Sirtinol Supresses Trophozoites Proliferation and Encystation of Acanthamoeba via Inhibition of Sirtuin Family Protein

The encystation of Acanthamoeba leads to the development of metabolically inactive and dormant cysts from vegetative trophozoites under unfavorable conditions. These cysts are highly resistant to anti-Acanthamoeba drugs and biocides. Therefore, the inhibition of encystation would be more effective in treating Acanthamoeba infection. In our previous study, a sirtuin family protein—Acanthamoeba silent-information regulator 2-like protein (AcSir2)—was identified, and its expression was discovered to be critical for Acanthamoeba castellanii proliferation and encystation. In this study, to develop Acanthamoeba sirtuin inhibitors, we examine the effects of sirtinol, a sirtuin inhibitor, on trophozoite growth and encystation. Sirtinol inhibited A. castellanii trophozoites proliferation (IC50=61.24 μM). The encystation rate of cells treated with sirtinol significantly decreased to 39.8% (200 μM sirtinol) after 24 hr of incubation compared to controls. In AcSir2-overexpressing cells, the transcriptional level of cyst-specific cysteine protease (CSCP), an Acanthamoeba cysteine protease involved in the encysting process, was 11.6- and 88.6-fold higher at 48 and 72 hr after induction of encystation compared to control. However, sirtinol suppresses CSCP transcription, resulting that the undegraded organelles and large molecules remained in sirtinol-treated cells during encystation. These results indicated that sirtinol sufficiently inhibited trophozoite proliferation and encystation, and can be used to treat Acanthamoeba infections.

Characterization of a Peptide Antibody Specific to the Adenylyl Cyclase-Associated Protein of Acanthamoeba castellanii

Acanthamoeba keratitis (AK) is a rare infectious disease and accurate diagnosis has remained arduous as clinical manifestations of AK were similar to keratitis of viral, bacterial, or fungal origins. In this study, we described the production of a polyclonal peptide antibody against the adenylyl cyclase-associated protein (ACAP) of A. castellanii, and evaluated its differential diagnostic potential. Enzyme-linked immunosorbent assay revealed high titers of A. castellanii-specific IgG and IgA antibodies being present in low dilutions of immunized rabbit serum. Western blot analysis revealed that the ACAP antibody specifically interacted with A. castellanii, while not interacting with human corneal epithelial (HCE) cells and other causes of keratitis such as Fusarium solani, Pseudomonas aeruginosa, and Staphylococcus aureus. Immunocytochemistry (ICC) results confirmed the specific detection of trophozoites and cysts of A. castellanii co-cultured with HCE cells. The ACAP antibody also specifically interacted with the trophozoites and cysts of 5 other Acanthamoeba species. These results indicate that the ACAP antibody of A. castellanii can specifically detect multiple AK-causing members belonging to the genus Acanthamoeba and may be useful for differentially diagnosing Acanthamoeba infections.

Specific Detection of Acanthamoeba species using Polyclonal Peptide Antibody Targeting the Periplasmic Binding Protein of A. castellanii

Acanthamoeba keratitis (AK) is a rare ocular disease, but it is a painful and sight-threatening infectious disease. Early diagnosis and adequate treatment are necessary to prevent serious complications. While AK is frequently diagnosis via several PCR assays or Acanthamoeba-specific antibodies, a more specific and effective diagnostic method is required. This study described the production of a polyclonal peptide antibody against the periplasmic binding protein (PBP) of A. castellanii and investigated its diagnostic potential. Western blot analysis showed that the PBP antibody specifically reacted with the cell lysates of A. castellanii. However, the PBP antibody did not interact with human corneal epithelial (HCE) cells and the other 3 major causative agents of keratitis. Immunocytochemistry (ICC) results revealed the specific detection of A. castellanii trophozoites and cysts by PBP antibodies when A. castellanii were co-cultured with HCE cells. PBP antibody specificity was further confirmed by co-culture of A. castellanii trophozoites with F. solani, S. aureus, and P. aeruginosa via ICC. The PBP antibody specifically reacted with the trophozoites and cysts of A. polyphaga, A. hatchetti, A. culbertsoni, A. royreba, and A. healyi, thus demonstrated its genus-specific nature. These results showed that the PBP polyclonal peptide antibody of A. castellanii could specifically detect several species of Acanthamoeba, contributing to the development of an effective antibody-based AK diagnostics.

