Toshio Kishimoto

Clinical Evaluation of Anti-Tuberculous Glycolipid Immunoglobulin G Antibody Assay for Rapid Serodiagnosis of Pulmonary Tuberculosis

ABSTRACT Previously we reported the development of a highly sensitive enzyme-linked immunosorbent assay specific for anti-tuberculous glycolipid (anti-TBGL) for the rapid serodiagnosis of tuberculosis. In this study, the usefulness of an anti-TBGL antibody assay kit for rapid serodiagnosis was evaluated in a controlled multicenter study. Antibody titers in sera from 318 patients with active pulmonary tuberculosis (216 positive for Mycobacterium tuberculosis in smear and/or culture tests and 102 smear and culture negative and clinically diagnosed), 58 patients with old tuberculosis, 177 patients with other respiratory diseases, 156 patients with nonrespiratory diseases, and 454 healthy subjects were examined. Sera from 256 younger healthy subjects from among the 454 healthy subjects were examined as a control. When the cutoff point of anti-TBGL antibody titer was determined as 2.0 U/ml, the sensitivity for active tuberculosis patients was 81.1% and the specificity was 95.7%. Sensitivity in patients with smear-negative and culture-negative active pulmonary tuberculosis was 73.5%. Even in patients with noncavitary minimally advanced lesions, the positivity rate (60.0%) and the antibody titer (4.6 ± 9.4 U/ml) were significantly higher than those in the healthy group. These results indicate that this assay using anti-TBGL antibody is useful for the rapid serodiagnosis of active pulmonary tuberculosis.

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.

In vitro and in vivo antichlamydial activities of newly developed quinolone antimicrobial agents.

The in vitro and in vivo activities of three newly developed quinolone antimicrobial agents (sparfloxacin, tosufloxacin, and OPC-17116) were investigated. All three agents showed potent in vitro activities against Chlamydia psittaci, C. trachomatis, and C. pneumoniae with MICs that ranged from 0.031 to 0.125 micrograms/ml. These values were higher than those of minocycline (0.0075 to 0.015 micrograms/ml) but lower than those of erythromycin (0.25 to 0.5 micrograms/ml) and ofloxacin and ciprofloxacin (0.5 to 1.0 micrograms/ml). Mice were challenged with 10(5) inclusion-forming units of C. psittaci each by nasal instillation. All untreated control animals died within 7 days. The survival rates of mice treated with 40 mg of sparfloxacin, OPC-17116, or tosufloxacin per kg of body weight every 12 h for 7 days were 73, 73, and 60%, respectively, 7 days after the challenge. The survival rate of mice treated with ofloxacin at the same dosage was 53%. On the basis of the above results, we concluded that these three new quinolones might be useful in the treatment of chlamydial respiratory infections.

First Detection of Rickettsia in Soft‐Bodied Ticks Associated with Seabirds, Japan

Rickettsia was first detected in seabird soft‐bodied ticks, Carios capensis and C. sawaii in Japan. According to sequence analysis, Rickettsia in Japan was identical to Rickettsia scc31 in C. capensis in the U.S.A. This suggested that an environmental circulation had consisted among microorganisms, ticks and long distance migratory seabirds around the Pacific Ocean.

Seroprevalence of Coxiella burnetii Infection in Dairy Cattle and Non-symptomatic People for Routine Health Screening in Korea

We report results on the seroprevalence of antibodies to Coxiella burnetii in cattle and healthy people in Korea. Upon agreement with dairy owners, serum samples from 414 dairy cattle were collected between March and June 2001 and samples from 205 people for health screening were collected between April and December 2002. The sera were analyzed for the presence of anti-C. burnetii phase II antibodies using an indirect microimmunofluorescence test; strong fluorescence at a 1:32 dilution was regarded as positive. The overall seroprevalence of C. burnetii in cattle in Korea was 25.6%, with regional variation from 8.9 to 59.3%. Of the positive serum samples, 75.5% had antibody titers ≥1:256. By contrast, only 1.5% of people in a rural area were seropositive, and most of the positive samples had low antibody titers. In conclusion, this study showed that relatively high seropositivity of C. burnetii in dairy cattle, accordingly, the studies on the high-risk groups are needed to evaluate the seroprevalence for this organism in Korea.

