Outbreaks of Campylobacteriosis Caused by Drinking Raw Milk in Japan: Evidence of Relationship Between Milk and Patients by Using Whole Genome Sequencing.

Raw milk may contain some infectious bacteria and usually requires pasteurization before drinking. In this study, we report rare outbreaks of campylobacteriosis associated with raw milk in Japan, and the application of whole genome sequencing (WGS) to studies on foodborne diseases. In August 2018, there were three outbreaks of campylobacteriosis, presumably caused by the consumption of unpasteurized raw milk, derived from the same farm; thus, these three outbreaks seemed to be associated with a single contaminant at the farm. Therefore, we analyzed Campylobacter jejuni isolates obtained at the three locations using several genetic methods. The sequence type of each isolate, revealed by multilocus sequence typing, was ST-61, and the profile determined using pulsed-field gel electrophoresis was the same; however, neither method could distinguish these from previously obtained strains. Subsequently, we performed WGS and single nucleotide variant (SNV) analysis that provided evidence of clonality, indicating that C. jejuni contamination was attributed to the farm. As in this study, evidence suggests that SNV analysis provides molecular biological support in cases with sufficient epidemiological information. Hence, similar analytical methods may be used in other sporadic cases to elucidate the relevance of the cases.

Development and Effective Utilization of a Rapid Multiplex Real-Time PCR of Diarrheagenic Escherichia coli for Food Poisoning Cases.

Diarrheagenic Escherichia coli (DEC) causes diarrheal symptoms in humans. The comprehensive detection of DEC from feces using SYBR Green real-time PCR assay requires multiple runs. Moreover, PCR screening can have discrepancies related to the conformance between the results from PCR screening and culturing. We aimed to develop a real-time PCR for the comprehensive testing of DEC for diagnostic support that can be used in any general laboratory and proposed its effective utilization. We tested specificity for the designed primer sets using 100 strains. Moreover, screening and isolation of DEC were performed using the proposed multiplex real-time PCR system for 308 fecal samples collected from 37 food poisoning incidences that occurred in Gifu Prefecture, Japan from 2017 to 2019. Furthermore, the factor of discrepant results between PCR screening and culturing was analyzed by quantifying the number of DEC cell and whole E. coli cell using real-time PCR for 47 PCR screening-positive fecal samples. The results obtained from the developed multiplex real-time PCR system were in 99% concordance with those from the conventional techniques. A total of 49 fecal samples were detected with virulence genes for the screening. Of the samples which were positive with virulence genes by PCR screening, 38.3% could not be detected from the strain for bacterial culture. We found that the culturing positive samples were significantly high in numbers for the DEC cells, but no significant difference was noted in the whole E. coli cells with culturing negative samples. The multiplex real-time PCR developed in this study was found to be rapid and practical for DEC testing. The PCR screening for DEC using this method can provide rapid information toward the diagnostic support of DEC infection.

Identification of N-acyl-N-indanyl-α-phenylglycinamides as selective TRPM8 antagonists designed to mitigate the risk of adverse effects.

Transient receptor potential melastatin 8 (TRPM8), a temperature-sensitive ion channel responsible for detecting cold, is an attractive molecular target for the treatment of pain and other disorders. We have previously discovered a selective TRPM8 antagonist, KPR-2579, which inhibited bladder afferent hyperactivity induced by acetic acid instillation into the bladder. However, additional studies have revealed potential adverse effects with KPR-2579, such as the formation of a reactive metabolite, CYP3A4 induction, and convulsions. In this report, we describe the optimization of α-phenylglycinamide derivatives to mitigate the risk of these adverse effects. The optimal compound 13x exhibited potent inhibition against icilin-induced wet-dog shakes and cold-induced frequent voiding in rats, with a wide safety margin against the potential side effects.

Rapid Serotyping of Salmonella Isolates Based on Single Nucleotide Polymorphism-Like Sequence Profiles of a Salmonella-Specific Gene.

