Toshikatsu Hagiwara

Chlamydia pneumoniae Serology: Interlaboratory Variation in Microimmunofluorescence Assay Results

The lack of standardization in chlamydia serology has made interpretation of published data difficult. This study was initiated to determine the extent of interlaboratory variation of microimmunofluorescence (MIF) test results for the serodiagnosis of Chlamydia pneumoniae infections. Identical panels of 22 sera were sent to 14 laboratories in eight countries for the determination of IgG and IgM antibodies by MIF. Although there was extensive variation in the numeric titer values, the overall percentage agreement with the reference standard titers from the University of Washington was 80%. For results by serodiagnostic category, the best agreement was for four-fold rise in IgG titers, while the lowest agreement was for negative or low IgG titers. Agreement for IgM titers was 50%-95%. Four laboratories failed to discern false-positive IgM titers possibly because of the presence of rheumatoid factor. Further studies are underway to determine the source of interlaboratory variation for the MIF test.

Differentiation of Chlamydia species by combined use of polymerase chain reaction and restriction endonuclease analysis.

To differentiate Chlamydia spp., a primer pair designed to generate a genus‐specific region of the major outer membrane protein (MOMP) gene was used in a PCR to amplify a single DNA fragment of 245‐259 bp. In the PCR, the expected single DNA fragment was amplified from strains of Chlamydia trachomatis, C. psittaci, C. pneumoniae and C. pecorum, respectively. By restriction endonuclease analysis with AluI and PvuII, the amplified products exhibited four distinct patterns, corresponding to the four species. It is, therefore, concluded that one‐step PCR followed by restriction endonuclease analysis as described in this study could be a valuable method for the detection and differentiation of Chlamydia species.

Serological Investigation of Bartonella henselae Infections in Clinically Cat‐Scratch Disease‐Suspected Patients, Patients with Cardiovascular Diseases, and Healthy Veterinary Students in Japan

Seroprevalence of Bartonella henselae was investigated in Japan in 48 individuals clinically suspected of having cat‐scratch disease (CSD), 159 patients with cardiovascular diseases, and 129 healthy veterinary students. Of 48 CSD‐suspected patients examined, 19 (39.6%) were positive for B. henselae‐IgG and 4 (8.3%) for B. henselae‐IgM. Of 159 patients with cardiovascular diseases, 5 (3.1%) were positive for B. henselae‐IgG. In healthy veterinary students, 14 of 129 (10.9%) were positive for B. henselae‐IgG and 1 (0.8%) for B. henselae‐IgM. The positive rates of B. henselae‐IgG and ‐IgM in CSD‐suspected patients were significantly higher than in other sources. Most CSD‐suspected and healthy individuals who were positive for B. henselae antibody had had some contacts with cats. In CSD‐suspected patients, the B. henselae positive rate in females was significantly higher than in males, and high seropositive rates to B. henselae were found in younger age groups.

Evaluation of a new amplified enzyme immunoassay (EIA) for the detection of Chlamydia trachomatis in male urine, female endocervical swab, and patient obtained vaginal swab specimens.

Aims—To compare the performance of a new generation dual amplified enzyme immunoassay (EIA) with a molecular method for the diagnosis of Chlamydia trachomatis, using a range of urogenital samples, and to assess the reliability of testing self collected vaginal specimens compared with clinician collected vaginal specimens. Methods—Two population groups were tested. For the first population group, first void urine samples were collected from 193 male patients with urethritis, and endocervical swabs were collected from 187 high risk commercial sex workers. All urine and endocervical specimens were tested by a conventional assay (IDEIA chlamydia), a new generation amplified immunoassay (IDEIA PCE chlamydia), and the Amplicor polymerase chain reaction (PCR). Discrepant results obtained among the three sample types were confirmed using a nested PCR test with a different plasmid target region. For the second population group, four swab specimens, including one patient obtained vaginal swab, two clinician obtained endocervical swabs, and one clinician obtained vaginal swab, were collected from 91 high risk sex workers. Self collected and clinician collected vaginal swabs were tested by IDEIA PCE chlamydia. Clinician obtained endocervical swabs were assayed by IDEIA PCE chlamydia and Amplicor PCR. Results—The performance of the IDEIA PCE chlamydia test was comparable to that of the Amplicor PCR test when male urine and female endocervical swab specimens were analysed. The relative sensitivities of IDEIA, IDEIA PCE, and Amplicor PCR on male first void urine specimens were 79.3%, 91.4%, and 100%, respectively. The relative sensitivities of the three tests on female endocervical specimens were 85.0%, 95.0%, and 100%, respectively. The positivity rates for patient collected vaginal specimens and clinician collected vaginal specimens by IDEIA PCE were 25.2% and 23.1%, respectively, whereas those for clinician collected endocervical swabs by PCR and IDEIA PCE were both 27.5%. Conclusions—IDEIA PCE chlamydia is a lower cost but sensitive alternative test to PCR for testing male urine samples and female endocervical swabs. In addition, self collected or clinician collected vaginal specimens tested by IDEIA PCE chlamydia are a reliable alternative to analysing endocervical specimens.