Differentially Expressed Gene Profile of Acanthamoeba castellanii Induced by an Endosymbiont Legionella pneumophila

Legionella pneumophila is an opportunistic pathogen that survives and proliferates within protists such as Acanthamoeba spp. in environment. However, intracellular pathogenic endosymbiosis and its implications within Acanthamoeba spp. remain poorly understood. In this study, RNA sequencing analysis was used to investigate transcriptional changes in A. castellanii in response to L. pneumophila infection. Based on RNA sequencing data, we identified 1,211 upregulated genes and 1,131 downregulated genes in A. castellanii infected with L. pneumophila for 12 hr. After 24 hr, 1,321 upregulated genes and 1,379 downregulated genes were identified. Gene ontology (GO) analysis revealed that L. pneumophila endosymbiosis enhanced hydrolase activity, catalytic activity, and DNA binding while reducing oxidoreductase activity in the molecular function (MF) domain. In particular, multiple genes associated with the GO term ‘integral component of membrane’ were downregulated during endosymbiosis. The endosymbiont also induced differential expression of various methyltransferases and acetyltransferases in A. castellanii. Findings herein are may significantly contribute to understanding endosymbiosis of L. pneumophila within A. castellanii.

Exploring medical educators’ readiness and the priority of their educational needs for online teaching / 한국의학교육

Purpose@#This study investigated medical educators’ readiness for online teaching by exploring their perceived ability and importance of online teaching competencies and identified the highest priority of their educational needs. @*Methods@#In this study, 144 medical education faculty members from a university were invited to participate. The faculty online teaching readiness scale was virtually distributed at the end of the spring semester of 2020 and 38 faculty members responded for 2 weeks. The collected data were analyzed with descriptive statistics, paired t-tests, Borich Needs Assessment, and the Locus for Focus model. @*Results@#The overall average perceived ability was 2.76, while the overall average perceived importance was 3.36. The course design and the technical competency categories showed the highest and lowest educational needs, respectively. Five competencies were given the highest priority of educational needs. @*Conclusion@#The results revealed that the medical educators are not ready for online teaching; thus, urgent educational needs for online teaching competencies exist.

Clinical performance of medical students in Korea in a whole-task emergency station in the objective structured clinical examination with a standardized patient complaining of palpitations / 보건의료교육평가

This study assessed the clinical performance of 150 third-year medicalstudents in Busan, Korea in a whole-task emergency objective structured clinical examination station that simulated a patient with palpitations visiting the emergency department. The examination was conducted from November 25 to 27, 2019. Clinical performance was assessed as the number and percentage of students who performed history-taking (HT), a physical examination (PE), an electrocardiography (ECG) study, patient education (Ed), and clinical reasoning (CR), which were items on the checklist. It was found that 18.0% of students checked the patient’s pulse, 51.3% completed an ECG study, and 57.9% explained the results to the patient. A sizable proportion (38.0%) of students did not even attempt an ECG study. In a whole-task emergency station, students showed good performance on HT and CR, but unsatisfactory results for PE, ECG study, and Ed. Clinical skills educational programs for subjected student should focus more on PE, timely diagnostic tests, and sufficient Ed.

Clinical performance of medical students in Korea in a whole-task emergency station in the objective structured clinical examination with a standardized patient complaining of palpitations / 보건의료교육평가

This study assessed the clinical performance of 150 third-year medicalstudents in Busan, Korea in a whole-task emergency objective structured clinical examination station that simulated a patient with palpitations visiting the emergency department. The examination was conducted from November 25 to 27, 2019. Clinical performance was assessed as the number and percentage of students who performed history-taking (HT), a physical examination (PE), an electrocardiography (ECG) study, patient education (Ed), and clinical reasoning (CR), which were items on the checklist. It was found that 18.0% of students checked the patient’s pulse, 51.3% completed an ECG study, and 57.9% explained the results to the patient. A sizable proportion (38.0%) of students did not even attempt an ECG study. In a whole-task emergency station, students showed good performance on HT and CR, but unsatisfactory results for PE, ECG study, and Ed. Clinical skills educational programs for subjected student should focus more on PE, timely diagnostic tests, and sufficient Ed.

