A human respiratory tract-associated bacterium with an extremely small genome.

Recent advances in culture-independent microbiological analyses have greatly expanded our understanding of the diversity of unculturable microbes. However, human pathogenic bacteria differing significantly from known taxa have rarely been discovered. Here, we present the complete genome sequence of an uncultured bacterium detected in human respiratory tract named IOLA, which was determined by developing a protocol to selectively amplify extremely AT-rich genomes. The IOLA genome is 303,838 bp in size with a 20.7% GC content, making it the smallest and most AT-rich genome among known human-associated bacterial genomes to our best knowledge and comparable to those of insect endosymbionts. While IOLA belongs to order Rickettsiales (mostly intracellular parasites), the gene content suggests an epicellular parasitic lifestyle. Surveillance of clinical samples provides evidence that IOLA can be predominantly detected in patients with respiratory bacterial infections and can persist for at least 15 months in the respiratory tract, suggesting that IOLA is a human respiratory tract-associated bacterium.

Effects of Breastfeeding on Stress Measured by Saliva Cortisol Level and Perceived Stress.

Purpose: The effects of breastfeeding on postpartum depression symptoms and stress using physiological measures require investigation. Background: Breastfeeding suppresses the secretion of cortisol. Oxytocin levels correlate negatively with symptoms of postpartum depression. Aim: To investigate the effects of breastfeeding on stress and postpartum depression. Methods: We examined 79 breastfeeding women using the Edinburgh Postnatal Depression Scale, the Perceived Stress Scale-10, and the Breastfeeding Self-Efficacy Scale, and measured the salivary cortisol levels before and after breastfeeding. Findings: There was a negative correlation between the duration of suckling and changes in salivary cortisol levels following breastfeeding (r s = -0.333, p < 0.05). Salivary cortisol levels immediately following breastfeeding were significantly lower compared to mothers who used mixed feeding methods (p < 0.001). Breastfeeding mothers had lower perceived stress than mothers using mixed feeding methods (ß = -0.260, p < 0.05). There was no association between breastfeeding and postpartum depression; however, there was an association between postpartum depression and perceived stress (ß = 0.526, p < 0.001). Conclusion: Salivary cortisol levels significantly decreased following breastfeeding, with longer suckling times correlating with lower cortisol levels. Breastfeeding reduced stress and increased breastfeeding self-efficacy.

Changes of intravaginal microbiota and inflammation after self-replacement ring pessary therapy compared to continuous ring pessary usage for pelvic organ prolapse.

AIM: Continuous usage of a ring pessary for pelvic organ prolapse may cause a disturbance of intravaginal microbiota and intravaginal mucosal damage. To avoid the side effects of continuous ring pessary therapy, daily self-replacement of the ring pessary is recommended. The purpose of this study is to evaluate the outcomes of self-replacement versus continuous ring usage, by analysing clinical findings and intravaginal microbiota. METHODS: Thirty seven patients who managed self-replacement ring pessary therapy and 13 patients with continuous ring pessary therapy participated in this study. The clinical symptoms were checked at 1 month after the initial ring pessary insertion. The changes in the intravaginal microbiota were evaluated by conventional methods, i.e. pH in the vagina, Lactobacillary grade, Nugent score, inflammatory cell counts, and culture-based bacterial detection methods. In addition, our clone library method using 16S rRNA sequencing of vaginal fluid was performed. RESULTS: Patients were divided into four groups: self-replacement pre/post-menopause and continuous and pre/post-menopause. Five patients of the self-replacement group (n = 37) and all patients of the continuous use group (n = 13) complained of increased discharge. However, both the conventional methods and the clone library method revealed that the number of the self-replacement group patients who had abnormal intravaginal microbiota were not significantly different from that of the continuous use group. CONCLUSION: Daily self-replacement ring pessary therapy prevented adverse clinical symptoms. However, abnormal intravaginal microbiota was frequently observed during self-replacement of ring pessary therapy as with continuous usage. Regardless, pelvic examinations should be performed routinely.

Inactivation of human and avian influenza viruses by potassium oleate of natural soap component through exothermic interaction.