Although serotyping is the most important method of identification of taxonomy in Salmonella, conventional serotype determination with a complete set of antisera is time consuming and laborious. Recently, rapid serotyping procedures with polymerase chain reaction (PCR) have been developed. In this study, we established a novel PCR-based rapid serotyping method that employs a unique target gene. Alignment study of Salmonella-specific gene (Salmonella enterotoxin [stn]) revealed a correlation between the stn gene sequence and the serotype of the organism. In 750 bp of stn gene, 55 nucleotides indicated single nucleotide polymorphism (SNP)-like polymorphism, and the correlation between the SNP-like polymorphism and the serotype of the organism suggests that SNP-like sequences in stn gene can serve as an index for serotyping. To develop a rapid serotyping method based on the SNP-like polymorphism, we selected serotype-associated 12 SNP-like sites in the stn gene and established a method based on high-resolution melting (HRM) and PCR, which identifies nucleotides at SNP-like sites within 1.5 h. This newly established rapid serotyping procedure (stn-HRM) could identify nine serotypes, including the frequently isolated serovar Enteritidis. These nine serotypes cover 64.3% of cases of Salmonella, as reported by the World Health Organization/Global Foodborne Infection Network (WHO/GFN) Country Databank from 2001 to 2010. In this study, we employed a unique target gene, stn, which is completely independent of the genes that were targeted in previously reported rapid serotyping procedures. Therefore, the results obtained by our newly developed stn-HRM procedure are independent of the results obtained by other procedures. Besides, stn-HRM can ensure accurate identification of the bacterial species as stn is a Salmonella-specific gene. It is expected that the combination of newly constructed stn-HRM and previously reported procedures could further improve the credibility of Salmonella isolate serotyping.

Electron Microscopy Revealed Massive Lipid Droplets in Cardiomyocytes in a Patient with Cardiogenic Shock Following a Fulminant Type 1 Diabetes Mellitus.

A 52-year-old man with consciousness disorder following a 2-day history of general fatigue, diarrhea, vomiting and excessive thirst was admitted to our hospital. Severe hyperglycemia (1,739 mg/dL) with a slightly elevated HbA1c level (6.9%), ketonuria and low C-peptide level (0.07 ng/mL) confirmed the diagnosis of fulminant type 1 diabetes mellitus (FT1DM). Following sudden unexplained cardiogenic shock shortly after the initiation of insulin therapy with no evidence of myocardial ischemia assessed by coronary angiography, the patient was supported with percutaneous venoarterial extracorporeal membrane oxygenation. Electron microscopic analysis of the myocardium revealed massive lipid droplets without the infiltration of inflammatory cells. His left ventricular function began to recover during the following days and returned to a normal level on day 14. Currently, the impact of FT1DM on intramyocardial lipid deposition is poorly understood. However, this case suggests that even short-term exposure to high concentrations of glucose can be responsible for lipotoxicity followed by severe cardiac dysfunction.

[A Case of Primary Intrasellar Chondroid Chordoma].

OBJECTIVES: Intracranial chordomas are thought to arise from remnants of the notochord and usually occur at the parasellar region. We present a case of a primary intrasellar chondroid chordoma in a patient who was initially diagnosed with a pituitary adenoma. CASE: A 77-year-old woman had a history of two surgeries for a tumor in the sella turcica(17 months after the 1st surgery). On initial MRI, the intrasellar mass showed low signal intensity on T1WI, very high signal intensity on T2WI, and inhomogeneous enhancement. On bone reconstructive CT, the sellar floor was thin, and no abnormalities were observed at the top of the clivus. Transsphenoidal surgery was performed. The pathological diagnosis was pituitary adenoma in both cases. Seventy-two months after the 1st(31 months after the 2nd)surgery, she developed right-sided oculomotor and abducent nerve palsies again. Since recurrence occurred during the relatively short period, the surgical specimens obtained from the 1st and 2nd surgeries were reexamined. Reexamination of the previously obtained specimen demonstrated areas of chondroid tissue that were embedded in a mucoid stroma and tumor cells that were composed of round or pleomorphic nuclei with vacuolated cytoplasm(physaliphorus cells)that were compatible with chondroid chordoma. The third surgery was performed. Postoperatively, her symptoms improved, and cyber knife therapy was administered for the residual part of the tumor. CONCLUSIONS: Although intrasellar chondroid chordomas are extremely rare, they should be considered in the differential diagnosis of tumors located in the sella turcica.