Comparison of Proteins Secreted into Extracellular Space of Pathogenic and Non-pathogenic Acanthamoeba castellanii

Pathogenic Acanthamoeba spp. cause granulomatous amoebic encephalitis and keratitis. Acanthamoeba keratitis (AK) is a rare but serious ocular infection that can result in permanent visual impairment or blindness. However, pathogenic factors of AK remain unclear and treatment for AK is arduous. Expression levels of proteins secreted into extracellular space were compared between A. castellanii pathogenic (ACP) and non-pathogenic strains. Two-dimensional polyacrylamide gel electrophoresis revealed 123 differentially expressed proteins, including 34 increased proteins, 7 qualitative increased proteins, 65 decreased proteins, and 17 qualitative decreased proteins in ACP strain. Twenty protein spots with greater than 5-fold increase in ACP strain were analyzed by liquid chromatography triple quadrupole mass spectrometry. These proteins showed similarity each to inosine-uridine preferring nucleoside hydrolase, carboxylesterase, oxygen-dependent choline dehydrogenase, periplasmic-binding protein proteinases and hypothetical proteins. These proteins expressed higher in ACP may provide some information to understand pathogenicity of Acanthamoeba.

Effect of 2, 6-Dichlorobenzonitrile on Amoebicidal Activity of Multipurpose Contact Lens Disinfecting Solutions

Multipurpose contact lens disinfecting solutions (MPDS) are widely used to cleanse and disinfect microorganisms. However, disinfection efficacy of these MPDS against Acanthamoeba cyst remain insufficient. 2, 6-dichlorobenzonitrile (DCB), a cellulose synthesis inhibitor, is capable of increasing the amoebical effect against Acanthamoeba by inhibiting its encystation. In this study, we investigated the possibility of DCB as a disinfecting agent to improve the amoebicidal activity of MPDS against Acanthamoeba cyst. Eight commercial MPDS (from a to h) were assessed, all of which displayed insufficient amoebicidal activity against the mature cysts. Solution e, f, and h showed strong amoebicidal effect on the immature cysts. Amoebicidal efficacy against mature cysts remained inadequate even when the 8 MPDS were combined with 100 μM DCB. However, 4 kinds of MPDS (solution d, e, f, and h) including 100 μM DCB demonstrated strong amoebicidal activity against the immature cysts. The amoebicidal activity of solution d was increased by addition of DCB. Cytotoxicity was absent in human corneal epithelial cells treated with either DCB or mixture of DCB with MPDS. These results suggested that DCB can enhance the amoebicical activity of MPDS against Acanthamoeba immature cyst in vitro.

Authenticity, acceptability, and feasibility of a hybrid gynecology station for the Papanicolaou test as part of a clinical skills examination in Korea / 보건의료교육평가

PURPOSE: The objective of this study was to evaluate the authenticity, acceptability, and feasibility of a hybrid station that combined a standardized patient encounter and a simulated Papanicolaou test. METHODS: We introduced a hybrid station in the routine clinical skills examination (CSE) for 335 third-year medical students at 4 universities in Korea from December 1 to December 3, 2014. After the tests, we conducted an anonymous survey on the authenticity, acceptability, and feasibility of the hybrid station. RESULTS: A total of 334 medical students and 17 professors completed the survey. A majority of the students (71.6%) and professors (82.4%) agreed that the hybrid station was more authentic than the standard CSE. Over 60 percent of the students and professors responded that the station was acceptable for assessing the students' competence. Most of the students (75.2%) and professors (82.4%) assessed the required tasks as being feasible after reading the instructions. CONCLUSION: Our results showed that the hybrid CSE station was a highly authentic, acceptable, and feasible way to assess medical students' performance.