Assay of Specific Anti-Chlamydia pneumoniae Antibodies by ELISA Method

We measured anti-Chlamydia pneumoniae (C. pneumoniae) specific antibody titers by means of a newly-developed enzyme-linked immunosorbent assay (ELISA) method using an anti-C. pneumoniae specific antibody detection reagent. The clinical usefulness of this method was hereby evaluated. The IgG, IgA and IgM titers in 418 serum specimens obtained from patients with respiratory tract infections were measured by this new ELISA method, and the results were compared with the titers determined for the same specimens with the micro immunofluorescence (Micro-IF) method. The results showed good correlation coefficients for IgG, IgA and IgM. The two assay methods showed high agreement rates for positivity and for negativity. Specimens which did not yield the same results with the ELISA method and the Micro-IF method were subjected to analysis by the Western blot method, and the rates of agreement with the ELISA results were high. In addition, the child (0 approximately 15 yrs old; n = 122) and adult (16 approximately 90 yrs old; n = 133) cases were classified on the basis of being antigen-positive or antigen-negative at the initial examination, and their antibody-positive rates were determined. The adults showed no statistically significant differences in the antibody-positive rates for either IgG or IgA antibodies as a function of the pretreatment antigen status. However, the children showed statistically significant (p < 0.001) differences in the antibody-positive rates for both IgG and IgA antibodies as a function of the antigen status in the antigen-positive group compared with the rates in the antigen-negative group. Furthermore, the IgM-positive rates for the children were high in the antigen-positive group compared with the rates in the antigen-negative group, and the difference was statistically significant (p < 0.001). The IgM-positive rates in the adults were also significantly (p < 0.05) different between the antigen-positive group and the antigen-negative group. The Micro-IF method was applied to 34 specimens from antigen-positive patients, and 22 specimens were found to show an IgG titer of > or = 512 or an IgM titer of > or = 16. The diagnoses of these patients were acute respiratory disease in sixteen, pneumonia in four. Application of the ELISA-method to those 22 specimens showed all of them to exhibit IgG absorbance of > or = 0.6 and IgA absorbance of 0.2. The results described above indicate the clinical usefulness of our new ELISA method for the detection of antibodies specific for C. pneumoniae. The significance of this ELISA method for serological diagnosis of C. pneumoniae infections and the criteria for diagnosis of acute infections were also discussed.

Molecular analyses of a potentially novel Anaplasma species closely related to Anaplasma phagocytophilum detected in sika deer (Cervus nippon yesoensis) in Japan

An Anaplasma species closely related to Anaplasma phagocytophilum detected in sika deer in Hokkaido, Japan was molecularly analyzed using 16S rRNA, citrate synthase (gltA), and heat-shock operon (groEL) gene sequences. Genome walking was performed to determine its complete gltA and groEL sequences (1233 bp and 1650 bp, respectively). Percent identities to the closest A. phagocytophilum sequences from the US and European strains were 98.6-98.8%, 76.5%, and 80.3-80.8% for 16S rRNA, gltA, and groEL genes, respectively. For deduced amino acid sequences, percent identities to the closest A. phagocytophilum sequences were 66.7% and 97.6% for gltA and groEL genes, respectively. Phylogenetic analyses revealed divergence from any known A. phagocytophilum strain. The lower identities and the divergent phylogenetic position of the Anaplasma sp. detected from sika deer in Japan with established A. phagocytophilum strains provide evidence of its potential novelty.

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.

Presence of a novel ehrlichia sp. in ixodes granulatus found in okinawa, japan

Ehrlichia‐specific DNA fragments of Ehrlichia omp‐1 and groEL genes were found in two I. granulatus ticks which had been collected from wild small mammals in a subtropical zone in Japan. The DNA sequences of groEL and 16SrDNA of the suspected Ehrlichia were clustered into a group of E. chaffeensis, E. muris, and Ehrlichia sp. HF565 found in I. ovatus, but were distinctly different. Therefore the Ehrlichia strain was designated as a novel Ehrlichia sp. 360. The Ehrlichia sp. 360 was detected in I. granulatus but not in any other ticks. This suggests that I. granulatus is a probable vector of Ehrlichia sp. 360 in Japan.

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.