Diagnostic Challenges in Pulmonary Embolism in Young Adults: Thrombosis Associated With Cytomegalovirus and Mycoplasma pneumoniae.

A 23-year-old man presented with a fever, shaking chills, headaches, nausea, and a dry cough. Investigations showed lymphocytic leukocytosis with atypical lymphocytes in a blood smear. Liver function test results, D-dimer concentrations, and fibrin degradation product concentrations were greatly elevated. Computed tomography of the whole body with contrast showed hepatosplenomegaly with splenic infarction and bilateral pulmonary embolism without deep vein thrombosis. Cytomegalovirus (CMV) immunoglobulin M, and serum CMV pp65 antigenemia were positive, and serum Mycoplasma pneumoniae (M. pneumoniae) antibody was also highly positive. These results suggested the diagnosis of co-infection of CMV and M. pneumoniae complicated by systemic arteriovenous thrombosis, which resulted in pulmonary embolism and splenic infarction. After he started edoxaban tosilate hydrate for the thrombosis, his symptoms resolved in a few days. To the best of our knowledge, this is the first case of co-infection of CMV and M. pneumoniae leading to pulmonary embolism and splenic infarction.

Eosinophil-mediated inflammation in the absence of eosinophilia.

The increase of eosinophil levels is a hallmark of type-2 inflammation. Blood eosinophil counts act as a convenient biomarker for asthma phenotyping and the selection of biologics, and they are even used as a prognostic factor for severe coronavirus disease 2019. However, the circulating eosinophil count does not always reflect tissue eosinophilia and vice versa. The mismatch of blood and tissue eosinophilia can be seen in various clinical settings. For example, blood eosinophil levels in patients with acute eosinophilic pneumonia are often within normal range despite the marked symptoms and increased number of eosinophils in bronchoalveolar lavage fluid. Histological studies using immunostaining for eosinophil granule proteins have revealed the extracellular deposition of granule proteins coincident with pathological conditions, even in the absence of a significant eosinophil infiltrate. The marked deposition of eosinophil granule proteins in tissue is often associated with cytolytic degranulation. Recent studies have indicated that extracellular trap cell death (ETosis) is a major mechanism of cytolysis. Cytolytic ETosis is a total cell degranulation in which cytoplasmic and nuclear contents, including DNA and histones that act as alarmins, are also released. In the present review, eosinophil-mediated inflammation in such mismatch conditions is discussed.

Clonal hematopoiesis in adult pure red cell aplasia.

Idiopathic pure red cell aplasia (PRCA) and secondary PRCA associated with thymoma and large granular lymphocyte leukemia are generally considered to be immune-mediated. The PRCA2004/2006 study showed that poor responses to immunosuppression and anemia relapse were associated with death. PRCA may represent the prodrome to MDS. Thus, clonal hematopoiesis may be responsible for treatment failure. We investigated gene mutations in myeloid neoplasm-associated genes in acquired PRCA. We identified 21 mutations affecting amino acid sequences in 11 of the 38 adult PRCA patients (28.9%) using stringent filtering of the error-prone sequences and SNPs. Four PRCA patients showed 7 driver mutations in TET2, DNMT3A and KDM6A, and 2 PRCA patients carried multiple mutations in TET2. Five PRCA patients had mutations with high VAFs exceeding 0.3. These results suggest that clonal hematopoiesis by stem/progenitor cells might be related to the pathophysiology of chronic PRCA in certain adult patients.

How to detect eosinophil ETosis (EETosis) and extracellular traps.

Eosinophils are short-lived and comprise only a small population of circulating leukocytes; however, they play surprisingly multifunctional roles in homeostasis and various diseases including allergy and infection. Recent research has shed light on active cytolytic eosinophil cell death that releases eosinophil extracellular traps (EETs) and total cellular contents, namely eosinophil extracellular trap cell death (EETosis). The pathological contribution of EETosis was made more cogent by recent findings that a classical pathological finding of eosinophilic inflammation, that of Charcot-Leyden crystals, is closely associated with EETosis. Currently no gold standard methods to identify EETosis exist, but "an active eosinophil lysis that releases cell-free granules and net-like chromatin structure" appears to be a common feature of EETosis. In this review, we describe several approaches that visualize EETs/EETosis in clinical samples and in vitro studies using isolated human eosinophils. EETs/EETosis can be observed using simple chemical or fluorescence staining, immunostaining, and electron microscopy, although it is noteworthy that visualization of EETs is greatly changed by sample preparation including the extracellular space of EETotic cells and shear flow. Considering the multiple aspects of biological significance, further study into EETs/EETosis is warranted to give a detailed understanding of the roles played in homeostasis and disease pathogenesis.