An influenza epidemic is still a problem despite the development of vaccines and anti-influenza drugs. Preventive measures such as handwashing are fundamental and important for counteracting influenza virus infection. In this study, we clarified the anti-influenza virus effects of surfactants, which are the main components of hand soaps for hand washing: potassium oleate (C18:1), sodium laureth sulfate (LES) and sodium lauryl sulfate (SDS). For a human influenza virus strain (H3N2), C18:1 reduced the infectivity by 4 logs or more, whereas LES and SDS reduced the infectivity by 1 log or less. Similar results were obtained when an avian influenza virus strain (H5N3) was used. The interaction between the surfactant and virus was then investigated by isothermal titration calorimetry. The LES-virus system showed a positive value of enthalpy changes (ΔH), meaning an exothermic interaction that indicated a hydrophobic interaction. In contrast, both the C18:1-virus system and the SDS-virus system showed negative values of ΔH, meaning an endothermic interaction that indicated an electrical interaction. The ΔH value of the C18:1-virus system was much higher than that of the SDS-virus system. A mixture of C18:1 and HA proteins similarly showed negative values of ΔH. These results indicate that influenza virus inactivation by a hydrophobic interaction of a surfactant with the viral envelope is insufficient to prevent infection, whereas inactivation by an electrical interaction of a surfactant with HA proteins is sufficient to prevent influenza virus infection.

Determining the Possible Etiology of Hospital-Acquired Pneumonia Using a Clone Library Analysis in Japan.

Obtaining precise etiological information regarding causative bacteria is important for the proper use of antimicrobials in hospital-acquired pneumonia (HAP), which is associated with a high rate of mortality. The aim of this study was to comparatively investigate the bacterial diversity in bronchoalveolar lavage fluid (BALF) in Japanese patients with HAP by the clone library method using the 16S rRNA gene. This study included Japanese patients with HAP who were treated at our hospital and referring hospitals. BALF specimens were obtained from pneumonia lesions identified on chest radiographs and/or computed tomography. Sputum specimens were also evaluated in patients with sputum production. Sixty-eight patients were ultimately enrolled. BALF cultivation revealed bacterial positivity in 53 of 68 (77.9%) patients, and Staphylococcus aureus (30.9%) was the most frequently isolated, followed by Pseudomonas aeruginosa (16.2%), and Escherichia coli (10.3%). In contrast, the clone library analysis identified the presence of some bacterial phenotype in 65 of 68 (95.6%) patients, and streptococci (16.2%), Corynebacterium species (11.8%), anaerobes (10.3%) were frequently detected as the predominant phylotypes. Both methods tended to detect S. aureus, Klebsiella pneumoniae, and E. coli in patients with late-onset pneumonia. In addition, the cases that phylotypes of S. aureus and P. aeruginosa were found to account for > 5% of the bacterial flora of each case were 42.9% and 72.7%, respectively. These results indicate that attention should be paid to the roles of gram-positive bacilli such as streptococci, Corynebacterium species and anaerobes, in addition to Gram-negative bacilli, in the pathogenesis of HAP.

Molecular Approaches to Studying Microbial Communities: Targeting the 16S Ribosomal RNA Gene.

Culture-independent methods to detect microorganisms have been developed in parallel with traditional culture-based methods ever since the classification of bacteria based on 16S rRNA gene sequences was advocated in the 1970s. The development and the prevalence of culture-independent molecular technologies have provided revolutionary progress in microbial studies. The development of these technologies contributes significantly to the research of microorganisms that cannot be detected by traditional methods such as culture-dependent methods.Many molecular methods targeting the 16S rRNA gene, such as fluorescence in situ hybridization (FISH), quantitative PCR, terminal restriction fragment length polymorphism (T-RFLP), denaturing-gradient gel electrophoresis (DGGE), clone library analysis, and next-generation DNA sequencing (NGS) technologies, have been applied to various microbial studies. Notably, the advent of NGS technologies enabled a large-scale research of the bacterial community. Many recent studies using the NGS technologies have revealed that a larger number of bacteria and taxa than previously thought inhabit various parts of the human body and various places on the earth. The principles and characteristics of each molecular method are different, and each method possesses individual advantages; for example target specificity, comprehensiveness, rapidness, and cost efficiency. Therefore it is important that the methods used in studies are suitable for the objective and materials. Herein, we highlights molecular approaches targeting the 16S rRNA gene in bacterial community analysis, and focuses on the advantages and limitations of each technology.

The Importance of Obligate Anaerobes and the Streptococcus anginosus Group in Pulmonary Abscess: A Clone Library Analysis Using Bronchoalveolar Lavage Fluid.