Biphasic low-grade nasopharyngeal papillary adenocarcinoma: a case report and literature review.

BACKGROUND: Low-grade nasopharyngeal papillary adenocarcinoma (LGNPPA) is distinctly rare. We report a patient with a uniquely biphasic LGNPPA; additionally, we review similar tumors reported in the literature. CASE PRESENTATION: A 56-year-old man presented with an asymptomatic pedunculated tumor in the vault of the nasopharynx, at the junction of the nasal septum and the roof, which was discovered during screening for laryngeal cancer. To obtain a definitive diagnosis, the patient underwent endoscopic endonasal surgery under general anesthesia. Immunohistochemical analysis of the tumor revealed it to be an LGNPPA with a prominent spindle cell component. CONCLUSION: To our knowledge, this is the fourth reported LGNPPA exhibiting a spindle cell component and the second with a prominent pathological condition. The prognosis of LGNPPA is usually excellent. Therefore, it is important for clinicians to scrutinize the lesion's pathology to avoid unnecessary, disfiguring surgery.

[A Case of Primary Extradural Meningioma Located at the High Convexity:A Case Report and Review of the Literature].

OBJECTIVES: Primary meningiomas arising outside the intracranial component are rather rare and have been termed primary extradural meningiomas(PEMs). We present a case of an intraosseous-type PEM occurring at a high convexity location and discuss the clinical characteristics of PEMs. CASE: An 80-year-old woman presented with a soft and painless subcutaneous mass of approximately 10 cm in diameter in the right parietal region, which had appeared 1 year previously. Mild cognitive dysfunction and left hemiparesis were observed upon admission. A skull radiograph and a bone window computed tomography scan revealed an osteolytic lesion at the above-mentioned site. Magnetic resonance imaging indicated that the mass was inhomogeneously enhanced and seemed to extend through the skull defect both intra- and extra-cranially. The preoperative diagnosis was a metastatic skull bone tumor due to the patient's history of breast cancer. During surgery, the tumor was found to be solid and had expanded through both the inner and outer tables of the skull, destroyed the inner table at one location, and perforated into the subdural space via the thinned, but not infiltrated, dura mater. The tumor was removed along with a wide margin of surrounding healthy bone and a cranioplasty was performed using a titanium plate. The histopathological diagnosis was atypical meningioma(World Health Organization grade II). Postoperatively, no adjuvant therapies(radiation and/or chemotherapy)were administered and the patient was reported to be well at 8 months post-surgery with no evidence of tumor recurrence. CONCLUSIONS: Although PEMs are rather rare, clinicians should consider the differential diagnosis of osteolytic skull vault tumors.

Synthesis and optimization of novel α-phenylglycinamides as selective TRPM8 antagonists.

Transient receptor potential melastatin 8 (TRPM8) is activated by innocuous cold and chemical substances, and antagonists of this channel have been considered to be effective for pain and urinary diseases. N-(3-aminopropyl)-2-{[(3-methylphenyl)methyl]oxy}-N-(2-thienylmethyl)benzamide hydrochloride (AMTB), a TRPM8 antagonist, was proposed to be effective for overactive bladder and painful bladder syndrome; however, there is a potential risk of low blood pressure. We report herein the synthesis and structure-activity relationships of novel phenylglycine derivatives that led to the identification of KPR-2579 (20l), a TRPM8 selective antagonist. KPR-2579 reduced the number of icilin-induced wet-dog shakes and rhythmic bladder contraction in rats, with no negative cardiovascular effects at the effective dose.

[A Case of Multiple Atherosclerotic Fusiform Cerebral Aneurysms with Repeated Subarachnoid Hemorrhage].

