Prevalence and molecular characterization of G1P[8] human rotaviruses possessing DS-1-like VP6, NSP4, and NSP5/6 in Japan.

During a survey of human rotaviruses in Okayama Prefecture, Japan in the 2011-2012 rotavirus season (between September 2011 and August 2012), G3P[8] was found to be a predominant genotype overall. However, G1P[8] emerged in the latter half of the season. To clarify the genetic background of the G1P[8] strains, the VP7, VP4, VP6, NSP4, and NSP5/6 genes of the strains were sequenced and genotyped. As a result, it was demonstrated that the strains with two different genotype constellations (G1-P[8]-I1-E1-H1 and G1-P[8]-I2-E2-H2) prevailed in the season. The G1P[8] strains possessing the DS-1-like VP6, NSP4, and NSP5/6 genes (the DS-1-like G1P[8] strains), which should reveal a short electropherotype, were originated from possible intergenogroup reassortment events. The DS-1-like G1P[8] strains accounted for 74.1% of all G1P[8] strains and were detected continuously throughout the season but not in the preceding season, indicating the possibility of new introduction and rapid spreading of these strains in the 2011-2012 season. The results suggest that the intergenogroup reassortants, considered generally unstable, can spread rapidly and become relevant.

Human granulocytic Anaplasmosis, Japan.

We retrospectively confirmed 2 cases of human Anaplasma phagocytophilum infection. Patient blood samples contained unique p44/msp2 for the pathogen, and antibodies bound to A. phagocytophilum antigens propagated in THP-1 rather than HL60 cells. Unless both cell lines are used for serodiagnosis of rickettsiosis-like infections, cases of human granulocytic anaplasmosis could go undetected.

Molecular characterization of OP354-like P[8] (P[8]b subtype) human rotaviruses A species isolated in Japan.

OP354-like P[8] (P[8]b subtype) species A rotaviruses (RVAs) were isolated first time in Japan during a RVA survey in Okayama Prefecture between 2006 and 2009. Two of 236 RVA-positive samples were identified as G1P[8]b by reverse transcription polymerase chain reaction. P[8]b strains (RVA/human-wt/JPN/OH1998/2008/G1P[8]b and RVA/human-wt/JPN/OH2024/2008/G1P[8]b) were isolated only in May, 2008 and both patients infected with P[8]b viruses lived in the same city, suggesting that the prevalence of P[8]b RVAs is limited considerably in Okayama Prefecture. Molecular analysis of four genes (VP4, VP6, VP7, and NSP4 genes) of Japanese P[8]b strains revealed that the VP4 genes of these strains were related closely to those of Southeast Asian and Indian P[8]b strains. In contrast, the VP6, VP7, and NSP4 genes of Japanese P[8]b strains were highly homologous to G1P[8]a strains prevalent in the same area. These results suggest that the Japanese P[8]b strain may be a result of reassortment events between Japanese G1P[8]a viruses and unidentified Asian viruses possessing the P[8]b VP4 gene.

Human Rickettsia heilongjiangensis infection, Japan.

A case of Rickettsia heilongjiangensis infection in Japan was identified in a 35-year-old man who had rash, fever, and eschars. Serum contained R. heilongjiangensis antibodies, and eschars contained R. heilongjiangensis DNA. R. heilongjiangensis was also isolated from ticks in the suspected geographic area of infection.

Epidemiology of Chlamydophila caviae-like Chlamydia isolated from urethra and uterine cervix.

In 2000, chlamydial strains OK133 and OK135 were isolated from 2 female patients with cervicitis. These strains were unresponsive to commercially available PCR and LCR test kits for the diagnosis of Chlamydia trachomatis infection, and their phenotypic characteristics were very similar. The OK135 nucleotide sequence in MOMP-VD2 gene closely resembled that of Chlamydophila caviae GPIC. A similar strain was isolated in 2003 from a male patient OKM2 with urethritis, from which the strain SC10-6 was cloned by the plaque purification method. The nucleotide sequence of the entire MOMP gene of SC10-6 was exactly the same as that of OK135. Thus, the strains OK135 and SC10-6, together with OK133, have been called C. caviae-like Chlamydia. We designed primers for nested PCR assay, the product of which showed a single-band 311-bp fragment, to detect C. caviae-like Chlamydia. Of swab specimens obtained from 202 patients from 2003 to 2006 (119 male and 83 female patients), 18 specimens (8.9%) from 14 male and 4 female patients were positive, suggesting that C. caviae-like Chlamydia infection is rather common. Thus far, it has not been determined whether C. caviae-like Chlamydia is pathogenic for humans.