BACKGROUND: Appropriate antibiotic use reduces the mortality of patients with lung abscess; however, 40-60% of the bacterial etiologies in these patients have remained unknown with the culture methods. Obligate anaerobes and the Streptococcus anginosus group are common pathogens in lung abscess, but a precise evaluation of these bacteria by ordinary culture methods seems to be difficult due to upper respiratory tract contamination. OBJECTIVES: The aim of this study was to assess the microbiota of lung abscess by bronchoalveolar lavage fluid (BALF) using the molecular method in comparison to culture methods. METHODS: BALF samples obtained from the affected lesions and sputum samples of 59 patients with lung abscess were evaluated. The microbiota in BALF was analyzed according to the molecular method using the 16S ribosomal RNA gene. RESULTS: Fifty-six of the 59 BALF samples were positive in polymerase chain reaction analysis. Fusobacterium spp. (23.7%) were most frequently detected, followed by the S. anginosus group (15.3%), as the predominant phylotypes. Obligate anaerobes were detected in 42.4% of the BALF specimens as the predominant phylotypes, whereas the detection rate of obligate anaerobes was 13.6% by culture methods using BALF. In addition, the detection rate among those patients in whom the phylotype of obligate anaerobes was detected in >5% of the lung microbiota according to the molecular method was 86.5% in the 'mixed-bacterial' infection group. CONCLUSIONS: The findings by the molecular method suggest that obligate anaerobes play important roles in the pathogenesis of lung abscess and provide additional bacterial information regarding conventional culture methods.

The significance of oral streptococci in patients with pneumonia with risk factors for aspiration: the bacterial floral analysis of 16S ribosomal RNA gene using bronchoalveolar lavage fluid.

BACKGROUND: Aspiration pneumonia has been a growing interest in an aging population. Anaerobes are important pathogens, however, the etiology of aspiration pneumonia is not fully understood. In addition, the relationship between the patient clinical characteristics and the causative pathogens in pneumonia patients with aspiration risk factors are unclear. To evaluate the relationship between the patient clinical characteristics with risk factors for aspiration and bacterial flora in bronchoalveolar lavage fluid (BALF) in pneumonia patients, the bacterial floral analysis of 16S ribosomal RNA gene was applied in addition to cultivation methods in BALF samples. METHODS: From April 2010 to February 2014, BALF samples were obtained from the affected lesions of pneumonia via bronchoscopy, and were evaluated by the bacterial floral analysis of 16S rRNA gene in addition to cultivation methods in patients with community-acquired pneumonia (CAP) and healthcare-associated pneumonia (HCAP). Factors associated with aspiration risks in these patients were analyzed. RESULTS: A total of 177 (CAP 83, HCAP 94) patients were enrolled. According to the results of the bacterial floral analysis, detection rate of oral streptococci as the most detected bacterial phylotypes in BALF was significantly higher in patients with aspiration risks (31.0 %) than in patients without aspiration risks (14.7 %) (P = 0.009). In addition, the percentages of oral streptococci in each BALF sample were significantly higher in patients with aspiration risks (26.6 ± 32.0 %) than in patients without aspiration risks (13.8 ± 25.3 %) (P = 0.002). A multiple linear regression analysis showed that an Eastern Cooperative Oncology Group (ECOG) performance status (PS) of ≥3, the presence of comorbidities, and a history of pneumonia within a previous year were significantly associated with a detection of oral streptococci in BALF. CONCLUSIONS: The bacterial floral analysis of 16S rRNA gene revealed that oral streptococci were mostly detected as the most detected bacterial phylotypes in BALF samples in CAP and HCAP patients with aspiration risks, especially in those with a poor ECOG-PS or a history of pneumonia.

Overexpression of Peroxiredoxin 4 Affects Intestinal Function in a Dietary Mouse Model of Nonalcoholic Fatty Liver Disease.