Pure fusiform aneurysms without dissection are considered to have a low risk of rupture. Furthermore, aneurysm wall with atherosclerotic change tends to be less susceptible to rupture. We present a rare case of multiple atherosclerotic fusiform aneurysms associated with repeated subarachnoid hemorrhage(SAH)during a 9-year observation period. A 34-year-old woman was admitted to our hospital because of SAH. SAH with a temporal hematoma due to a fusiform aneurysm of the right middle cerebral artery(MCA)was detected using computed tomography(CT). Associated fusiform aneurysms were observed in the left posterior cerebral artery(PCA)and the left MCA. The right MCA aneurysm was occluded with angioplastic clipping. Eight years after the first onset, angioplastic clipping was performed on the other two fusiform aneurysms because of their growth. Intraoperative findings showed atherosclerotic change in the parent artery wall, similar to the right MCA aneurysms. The right MCA aneurysm ruptured again 1 year later. The aneurysm was treated with proximal occlusion combined with a bypass from the occipital artery to the distal MCA. The patient was in a stable state, although, she was disabled because of SAH damage. The effects of atherosclerotic fusiform aneurysms, which are rarely encountered, are not well known. Some studies have reported the risk of hemorrhage from these aneurysms. This case suggests the necessity of long-term follow-up for the prediction of aneurysm growth and bleeding. Moreover, proximal occlusion combined with an external bypass is better for the treatment of this type of aneurysm because angioplastic clipping is not curative.

[A Case of HER2-Positive Inflammatory Breast Cancer for Which Preoperative Chemotherapy with Pertuzumab Resulted in a Pathological Complete Response].

A 52-year-old woman presented with redness and swelling with a peau d'orange appearance in the whole right breast. Ultrasound revealed elevated subcutaneus fat density and a diffuse hypoechoic area. She was diagnosed with inflammatory breast cancer(T4dN2M0, Stage III B of the HER2 subtype). After 4 courses of EC treatment as primary systemic therapy, the hypoechoic area was still present. Subsequent chemotherapy with pertuzumab, trastuzumab, and docetaxel was effective, as hypoechoic area was not observed on ultrasound. She underwent mastectomy and axillary dissection, and pathological examination revealed pCR. At present, 2 years after surgery, the patient is alive with no reccurence.

Pupillary Size and Light Reflex in Premature Infants.

The authors examined pupillary light reflexes to both red and blue stimuli using infrared video-pupillography in premature infants. Pupillary light reflexes were obtained in response to 470 nm blue and 635 nm red stimuli with varying intensities of 10 cd/m2 and 100 cd/m2. Red stimuli did not evoke a pupillary response in premature infants. However, blue stimuli produced a pupillary reflex, and the extent of the reaction was dependent on the intensity of the stimulus. These results suggest that in premature infants, the origin of light reflex may be mediated predominately by melanopsin-containing retinal ganglion cells.

Enhanced Depth Imaging of Central Laminar Thickness in Optic Neuropathy: Comparison with Normal Eyes.

The purpose of this study was to compare central laminar thickness (LT) among patients with glaucomatous optic neuropathy (GON), patients with non-GON, and normal subjects using enhanced-depth imaging optical coherence tomography (EDI-OCT). Enrolled were 57 patients (n = 64 eyes), including 30 women and 27 men. Three groups were identified: GON (n = 18 eyes), non-GON (n = 16 eyes), and control (n = 30 eyes). The GON group comprised eyes with primary open-angle glaucoma (POAG) (n = 9) and normal-tension glaucoma (NTG) (n = 9). The non-GON group comprised eyes with demyelinating optic neuritis (n = 9), anterior ischemic optic neuropathy (AION) (n = 2), compressive ON (n = 2), Leber hereditary ON (n = 2), and traumatic ON (n = 1). GON and non-GON groups were further divided into mild, moderate, and severe subgroups. Inclusion in the GON group was based on mean deviations (MDs) of visual fields; inclusion in the non-GON group was based on critical flicker frequency (CFF) responses. Intraclass correlation coefficients (ICCs) were used to verify reproducibility of measurements. LTs of GON and non-GON group eyes were thinner than those of control group eyes (p < 0.01); LTs of GON group eyes were thinner than those of non-GON group eyes (p = 0.01). LTs of severe GON subgroup eyes were thinner than those of moderate and mild GON subgroup eyes (p < 0.001; p = 0.024, respectively). LTs of severe non-GON subgroup eyes were thinner than those of mild non-GON subgroup eyes (p = 0.002). These results show that EDI-OCT is valuable for documenting structural abnormalities in optic neuropathy (ON).