Diagnostic assay for Rickettsia japonica.

We developed a specific and rapid detection system for Rickettsia japonica and R. heilongjiangensis, the causative agents of spotted fever, using a TaqMan minor groove binder probe for a particular open reading frame (ORF) identified by the R. japonica genome project. The target ORF was present only in R. japonica-related strains.

Assay of Chlamydia pneumoniae-specific IgM antibodies by ELISA method--reduction of non-specific reaction and resetting of serological criteria by measuring IgM antibodies--.

In the present study, we tried to reduce the non-specific reactions for measuring anti-Chlamydia pneumoniae IgM antibodies by the ELISA kit of HITAZYME C. pneumoniae Ab-IgM (HITAZYME IgM) by using a new absorbent. We also tried to reset the IgM cut-off index (ID) of HITAZYME IgM by testing serum samples from healthy children and healthy adults with no respiratory symptoms. The results suggest that the use of the new absorbent (anti-human IgG antibodies) may reduce the non-specific reactions by rheumatoid factor and anti-nuclear antibodies, and that the setting of the higher cut-off ID (2.00), calculated as the mean ID+3SD of the serum samples from healthy children and healthy adults, respectively, would improve the specificity of IgM during the measurement by HITAZYME IgM.

[Psittacosis outbreak at an avian exhibition].

Psittacosis outbreak due to Chlamydophila psittaci occurred among staff members at an avian exhibition of nearly 1,000 birds in Kobe, Japan, in December 2005. Staff members not trained about zoonosis or psittacosis used little protective attire such as masks and gloves when caring for their discharges. Two of 67 staff members contracted psittacosis pneumonia. Additional two suffered from pneumonia and 19 reported symptoms such as fever and cough, although none were diagnosed with psittacosis. The roughly 970 birds were kept without quarantine and identified by leg bands. Doxycycline administrated in drinking water and food failed to eradicate chlamydia, so all birds were captured, identified by leg band, and tested for chlamydia by PCR. Six were found to carry large amounts of chlamydia. Major outer membrane protein (MOMP) DNA sequence of chlamydia in a patient's bronchoalveolar lavage fluid (BALF) was identical to that derived from a channel-billed toucan kept in a closed aviary, and staff members may have been infected by inhaling excrement while working in the aviary. The MOMP DNA sequence was useful in comparing strains. We review the difficulty of diagnosing psittacosis and the knowledge and infection control measures required against it.

Chlamydophilal antigens induce foam cell formation via c-Jun NH2-terminal kinase.

Chlamydophila pneumoniae is known to be associated with atherosclerosis. Recent studies have reported that components of Chlamydophila pneumoniae (chlamydophilal antigens) induce foam cell formation in macrophages. However, the mechanism of foam cell formation induced by chlamydophilal antigens has yet to be elucidated. In this paper, we first found that mitogen-activated protein kinases including extracellular signal-regulated kinase, p38 and c-Jun NH2 terminal kinase are phosphorylated after stimulation by chlamydophilal antigens. We then showed that chlamydophilal antigens induce foam cell formation mainly via c-Jun NH2 terminal kinase. Finally, we demonstrated that foam cell formation and phosphorylation of mitogen-activated protein kinases induced by chlamydophilal antigens are mainly recognized through Toll-like receptor 2. These results collectively indicated that chlamydophilal antigens induce foam cell formation mainly via Toll-like receptor 2 and c-Jun NH2 terminal kinase.

Longitudinal epidemiology of Chlamydia trachomatis serovars in female patients in Japan.