High-throughput sequencing of IgG B-cell receptors reveals frequent usage of the rearranged IGHV4-28/IGHJ4 gene in primary immune thrombocytopenia.

Primary immune thrombocytopenia (ITP) is an acquired form of thrombocytopenia caused by IgG anti-platelet autoantibodies and represents an organ-specific autoimmune disorder. Although the glycoprotein (GP)IIb/IIIa and GPIb/IX have been shown to be targets for autoantibodies, the antigen specificity of autoantibodies is not fully elucidated. To identify the characteristics of IgG B-cell receptor (BCR) repertoires in ITP, we took advantage of adaptor-ligation PCR and high-throughput DNA sequencing methods for analyzing the clone-based repertoires of IgG-expressing peripheral blood B cells. A total of 2,009,943 in-frame and 315,469 unique reads for IGH (immunoglobulin heavy) were obtained from twenty blood samples. Comparison of the IGHV repertoires between patients and controls revealed an increased usage of IGHV4-28 in ITP patients. One hundred eighty-six distinct IGHV4-28-carrying sequences were identified in ITP patients and the majority of these clones used an IGHJ4 segment. The IGHV4-28/IGHJ4-carrying B-cell clones were found in all ITP patients. Oligoclonal expansions of IGHV4-28/IGHJ4-carrying B cells were accompanied by multiple related clones with single amino substitution in the CDR3 region suggesting somatic hypermutation. Taken together, the expansion of IGHV4-28/IGHJ4-carrying IgG-expressing B cells in ITP may be the result of certain antigenic pressure and may provide a clue for the immune pathophysiology of ITP.

Left ventricular assist device-associated endocarditis involving multiple clones of Staphylococcus aureus with distinct antimicrobial susceptibility patterns.

The case of a patient with left ventricular assist device (LVAD)-associated endocarditis involving multiple clones of Staphylococcus aureus is presented. Different clones with distinct colony morphology were identified from blood cultures collected on the same day and showed diverse antimicrobial resistance patterns. In addition, a difference in antimicrobial susceptibility was observed even within an identical clone recovered 400 days apart due to the loss of SCCmec for methicillin and modification of the 23S rRNA target site for linezolid during a long-term treatment course.

Anti-drug Antibodies Against a Novel Humanized Anti-CD20 Antibody Impair Its Therapeutic Effect on Primary Biliary Cholangitis in Human CD20- and FcγR-Expressing Mice.

There is considerable interest in expanding B cell-targeted therapies in human autoimmune diseases. However, clinical trials in human primary biliary cholangitis (PBC) using a chimeric antibody against human CD20 (hCD20) have showed limited efficacy. Two potential explanations for these disappointing results are the appearance of anti-drug antibodies (ADAs) and the high frequency of patients with moderate PBC or patients who had failed ursodeoxycholic acid treatment. Here, we studied a novel humanized IgG1 antibody against hCD20 and explored its efficacy in early stage PBC using a well-defined murine model. We developed a unique murine model consisting of dnTGF-ßRII mice expressing hCD20 and human Fcγ receptors (hFcγRs). Beginning at 4-6 weeks of age, equivalent to stage I/II human PBC, female mice were given weekly injections of an anti-hCD20 antibody (TKM-011) or vehicle control, and monitored for liver histology as well as a broad panel of immunological readouts. After 16 weeks' treatment, we observed a significant reduction in portal inflammation, a decrease in liver-infiltrating mononuclear cells as well as a reduction in liver CD8+ T cells. Importantly, direct correlations between numbers of liver non-B cells and B cells (r = 0.7426, p = 0.0006) and between numbers of liver memory CD8+ T cells and B cells (r = 0.6423, p = 0.0054) were apparent. Accompanying these changes was a dramatic reduction in anti-mitochondrial antibodies (AMAs), interleukin (IL)-12p40 and IL-5, and elevated levels of the anti-inflammatory chemokine CXCL1/KC. In mice that developed ADAs, clinical improvements were less pronounced. Sustained treatment with B cell-targeted therapies may broadly inhibit effector pathways in PBC, but may need to be administered early in the natural history of PBC.