Medical students' clinical performance of dealing with patients in the context of domestic violence / 한국의학교육

PURPOSE: The aim of this study was to inquire about the clinical performance and determine the performance pattern of medical students in standardized patient (SP) based examinations of domestic violence (DV). METHODS: The clinical performance sores in DV station with SP of third-year (n=111, in 2014) and 4th-year (n=143, in 2016) medical students of five universities in the Busan-Gyeongnam Clinical Skills Examination Consortium were subjected in this study. The scenarios and checklists of DV cases were developed by the case development committee of the consortium. The students’ performance was compared with other stations encountered in SP. The items of the checklists were categorized to determine the performance pattern of students investigating DV into six domains: disclosure strategy (D), DV related history taking (H), checking the perpetrator’s psychosocial state (P), checking the victim’s condition (V), negotiating and persuading the interviewee (N), and providing information about DV (I). RESULTS: Medical students showed poorer performance in DV stations than in the other stations with SP in the same examination. Most students did confirm the perpetrator and commented on confidentiality but ignored the perpetrator’s state and patient’s physical and psychological condition. The students performed well in the domains of D, H, and I but performed poorly in domains P, V, and N. CONCLUSION: Medical students showed poor clinical performance in the DV station. They performed an ‘event oriented interview’ rather than ‘patient centered’ communication. An integrated educational program of DV should be set to improve students’ clinical performance.

Authenticity, acceptability, and feasibility of a hybrid gynecology station for the Papanicolaou test as part of a clinical skills examination in Korea / 보건의료교육평가

PURPOSE@#The objective of this study was to evaluate the authenticity, acceptability, and feasibility of a hybrid station that combined a standardized patient encounter and a simulated Papanicolaou test.@*METHODS@#We introduced a hybrid station in the routine clinical skills examination (CSE) for 335 third-year medical students at 4 universities in Korea from December 1 to December 3, 2014. After the tests, we conducted an anonymous survey on the authenticity, acceptability, and feasibility of the hybrid station.@*RESULTS@#A total of 334 medical students and 17 professors completed the survey. A majority of the students (71.6%) and professors (82.4%) agreed that the hybrid station was more authentic than the standard CSE. Over 60 percent of the students and professors responded that the station was acceptable for assessing the students' competence. Most of the students (75.2%) and professors (82.4%) assessed the required tasks as being feasible after reading the instructions.@*CONCLUSION@#Our results showed that the hybrid CSE station was a highly authentic, acceptable, and feasible way to assess medical students' performance.

Identification and Characterization of Protein Arginine Methyltransferase 1 in Acanthamoeba castellanii

Protein arginine methyltransferase (PRMT) is an important epigenetic regulator in eukaryotic cells. During encystation, an essential process for Acanthamoeba survival, the expression of a lot of genes involved in the encystation process has to be regulated in order to be induced or inhibited. However, the regulation mechanism of these genes is yet unknown. In this study, the full-length 1,059 bp cDNA sequence of Acanthamoeba castellanii PRMT1 (AcPRMT1) was cloned for the first time. The AcPRMT1 protein comprised of 352 amino acids with a SAM-dependent methyltransferase PRMT-type domain. The expression level of AcPRMT1 was highly increased during encystation of A. castellanii. The EGFP-AcPRMT1 fusion protein was distributed over the cytoplasm, but it was mainly localized in the nucleus of Acanthamoeba. Knock down of AcPRMT1 by synthetic siRNA with a complementary sequence failed to form mature cysts. These findings suggested that AcPRMT1 plays a critical role in the regulation of encystation of A. castellanii. The target gene of AcPRMT1 regulation and the detailed mechanisms need to be investigated by further studies.

DNA Methylation of Gene Expression in Acanthamoeba castellanii Encystation

Encystation mediating cyst specific cysteine proteinase (CSCP) of Acanthamoeba castellanii is expressed remarkably during encystation. However, the molecular mechanism involved in the regulation of CSCP gene expression remains unclear. In this study, we focused on epigenetic regulation of gene expression during encystation of Acanthamoeba. To evaluate methylation as a potential mechanism involved in the regulation of CSCP expression, we first investigated the correlation between promoter methylation status of CSCP gene and its expression. A 2,878 bp of promoter sequence of CSCP gene was amplified by PCR. Three CpG islands (island 1–3) were detected in this sequence using bioinformatics tools. Methylation of CpG island in trophozoites and cysts was measured by bisulfite sequence PCR. CSCP promoter methylation of CpG island 1 (1,633 bp) was found in 8.2% of trophozoites and 7.3% of cysts. Methylation of CpG island 2 (625 bp) was observed in 4.2% of trophozoites and 5.8% of cysts. Methylation of CpG island 3 (367 bp) in trophozoites and cysts was both 3.6%. These results suggest that DNA methylation system is present in CSCP gene expression of Acanthamoeba. In addition, the expression of encystation mediating CSCP is correlated with promoter CpG island 1 hypomethylation.