Epstein-barr virus diversity in immunocompetent healthy persons: reassessment of the distribution of genetic variants.

Epstein-Barr virus (EBV) has many strains; however, it remains unclear whether a causal relationship exists between different regions and viral genetic variants in healthy persons. This study was designed to examine the relationship between EBV strains in tonsils and adenoids and peripheral blood lymphocytes of the same individuals using different measurements of EBV strain polymorphism. This study examined whether EBV contains two or three copies of a tandem repeat sequence in the first intron of the BZLF-1 gene. The genotype of the virus from P3HR-1, designated Z*, yielded a 415-bp product, and this was distinguished from the smaller, 386-bp product obtained with the B95-8 virus, designated the Z genotype. Simultaneous sequence infections with Z and Z* genotypes were also detected in one of the tonsils examined, suggesting that more than one strain or variant of EBV genotype may be present in a specimen from the same subject. Co-infection with Z and Z* was recognized in two subjects, so variation of the EBV gene may be seen in at least two different strains of EBV. It was seen that Z and Z* strain-infected cells are constantly in flux through lymph nodes and/or the blood stream in healthy persons; therefore, these results indicated that EBV genome variants probably show no specific tissue distribution.

Secondary IOL implantation for an aphakic patient with congenital cataract living in Bonin Islands; follow-up study.

Introduction: We previously reported a case of a patient with bilateral congenital cataract identified in the Ogasawara village, Bonin Islands, Japan, on a visit by an ophthalmologist and describe its course over 17 years from initial surgery. Here, we report on a secondary intraocular lens (IOL) implantation that was subsequently performed at 22 years and 5 months of age. Methods: After cataract surgery at 7 months, the aphakic patient began amblyopia treatment using extended-wear soft contact lenses (SCLs). After 10 years of age, SCLs were chosen to achieve crossed monovision considering the cosmetic appearance when facing other people. At 22 years of age, a secondary IOL implantation was performed. Secondary implant in the patient considered appearance and postoperative vision without glasses, we selected the crossed monovision method using a monofocal IOL. The postoperative targeted refraction for the right (non-dominant eye) and left eyes (dominant eye) planed - 0.33D and - 2.25D, respectively. Results: At 3 months after surgery, the corrected distance visual acuity (CDVA) with IOLs for the right and left eyes was 20/16 and 20/60, respectively, and the binocular visual acuity was 20/16 for distant vision and 20/25 for near vision. The CDVA for the right eye was: 20/13 × IOL = sph-0.25D and that for the left eye was: 20/13 × IOL = sph -1.75D D/cyl -0.50D/Ax170°. Although the exotropia was complicated by dissociated horizontal deviation and dissociated vertical deviation, there were no significant changes in ocular position before and after surgery. The patient was satisfied with achieving independence from spectacles and SCLs. Conclusion: The use of the crossed monovision method with monofocal IOLs in this patient and defective binocular function created a visual environment with no inconvenience in everyday life after secondary IOL implantation. In terms of secondary implant after amblyopia treatment, the IOL type or postoperative targeted refraction must be chosen to maintain or improve the visual environment obtained with the amblyopia treatment.

A case of pyloric gland adenoma with high-grade dysplasia in the duodenum arising from heterotopic gastric mucosa observed over 5 years.

Pyloric gland adenoma (PGA) in the duodenum is a rare gastric phenotype duodenal neoplasm. Although heterotopic gastric mucosa in the duodenum has been recognized as a benign lesion, it is a potential precursor of PGA and gastric phenotype adenocarcinoma. Herein, we present a case follow-up of endoscopic and histological changes in the PGA in the duodenum from low-grade to high-grade dysplasia. PGA was considered to arise from the heterotopic gastric mucosa, because the heterotopic gastric mucosa was observed in the initial examination. It is difficult to distinguish heterotopic gastric mucosa from PGAs, both endoscopically and histologically. This increase in size may be useful for their differentiation. Therefore, endoscopists should not underestimate the growth of the heterotopic gastric mucosa compared to that in the previous examination.

