In addition to tube drains, pleural empyema is treated with antibiotics and anti-inflammatory drugs. We aimed to evaluate the anti-inflammatory activity of roflumilast combined with linezolid in a rat model of pleural empyema induced by Staphylococcus aureus. A total of 40 rats were divided into 7 groups: sham (n = 4), S. aureus inoculation (n = 6), S. aureus + 10 mg/kg linezolid (n = 6), S. aureus + 5 mg/kg roflumilast (n = 6), S. aureus + 10 mg/kg linezolid + 5 mg/kg roflumilast (n = 6), S. aureus + 10 mg/kg roflumilast (n = 6), and S. aureus + 10 mg/kg linezolid + 10 mg/kg roflumilast (n = 6). Animals were administered linezolid 1 h before and 12 h after inoculation with S. aureus. Roflumilast was administered orally as a single dose 30 min before inoculation with S. aureus. Compared to linezolid treatment alone, linezolid combined with 5 mg/kg roflumilast significantly improved TNF-α, IL-1β, vasodilation/congestion, and tissue/pleural polynuclear leukocyte (PNL) infiltration (p < 0.05). Linezolid combined with 10 mg/kg roflumilast also provided a significant improvement in TNF-α, IL-1β, IL-6, endothelin-1, vasodilation/congestion, mesothelial cell damage, lung tissue PNL, and pleural PNL compared to linezolid alone (p < 0.05). Due to its anti-inflammatory effects and significant impact on recovery, roflumilast can be used in conjunction with antibiotherapy for the treatment of pleural empyema.
In this study, umesu phenolics were purified from the salt extracts of Japanese apricot (Nanko-mume cultivar of Prunus mume Sieb. et Zucc.). Characterization of umesu phenolics revealed that, when added to the culture media of the infected cells, they inhibited the multiplication of influenza and many other RNA and DNA viruses. In addition to these antiviral activities, the phenolics significantly decreased the plating efficiency of influenza virus, if present in the virus inoculum. More drastic effects were observed in terms of virucidal activity; the infectivity of several strains of influenza viruses decreased less than 0.001 when they were incubated with 4 mg/ml phenolics at 30 ℃ for 5 min. The virucidal activity of phenolics was found to be more remarkable in acidic conditions; however, the activity was not merely a result of the acidity of the phenolics. These results clearly support the antiviral and virucidal activities of the umesu phenolics against influenza viruses and suggest their potential pharmacological usefulness as disinfectants or preventive medicine against superficial infections, such as the respiratory infections.
The aim of this study was to explore whether there was any specific genotype responsible for the high prevalence of Mycoplasma pneumoniae infection in children. A total of 247 M. pneumoniae-DNA positive clinical specimens including 200 from children and 47 from adults, collected in Beijing, China, during the same period, were analyzed. We performed P1-restriction fragment length polymorphism analysis (RFLP), multi-locus variable number tandem repeat analysis (MLVA) and detected the macrolide resistance-associated mutations in 23S rRNA of the clinical specimens. In the present study, we observed P1 genotype 1 and MLVA type M4-5-7-2 accounted for the majority of the cases across all ages in Beijing. Macrolide resistance-associated mutants of M. pneumoniae were also at a high level with 90.5% (181/200) in children and 76.6% (36/47) in adults. However, more diverse genotypes and a higher prevalence of macrolide resistance-associated mutations were found in the pediatric specimens. Further investigations are warranted to help to explain the difference of morbidity and molecular characteristics across the demographic spectrum.
Rabies as an endemic disease in most Asian and African countries, especially in remote areas, and requires a reliable diagnostic method. This study aimed to develop a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for rapid detection of rabies virus RNA in the brain samples, compared to SYBR Green real time RT-PCR test as a molecular technique and direct fluorescent antibody test as a serological method. In this study, RT-LAMP was developed to diagnose rabies. Six primers were designed based on the nucleoprotein (N) of rabies virus. The sensitivity and specificity of SYBR Green real-time RT-PCR and RT-LAMP methods were also determined.