A pilot study on the evaluation of medical student documentation: assessment of SOAP notes / 한국의학교육

PURPOSE: The purpose of this study was evaluation of the current status of medical students' documentation of patient medical records METHODS: We checked the completeness, appropriateness, and accuracy of 95 Subjective-Objective-Assessment-Plan (SOAP) notes documented by third-year medical students who participated in clinical skill tests on December 1, 2014. Students were required to complete the SOAP note within 15 minutes of an standard patient (SP)-encounter with a SP complaining rhinorrhea and warring about meningitis. RESULTS: Of the 95 SOAP notes reviewed, 36.8% were not signed. Only 27.4% documented the patient's symptoms under the Objective component, although all students completed the Subjective notes appropriately. A possible diagnosis was assessed by 94.7% students. Plans were described in 94.7% of the SOAP notes. Over half the students planned workups (56.7%) for diagnosis and treatment (52.6%). Accurate documentation of the symptoms, physical findings, diagnoses, and plans were provided in 78.9%, 9.5%, 62.1%, and 38.0% notes, respectively. CONCLUSION: Our results showed that third-year medical students' SOAP notes were not complete, appropriate, or accurate. The most significant problems with completeness were the omission of students' signatures, and inappropriate documentation of the physical examinations conducted. An education and assessment program for complete and accurate medical recording has to be developed.

Prevalence of Trichomonas vaginalis in Women Visiting 2 Obstetrics and Gynecology Clinics in Daegu, South Korea

This study explored epidemiological trends in trichomoniasis in Daegu, South Korea. Wet mount microscopy, PCR, and multiplex PCR were used to test for Trichomonas vaginalis in vaginal swab samples obtained from 621 women visiting 2 clinics in Daegu. Of the 621 women tested, microscopy detected T. vaginalis in 4 (0.6%) patients, PCR detected T. vaginalis in 19 (3.0%) patients, and multiplex PCR detected T. vaginalis in 12 (1.9%) patients. Testing via PCR demonstrated high sensitivity and high negative predictive value for T. vaginalis. Among the 19 women who tested positive for T. vaginalis according to PCR, 94.7% (18/19) reported vaginal signs and symptoms. Notably, more than 50% of T. vaginalis infections occurred in females younger than 30 years old, and 58% were unmarried. Multiplex PCR, which simultaneously detects pathogens from various sexually transmitted infections, revealed that 91.7% (11/12) of patients were infected with 2 or more pathogens. Mycoplasma hominis was the most prevalent co-infection pathogen with T. vaginalis, followed by Ureaplasma urealyticum and Chlamydia trachomatis. Our results indicate that PCR and multiplex PCR are the most sensitive tools for T. vaginalis diagnosis, rather than microscopy which has been routinely used to detect T. vaginalis infections in South Korea. Therefore, clinicians should take note of the high prevalence of T. vaginalis infections among adolescent and young women in order to prevent persistent infection and transmission of this disease.

Efficacy of Korean Multipurpose Contact Lens Disinfecting Solutions against Acanthamoeba castellanii

Acanthamoeba keratitis has been increasing in recent years. Main risk factors are contact lens wear and their cleaning solutions. Most contact lens wearers use multipurpose disinfecting solutions (MPDS) for cleansing and disinfecting microorganisms because of its convenience. We determined amoebicidal effects of MPDS made in Korea and their cytotoxicity on human corneal epithelium cells. Fifteen commercial MPDS (A to O) were tested for their amoebicidal effects on Acanthamoeba castellanii trophozoites and cysts by using a most probable number (MPN) technique. Among them, 7 kinds of MPDS showed little or no amoebicidal effects for 24 hr exposure. Solutions A, B, G, H, L, and O showed positive amoebicidal effects, and solutions M and N killed almost all trophozoites and cysts after 24 hr exposure. However, 50%-N solution showed 56% cytotoxicity on human corneal epithelial cells within 4 hr exposure, and 50%-O solution also showed 62% cytotoxicity on human cells within 4 hr exposure. Solution A did not show any cytotoxicity on human cells. These results revealed that most MPDS made in Korea were ineffective to kill Acanthamoeba. The solutions having amoebicidal activity also showed high levels of cytotoxicity on human corneal epithelial cells. New formulations for improved MPDS that are amoebicidal but safe for host cells are needed to prevent Acanthamoeba keratitis.