The objective of this study was to clarify the longitudinal epidemiology of Chlamydia trachomatis serovars in Japan. A total of 339 endocervical swab specimens obtained from female patients who attended the Department of Obstetrics and Gynecology, Saitama Medical School, were used. Positive specimens of either transport medium of IDEIA Chlamydia (1st group, from 1999 to 2001), or DNA extract of Cobas Amplicor STD-1 Chlamydia trachomatis (2nd group, from 2003 to 2005) were used for serotyping. Typing of C. trachomatis serovars in DNA extracts was performed by polymerase chain reaction-restriction fragment length polymorphism. Ten serovars, A, B, D, E, F, G, H, I, J and K, were identified in the 1st group, and serovar E was most frequently identified (27.6%). In the 2nd group, nine serovars, B, C, D, E, F, G, H, I and K, were identified, and serovar D was most frequently identified (24.7%). Serovars B and Ba were significantly more common around 2000 and the mid-1990s (from 1993 to 1996), respectively. Numbers of serovar I increased significantly during the research period. In addition, serovar I was more frequent in the 2nd group than in the 1st group in women aged 20-29 years. There were no significant differences of serovar distribution between pregnant and non-pregnant women.

Outbreak of Chlamydia pneumoniae infection in a Japanese nursing home, 1999-2000.

OBJECTIVE: To identify risk factors for infection and severe illness due to Chlamydia pneumoniae. METHODS: To identify risk factors for infection, we conducted a case-control study among nursing home residents who had onset of symptoms during December 1, 1999, to February 20, 2000. To identify risk factors for severe illness among nursing home residents, we conducted a retrospective cohort study. SETTING: A nursing home providing long-term and day care services for elderly patients in Japan.Participants. Fifty-nine residents and 41 staff members of a nursing home. RESULTS: The attack rates for respiratory illness were 53% (31 of 59) among residents and 22% (9 of 41) among staff. Infection was confirmed in 15 resident and 2 staff case patients by isolation of C. pneumoniae from nasal swab specimens. Fifteen resident case patients developed severe illness (ie, bronchitis, pneumonia, and hypoxia); one case patient died. The median age of resident case patients was 87 years. We could identify neither the source of the outbreak nor significant risk factors for infection and severe illness in residents. However, residents with a higher level of physical activity were more likely to become infected, whereas older residents (aged more than 85 years) and those with a lower level of physical activity were more likely to develop severe illness (P>.05). Contact with residents was a risk factor for infection in staff (relative risk, undefined; P=.04). CONCLUSIONS: C. pneumoniae can cause large outbreaks of infection and severe illness among elderly persons, and its transmission is likely to be enhanced by close contacts among people in nursing homes. Therefore, early detection of an outbreak by means of better surveillance, and subsequent isolation of patients, may be effective control measures.

Outbreak of respiratory tract infections on an islet in Korea: possible Chlamydia pneumoniae infection.

In March 2004, we experienced an outbreak of Chlamydia pneumoniae infection on an islet of Korea. In order to assess the significance of the epidemic, we performed a mass examination of 137 students (7-16 years old; male, 69; female, 58) at a school. The examination consisted of a questionnaire inquiring about respiratory symptoms, a serum antibody test for C. pneumoniae using a microimmunofluorescence (MIF) method and enzyme-linked immunosorbent assay (ELISA), and nasopharyngeal swab tests to detect of the organism by specific PCR and cell culture. The results demonstrated that 72 (58.3%) of the students had respiratory symptoms such as rhinorrhea, a sore throat, and/or cough or fever. The PCR positivity of acute-phase patients was 63% (12/19) and PCR positivity using the culture sample was 94% (18/19). However, the existence of the organism was not confirmed fluorescein isothiocyanate (FITC). ELISA, one of the serological methods utilized, demonstrated, in the same patients, 48% (13/27) positive IgM antibodies at the acute phase of the outbreak, and 16% (3/19) positive IgM antibodies during the convalescent phase. The index value (ID) 3.0 for single-sera IgG was 19% (5/27) and that for IgA was 4% (1/27) at the acute phase; the corresponding percentages in the convalescent phase were 11% (2/19) and 5% (1/19), respectively. However, as regards paired sera, no patient demonstrated a 1.35 ELISA ID value at 2 weeks, or an increased value of 1.0 at 8 weeks after the onset of the outbreak. In the MIF experiment, the percent positivity of unpaired IgM from the acute phase was 58% (11/19). At convalescent phase, this percentage was 47% (9/19); however, the positivity of paired serum IgG was 26% (5/19). In the same sample, the percentage of positive cases demonstrated by both ELISA and MIF approaches for single IgM was 37% (7/19) at the acute phase and 11% (2/19) at the convalescent phase. We were unable to isolate C. pneumoniae by cell culture, but we did obtain sufficient serological and PCR data to consider C. pneumoniae as the causative agent of the outbreak. Meaningful results were acquired in terms of serology, and were compared to the healthy population in Korea. Although it remains necessary to investigate the possibility of co-infection and to determine whether or not this outbreak coincides with the prevalence of influenza, it was unequivocally concluded that this outbreak of C. pneumoniae infection has occurred on an islet of Korea.

