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We explored the bacteria present in the vaginal microbiota facilitating the prevalence of Chlamydia trachomatis in women visiting a community hospital in Sapporo, Japan, by amplicon sequencing. A total of 273 cervical swab samples were collected, and bacterial vaginosis was evaluated in all specimens by assessment of the Nugent score. In 16 of the samples, bacterial 16S rDNA could not be detected and they were therefore omitted from subsequent experiments (n = 257). A significant negative correlation was observed between the Nugent scores and the amount of Lactobacillus 16S rDNA. Among the 257 samples, chlamydial plasmid was detected in 20 samples and was used for amplicon sequencing. No significant association between the Nugent score and the prevalence of C. trachomatis was detected. Based on the results of chlamydial plasmid detection and the Nugent score, chlamydia-negative samples (n = 27) were randomly selected. Finally, the number of operational taxonomic units (OTUs) obtained from amplicon sequencing was compared between chlamydia-positive (n = 20) and -negative samples (n = 27), revealing that a significant difference was only detected for the OTU numbers of Enterobacteriaceae between the C. trachomatis-positive and -negative groups. However, almost all of the samples utilized for amplicon sequencing failed to grow on MacConkey agar plates and produce indole. Taken together, we concluded that traces of bacteria, not live bacteria, belonging to the Enterobacteriaceae indicated the flow of bacteria through the anogenital route along with gut indole, and the resulting impact on the prevalence of C. trachomatis in the cervicogenital tract of women in Japan.

Although sexually transmitted disease due to Chlamydia trachomatis occurs similarly in both men and women, the female urogenital tract differs from that of males anatomically and physiologically, possibly leading to specific polymorphisms of the bacterial surface molecules. In the present study, we therefore characterized polymorphic features in a high-definition phylogenetic marker, polymorphic outer membrane protein (Pmp) F of C. trachomatis strains isolated from male urogenital tracts in Japan (Category: Japan-males, n = 12), when compared with those isolated from female cervical ducts in Japan (Category: Japan-females, n = 11), female cervical ducts in the other country (Category: Ref-females, n = 12) or homosexual male rectums in the other country (Category: Ref-males, n = 7), by general bioinformatics analysis tool with MAFFT software. As a result, phylogenetic reconstruction of the PmpF amino acid sequences showing three distinct clusters revealed that the Japan-males were limited into cluster 1 and 2, although there were only four clusters even though including an outgroup. Meanwhile, the phylogenetic distance values of PmpF passenger domain without hinge region, but not its full-length sequence, showed that the Japan-males were more stable and displayed less diversity when compared with the other categories, supported by the sequence conservation features. Thus, PmpF passenger domain is a useful phylogenetic maker, and the phylogenic features indicate that C. trachomatis strains isolated from male urogenital tracts in Japan may be unique, suggesting an adaptation depending on selective pressure, such as the presence or absence of microbial flora, furthermore possibly connecting to sexual differentiation.

We present herein our first experience with a case of thanatophoric dysplasia (type I) that was diagnosed during the second trimester using three- and four-dimensional HDlive ultrasonography. The HDlive rendering mode clearly showed the anatomical features of thanatophoric dysplasia: external malformations and skeletal abnormalities, including extremely short limbs, flattened vertebral bodies, and short horizontal ribs, among others. HDlive can provide valuable, highly realistic images for the differential diagnosis of skeletal dysplasia. It may also play an important complementary role when conventional two- and three-dimensional ultrasonography does not provide sufficient definition.

This study aimed to show reconstruction of the fetal stomach using the HDlive rendering mode in ultrasound. Seventeen healthy singleton fetuses at 18–34 weeks’ gestational age were observed using the HDlive rendering mode of ultrasound in utero. In all of the fetuses, we identified specific spatial structures, including macroscopic anatomical features (e.g., the pyrous, cardia, fundus, and great curvature) of the fetal stomach, using the HDlive rendering mode. In particular, HDlive rendering images showed remarkably fine details that appeared as if they were being viewed under an endoscope, with visible rugal folds after 27 weeks’ gestational age. Our study suggests that the HDlive rendering mode can be used as an additional method for evaluating the fetal stomach. The HDlive rendering mode shows detailed 3D structural images and anatomically realistic images of the fetal stomach. This technique may be effective in prenatal diagnosis for examining detailed information of fetal organs.