Urine Xanthine Crystals in Tumor Lysis Syndrome.

Urine xanthine crystals are remarkably rare but can be observed by routine urine microscopy. We report the results of a 67-year-old man with T-cell-prolymphocytic leukemia whose urine contained xanthine crystals after chemotherapy and prophylactic administration of febuxostat. Accumulation of xanthine was due to tumor lysis syndrome causing a massive release of DNA. The metabolized DNA caused an increase of xanthine, which was not readily converted to uric acid by xanthine oxidase because of febuxostat inhibition of this enzyme.

Molecular epidemiology of Klebsiella pneumoniae K1 and K2 isolates in Japan.

Although severe infections caused by hypervirulent Klebsiella pneumoniae isolates, such as K1 isolates belonging to sequence type (ST) 23, have been a significant problem in Asian countries, epidemiology of these isolates in Japan remains unclear. We performed a nationwide molecular epidemiological study of K. pneumoniae K1 and K2 isolates in Japan. Of the 259K. pneumoniae isolates collected, 14 and 16 isolates were identified as capsular genotypes K1 and K2, respectively. All K1 isolates were ST23 or its closely related clones and showed high genetic similarity by pulsed-field gel electrophoresis (PFGE) and the DiversiLab system (DL). K2 isolates, belonging to ST14, ST25, ST65, ST86, and ST110, were more genetically diverse than K1 isolates. Isolates belonging to a specific ST showed identical virulence gene profiles with a few exceptions. PFGE and DL results using K1 and K2 isolates were generally in agreement.

ICAM-1 upregulation is not required for retinoic acid-induced human eosinophil survival.

Active metabolites of vitamin A, retinoic acids (RAs), are known to play critical roles in mucosal immune responses and dramatically inhibit human eosinophil apoptosis, but the detailed mechanisms have not been elucidated. We previously screened for ICAM-1 (CD54) upregulation in RA-stimulated human eosinophils by gene microarray analysis. As ICAM-1 induction and activation were observed to have a role in maintenance of eosinophil survival, we tested the hypothesis that RAs prolong eosinophil survival through ICAM-1 outside-in signaling. Blood-derived isolated eosinophils cultured with 9-cis RA and all-trans RA showed significant upregulation of ICAM-1 mRNA and cell surface expression. TTNPB, a retinoic acid receptor agonist, also induced ICAM-1 expression, while HX630, a retinoid X receptor agonist, did not. Furthermore, an RAR antagonist, HX531, completely inhibited the effect of RAs. Upregulated ICAM-1 was associated with altered kinetics of Akt, ERK, and p38 MAP kinase phosphorylation through ICAM-1 cross-linking, but an ICAM-1-blocking antibody did not affect RA-mediated cell survival. These findings indicate that RAs induce functional ICAM-1 expression through RARs, but the induced ICAM-1 does not contribute to prolongation of eosinophil survival.

The effect of hepatocyte growth factor on secretory functions in human eosinophils.

Hepatocyte growth factor (HGF), originally identified as a potent mitogen for mature hepatocytes, is now recognized as a humoral mediator in inflammatory and immune responses. Previous studies indicated that HGF negatively regulated allergic airway inflammation. In view of eosinophils playing a role in the pathogenesis of asthma, especially in airway remodeling as a rich source of pro-fibrogenic mediators, the effects of HGF on the different types of eosinophil secretory functions were examined in this study. We found that HGF significantly inhibited IL-5-induced secretion of TGF-ß and VEGF from human eosinophils. The inhibitory effect is not associated with TGF-ß transcription; rather, it is associated with ultrastructural granule emptying and loss of intracellular TGF-ß contents, indicating HGF inhibits the process of piecemeal degranulation. The effect of HGF on extracellular trap cell death (ETosis) that mediates cytolytic degranulation was also investigated; however, immobilized IgG- or phorbol myristate acetate-induced ETosis was only minimally attenuated by HGF. These results reveal the effect of HGF on the distinct pathways of eosinophil secretory functions and also provide novel insights into the role of HGF in the pathogenesis of allergic inflammation.