RT-LAMP was optimized at 58 ℃ for 60 min. The sensitivity and specificity of RT-LAMP and SYBR Green real-time RT-PCR were 91.2% and 84.2%, and 94.12% and 88.9%, respectively. The slight difference between the sensitivity and specificity of RT-LAMP and that of SYBR Green Real-Time RT-PCR demonstrated that RT-LAMP could be used as a reliable and cost-effective method for the diagnosis of rabies.
Rotavirus A (RVA) has been detected in patients with gastroenteritis even after vaccine introduction in Japan. To investigate circulating RVA strains, RVA-positive stool specimens obtained in Tokyo in 2017 and 2018 were analyzed using next-generation sequencing. A total of 50 and 21 RVA samples were obtained in 2017 and 2018, respectively. In 2017, G2P (40.0%) was the most prevalent strain, followed by G3P (DS-1-like) (28.0%), G8P (10.0%), G3P (Wa-like) (8.0%), G9P-E1 (8.0%), and mixed infection (6.0%). In 2018, G3P (DS-1-like) (28.6%) and G9P-E2 (28.6%) were the most prevalent strains, followed by G9P-E1 (19.0%), G2P (9.5%), G8P (9.5%), and mixed infection (4.8%). Six G9P-E2 strains detected in 2018 showed an atypical genotype constellation (G9P-I1-R1-C1-M1-A1-N1-T1-E2-H1), which had not been reported previously. Phylogenetic analyses suggested that the RVA virus was generated by inter-genogroup reassortment between commonly circulating G9P and G2P strains in Japan. The G9P strain seemed to be reassorted with only the NSP4 gene of the E2 genotype of the G2P strain. Since this newly-emerged G9P-E2 virus was detected in different locations in Tokyo, the virus appears to have already begun to spread to a wider area.
A 10-year-old girl was confirmed, by laboratory tests, to be infected with the H5N6 subtype of the highly pathogenic avian influenza virus in Suzhou city in November, 2018 (JSSZ01). The hemagglutinin (HA) gene of this H5N6 virus belonged to the 184.108.40.206 H5 clade, and the HA linkage peptide of the JSSZ01 strain was RERRRKR↓GLF with multiple basic amino acids. Q226L and G228S mutations were not observed, but S128P, S137A, and T160A substitutions were identified in the receptor binding sites. The resistance mutation D198N in the neuraminidase (NA) protein was also identified in this strain. Additionally, an 11 amino acid (AA positions 59–69) deletion was identified in the NA stalk region. Most genes of JSSZ01 exhibited highest identity with previously characterized H5N6 viruses, but its PA segment sequence was highly similar to previously identified avian H3 viruses (Accession Number: EPI596567 or EPI590058), indicating that JSSZ01 may be created by gene reassortment.
Human gnathostomiasis, which is endemic in Mexico, is a worldwide health concern. It is mainly caused by the consumption of raw or insufficiently cooked fish containing the advanced third-stage larvae (AL3A) of Gnathostoma species. The diagnosis of gnathostomiasis is based on epidemiological surveys and immunological diagnostic tests. When a larva is recovered, the species can be identified by molecular techniques. Polymerase chain reaction (PCR) amplification of the second internal transcription spacer (ITS-2) is useful to identify nematode species, including Gnathostoma species. This study aims to develop a duplex-PCR amplification method of the ITS-2 region to differentiate between the Gnathostoma binucleatum and G. turgidum parasites that coexist in the same endemic area, as well as to identify the Gnathostoma larvae recovered from the biopsies of two gnathostomiasis patients from Sinaloa, Mexico. The duplex PCR established based on the ITS-2 sequence showed that the length of the amplicons was 321 bp for G. binucleatum and 226 bp for G. turgidum. The amplicons from the AL3A of both patients were 321 bp. Furthermore, the length and composition of these amplicons were identical to those deposited in GenBank as G. binucleatum (accession No. JF919679), corroborating our previous morphological finding that G. binucleatum is the etiological agent for human gnathostomiasis in the endemic area of Sinaloa, Mexico.