An antiinflammatory drug biphenylylacetic acid (BPAA) as a model drug was selectively conjugated onto one of the primary hydroxyl groups of alpha-, beta-, and gamma-cyclodextrins (CyDs) through an ester or amide linkage, and the in vivo drug release behavior of these prodrugs in rat gastrointestinal tracts after oral administration was investigated. The CyD prodrugs were stable in rat stomach and small intestine and negligibly absorbed from these tracts. Three to six hours after the oral administration, most of the prodrugs had moved to the cecum and colon. The alpha- and gamma-CyD amide prodrugs were hydrolyzed to the maltose conjugate in the cecum and colon, and these prodrugs and the conjugate were negligibly absorbed. On the other hand, the alpha- and gamma-CyD ester prodrugs produced BPAA in the cecum and colon, and BPAA appeared in the blood after 3-6 h. Both beta-CyD amide and ester prodrugs released only small or negligible amounts of the maltose conjugate or BPAA in the cecum and colon, within 24 h, probably due to the low solubility in the biological media. Further, the antiinflammatory effect of the gamma-CyD ester prodrug was evaluated using the model of carageenan-induced acute edema in rat paw and compared with those of BPAA alone and the BPAA/beta-CyD complex prepared by the kneading method in a molar ratio of 1:1. In the case of the beta-CyD complex, a rapid antiinflammatory response was observed, compared to BPAA alone, because the drug is mainly absorbed from the small intestine after a fast dissolution of the complex. In sharp contrast, the gamma-CyD ester prodrug needed a fairly long lag time to exhibit the drug activity, because BPAA is produced after the prodrug had reached the cecum and colon. The present results clearly suggest that the CyD prodrug approach can provide a versatile means for constructions of not only colon-specific delivery systems but also delayed-release system of certain drugs.

Cyclodextrins (CyDs) are known to be fermented to small saccharides by colonic microflora, whereas they are only slightly hydrolyzable and thus are not easily absorbed in the stomach and small intestine. This property of CyDs is particularly useful for colon-specific delivery of drugs. In this study, an antiinflammatory 4-biphenylylacetic acid (BPAA) was selectively conjugated onto one of the primary hydroxyl groups of α-, β-, and γ-CyDs through an ester or amide linkage, 6A-O-[(4-biphenylyl)acetyl]-α-, -β-, and -γ-CyDs (1−3) and 6A-deoxy-6A-[[(4-biphenylyl)acetyl]amino]-α-, -β-, and -γ-CyDs (4−6). In rat cecal and colonic contents (10%, w/v), 1 and 3 released more than 95% of BPAA within 1−2 h, and 2 released about 50% of the drug within 12 h. The amide prodrugs, 4−6, did not release BPAA in the cecal contents, but gave BPAA/maltose or BPAA/triose conjugates linked through an amide bond. On the other hand, these prodrugs were found to be stable in the contents of rat stomachs and small intestines, in int...

Background Although Chlamydia trachomatis is the most commonly reported pathogen that causes urogenital infection such as urethritis or cervicitis, Ureaplasma parvum and Ureaplasma urealyticum, which are commensals in the genital tract, have also now been recognized as contributors to urogenital infection. However, whether the presence of either U. parvum or U. urealyticum is related to that of C. trachomatis in the urogenital tract remains unknown. We therefore attempted to estimate by PCR the prevalence of C. trachomatis, U. parvum and U. urealyticum in endocervical samples obtained from healthy women attending their first prenatal visit in Sapporo, Japan. Methods The samples were taken from 303 apparently healthy women, and the extracted DNAs (n = 280) were used for PCR detection targeting C. trachomatis, U. parvum and U. urealyticum. Statistical analysis of the data was performed by Fisher's exact test. Results PCR detection revealed that the prevalence of C. trachomatis, U. parvum and U. urealyticum was 14.3% (40/280), 41.7% (117/280) and 8.9% (25/280), respectively. C. trachomatis ompA genotype D was most frequently identified. Surprisingly, either C. trachomatis or Ureaplasma spp. was detected in almost half of the healthy women. Mixed infection of C. trachomatis with either U. parvum or U. urealyticum was also observed in 9.2% (26/280) of the women. There was a significant association between C. trachomatis and either U. parvum (p = 0.023) or Ureaplasma total (p = 0.013), but not U. urealyticum (p = 0.275). Conclusion This study demonstrated that the presence of Ureaplasma had a significant effect on the presence of C. trachomatis in the genital tract of healthy women, suggesting that mixed infection is an important factor in bacterial pathogenesis in the genital tract.

