Surveillance of adenovirus D in patients with epidemic keratoconjunctivitis from Fukui Prefecture, Japan, 1995-2010.

Human adenoviruses species D (HAdV-D) are known to cause severe epidemic keratoconjunctivitis. However, the isolation rate of HAdV-D is not high, because HAdV-D is usually slow to propagate. Although new types of HAdV-D have been reported, accurate surveillance has not been performed because of difficulties in culturing the viruses and lack of a practical identification method. In this study, HAdV-Ds were detected and identified from patients with epidemic keratoconjunctivitis in the Fukui Prefecture during 1995-2010 by PCR, loop-mediated isothermal amplification (LAMP) of DNA, and conventional virus isolation and neutralization tests. All samples were subjected to culture and PCR and LAMP. A total of 124 strains of HAdV-D were detected from 157 patients with epidemic keratoconjunctivitis. The strains consisted of the following types: D8 (n = 8), D19 (n = 4), D37 (n = 40), D53 (n = 5), D54 (n = 66), and D56 (n = 1). Among these, D53, D54, and D56 are new types that have been reported recently. The results of this study demonstrated that new types of HAdV-D caused epidemic keratoconjunctivitis during 1995-2010, and included an outbreak of keratoconjunctivitis caused by HAdV-D54. The LAMP method was able to detect and identify HAdV-D53 and HAdV-D54 in 1 hr, and may therefore be applicable for use at the bedside.

Multilocus sequence typing implicates rodents as the main reservoir host of human-pathogenic Borrelia garinii in Japan.

Multilocus sequence typing of Borrelia garinii isolates from humans and comparison with rodent and tick isolates were performed. Fifty-nine isolates were divided into two phylogenetic groups, and an association was detected between clinical and rodent isolates, suggesting that, in Japan, human-pathogenic B. garinii comes from rodents via ticks.

[Mutations of gyrA gene and parC gene in fluoroquinolone-resistant Escherichia coli isolates from sporadic diarrheal cases].

We studied 107 isolates of Escherichia coli O153 from sporadic diarrhea cases in Fukui, Toyama, Aichi, and Saga prefectures from 1991 to 2005 for antimicrobial susceptibility and mechanisms of fluoroquinolone resistance, based on standard disk diffusion. Of 12 drugs tested, ampicillin displayed resistance to 72.9% of isolates, streptomycin to 48.6%, tetracycline to 46.7%, sulfisoxazole to 46.7%, trimethoprim/sulfamethoxazole to 29.9%, nalidixic acid (NA) to 29.9%, and ciprofloxacin (CPFX) to 24.3%. Ten of 32 isolates resistant to 3-6 drugs and 16 of 18 isolates resistant to 7-10 drugs were resistant both to NA and CPFX. Mutations of amino acid in quinolone resistance-determining regions of gyrA and parC genes were detected in 24 isolates resistant both to NA and CPFX, and in 1 isolate resistant to NA. The former possessed a combination of double substitution (S83L and D87L) in GyrA and a single substitution (S80I) in ParC. Some 12 of 24 isolates possessed another single substitution (E84V or E84G or A108T) in ParC. The 25 isolates were classified into 4 types as follows. 1 isolate as type 1: GyrA (S83L) and ParC (S80I); 12 isolates as type 2: GyrA (S83L and D87N) and ParC (S80I); 8 isolates as type 3: GyrA (S83L and D87N) and ParC (S80I and E84G/S80R and E84V); and 4 isolate as type 4: GyrA (S83L and D87N) and ParC (S80I and A108T). In the relationship between amino acid mutations and minimal inhibitory concentrations (MIC) of fluoroquinolone, MICs of CPFX, ofloxacin, and norfloxacin showed 1microg/mL, 2microg/mL and 8microg/mL in type 1; 8 approximately 32microg/mL, 8 approximately 32microg/mL and 16 approximately 256microg/mL in type 2; and 32 approximately 256microg/mL' 32 approximately 128microg/mL and 128-->512microg/ mL in types 3 and 4. These results suggest that most of multiple-antimicrobial-resitant E. coli O153 isolates from sporadic diarrhea cases were resistant to fluoroquinolones and possessed mutations at gyrA and parC genes associated with fluoroquinolone resistance.

Molecular evidence of the dispersal of Lyme disease Borrelia from the Asian Continent to Japan via migratory birds.

Based on specific sequencing, we found that a Borrelia garinii strain from a rodent in Fukui Prefecture, Japan was highly similar to the unique Borrelia strains (pattern R'/R) isolated in northeastern China and Korea, and to strains from ticks feeding on migratory birds in Fukui Prefecture. These findings indicate that the Borrelia with this unique pattern may be locally naturalized to the epizootic transmission cycle in Japan, and that Borrelia is dispersed from the Asian Continent to Japan via migratory birds.