Acute Chlamydia pneumoniae infection with heat-shock-protein-60-related response in patients with acute coronary syndrome.

Recent evidence has suggested an association between Chlamydia pneumoniae infection and coronary atherosclerosis. A significant association has also been detected between heat shock protein (HSP) 60 antibody and the severity of coronary atherosclerosis. The aim of this study was to define the relationship between instability of ischemic heart disease (IHD) and serum levels of HSP60 and C. pneumoniae antibodies. Blood samples for the measurement of serum antibody titers were obtained from 1131 patients with ischemic heart disease (65+/-9 years; male/female, 828/303) and 127 non-IHD controls with normal coronary arteries (64+/-9 years; male/female, 60/67) on the day of cardiac catheterization. The serum levels of anti-human HSP60 IgG antibody and anti-chlamydial IgM, but not IgG or IgA, antibody were significantly higher in ACS patients than in stable IHD patients or controls. These results suggest that acute C. pneumoniae infection with HSP60-related immunological responses may contribute to the pathophysiology of acute coronary syndromes.

Chlamydia pneumoniae growth inhibition in human monocytic THP-1 cells and human epithelial HEp-2 cells by a novel phenoxazine derivative.

In this study the effects of 2-amino-phenoxazine-3-one (phenoxazine derivate, Phx-3) on Chlamydia (Chlamydophila) pneumoniae growth in human monocytic THP-1 cells as well as human epithelial HEp-2 cells were examined. Cells were infected with bacteria at an m.o.i. of 10 by centrifugation. After washing to remove any remaining bacteria, the cells were incubated with or without Phx-3 in the presence or absence of tryptophan for 72 h. The bacteria in cells were assessed by staining of chlamydial inclusions with FITC-labelled anti-chlamydial antibody, electron microscopic analysis, real-time RT-PCR specific for C. pneumoniae 16S rRNA and propagation on HEp-2 cells. Treatment with Phx-3 significantly inhibited growth of C. pneumoniae in THP-1 and HEp-2 cells. A decrease in the number of bacterial 16S rRNA transcripts was also confirmed in both cell lines by real-time RT-PCR. Electron microscopic studies revealed that treatment with Phx-3 induces bacterial destruction in most of the inclusion bodies in these cells. Addition of tryptophan to the culture slightly blocked the growth inhibition of C. pneumoniae by Phx-3. The reagents did not show any cytotoxicity to the cells at the concentrations used. The results suggest that Phx-3 inhibits C. pneumoniae replication in human monocytic cells as well as epithelial cells, partially depending on the tryptophan-metabolic pathway of host cells. Thus, Phx-3 might be a useful compound for controlling C. pneumoniae growth in cells and may be an alternative conventional therapy.

Prevalence of antibodies against Simkania negevensis in a healthy Japanese population determined by the microimmunofluorescence test.