Antimicrobial resistance profiles of Campylobacter jejuni and Salmonella spp. isolated from enteritis patients in Japan.

Understanding the antimicrobial resistance of Campylobacter jejuni and Salmonella spp. isolated from patients with enteritis will aid in therapeutic decision-making. This study aimed to characterize C. jejuni and Salmonella spp. isolates from patients with enteritis. For C. jejuni, the resistance rates against ampicillin, tetracycline, and ciprofloxacin were 17.2%, 23.8%, and 46.4%, respectively. All the C. jejuni isolates were susceptible to erythromycin, which is recommended as a first-choice antimicrobial if Campylobacter enteritis is strongly suspected. C. jejuni was classified into 64 sequence types (STs), and the five major STs were ST22, ST354, ST21, ST918, and ST50. The ciprofloxacin-resistance rate of ST22 was 85.7%. For Salmonella, the resistance rates against ampicillin, cefotaxime, streptomycin, kanamycin, tetracycline, and nalidixic acid were 14.7%, 2.0%, 57.8%, 10.8%, 16.7%, and 11.8%, respectively. All the Salmonella spp. isolates were susceptible to ciprofloxacin. Therefore, fluoroquinolones are the recommended antimicrobials against Salmonella enteritis. S. Thompson, S. Enteritidis, and S. Schwarzengrund were the three most prevalent serotypes. The two cefotaxime-resistant isolates were serotyped as S. Typhimurium and were found to harbor blaCMY-2. The results of this study would help select antimicrobials for treating patients with Campylobacter and Salmonella enteritis.

A Salmonella enterica Serovar Oranienburg Clone Caused a Cluster of Bacteremia Cases in Persons With No Recognizable Underlying Diseases in Japan.

Background: Salmonella enterica subspecies enterica serovar Oranienburg (SO) is a foodborne pathogen but rarely causes systemic infections such as bacteremia. Between July and September 2018, bacteremia cases caused by SO were identified in 12 persons without any underlying medical conditions in the southern Kyushu area of Japan. Methods: Randomly amplified polymorphic DNA (RAPD) analysis was performed to investigate the genetic similarity of the 12 bacteremia-related strains and other Japanese isolates. Furthermore, a series of whole-genome sequence (WGS)-based phylogenetic analyses was performed with a global SO strain set (n = 1648). Results: The resolution power of RAPD was insufficient to investigate the genetic similarity between the bacteremia-related strains and other strains. WGS-based phylogenetic analyses revealed that the bacteremia-related strains formed a tight cluster along with 2 strains isolated from asymptomatic carriers in 2018 in the same area, with a maximum within-cluster single-nucleotide polymorphism (SNP) distance of 11. While several strains isolated in the United States and the United Kingdom were found to be closely related to the bacteremia-related strains, 2 strains isolated in 2016 in the southern Kyushu area were most closely related, with SNP distances of 4-11 and 5-10, and had the same plasmids as the bacteremia-related strains. Conclusions: The 12 bacteremia cases identified were caused by a single SO clone. As none of the bacteremia patients had any underlying diseases, this clone may be prone to cause bacteremia. Although further analyses are required to understand its virulence, particular attention should be given to this clone and its close relatives in the surveillance of nontyphoidal salmonellae.

Detection and molecular characterization of hepatitis E virus in clinical, environmental and putative animal sources.

Putative animal reservoirs and environmental samples were studied to investigate potential routes of transmission for indigenous hepatitis E virus (HEV) infection in Hokkaido, Japan. A total of 468 liver samples and 954 environmental samples were collected from 2003 to 2011 for this study. Four swine livers (1 %) were positive for HEV RNA; two strains belonged to genotype 3 and the other two strains were genotype 4. Genotype 3 HEV was detected in a sewage sample and a seawater sample. HEV strains derived from swine liver, seawater and raw sewage samples shared 93-100 % sequence similarity with human HEV strains.