BACKGROUND: Accumulating evidence has shown that methionine- and choline-deficient high fat (MCD+HF) diet induces the development of nonalcoholic fatty liver disease (NAFLD), in which elevated reactive oxygen species play a crucial role. We have reported that peroxiredoxin 4 (PRDX4), a unique secretory member of the PRDX antioxidant family, protects against NAFLD progression. However, the detailed mechanism and potential effects on the intestinal function still remain unclear. METHODS & RESULTS: Two weeks after feeding mice a MCD+HF diet, the livers of human PRDX4 transgenic (Tg) mice exhibited significant suppression in the development of NAFLD compared with wild-type (WT) mice. The serum thiobarbituric acid reactive substances levels were significantly lower in Tg mice. In contrast, the Tg small intestine with PRDX4 overexpression showed more suppressed shortening of total length and villi height, and more accumulation of lipid in the jejunum, along with lower levels of dihydroethidium binding. The enterocytes exhibited fewer apoptotic but more proliferating cells, and inflammation was reduced in the mucosa. Furthermore, the small intestine of Tg mice had significantly higher expression of cholesterol absorption-regulatory factors, including liver X receptor-α, but lower expression of microsomal triglyceride-transfer protein. CONCLUSION: Our present data provide the first evidence of the beneficial effects of PRDX4 on intestinal function in the reduction of the severity of NAFLD, by ameliorating oxidative stress-induced local and systemic injury. We can suggest that both liver and intestine are spared, to some degree, by the antioxidant properties of PRDX4.

Clinical impact of methicillin-resistant staphylococcus aureus on bacterial pneumonia: cultivation and 16S ribosomal RNA gene analysis of bronchoalveolar lavage fluid.

BACKGROUND: Determining whether methicillin-resistant Staphylococcus aureus (MRSA) is a true causative pathogen or reflective of colonization when MRSA is cultured from the respiratory tract remains important in treating patients with pneumonia. METHODS: We evaluated the bacterial microbiota in bronchoalveolar lavage fluid (BALF) using the clone library method with a 16S ribosomal RNA (rRNA) gene analysis in 42 patients from a pneumonia registry who had MRSA cultured from their sputum or BALF samples. Patients were divided into two groups: those treated with (Group A) or without (Group B) anti-MRSA agents, and their clinical features were compared. RESULTS: Among 248 patients with pneumonia, 42 patients who had MRSA cultured from the respiratory tract were analyzed (Group A: 13 patients, Group B: 29 patients). No clones of S. aureus were detected in the BALF of 20 out of 42 patients. Twenty-eight of 29 patients in Group B showed favorable clinical outcomes, indicating that these patients had non-MRSA pneumonia. Using a microflora analysis of the BALF, the S. aureus phylotype was predominant in 5 of 28 (17.9%) patients among the detected bacterial phylotypes, but a minor population (the percentage of clones ≤ 10%) in 19 (67.9%) of 28 patients. A statistical analysis revealed no positive relationship between the percentage of clones of the S. aureus phylotype and risk factors of MRSA pneumonia. CONCLUSIONS: The molecular method using BALF specimens suggests that conventional cultivation method results may mislead true causative pathogens, especially in patients with MRSA pneumonia. Further studies are necessary to elucidate these clinically important issues.

The first report: An analysis of bacterial flora of the first voided urine specimens of patients with male urethritis using the 16S ribosomal RNA gene-based clone library method.

OBJECTIVES: To analyse the bacterial flora of urine from patients with male urethritis using the clone library method. METHODS: Urine specimens from patients with urethritis were used. The bacterial flora was analysed according to the 16S ribosomal RNA gene-based clone library method. In addition, Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma urealyticum or Ureaplasma parvum were detected by the conventional PCR methods (TMA or real-time PCR) and data from the clone library and conventional PCR methods were compared. RESULTS: Among 58 urine specimens, 38 were successfully analysed using the clone library method. From the specimens, 2427 clones were evaluated and 95 bacterial phylotypes were detected. N. gonorrhoeae was detected from 6 specimens and as the predominant bacterial species in 5 specimens. M. genitalium was detected from 5 specimens and as the predominant bacterial species in 3 specimens. C. trachomatis was detected from 15 specimens using the TMA method, but was detected from only 1 specimen using the clone library method. U. parvum was detected from only 2 specimens using the clone library method. In addition, Haemophilus influenzae and Neisseria meningitidis were also detected in 8 and 1 specimens, respectively. Gardnerella vaginalis, which is a potential pathogen for bacterial vaginitis in women, was detected in 10 specimens. CONCLUSIONS: The clone library method can detect the occupancy rate of each bacteria species among the bacterial flora and may be a new method for bacterial analyses in male urethritis.

Successful Additional Corticosteroid Treatment in a Patient with Mycoplasma pneumoniae Pneumonia in whom a Monobacterial Infection was Confirmed by a Molecular Method Using Bronchoalveolar Lavage Fluid.