Haemophilus influenzae type b (Hib) causes several invasive infections such as meningitis, septic arthritis, and pneumonia, especially in children below 5 years old. Despite the availability of Hib vaccines against Hib infection, seroepidemiological surveys of Hib infections have not yet been systematically conducted in Japan. We analyzed 1,338 serum samples, provided by the National Serum Reference Bank of the National Institute of Infectious Diseases (Tokyo, Japan), from 0- to 5-year-old children. Anti-polyribosylribitol phosphate (PRP) immunoglobulin G (IgG) antibody levels against Hib were determined using an anti-H. influenzae IgG enzyme immunoassay kit. In a total of 1,168 (87.3%) serum samples from children, anti-PRP IgG antibody levels were ≥ 0.15 μg/mL, providing natural immunity. Titers expected to provide long-term protection (≥ 1 μg/mL) were increased from 2.7% to 51.6% in children < 1 year old after the introduction of Hib vaccine (1980, 5.3%; 1995, 2.7%; 2010, 22%; 2012, 51.6%). Our data confirmed that the introduction of Hib vaccination in children below 5 years old increased the proportion of children having high anti-PRP IgG antibody levels, ensuring long-term protection against Hib.
We previously reported a hospital-based epidemiological study on enterovirus (EV)-D68 infection among children during the autumn of 2015, which indirectly inferred an outbreak in Sendai, Japan. In this study, stocked sera of children (aged 0–6 years; without symptoms of infectious diseases) in the Sendai community collected during 4 periods (1 year before, 6 months before, immediately after, and 1 year after the possible outbreak period) were analyzed using the neutralization antibody titer assay to determine community children’s immunity levels against EV-D68 infection. The immunity levels were confirmed to have increased during the possible outbreak period and to have gradually waned over 1 year without another outbreak. These results provide background information supporting the results of our previous hospital-based surveillance study.
Annually, more than 1.2 million travelers from other countries visit the Maldives for sightseeing, business, and honeymoon. In 2018, the largest dengue fever outbreak occurred, affecting more than 3,200 people. During this outbreak, we encountered a newly married Japanese couple returning from the Maldives on their honeymoon in October 2018, both were infected by the dengue virus type 2 during the travel. The number of imported dengue fever cases from the Maldives may increase; hence, physicians should stay up to date on dengue outbreak information worldwide.
We screened for the presence of Borrelia spp. in ticks collected from vegetation by flagging and from wild animals between May 2017 and November 2018 in Fukuoka, located in the northern Kyushu region of Japan. A total of 1,601 ticks were collected and separated based on morphology into nine species, namely Ixodes turdus, I. ovatus, Amblyomma testudinarium, Haemaphysalis flava, H. formosensis, H. kitaokai, H. longicornis, H. hystricis, and H. megaspinosa. The ticks were segregated into 561 pools and nested PCR was used to detect borrelial DNA. Borrelia turdi and Borrelia sp. HM were identified in two of the 561 pools. This is the first report of the presence of the Lyme disease group of Borrelia and of the relapsing fever group of Borrelia in Fukuoka, Japan.
In recent years, the prevalence of invasive Streptococcus dysgalactiae subsp. equisimilis (SDSE) infections has increased gradually throughout the world, including Japan. Here, we report the case of an abdominal aortic graft infection caused by stG485.0, ST29 SDSE in an elderly patient with diabetes. The patient was an 86-year-old man who had undergone surgery 10 years ago for treating a non-infected abdominal aortic aneurysm using a bifurcated graft. He was referred to our hospital after being suspected of having an abdominal aortic graft infection based on computed tomography (CT) scans. He underwent surgery to drain the pus that had accumulated between the aneurysm and graft. Although blood cultures were negative, the surgical specimen culture was positive for a β-hemolytic group G streptococci, which was subsequently identified as SDSE using 16S ribosomal RNA sequencing. Genetic relationships deduced from emm and multilocus sequence typing revealed the isolate to be types stG485.0 and ST29, respectively. Although aortic aneurysm graft infection has a poor prognosis, we successfully rescued the patient through prompt surgery and identification of the responsible pathogen. This case indicates that attention must be paid toward possible SDSE infections in the field of vascular surgery.
