Usefulness of a new immunochromatographic assay using fluorescent silica nanoparticles for serodiagnosis of Thai patients with amebiasis.

A fluorescence immunochromatography (FIC) kit was developed recently using fluorescent silica nanoparticles coated with a recombinant C-terminal fragment of the surface lectin intermediate subunit (C-Igl) of Entamoeba histolytica to establish rapid serodiagnosis of amebiasis. We further evaluated the system using serum samples from 52 Thai patients with amebiasis. Of the patients, 50 (96%) tested positive using FIC. The samples were also tested using enzyme-linked immunosorbent assay (ELISA) with C-Igl as the antigen. Two samples were negative on ELISA but positive on FIC. The correlation coefficient between the fluorescence intensity using FIC and the optical density value using ELISA was 0.5390, indicating a moderate correlation between the two tests. Serum samples from 20 patients with malaria and 22 patients with Clostridioides difficile infection were also tested using FIC. The false-positive rates were 4/20 (20%) and 1/22 (4%) in patients with malaria and C. difficile infection, respectively. Combining the data from the present study with our previous study, the sensitivity and specificity of FIC were determined to be 98.5% and 95.2%, respectively. The results of the 50 samples were studied using a fluorescence scope and a fluorescence intensity reader, and the findings were compared. Disagreements were found in only two samples showing near-borderline fluorescence intensity, indicating that the use of scope was adequate for judging the results. These results demonstrate that FIC is a simple and rapid test for the serodiagnosis of amebiasis.

Immune and allergenic effects of the microalga Coccomyxa sp. strain KJ in healthy humans: A pilot study.

BACKGROUND: The Coccomyxa sp. strain KJ (Coccomyxa KJ), a microalga found in Japan, has a potential function in controlling viral infections. Recently, its dry powder has been marketed as a health food product. OBJECTIVES: This pilot study investigated the effects of Coccomyxa KJ powder tablet intake on allergic reactions and immune functions in healthy participants. MATERIAL AND METHODS: Nine healthy volunteers (4 males and 5 females) who expressed interest in foods containing Coccomyxa KJ, and were willing to undergo blood tests, were recruited. Each individual was asked to take 2 Coccomyxa KJ powder tablets (0.3 g) before breakfast once a day for 4 weeks. The salivary immunoglobulin A (IgA) level and blood parameters (white blood cell (WBC) count, eosinophil and lymphocyte counts and percentages, natural killer (NK) cell activity, interleukin (IL)-6 level, and T helper (Th)1/Th2 cell ratio) were evaluated at baseline and weeks 2 and 4. RESULTS: The 4-week intake of Coccomyxa KJ did not affect salivary IgA levels, WBC count, eosinophil and lymphocyte counts and percentages, or the Th1/Th2 ratio. There were significant differences in the NK cell activity after 4 weeks, with an average increase of 11.78 (95% confidence interval (95% CI): 6.80-16.76). None of the patients experienced adverse reactions during or after the study. CONCLUSIONS: Long-term Coccomyxa KJ intake improved NK cell activity without causing adverse effects on the indicators of local immunity, systemic inflammation and immune response balance. This study suggests that Coccomyxa KJ powder tablets can induce beneficial immune modifications without causing any adverse effects.

Ultrasonographic Characteristics in the Fingers and Other Superficial Glomus Tumours.

Glomus tumours are painful superficial tumours, and ultrasonography is an extremely useful and noninvasive diagnostic technique for superficial organs. In this study, we retrospectively examined glomus tumours using ultrasonography. Among 18 patients histopathologically diagnosed with glomus tumours via ultrasonography, we observed five different development sites: subungual areas or those surrounding the nail bed (12), other areas on the finger surface (3), abdominal wall (1), upper arm (1), and forearm (1). The ultrasonographic images revealed significant differences in tumour size, indicating that tumours on other body surfaces tended to be smaller than those on patients' fingers (p < 0.01). The depth/width ratios of tumours on the other body surfaces were significantly higher than those on the fingers (p < 0.05). The tumours showed a regular shape (72.2%) and clear border (100%). Furthermore, most tumours were low-echo tumours with a diameter of up to 15 mm, clear margins, and no lateral shadows. Abundant blood flow and vessels in and out of the tumours were also observed. In conclusion, our study describes the ultrasonographic characteristics of glomus tumours and reveals that they cannot be ruled out when diagnosing small painful subcutaneous tumours.

A pilot study of on-site evaluation as external quality assessment for ISO 15189 accreditation of laboratories performing next-generation sequencing oncology tests.