Recently, two new kits, HITAZYME (Hitachi Chemical Co., Ltd.) and SERO IPALISA (Savyon Diagnostics, Ltd.), for the assay of anti-C. trachomatis antibodies by the enzyme immunoassay (EIA) method have been developed and put into clinical application. In the study reported here, the authors investigated the clinical usefulness of these assay kits, together with the IPAzyme and micro-IF test, in the diagnosis of cases of urogenital tract C. trachomatis infections. 1. The positive rates for IgA antibodies, which are considered to be an indicator of active infection, obtained with the HITAZYME and SERO IPALISA kits in the 82 antigen-positive cases were significantly (p < 0.005) higher than the rates obtained with the IPAzyme and micro-IF test. These results showed the usefulness of the HITAZYME and SERO IPALISA kits for detecting C. trachomatis infections. 2. A comparison was made of the assay results obtained with the HITAZYME and SERO IPALISA kits, and it was found that there was a large number of cases (142) that tested negative for IgA antibodies with the HITAZYME but positive with the SERO IPALISA kit. We carried out a confirmatory test on the specimens of cases for which the results obtained with the HITAZYME and SERO IPALISA kits were not in agreement. This test employed the Western blotting method using COMC (the antigen extracted from EB of C. trachomatis strain L2 and used in the HITAZYME kit) and whole EB of C. trachomatis strain L2 (the antigen used in the SERO IPALISA kit). The results showed a significantly higher degree of agreement between the HITAZYME kit data and the Western blotting data than between the SERO IPALISA kit data and the Western blotting data. 3. In addition, with the objective of investigating the existence of cross reactivity with anti-C. pneumoniae antibodies, we performed Western blotting using as the antigen crude whole EB of C. pneumoniae strain TW-183. The results showed that anti-C. pneumoniae antibodies were detected in 25 of 35 (71.4%) cases that were negative with C. trachomatis antigen and the HITAZYME kit and positive with the SERO IPALISA kit. These findings indicate a strong possibility that these cases positive with the SERO IPALISA kit are due to a cross reaction with anti-C. pneumoniae antibodies.(ABSTRACT TRUNCATED AT 400 WORDS)

We evaluated the clinical usefulness of a new EIA kit using a monoclonal antibody, IDEIA CHLAMYDIA (IDEIA, Novo Nordisk), for detection of C. trachomatis antigen from the genital tracts of male and female cases. The results were compared with those by Chlamydiazyme (Abbott). 1. C. trachomatis antigen detection by the IDEIA and Chlamydiazyme tests before treatment; IDEIA has a significantly higher detection rate (38.0%, 105/276) than Chlamydiazyme (29.8%, 80/276), for C. trachomatis antigen from urethral smears of 276 male patients with urethritis. In 646 female cases, including cervicitis and so on, IDEIA detected C. trachomatis antigen from cervical smears in 14.5% (94/648) of the total, while Chlamydiazyme did so in 11.9% (77/648). When considering the different results using IDEIA and Chlamydiazyme, approximately 20% of the IDEIA-positive cases were Chlamydiazyme-negative. However, when IDEIA was negative, less than 1% showed Chlamydia-positive. 2. C. trachomatis antigen detection during and after treatment; We studied the clinical courses of 14 male urethritis and 8 female cervicitis cases who had had positive results with both IDEIA and Chlamydiazyme before treatment. Two of the 14 urethritis cases showed positive results with IDEIA, but not with Chlamydiazyme after either 7 or 14 days treatment by an antimicrobial agent. These two also had symptoms indicating persistent urethritis. One of the 8 female cervicitis cases showed a positive result with IDEIA but not with Chlamydiazyme after 7 days treatment by an antimicrobial agent, and this case also had symptoms indicating persistent cervicitis. Thus, these clinical findings suggest that IDEIA can detect even a small quantity of antigen soon after treatment, but Chlamydiazyme can not. In conclusion, IDEIA has a higher sensitivity than Chlamydiazyme, in the detection of C. trachomatis antigen, suggesting that IDEIA is more useful.