Babesia divergens is the major causal agent of zoonotic human babesiosis across Europe. Previously, we reported the detection of a B. divergens Asia lineage in wild sika deer (Cervus nippon) in Japan which was genetically closely related to the European B. divergens. To further elucidate its etiology, we conducted a large epidemiological survey by combining lineage-specific PCR system and blood direct PCR. The infection rate of the Asia lineage was 6.6% (116/1,747) throughout Japan, where Hokkaido (45%), Nagano (17%), Iwate (12%), Gunma (11%), and Yamanashi (11%) were highly enzootic (> 10%) among the 30 prefectures examined. European B. divergens was not detected. A geographical information system (GIS) map revealed dense populations of PCR-positive deer in the mountains including the Japanese Alps in eastern Honshu, and Hokkaido. These areas markedly overlapped with the major habitats of Ixodes persulcatus, a principal tick vector responsible for the transmission of the Asia lineage. Other areas in southern Japan including Miyazaki, Kagoshima, and Shimane Prefectures, where positive sika deer were sporadically detected, may be habitats for other tick species involved in the enzootic cycle as I. persulcatus were scarce. The rise in human babesiosis cases is occasionally attributed to healthy blood donors who were unaware of tick bites and Babesia infection. Therefore, there is an urgent need to investigate whether infections in humans have occurred in Japan.
Panton-Valentine leukocidin (PVL) is a causative agent of lethal necrotizing pneumonia and is associated with epidemic strains of community-acquired (CA) methicillin-resistant Staphylococcus aureus (MRSA). PVL-producing strains have rarely been isolated in Japan. However, PVL-positive CA-MRSA has been isolated much more frequently in recent years. To investigate the relevance of pvl genes (lukS/F-PV) and clinical traits in epidemic S. aureus strains, we genotyped four PVL-positive CA-MRSA strains isolated from patients with skin and soft tissue infections and measured their susceptibility to antibiotics. Three of the isolates matched the genotype of the USA300 clone, which has predominantly been isolated in the USA. The remaining strain matched the ST217 genotype, and its spa type was identical to that of PVL-positive strains previously reported in India and China. Abscess drainage was necessary in all cases, and deep cutaneous ulcers were formed in three out of four cases regardless of the genotype. The ST217 genotype strain was resistant to clindamycin, in addition to quinolones, macrolides, and aminoglycosides. Thus, diagnostic determination of lukS/F-PV should be used as a guide for selecting the treatment regimen.
Using multiplex real-time PCR, 960 fecal samples collected from poultry, cattle, and patients with diarrhea in Bangladesh were screened for diarrheagenic Escherichia coli (DEC). The invasion-related gene virB showed the highest prevalence in human patients (41%) and was shown to be positively correlated first with afaB with regards to diffuse adhesion and second with aggR with regards to aggregative adhesion. These three genes were specific to human patients. In contrast, the Shiga toxin genes stx1 (57%) and stx2 (40%) were prevalent in cattle samples. The eae gene, which is associated with attaching and effacing lesion formation, and the elt and est genes, which are associated with enterotoxins, were detected from all three sample sources. Heat map construction and hierarchical clustering assigned the samples into five different clusters, with the patient samples positive for virB and afaB being placed together in one cluster. Although the detection of virulence genes cannot be a direct indication of the distribution of diarrheagenic organisms, their detection suggests that Shigella spp. or enteroinvasive E. coli are the most prevalent diarrheagenic bacteria in Bangladesh and that diffusely adherent E. coli is concomitantly present with these bacteria. eae-possessing organisms in patients may come from cattle and poultry sources. The small number of stx-positive patients could be explained by the small number of animal samples that were positive for both eae and stx.