BACKGROUND: As next-generation sequencing (NGS) oncology tests vary by platform, application, and target of genes, specific methods for external quality assessment (EQA) have not been universally applied. Hence, we have attempted to implement on-site evaluation as EQA in the accreditation program under ISO 15189 for laboratories that perform NGS oncology tests. METHODS: A total of 10 laboratories that performed NGS oncology tests were enrolled. Two types of EQA samples were prepared (Acrometrix Oncology Hotspot Control DNA and OncoSpan gDNA DNA samples), and the variant allele frequency of targeted genes was assigned. The samples were subjected to NGS oncology tests in participant laboratories according to their routine protocols. Based on the result reports, auditors visited the participant laboratories to perform on-site evaluations and provided feedback regarding possible laboratory process improvement. RESULTS: The participant laboratories identified the targeted variants in the Acrometrix Oncology Hotspot Control DNA and OncoSpan gDNA samples with a success rate of 31-100% and 9.5-100%, respectively, compared with reference information, depending on their sequencing systems, and reported a few lower-variant allele frequencies. Six of the eight evaluated laboratories failed to report at least three pathogenic variants due to errors in wet-lab and/or dry-lab processes. Based on the feedback reports and self-assessment, auditors and laboratory staff discussed potential improvements to processes during on-site evaluations for laboratory accreditations. CONCLUSIONS: On-site evaluation as EQA for NGS oncology tests in the laboratory accreditation program under ISO 15189 was successfully implemented and proved applicable to a broad spectrum of NGS tests.

Methods of Cleaning Taps to Prevent Hospital-Associated Infections: An Environmental Survey-Based Study.

In hospitals, outbreaks can occur due to pathogens accumulating in the areas around the wards' washbasins. Carbapenem-resistant Enterobacterales (CRE) was detected in an environmental survey in the high-care unit of a university hospital in Isehara, Japan, and effective cleaning methods were investigated. This study investigated methods of cleaning taps using commonly used detergents and disinfectants, and it assessed their effectiveness in removing hard scale and pathogens, including CRE. The taps were cleaned using various methods and cleaning agents, including environmentally neutral detergent, citric acid, baking soda, cleanser, 80% ethanol, 0.1% sodium hypochlorite, and a phosphoric acid-based environmental detergent (Space Shot). The cleaning effect was assessed based on the agent's effectiveness at removing hard scale from taps. Biofilms and scale were identified on taps, and several bacterial species were cultured. Only phosphoric acid-based detergent was effective at removing hard scale. After cleaning with the phosphoric acid-based detergent, the bacterial count decreased, and no CRE or other pathogens were detected. These results provide a reference for other facilities considering introducing this cleaning method.

Electrical biosensing system utilizing ion-producing enzymes conjugated with aptamers for the sensing of severe acute respiratory syndrome coronavirus 2.

Viral outbreaks, which include the ongoing coronavirus disease 2019 (COVID-19) pandemic provoked by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), are a major global crisis that enormously threaten human health and social activities worldwide. Consequently, the rapid and repeated treatment and isolation of these viruses to control their spread are crucial to address the COVID-19 pandemic and future epidemics of novel emerging viruses. The application of cost-efficient, rapid, and easy-to-operate detection devices with miniaturized footprints as a substitute for the conventional optic-based polymerase chain reaction (PCR) and immunoassay tests is critical. In this context, semiconductor-based electrical biosensors are attractive sensing platforms for signal readout. Therefore, this study aimed to examine the electrical sensing of patient-derived SARS-CoV-2 samples by harnessing the activity of DNA aptamers directed against spike proteins on viral surfaces. We obtained rapid and sensitive virus detection beyond the Debye length limitation by exploiting aptamers coupled with alkaline phosphatases, which catalytically generate free hydrogen ions which can readily be measured on pH meters or ion-sensitive field-effect transistors. Furthermore, we demonstrated the detection of the viruses of approximately 100 copies/µL in 10 min, surpassing the capability of typical immunochromatographic assays. Therefore, our newly developed technology has great potential for point-of-care testing not only for SARS-CoV-2, but also for other types of pathogens and biomolecules.

Impact of ward pharmacist-led antimicrobial stewardship in intensive care units.