A 23-year-old Japanese woman was admitted to A hospital due to pneumonia. IgM for Mycoplasma pneumoniae was positive, and the patient was treated with imipenem/cilastatin, clindamycin, pazufloxacin and minocycline. However, both the chest radiological findings and the symptoms became exacerbated, and she was therefore transferred to our hospital. The bronchoalveolar lavage fluid was obtained, and a 16S rRNA gene sequencing analysis revealed a monobacterial infection of Mycoplasma pneumoniae. Therefore, corticosteroid treatment in addition to minocycline was administered, and the patients symptoms, laboratory data and chest radiographs improved. Corticosteroid therapy may therefore be considered for patients with refractory M. pneumoniae pneumonia.

Implantable cardioverter defibrillator infection due to Mycobacterium mageritense.

Rapidly growing non-tuberculous mycobacteria (RGM) are usually detected in blood cultures after 4-5 days of incubation, so it is important to differentiate RGM from contamination of commensal organisms on human skin. We report an unusual case of Mycobacterium mageritense bacteremia and infection of an implantable cardioverter defibrillator originally misidentified as Corynebacterium spp. or Nocardia spp. in gram-stained smears. 16S rRNA gene sequencing had utility in the definitive identification of isolates. We should be aware that RGM infection may exist in repeated implantable device infections.

Effects of pelvic organ prolapse ring pessary therapy on intravaginal microbial flora.

INTRODUCTION AND HYPOTHESIS: The purpose of this paper is to report our evaluation of changes in intravaginal microbial flora after ring pessary therapy for pelvic organ prolapse (POP) using conventional and our clone library method. METHODS: Thirteen patients with POP who were fitted with a ring pessary participated in this longitudinal study that incorporates data from before and 1 month after beginning ring pessary therapy. Changes in intravaginal microbial flora were evaluated by conventional methods, i.e., vaginal pH, lactobacillary grade (LAC grade), Nugent score, and culture-based bacterial detection methods. In addition, we performed our clone library method using 16S ribosomal RNA (rRNA) sequencing of vaginal fluid. RESULTS: Conventional methods revealed that most patients had abnormal intravaginal microbial flora. Mean numbers of detected bacterial species by the culture-based and our clone library method were 3.1 (1-6) and 11.8 (1-25), respectively. Our clone library method showed that Lactobacillus spp. increased in four and decreased in two cases after ring pessary therapy but reappeared after therapy in two cases; no Lactobacillus spp. were detected in five cases. CONCLUSIONS: Our study showed that ring pessary therapy did not always disturb intravaginal microbial flora, especially for patients with Lactobacillus spp. prior to ring pessary insertion. Anaerobic circumstances in the vagina after therapy seem to have induced the growth of anaerobic bacteria.

Glycopeptidolipid of Mycobacterium smegmatis J15cs Affects Morphology and Survival in Host Cells.

Mycobacterium smegmatis has been widely used as a mycobacterial infection model. Unlike the M. smegmatis mc(2)155 strain, M. smegmatis J15cs strain has the advantage of surviving for one week in murine macrophages. In our previous report, we clarified that the J15cs strain has deleted apolar glycopeptidolipids (GPLs) in the cell wall, which may affect its morphology and survival in host cells. In this study, the gene causing the GPL deletion in the J15cs strain was identified. The mps1-2 gene (MSMEG_0400-0402) correlated with GPL biosynthesis. The J15cs strain had 18 bps deleted in the mps1 gene compared to that of the mc(2)155 strain. The mps1-complemented J15cs mutant restored the expression of GPLs. Although the J15cs strain produces a rough and dry colony, the colony morphology of this mps1-complement was smooth like the mc(2)155 strain. The length in the mps1-complemented J15cs mutant was shortened by the expression of GPLs. In addition, the GPL-restored J15cs mutant did not survive as long as the parent J15cs strain in the murine macrophage cell line J774.1 cells. The results are direct evidence that the deletion of GPLs in the J15cs strain affects bacterial size, morphology, and survival in host cells.

Possible role of anaerobes in the pathogenesis of nontuberculous mycobacterial infection.