Identification of Protein Arginine Methyltransferase 5 as a Regulator for Encystation of Acanthamoeba

Encystation is an essential process for Acanthamoeba survival under nutrient-limiting conditions and exposure to drugs. The expression of several genes has been observed to increase or decrease during encystation. Epigenetic processes involved in regulation of gene expression have been shown to play a role in several pathogenic parasites. In the present study, we identified the protein arginine methyltransferase 5 (PRMT5), a known epigenetic regulator, in Acanthamoeba castellanii. PRMT5 of A. castellanii (AcPRMT5) contained domains found in S-adenosylmethionine-dependent methyltransferases and in PRMT5 arginine-N-methyltransferase. Expression levels of AcPRMT5 were increased during encystation of A. castellanii. The EGFP-PRMT5 fusion protein was mainly localized in the nucleus of trophozoites. A. castellanii transfected with siRNA designed against AcPRMT5 failed to form mature cysts. The findings of this study lead to a better understanding of epigenetic mechanisms behind the regulation of encystation in cyst-forming pathogenic protozoa.

Assessing clinical reasoning abilities of medical students using clinical performance examination / 한국의학교육

PURPOSE: The purpose of this study is to investigate the reliability and validity of new clinical performance examination (CPX) for assessing clinical reasoning skills and evaluating clinical reasoning ability of the students. METHODS: Third-year medical school students (n=313) in Busan-Gyeongnam consortium in 2014 were included in the study. One of 12 stations was developed to assess clinical reasoning abilities. The scenario and checklists of the station were revised by six experts. Chief complaint of the case was rhinorrhea, accompanied by fever, headache, and vomiting. Checklists focused on identifying of the main problem and systematic approach to the problem. Students interviewed the patient and recorded subjective and objective findings, assessments, plans (SOAP) note for 15 minutes. Two professors assessed students simultaneously. We performed statistical analysis on their scores and survey. RESULTS: The Cronbach α of subject station was 0.878 and Cohen κ coefficient between graders was 0.785. Students agreed on CPX as an adequate tool to evaluate students' performance, but some graders argued that the CPX failed to secure its validity due to their lack of understanding the case. One hundred eight students (34.5%) identified essential problem early and only 58 (18.5%) performed systematic history taking and physical examination. One hundred seventy-three of them (55.3%) communicated correct diagnosis with the patient. Most of them had trouble in writing SOAP notes. CONCLUSION: To gain reliability and validity, interrater agreement should be secured. Students' clinical reasoning skills were not enough. Students need to be trained on problem identification, reasoning skills and accurate record-keeping.

Prevalence of Trichomonas vaginalis by PCR in Men Attending a Primary Care Urology Clinic in South Korea

Trichomonas vaginalis, a causative agent of trichomoniasis, may trigger symptomatic or asymptomatic nongonococcal urethritis and chronic prostatitis in men. Despite the availability of highly sensitive diagnostic tests, such as nucleic acid amplification tests, including PCR, few prospective studies present data on male T. vaginalis infection in South Korea. In the present study, the prevalence of T. vaginalis and associated clinical conditions were evaluated in 201 male patients from a primary care urology clinic in South Korea. The prevalence of T. vaginalis infection in our cohort was 4% (8/201) by PCR. T. vaginalis infection was common in men older than 40 years (median age, 52 years). Among the 8 Trichomonas-positive patients, 87.5% (7/8) had prostatic diseases, such as prostatitis and benign prostatic hyperplasia, and 25.0% (2/8) and 12.5% (1/8) were coinfected with Chlamydia trachomatis and Mycoplasma genitalium, respectively. Our results suggest that T. vaginalis infection is not rare in men attending primary care urology clinics in South Korea, especially in those older than 40 years, in whom it may explain the presence of prostatic disease. The possibility of T. vaginalis infection should be routinely considered in older male patients with prostatic diseases in South Korea.