In Vitro Inhibitory Effects of Hinokitiol on Proliferation of Chlamydia trachomatis

ABSTRACT The inhibitory effects of hinokitiol (β-thujaplicin) on Chlamydia trachomatis D/UW-3/Cx were shown by MIC, minimum lethal concentration (MLC), and preinoculation minimal microbicidal concentration assays using HeLa 229 cells. The MIC and the MLC were both 32 μg/ml. Further evaluation of hinokitiol as a topical agent against C. trachomatis is warranted.

In vitro analysis of the change in resistance of Chlamydia trachomatis under exposure to sub-MIC levofloxacin for a therapeutic term.

Background: While fluoroquinolone-resistant Chlamydia trachomatis strains have not been clinically isolated, they were isolated in an in vitro study recently. Methods: To determine whether C. trachomatis strains develop resistance under sub-MIC antibacterial exposure in a clinical therapeutic term, C. trachomatis strains were exposed to sub-MIC levofloxacin (LVFX) for about 2 weeks. The MIC of LVFX was measured and DNA fingerprinting was performed every 72 h by PCR using random primers. Results: There was almost no change in the MIC under exposure to 0.125 μg/ml LVFX. However, some mutational changes in DNA fingerprints developed. Conclusions: In clinical therapeutic terms, resistant strains of C. trachomatis will probably not develop, even if sub-MIC LVFX is employed.

Biosynthesized Tea Polyphenols Inactivate Chlamydia trachomatis In Vitro

ABSTRACT Biosynthesized tea polyphenols showed antichlamydial activity against Chlamydia trachomatis D/UW-3/Cx and L2/434/Bu using cell culture. The most active compounds were (−)-epigallocatechin gallate and (−)-epicatechin gallate, followed by (−)-epicatechin (EC). (+)-Epicatechin and (−)-epigallocatechin were intermediate. EC was the least toxic. These results warrant evaluation of tea polyphenols as topical antichlamydial agents.

Experimental feline toxoplasmosis.

The route of inoculation as well as stage of inoculated organism in its life cycle influenced the pathogenicity or infectivity of Toxoplasma in cats. All 6 cats administered orally with Toxoplasma cysts were infected without any clinical symptoms and excreted oocysts in the feces at the early stage of infection (5-12 days after administration). Of 3 cats administered orally with Toxoplasma oocysts, only one animal developed latent infection excreting oocysts in the feces at the end of 5th week after administration (38th, 40th day). On postmortem examination, none of infected animals showed abnormalities. Toxoplasma was recovered from the lung, striated musculatures and others. When cats were inoculated intraperitoneally with cysts, all of them developed fatal infection. Clinical signs were manifested by fever, anorexia, lethargy and dyspnea, which were similar to those of cats infected naturally. On postmortem examination, a large amount of fluid was found to have accumulated in the pleural and peritoneal cavities. Cloudy swelling of the liver and inflammatory edem of the lung were also observed. The level of SGOT and SGPT values was elevated significantly. None of animals inoculated intraperitoneally with cysts excreted oocysts in their feces though Toxoplasma was recovered from all tissues and organs of them. Intramusculally inoculation of cortisone acetate did not affect on relapsing or oocyst reproduction.

A case of pediatric Chlamydia pneumoniae infection and properties of the isolate

Chlamydia pneumoniae was isolated from the throat of a 2-year-old girl with upper respiratory illness. The isolate, Shizuoka-37, was stained with C. pneumoniae specific monoclonal antibody (RR402), as well as the genus specific antibody (Cultureset), but not with C. trachomatis specific monoclonal antibody (Micro-Trak). C. pneumoniae genome was amplified by polymerase chain reaction in the isolate. Elementary bodies (EB) of the isolate was round shaped by electron micrograph.

Monoclonal Antibodies to Herpes Simplex Viruses Antigens-Specificity and Utility in Rapid Serotyping of Clinical Isolates

Sixteen hybridomas secreting antibodies to HSV-1 and 22 hybridomas secreting antibodies to HSV-2 were derived from fusion of SP2/0 myeloma cells with spleen cells from BALB/c mice immunized with each respective virus. Four of the former 16 hybridomas and seven of the latter 22 hybridomas were subcloned and injected into pristane-primed mice to obtain high titers of monoclonal antibodies. Antigen specificity of these monoclonal antibodies were determined by the Western blotting (WB) assay. Two out of four monoclonal antibodies that showed selective reactivity for HSV-1 in IFA, reacted with HSV-1 specific proteins; #1 reacted with 100 KD and 70 KD proteins and #4 with a 150 KD protein, respectively, while the remaining two antibodies reacted only with a 50 KD protein that is type-common antigen. On the other hand, two out of seven antibodies which showed selective reactivity for HSV-2 in IFA, reacted with HSV-2 specific proteins: #5 with a 100 KD protein and #10 with three proteins of 30, 25, and 20 KD, and the other two antibodies reacted with a 50 KD protein that is a type-common antigen. The remaining three antibodies, two of which were found to be immunoglobulin type IgM, reacted with neither HSV-1 nor HSV-2 antigens in WB assay. In order to determine their utility in serotyping, 11 monoclonal antibodies were examined by IFA test for reactivity to cells that were infected with 20 HSV-1 or 16 HSV-2 isolates which had been typed by neutralization test.(ABSTRACT TRUNCATED AT 250 WORDS)