The first reported case of spotted fever in Fukui Prefecture, the northern part of central Japan.

A 53-year-old man visited Mt. Arashima-dake in Fukui Prefecture, and was infested by a tick-like organism. He visited a local clinic on July 12, 2004, complaining of high fever, general fatigue and rash. After several days without definite diagnosis, he was admitted to the Fukui Prefectural Hospital, where he was treated with minocycline hydrochloride for 10 days until recovery. His clinical symptoms on admission were high fever (39.6 degrees C), erythematous eruption, eschar on the right upper arm, and regional lymphoadenopathy. The epidemiological status and some clinical findings strongly suggested spotted fever (SF), and SF was confirmed based on the finding that his sera were reactive only to antigens of the SF group rickettsiae in the indirect immunoperoxidase analysis. This case is the first official report of SF rickettsiosis in Fukui Prefecture, the northern part of central Japan.

[Comparison of multidrug-resistant Salmonella enterica serovar Newport isolates from a patient and sewages in Fukui Prefecture].

From May 2003 to March 2004, nine strains of multidrug-resistant (MDR) Salmonella enterica serovar Newport were isolated from 5 sewages of 3 sewage treatment plants located in 2 different regions of Fukui Prefecture. Antimicrobial susceptibility testing identified 2 different MDR patterns (type I and II). Type I strains were resistant to ampicillin, tetracycline, streptomycin, chloramphenicol, amoxicillin/clavulanic acid, sulfisoxazole, cefotaxime, cephalothin, ceftriaxone, and ceftrazidime and type II strains were additionally resistant to sulfamethoxazole-trimethoprim and spectionomycin. Neither of kanamycin, fosfomycin, nalidixic acid, gentamycin, or ciprofloxacin resistance was observed. Type I and II strains were different in pulsed-field gel electrophoresis patterns and plasmid profiles, while they showed the same profiles with in each type. All the MDR strains in this study were proved to carry the blaCMY-2 gene by PCR and sequence analyses. Numbers of reports on MDR S. Newport with blaCMY-2 have been increasing in foreign countries such as the United States. The MDR patterns of the isolates in this study were similar to those of the United States. These properties of the type I strains were identical with that a strain isolated from a 7-year-old patient in Fukui Prefecture, in September 2003 which we previously reported. However, the patient lived in a region where no epidemiological associations with the sewage treatment plants were found. This suggests that the MDR strains might prevail in Fukui Prefecture. Domestic surveillance for MDR S. Newport would be need.

[Four serotypes highly positive for pathogenic factor-related genes found among Escherichia coli isolates from sporadic diarrheal cases in Fukui Prefecture].

To evaluate the prevalence of pathogenic factor-related genes in 964 O-serotype Escherichia coli isolated from sporadic diarrhea cases at 2 hospitals in Fukui during 1997 to 2001, we checked all of these strains for H-serotype and examined them for 4 pathogenic factor-related genes (LT, ST, stx and invE) by PCR. Of these strains, 409 except for most of 9 serotypes which are usually low prevalence of pathogenic factor-related genes, and additional 16 strains isolated from other hospitals in Fukui Prefecture were also examined for 3 pathogenic factor-related genes (eaeA, astA and aggR) by PCR. It was found that 4 serotypes, O6:H16, O25:HNM, O111:H21 and O126:H27, were highly positive for pathogenic factor-related genes; O6:H16 (11/12 strains) positive for LT, ST, astA, O 25:HNM (10/14 strains) positive for ST and astA, O111:H21 (22/22 strains) positive for astA or aggR, and O126:H27 (8/9) positive for astA and aggR genes. According to the drug susceptibility test, these serotypes as O6:H16, O25:HNM, O111:H21 and O126:H27 showed a significant resistance to some drugs in 7/12, 4/14, 21/22 and 9/9 strains, respectively. Such results indicate that both O111 and O126-serotypes are highly positive for pathogenic factor-related genes and also drug-resistant, namely this fact suggests that the O-serotyping as laboratory screening is one of the useful measures of clinical management. Additionally, the pulsedfied gel electrophoresis patterns found in each of the 4 serotypes mentioned revealed that a part of E. coli in Fukui might be derived from the same source of infection.

PCR detection of Anaplasma phagocytophilum and Borrelia burgdorferi in Ixodes persulcatus ticks in Mongolia.