Various outcomes of mortality, medical costs, and antimicrobial usage result from antimicrobial stewardship (AS) programmes. Here, we clarified the effects of AS implementation by a well-trained pharmacist in an open intensive care unit (open ICU) through a retrospective, comparative study of 5123 open ICU patients of Tokai University Hospital. The 12 months before and after AS implementation were considered the control and study periods, respectively. After AS implementation, the number of AS cases increased significantly. The period until the implementation of therapeutic drug monitoring was significantly shortened, and antimicrobial drug usage increased significantly. The methicillin-resistant Staphylococcus aureus (MRSA) detection rate decreased significantly. Earlier and more frequent AS implementation could enhance treatment effects, possibly decreasing the MRSA incidence. Despite active AS implementation, antimicrobial drug usage did not necessarily decrease. ICU pharmacists with experience in AS should take on leadership roles and implement active AS strategies in open ICU settings.

Usefulness of LacdiNAc-glycosylated Prostate-specific Antigen Density for Predicting Pathological Findings of Magnetic Resonance Imaging-transrectal Ultrasound Fusion Image-guided Prostate Biopsy for the Patients With Highest Prostate Imaging Reporting and Data System Category ≥3.

PURPOSE: This study aimed to evaluate the usefulness of the LDN-PSA (LacdiNAc-glycosylated-prostate specific antigen) in detecting clinically significant prostate cancer in patients suspected of having clinically significant prostate cancer on multiparametric magnetic resonance imaging. MATERIALS AND METHODS: Patients with prostate specific antigen levels ranging between 3.0 ng/mL and 20 ng/mL and suspicious lesions with PI-RADS (Prostate Imaging-Reporting and Data System) category ≥3 were included prospectively. The LDN-PSA was measured using an automated 2-step Wisteria floribunda agglutinin lectin-anti-prostate specific antigen antibody sandwich immunoassay. RESULTS: Two hundred four patients were included. Clinically significant prostate cancer was detected in 105 patients. On multivariable logistic regression analysis, prostate specific antigen density (OR 1.61, P = .010), LDN-PSAD (OR 1.04, P = .012), highest PI-RADS category (3 vs 4, 5; OR 14.5, P < .0001), and location of the lesion with highest PI-RADS category (transition zone vs peripheral zone) (OR 0.34, P = .009) were significant risk factors for detecting clinically significant prostate cancer. Among the patients with the highest PI-RADS category 3 (n=113), clinically significant prostate cancer was detected in 28 patients. On multivariable logistic regression analysis to predict the detection of clinically significant prostate cancer in patients with the highest PI-RADS category 3, age (OR 1.10, P = .026) and LDN-PSAD (OR 1.07, P < .0001) were risk factors for detecting clinically significant prostate cancer. CONCLUSIONS: LDN-PSAD would be a biomarker for detecting clinically significant prostate cancer in patients with prostate specific antigen levels ≤20 ng/mL and suspicious lesions with PI-RADS category ≥3. The use of LDN-PSAD as an adjunct to the use of prostate specific antigen levels would avoid unnecessary biopsies in patients with the highest PI-RADS category 3. Multi-institutional studies with large population are recommended.

Virucidal and Immunostimulating Activities of Monogalactosyl Diacylglyceride from Coccomyxa sp. KJ, a Green Microalga, against Murine Norovirus and Feline Calicivirus.

Human noroviruses are the most common pathogens causing acute gastroenteritis and may lead to more severe illnesses among immunosuppressed people, including elderly and organ transplant recipients. To date, there are no safe and effective vaccines or antiviral agents for norovirus infections. In the present study, we aimed to demonstrate the antiviral activity of monogalactosyl diacylglyceride (MGDG) isolated from a microalga, Coccomyxa sp. KJ, against murine norovirus (MNV) and feline calicivirus (FCV), the surrogates for human norovirus. MGDG showed virucidal activities against these viruses in a dose- and time-dependent manner-MGDG at 100 µg/mL reduced the infectivity of MNV and FCV to approximately 10% after 60 min incubation. In the animal experiments of MNV infection, intraoral administration of MGDG (1 mg/day) exerted a therapeutic effect by suppressing viral shedding in the feces and produced high neutralizing antibody titers in sera and feces. When MGDG was orally administered to immunocompromised mice treated with 5-fluorouracil, the compound exhibited earlier stopping of viral shedding and higher neutralizing antibody titers of sera than those in the control mice administered with distilled water. Thus, MGDG may offer a new therapeutic and prophylactic alternative against norovirus infections.