[Viewpoint: Skill Certifications for Japanese Medical Technologists].

With the development of medicine, the field of clinical laboratory medicine evolves rapidly, and it will be more specialized in the near future. Medical technologists are required to hone their skills and knowledge, in order to keep up with the evolution. In recent years, board certifications by several medical societies are considered to indicate the skills of medical technologists. The number of board-certified medical technologists in populated areas such as Tokyo, Kanagawa, Osaka, and Fukuoka is greater than in less populated areas such as Kyusyu and Tohoku. The rate of certified medical technologists among prefectures is the highest in Mie (10.1%), followed by Nagasaki (8.8%). Tokyo, Ishikawa, Kyoto, and Osaka have acquisition rates greater than 7%. In contrast, prefectures of Miyazaki, Kumamoto, Yamanashi, and Akita have low acquisition rates of less than 4%. Being certified is not only an opportunity for personal career advancement, but also a chance to improve the laboratory. More technologists are being certified in our laboratory, and we are encouraging a future increase in their number. However, there are some problems to be overcome. Assignment of competent staff and long-term and premeditated rotation are considered to be important for staff to find the work rewarding, and the laboratory to be trusted by physicians.

Worldwide Lineages of Clinical Pneumococci in a Japanese Teaching Hospital Identified by DiversiLab System.

Pneumococcal Molecular Epidemiology Network (PMEN) clones are representatives of worldwide-spreading pathogens. DiversiLab system, a repetitive PCR system, has been proposed as a less labor-and time-intensive genotyping platform alternative to conventional methods. However, the utility and analysis parameters of DiversiLab for identifying worldwide lineages was not established. To evaluate and optimize the performance of DiversiLab for identifying worldwide pneumococcal lineages, we examined 245 consecutive isolates of clinical Streptococcus pneumoniae from all age-group patients at a teaching hospital in Japan. The capsular swelling reaction of all isolates yielded 24 different serotypes. Intensive visual observation (VO) of DiversiLab band pattern difference divided all isolates into 73 clusters. Multilocus sequence typing (MLST) of representative 73 isolates from each VO cluster yielded 51 different STs. Among them, PMEN-related lineages accounted for 63% (46/73). Although the serotype of PMEN-related isolates was identical to that of the original PMEN clone in 70% (32/46), CC156-related PMEN lineages, namely Greece(6B)-22 and Colombia(23F)-26, harbored various capsular types discordant to the original PMEN clones. Regarding automated analysis, genotyping by extended Jaccard (XJ) with a 75% similarity index cutoff (SIC) showed the highest correlation with serotyping (adjusted Rand's coefficient, 0.528). Elevating the SIC for XJ to 85% increased the discriminatory power sufficient for distinguishing two major PMEN-related isolates of Taiwan(19F)-14 and Netherlands(3)-31. These results demonstrated a potential utility of DiversiLab for identifying worldwide lineage of pneumococcus. An optimized parameters of automated analysis should be useful especially for comparison for reference strains by "identification" function of DiversiLab.

Neurosyphilis Mimicking Ramsay Hunt Syndrome.

A 36-year-old man presented with facial nerve palsy, hearing loss, vertigo and headache. He was initially diagnosed with Ramsay Hunt syndrome and treated with a systemic steroid and valaciclovir; however, his symptoms deteriorated. Serum rapid plasma reagin (RPR) and treponema pallidum hemagglutination tests were positive. Cerebrospinal fluid analysis revealed an elevated white blood cell count and positive RPR, confirming the diagnosis of neurosyphilis. Penicillin G (PCG) was administered, and his facial nerve function and headache improved. However, left-side hearing loss worsened temporarily, which was assumed to be a Jarisch-Herxheimer reaction. Betamethasone was administered along with PCG, and he recovered completely.

Incorrect diagnosis of Clostridium difficile infection in a university hospital in Japan.

