination of I. nipponensis as a vector for HGA in Korea.

Scalp eschar and neck lymphadenopathy after tick bite (SENLAT) caused by Bartonella henselae in Korea: a case report.

BACKGROUND: Tick-borne lymphadenopathy (TIBOLA) is an infectious disease, mainly caused by species from the spotted fever group rickettsiae and is characterized by enlarged lymph nodes following a tick bite. Among cases of TIBOLA, a case of scalp eschar and neck lymphadenopathy after tick bite (SENLAT) is diagnosed when an eschar is present on the scalp, accompanied by peripheral lymphadenopathy (LAP). Only a few cases of SENLAT caused by Bartonella henselae have been reported. CASE PRESENTATION: A 58-year-old male sought medical advice while suffering from high fever and diarrhea. Three weeks before the visit, he had been hunting a water deer, and upon bringing the deer home discovered a tick on his scalp area. Symptoms occurred one week after hunting, and a lump was palpated on the right neck area 6 days after the onset of symptoms. Physical examination upon presentation confirmed an eschar-like lesion on the right scalp area, and cervical palpation revealed that the lymph nodes on the right side were non-painful and enlarged at 2.5 × 1.5 cm. Fine needle aspiration of the enlarged lymph nodes was performed, and results of nested PCR for the Bartonella internal transcribed spacer (ITS) confirmed B. henselae as the causative agent. CONCLUSION: With an isolated case of SENLAT and a confirmation of B. henselae in Korea, it is pertinent to raise awareness to physicians in other Asian countries that B. henselae could be a causative agent for SENLAT.

Usefulness of Nested Polymerase Chain Reaction with Clinical Specimens for Diagnosis of Leptospirosis: a Case Series and a Review of Literature.

A culture of the Leptospira species and the microscopic agglutination test (MAT) are considered as the reference standard for the diagnosis of leptospirosis, but both tests are imperfect for early diagnosis. We describe 4 patients diagnosed with leptospirosis using nested polymerase chain reaction (N-PCR) that targeted the 16S rRNA gene and the passive hemagglutination assay (PHA). In our 4 cases, Leptospira DNA in the urine, plasma, or cerebrospinal fluid (CSF), was detected by N-PCR in the early phase of leptospirosis, except in the sample from the buffy coat. Especially, case 3 showed that N-PCR with the urine and CSF was positive 8 days after symptom onset, but not for the plasma or buffy coat. We report 4 cases of leptospirosis that were diagnosed by N-PCR that targeted the 16S rRNA gene with urine, plasma, or CSF, but not the buffy coat. Three were cured by doxycycline but the case 4 was fatal. Detection of Leptospira DNA by PCR from the urine and CSF, in addition to plasma, may be helpful to confirm the diagnosis.

Recent Trends in Controlling the Enzymatic Browning of Fruit and Vegetable Products.

Enzymatic browning because of polyphenol oxidases (PPOs) contributes to the color quality of fruit and vegetable (FV) products. Physical and chemical methods have been developed to inhibit the activity of PPOs, and several synthetic chemical compounds are commonly being used as PPO inhibitors in FV products. Recently, there has been an emphasis on consumer-oriented innovations in the food industry. Consumers tend to urge the use of natural and environment-friendly PPO inhibitors. The purpose of this review is to summarize the mechanisms underlying the anti-browning action of chemical PPO inhibitors and current trends in the research on these inhibitors. Based on their mechanisms of action, chemical inhibitors can be categorized as antioxidants, reducing agents, chelating agents, acidulants, and/or mixed-type PPO inhibitors. Here, we focused on the food ingredients, dietary components, food by-products, and waste associated with anti-browning activity.