Virucidal effect of monogalactosyl diacylglyceride from a green microalga, Coccomyxa sp. KJ, against clinical isolates of SARS-CoV-2 as assessed by a plaque assay.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19) and is capable of human-to-human transmission and rapid global spread. Thus, the establishment of high-quality viral detection and quantification methods, and the development of anti-SARS-CoV-2 agents are critical. METHODS: Here, we present the rapid detection of infectious SARS-CoV-2 particles using a plaque assay with 0.5% agarose-ME (Medium Electroosmosis) as an overlay medium. RESULTS: The plaques were capable of detecting the virus within 36-40 h post-infection. In addition, we showed that a monogalactosyl diacylglyceride isolated from a microalga (Coccomyxa sp. KJ) could inactivate the clinical isolates of SARS-CoV-2 in a time- and concentration-dependent manner. CONCLUSIONS: These results would allow rapid quantification of the infectious virus titers and help develop more potent virucidal agents against SARS-CoV-2.

Nationwide external quality assessment of SARS-CoV-2 nucleic acid amplification tests in Japan.

OBJECTIVES: We conducted a nationwide external quality assessment (EQA) study of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleic acid amplification testing in Japan. METHODS: A total of 563 public health and private sector laboratories participated. The EQA samples comprised 6 RNA and full-process controls. RESULTS: The overall agreements were 99.3% and 97.9% for the RNA and full-process controls, respectively. A total of 530/563 (94.1%) laboratories reported correct results; public health laboratories had the highest accuracy. Thirty-three laboratories reported at least one incorrect result (26 laboratories of medical facilities, 5 commercial laboratories, 1 public health laboratory, and 1 other). Sixteen laboratories of medical facilities that used a fully automated assay system failed to detect the presence of the full-process control, due to inherent insufficiency in the limit of detection (LOD). Other causes of incorrect results included failure to ensure the LOD (n = 13), error in result judging or reporting (n = 3), and error in sample handling (n = 1). CONCLUSIONS: Performance was mostly dependent on the laboratory category and assay evaluation, particularly the LOD. Guidance should be developed based on these results, particularly in the phase of new entry into laboratory services for SARS-CoV-2.

Evaluation of the basic assay performance of the GeneSoc® rapid PCR testing system for detection of severe acute respiratory syndrome coronavirus 2.

In the ongoing coronavirus disease 2019 (COVID-19) pandemic, PCR has been widely used for screening patients displaying relevant symptoms. The rapid detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enables prompt diagnosis and the implementation of proper precautionary and isolation measures for the patient. In the present study, we aimed to evaluate the basic assay performance of an innovative PCR system, GeneSoC® (Kyorin Pharmaceutical Co. Ltd., Tokyo, Japan). A total of 1,445 clinical samples were submitted to the clinical laboratory, including confirmed or suspected cases of COVID-19, from February 13 to August 31. Specimen types included nasopharyngeal swabs. The sampling was performed several times for each patient every 2-7 days. Using this system, sequences specific for SARS-CoV-2 RNA could be detected in a sample within 10-15 min using the microfluidic thermal cycling technology. Analytical sensitivity studies showed that GeneSoC® could detect the target sequence of the viral envelope and RNA-dependent RNA-polymerase (RdRp) genes at 5 and 10 copies/µL, respectively. The precision of the GeneSoC® measurements using clinical isolates of the virus at a concentration of 103 copies/µL was favorable for both the genes; within-run repeatability and between-run reproducibility coefficient of variation values were less than 3% and 2%, respectively; and the reproducibility of inter-detection units was less than 5%. Method comparison by LightCycler® 480 showed the positive and negative agreement to be 100% [(174/174) and (1271/1271), respectively]. GeneSoC® proved to be a rapid and reliable detection system for the prompt diagnosis of symptomatic COVID-19 patients and could help reduce the spread of infections and facilitate more rapid treatment of infected patients.

Rapid profiling of drug-resistant bacteria using DNA-binding dyes and a nanopore-based DNA sequencer.

Spread of drug-resistant bacteria is a serious problem worldwide. We thus designed a new sequence-based protocol that can quickly identify bacterial compositions of clinical samples and their drug-resistance profiles simultaneously. Here we utilized propidium monoazide (PMA) that prohibits DNA amplifications from dead bacteria, and subjected the original and antibiotics-treated samples to 16S rRNA metagenome sequencing. We tested our protocol on bacterial mixtures, and observed that sequencing reads derived from drug-resistant bacteria were significantly increased compared with those from drug-sensitive bacteria when samples were treated by antibiotics. Our protocol is scalable and will be useful for quickly profiling drug-resistant bacteria.