Simkania negevensis has been associated with bronchiolitis in infants and community-acquired pneumonia in adults. Reports of exposure to this microorganism are only available from Israel, North America and Western Europe. Currently, no standard method for diagnosis of S. negevensis infection has been established nor have prevalence rates been shown in Japan. For the first time we demonstrated the ability of the microimmunofluorescence (MIF) test to detect S. negevensis-specific immunoglobulin G and exposure to S. negevensis in Japan. The positive rate in healthy volunteers was 4.3% (25/588), with rates increasing with age. Results indicate the usefulness of the MIF test as a serological method for detecting S. negevensis-specific antibodies. A standard serological test for infection with S. negevensis is needed.

Distribution of Chlamydia trachomatis serovars among female prostitutes and non-prostitutes in Thailand, and non-prostitutes in Japan during the mid-90s.

The distribution of Chlamydia trachomatis serovars in Thailand and Japan during the same period of the mid-90s was determined. Seventy-one C . trachomatis specimens isolated from female patients who visited the Venereal Diseases Center at Bangkok, Thailand in 1994 were used in this study. Of these, 56 patients were prostitutes. Forty-seven specimens obtained from female non-prostitutes who attended the Department of Obstetrics and Gynecology, Saitama Medical School, Japan during the period from 1993 to 1995 were also used in this study. DNA was extracted from these specimens and typing of C. trachomatis serovars was performed by the polymerase chain reaction-restriction fragment length polymorphism method. The identified serovars among prostitutes of Thailand (n = 56)/non-prostitutes of Thailand (n = 15)/non-prostitutes of Japan (n = 47) were as follows: Ba 1/0/2, D 8/1/15, E 11/2/8, F 16/9/8, G 4/0/7, H 3/2/3, I 1/0/1, J 3/0/0, and K 10/1/4. Serovar F was most prevalent (35.2%) in both prostitutes and non-prostitutes from Thailand, followed by serovar E (18.3%). On the other hand, serovar D was the most frequent serovar in non-prostitutes in Japan (31.9%) followed by serovars F (17.0%) and E (17.0%). A difference in the distribution of C. trachomatis serovars of Thailand and Japan was noted.

Detection of Rickettsia and Ehrlichia spp. in Ticks Associated with Exotic Reptiles and Amphibians Imported into Japan.

One of the major routes of transmission of rickettsial and ehrlichial diseases is via ticks that infest numerous host species, including humans. Besides mammals, reptiles and amphibians also carry ticks that may harbor Rickettsia and Ehrlichia strains that are pathogenic to humans. Furthermore, reptiles and amphibians are exempt from quarantine in Japan, thus facilitating the entry of parasites and pathogens to the country through import. Accordingly, in the current study, we examined the presence of Rickettsia and Ehrlichia spp. genes in ticks associated with reptiles and amphibians originating from outside Japan. Ninety-three ticks representing nine tick species (genera Amblyomma and Hyalomma) were isolated from at least 28 animals spanning 10 species and originating from 12 countries (Ghana, Jordan, Madagascar, Panama, Russia, Sri Lanka, Sudan, Suriname, Tanzania, Togo, Uzbekistan, and Zambia). None of the nine tick species are indigenous in Japan. The genes encoding the common rickettsial 17-kDa antigen, citrate synthase (gltA), and outer membrane protein A (ompA) were positively detected in 45.2% (42/93), 40.9% (38/93), and 23.7% (22/93) of the ticks, respectively, by polymerase chain reaction (PCR). The genes encoding ehrlichial heat shock protein (groEL) and major outer membrane protein (omp-1) were PCR-positive in 7.5% (7/93) and 2.2% (2/93) of the ticks, respectively. The p44 gene, which encodes the Anaplasma outer membrane protein, was not detected. Phylogenetic analysis showed that several of the rickettsial and ehrlichial sequences isolated in this study were highly similar to human pathogen genes, including agents not previously detected in Japan. These data demonstrate the global transportation of pathogenic Rickettsia and Ehrlichia through reptile- and amphibian-associated ticks. These imported animals have potential to transfer pathogens into human life. These results highlight the need to control the international transportation of known and potential pathogens carried by ticks in reptiles, amphibians, and other animals, in order to improve national and international public health.