Physicians often fail to suspect Clostridium difficile infection (CDI) and many microbiology laboratories use suboptimal diagnostic techniques. To estimate the extent of and reasons for incorrect diagnosis of CDI in Japan, we investigated toxigenic C. difficile isolated from all stool culture samples and clinical course. Over a 12-month period in 2010, all stool culture samples (n = 975) submitted from inpatients in a university hospital in Japan were cultured for C. difficile and routine microbiological testing was conducted. In total, 177 C. difficile isolates were recovered, and 127 isolates were toxigenic. Among the toxin-A-positive/toxin-B-positive isolates, 12 were also positive for the binary toxin gene. However, clinically important ribotypes, such as 027 and 078, were not identified. A total of 58 (45.7%) cases with toxigenic C. difficile had unformed stool, and the incidence CDI was 1.6 cases per 10,000 patient-days. Of these 58 cases, 40 were not diagnosed in routine testing due to a lack of clinical suspicion (24.1%, 14/58) or a negative C. difficile toxin assay result (44.8%, 26/58). A stool toxin assay was performed in 54 patients (78.2%, 54/69) who did not have unformed stool. The present study demonstrated that a significant number of CDI cases in Japan might be overlooked or misdiagnosed in clinical practice due to a lack of clinical suspicion and limitations of microbiological testing for CDI in Japan. Providing education to promote awareness of CDI among physicians is important to improve the accuracy of diagnosis in Japan.

A fatal infection caused by sequence type 398 methicillin-resistant Staphylococcus aureus carrying the Panton-Valentine leukocidin gene: A case report in Japan.

Methicillin-resistant Staphylococcus aureus (MRSA) has now been recognized as a common pathogen in the community. Sequence type (ST) 398 MRSA is generally considered as an emerging zoonotic agent spreading among livestock and personnel who have direct contact with animals, mainly in Europe. A 37-year-old Chinese woman receiving steroid therapy for systemic lupus erythematosus with general fatigue and myalgia was brought to the emergency department in critical condition. Her condition deteriorated despite aggressive management and she died on day 7. Her blood culture revealed ST398 MRSA-SCCmec V with Panton-Valentine Leukocidin (PVL) gene. This is the first case report of a fatal infection caused by this lineage. According to the results of molecular analyses, the isolate from this particular patient's blood was genetically close to a lineage detected in China, and is less likely to be related to an animal-associated lineage.

Molecular analysis of the integrons of metallo-ß-lactamase-producing Pseudomonas aeruginosa isolates collected by nationwide surveillance programs across Japan.

BACKGROUND: We investigate the evolving molecular epidemiology of metallo-ß-lactamase (MBL)-producing Pseudomonas aeruginosa isolates collected in a 100 institution, nationwide surveillance study in Japan from 2004 to 2006. RESULTS: MBL-producers were detected in 23/996 isolates (2.3%) in 2004 and 21/992 (2.1%) in 2006. Antimicrobial resistance (specifically, carbapenem resistance) rates between two periods did not differ significantly. MBL-producers were more prevalent in urinary tract isolates. bla IMP-1 group was the most predominant (38 isolates, 80%), followed by 3 bla IMP-7, 2 bla IMP-11 group, and 1 bla VIM-1. All MBL genes were identified in 16 different class 1 integrons, most of which were novel to INTEGRALL database. A total of 17 isolates of sequence type (ST) 235, a recognized worldwide drug-resistant lineage, were distributed in 5 geographic regions across Japan. ST235 isolates included a sublineage associated with In113-like integron. ST357 was identified in 14 isolates, 9 of which harboring a sole bla IMP-1 gene cassette (In994) were recovered from Chugoku region in 2004. ST357 isolates with bla IMP-11 group or ST235 with bla IMP-7 emerged in 2006. We also report for the first time the presence of novel fosI gene cassette in strains other than Mycobacterium spp. CONCLUSIONS: Our data give an important "snapshot" of the molecular characteristics and dynamics of MBL-producing lineages in P. aeruginosa in Japan. The significant association of specific genotypes and integrons implies that dissemination and transmission of the preexisting resistant lineage, rather than horizontal gene transfer in situ, might largely explain their endemicity.