Diagnostic Tests for COVID-19 and Their Accuracy: Important Information for Physicians Caring for Patients with Stroke.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SAR-CoV-2), which appeared at the end of 2019 and has spread rapidly worldwide. In Japan, the increasing number of people infected with SAR-CoV-2 is also a cause of concern for physicians managing stroke patients. From the perspective of viral transmission in the hospital, stroke physicians must determine whether patients who have been transported by emergency have confirmed or suspected COVID-19. For this reason, stroke physicians must also understand about the characteristics and accuracy of the test for COVID-19 diagnosis. This article describes the sensitivity of the clinical symptoms, imaging investigations such as chest radiography and chest CT, and accuracy of nucleic-acid amplification tests and antigen tests used in the diagnosis of COVID-19. However, it should be noted that the accuracy of specimen tests may change depending on the collection site, timing, and method, because positive results in these tested specimens depend on the viral loads. In performing medical treatment for stroke, high accuracy and rapid inspection for COVID-19 is desired, but this is not currently available. For acute stroke treatment, such as thrombectomy, we recommend that these emergency patients, who are suspected of COVID-19 by clinical symptoms and image investigations, should be treated with implementation of strict infection control against droplets, contact, and airborne transmission until the most sensitive polymerase chain reaction test result is confirmed as negative.

Sonographic appearance of syphilitic induration mimicking squamous cell carcinoma in the lower lip: a case report.

BACKGROUND: Syphilis is a sexually transmitted disease caused by the spirochete Treponema pallidum. Recently, its incidence has been increasing worldwide. We encountered a young woman who presented with induration mimicking squamous cell carcinoma in the lower lip, without major medical conditions. CASE PRESENTATION: A 25-year-old Japanese woman presented with a 1-month history of a painless induration in her lower lip. Because squamous cell carcinoma was suspected, a preoperation work up was performed, including laboratory tests, an ultrasonographic examination, and a biopsy. The ultrasonography findings showed an oval-shaped 17 × 11 × 12 mm tumor-like lesion with heterogeneous internal echo and an indistinct border. A pressure test and color Doppler sonography revealed that the lesion was soft with a very abundant blood flow. These findings suggested the possibility of underlying inflammatory causes rather than a neoplastic tumor. Serology tests for syphilis, including the anti-Treponema pallidum antibody and reactive rapid plasma reagin tests, were positive. The biopsy revealed no malignancy. Finally, she was diagnosed as having primary syphilis and treated with amoxicillin for 28 days. The rapid plasma reagin value gradually decreased and the initial induration in her lower lip disappeared. CONCLUSION: This case highlights the need for prompt examinations for possible underlying infective causes, such as syphilis, when seeing a painless induration with ulcer in the lip. Ultrasonography was helpful in the differential diagnosis of a tumor-like lesion and should be included in addition to syphilis serology tests, such as anti-Treponema pallidum antibody and rapid plasma reagin tests.

A Protocol of Infection Control for Mechanical Thrombectomy in Possible COVID-19 Patients: Tokai University COVID-19 Manual.

The crisis of the coronavirus disease (COVID-19) is causing damage to the social and medical community. However, extreme emergency neuro-interventions such as mechanical thrombectomy still require the healthcare workers to offer the appropriate treatment while preventing further spread of the infection. This article outlines the necessary steps in managing a possible COVID-19 patient starting from patient screening to personnel infection and environmental contamination measures.

A simple screening method for the diagnosis of chronic myeloid leukemia using the parameters of a complete blood count and differentials.

BACKGROUND: This study aimed to develop a simple and inexpensive method using the complete blood count (CBC) and differentials to screen for chronic myeloid leukemia (CML). METHODS: The receiver operating characteristic (ROC) curves of each CBC parameter, differential and the neutrophil alkaline phosphatase (NAP) score using CML and non-CML cases were generated to determine effective cut-off values. They were applied to the review of randomly-selected 45,608 samples for validation. RESULTS: The leukocyte count showed the highest area under the ROC curve (AUC) value (0.909) among the CBC parameters. In the absolute counts of differentials, the AUC was the highest in basophils (0.982), followed by immature granulocytes (IGs) (0.975), which had cut-off values of 0.43 × 109/L and 0.46 × 109/L, respectively. The AUC of the NAP score was 0.963 at a cut-off value 122. In the validation, the absolute basophil counts were elevated in 280 samples from 96 cases, including 22 CML cases. In contrast, the absolute IG counts were elevated in 1310 samples from 516 cases, including only 17 CML cases. Three newly-diagnosed CML cases whose data were analyzed sequentially at the CML onset consistently met the basophil criteria before the IG criteria. CONCLUSIONS: The absolute basophil count is effective for screening for CML.