A molecular epidemiological survey was conducted to identify the tick-borne disease agents Anaplasma phagocytophilum and Borrelia burgdorferi sensu lato in Selenge Province, Mongolia. The survey was in response to a suspected A. phagocytophilum infection in a patient. In 2012, a total of 129 questing Ixodes persulcatus adult ticks were sampled by flagging vegetation. A. phagocytophilum and Borrelia spp. were detected by PCR, targeting the 16S rDNA (rrs) and 5S-23S intergenic spacer region, respectively. Infection rates for A. phagocytophilum and B. burgdorferi sensu lato spp. were 6.2% and 55.0%, respectively. Six of the 129 ticks (4.9%) were coinfected with A. phagocytophilum and B. burgdorferi sensu lato. Among Borrelia spp., the highest prevalence rate was that for B. garinii 20047 type (26.3%), followed by B. afzelii (7.8%) and B. garinii NT29 type (7.0%). Furthermore, ticks were detected that were dually infected with B. afzelii and B. garinii 20047 type (7.8%) and B. garinii NT29 and 20047 types (6.2%).

Molecular epidemiology of human metapneumovirus from 2005 to 2011 in Fukui, Japan.

To investigate the molecular epidemiology of human metapneumovirus (HMPV) infections in acute respiratory infections (ARI), we performed genetic analysis of the F gene in HMPV from patients with ARI in Fukui Prefecture from August 2005 to July 2011. HMPV was detected in 53 of 741 nasopharyngeal swabs (7.2%). Phylogenetic analysis helped us assign 31 strains to subgroup A2, 1 strain to subgroup B1, and 21 strains to subgroup B2. The prevalence of HMPV was peaked between January and June. A high degree of nucleotide identity was seen among subgroup A2 strains (95.6-100%) and subgroup B2 strains (97.5-100%). In addition, no positively selected sites (substitutions) were found in the F gene in these HMPV strains. The results suggest that the prevalent HMPV strains in Fukui were associated with various ARI in Japan during the investigation period.

Multilocus sequence typing and DNA similarity analysis implicates that a Borrelia valaisiana-related sp. isolated in Japan is distinguishable from European B. valaisiana.

Lyme disease Borrelia spp. are transmitted by Ixodes ticks, and more than 10 species of borreliae have been identified around the world. Recently, another Borrelia sp. has been reported in Asia (Japan, Korea, China, Taiwan and Thailand) as Borrelia valaisiana-related sp. In the present study, we obtained and genetically characterized 19 B. valaisiana-related sp. strains from mammals and ticks. Genetic analyses showed that the Borrelia strains were distinct from B. valaisiana found in Europe. Multilocus sequence typing revealed that these Borrelia isolates formed a monophyletic group with B. yangtze strains in China. Some of the strains were isolated from the bladders of small mammals, and also two strains were experimentally confirmed to be infectious in C3H/HeN mice. We observed that the Borrelia sp. was maintained in the Ixodes granulatus tick after molting. These results suggested that small mammals and I. granulatus were possible reservoir hosts and the vector tick for the Borrelia sp., respectively. B. valaisiana, originally found in Europe, was transmitted mainly by I. ricinus, and birds were mainly thought to be reservoir hosts. Our results suggested that Japanese isolates of B. yangtze (formerly B. valaisiana-related sp.) were distinguishable from B. valaisiana according to the reservoir host and its vector tick. In this study, we also deposited borrelia strain Okinawa-CW62 into bioresource centers as a reference strain of B. yangtze(=DSM 24625, JCM 17189).

Survey of the vectorial competence of ticks in an endemic area of spotted fever group rickettsioses in Fukui Prefecture, Japan.

The prevalence of SFGR in ixodid ticks in the Mt. Arashima-dake area in the northern part of Fukui Prefecture was surveyed, because of strong suspicions that the first case identified in this Prefecture had become infected with R. helvetica in this region. The ticks identified consisted of three genera and six species; I.ovatus, I. persulcatus, I. monospinosus, H. flava, H. japonica and D. taiwanensis. Of all 222 ticks collected, only I. monospinosus ticks (8 of 32 examined) were positive for SFGR isolates, which were genetically identified as R. helvetica. Ticks (157 of all 222) positive for SFGR-DNA fragments consisted of I. monospinosus (14 of 32), I. persulcatus (11 of 55), I. ovatus (3 of 38), H. flava (5 of 21) and H. japonica (2 of 9). Of these, thirteen I. monospinosus, eight I. persulcatus, three I. ovatus, two H. flava and one H. japonica were identified by nucleotide sequences as positive for R. helvetica. DNA fragments from three H. flava and one H. japonica showed greater homology to R. japonica than to R. helvetica or R. asiatica. The present results indicate that it is most likely that the vector tick of R. helvetica infection in Fukui Prefecture is I. monospinosus.