BACKGROUND AND OBJECTIVE: Recent advances in cultivation-independent molecular biological modalities for detecting bacterial species have indicated that several bacterial species may play a role in the pathogenesis of certain infectious diseases. The aim of this study was to evaluate the role of bacterial flora in the pathogenesis of nontuberculous mycobacteriosis (NTM) using a bacterial floral analysis of bronchoalveolar lavage fluid (BALF) with 16S rRNA gene sequencing in patients with bronchiectasis. METHODS: Fifty-eight patients with bronchiectasis evaluated using chest computed tomography were enrolled. BALF obtained from the most affected lung lesions was evaluated using culture and culture-independent methodologies. Approximately 600 bp of the bacterial 16S rRNA gene (E341F-E907R) was amplified via polymerase chain reaction using universal primers, and clone libraries were constructed. Nucleotide sequences of 96 randomly chosen clones for each specimen were determined, and the homology was searched using a basic local alignment search tool to determine the bacterial phylotypes and their proportions (bacterial floral analysis) in each specimen. RESULTS: Twenty-nine patients with bronchiectasis were diagnosed with NTM based on culture-based methods using Ogawa medium. The molecular method showed a significantly high rate of anaerobes among the patients with NTM compared with that observed in the bronchiectasis patients without NTM. In addition, findings of collapse/consolidation were significantly related to the proportion of Prevotella species in the BALF samples determined using the molecular method (P < 0.001). CONCLUSION: Given the results of the present study, anaerobes may play an important role in the pathogenesis of bronchiectasis in patients with NTM.

Bacteriological assessment of healthcare-associated pneumonia using a clone library analysis.

BACKGROUND: The causative pathogens of healthcare-associated pneumonia (HCAP) remain controversial, and the use of conventional cultivation of sputum samples is occasionally inappropriate due to the potential for oral bacterial contamination. It is also sometimes difficult to determine whether methicillin-resistant Staphylococcus aureus (MRSA) is a true causative pathogen of HCAP. METHODS: We evaluated the bacterial diversity in bronchoalveolar lavage fluid (BALF) using molecular and cultivation methods in 82 HCAP patients. BALF specimens were obtained from the lesions of pneumonia using bronchoscopy. The bacterial flora was analyzed according to the clone library method using amplified fragments of the 16S ribosomal RNA gene with universal primers. In addition, sputum cultures and the above specimens were assessed. RESULTS: Eighty (97.6%) of the 82 BALF samples obtained from the patients with HCAP showed positive polymerase chain reaction results. The predominant phylotypes detected in the BALF in this study included bacteria common in cases of community- and hospital-acquired pneumonia. In addition, the phylotypes of streptococci and anaerobes were detected in 19 (23.2%) and 8 (9.8%) cases, respectively. In particular, phylotypes of streptococci were highly detected among the patients 75 of age or older. Staphylococcus aureus was cultured in 23 (28.0%) cases using conventional cultivation methods and detected in only 6 (7.3%) cases as predominant phylotypes according to the clone library method. CONCLUSIONS: The clone library analysis of BALF in the HCAP patients detected heterogeneous bacteria and a high incidence of streptococci compared with that observed using cultivation methods. In addition, the results of our study may indicate a lower incidence of MRSA than previously expected in HCAP patients.

Japanese men's success in altered fatherhood role in a foreign country.

This study explores the meaning of the lived experience of childbirth and parenting of Japanese men who became fathers in a foreign country. Japanese men have been raised to maintain very strict gender roles, excluding them from sharing with their wives the experience of childbirth and the day-to-day parenting of young children. The study employed a descriptive phenomenological approach with in-depth interviews. Participants included nine Japanese men born and raised in Japan who were living in Honolulu. Three theme categories emerged from the data: "making active efforts in preparation for childbirth in a foreign country"; "challenges in pregnancy, childbirth, child care, and as husbands or partners"; and "challenges in transition to parenthood." Japanese men successfully altered their transitional and authoritarian gender role to a family orientated social structure, under the influence of Western values, when living in foreign country. By spending more time with their new family, they acknowledged the processes of becoming a father. The ability to adapt their expectations of fatherhood in line with Western values was enhanced by the support of coworkers, their mature age, rich educational background, and the personal financial resources of the male participants in the study.

Refractory primary hepatic actinomycosis with direct infiltration to the diaphragm and thorax: the usefulness of contrast-enhanced ultrasonography.

An 80-year-old man was admitted to our hospital with a diagnosis of primary hepatic actinomycosis determined based on a percutaneous aspiration biopsy. The abscesses and state of liquefaction were easily and effectively visualized on contrast-enhanced ultrasonography. Ampicillin/sulbactam was administered; however, lesions of hepatic actinomycosis suddenly infiltrated the diaphragm and right thorax six months later. A drainage tube was inserted into the right thoracic space, and the pleural effusion gradually decreased. The patient received continuous antibiotic therapy for nearly two years and remained free of hepatic actinomycosis on follow-up more than one year later.