C. trachomatis antigen in first-voided urine sediments was detected by a new EIA kit using a monoclonal antibody, IDEIA CHLAMYDIA (IDEIA, Novo Nordisk), in males with urethritis and females with cervicitis. The result was compared with that by Chlamydiazyme (Abbott). 1. C. trachomatis antigen detection in male urethritis (285 cases) by the IDEIA test: The antigen detection rate was 37.9% (108/285) in urethral smears, and 33.7% (96/285) in first-voided urine sediments of the patients. The positive co-incidence rate between urethral smears and first-voided urine sediments was 82.4% (98/108). Thus, the detection of the antigen seems feasible in first-voided urine sediments. 2. Comparison of C. trachomatis antigen detection by the IDEIA and Chlamydiazyme tests: In 78 male cases with urethritis undergoing both tests, the rates of antigen detection from urethral smears and first-voided urine sediments were studied. The detection rate from urethral smears was 41.0% (32/78) for IDEIA, and 37.2% (29/78) for Chlamydiazyme. In first-voided urine sediments, the rate was 35.9% (28/78) for IDEIA and 24.4% (19/78) for Chlamydiazyme. In both specimens, the detection sensitivity was higher for IDEIA. 3. C. trachomatis antigen detection in chlamydial cervicitis (28 cases) by the IDEIA test: The antigen detection rate was 46.4% (13/28) in urethral smears and 60.7% (17/28) in first-voided urine sediments. The detection rate in first voided urine sediments was higher. Thus, in patients suspected of having chlamydial cervicitis, it seems necessary not only to search the antigen in cervical smears but also to study the first-voided urine sediments.(ABSTRACT TRUNCATED AT 250 WORDS)

We surveyed Chlamydial infection with Chlamydiazyme for 3010 pregnant housewives in Hokkaido. Four hundred and fourty six out of the 3010 pregnant housewives were also be retrospectively investigated for the influence of C. trachomatis on outcome of pregnancy and on their newborns. The results were as follows. 1) Of the 3010 pregnant housewives, 217 (7.2%) were C. trachomatis-antigen positive, when their endocervical specimens were tested. There was no difference in the positive rate of C. trachomatis among the six cities where our investigation was performed. 2) A high C. trachomatis-positive rate (21.9%) was achieved in the pregnant teen-aged housewives with a significant decrease as age increased. 3) As for placenta previa, threatened abortion, preterm delivery and small for date infants, the incidence was higher in the untreated C. trachomatis positive group than in the C. trachomatis negative group. 4) The weeks of gestation and birth weight of newborns in the untreated C. trachomatis positive group were significantly lower than those of the C. trachomatis negative group. 5) No statistical significance was found in the incidence of premature rupture of membrane, fatal distress, spontaneous abortion nor postpartum fever between the C. trachomatis negative group and the untreated C. trachomatis positive group. These results suggest that Chlamydial infection in pregnant housewives is widely spread in Hokkaido and gives some disadvantage to pregnancy outcome and newborns. Consequently, Chlamydial infection in pregnant women must be appropriately diagnosed and treated in the early stage of pregnancy.

Background. A urine-based enzyme-linked immunosorbent assay (ELISA) kit for detection of antibody to Helicobacter pylori has been developed in Japan. Urine samples can be obtained noninvasively and are easier and safer to handle than are serum samples. The aim of this study was to examine the clinical usefulness of this urine-based ELISA kit. Materials and Methods. A pair of random, single-void urine and serum samples was collected from each of 1,061 subjects, including 238 patients with gastroduodenal disease. The sensitivity and specificity of the urine-based ELISA was compared with those of three commercially available serum-based ELISA kits. For those patients with gastroduodenal disease, the urine- and serum-based ELISA results were also compared with those for other diagnostic methods using endoscopic biopsy specimens, such as culture, histology, and rapid urease tests. Results. Based on the three serum-based ELISA results, the sensitivity, specificity, and accuracy of the urine-based ELISA were 97.7%, 95.6%, and 96.8%, respectively. On the basis of the biopsy test results, the sensitivity (96.2%), specificity (78.9%), and accuracy (91.0%) of the urine-based ELISA were almost equivalent or superior to all three serum-based ELISAs tested. In addition, 10 of the 12 false-positive cases for urine-based ELISA were confirmed to be true positives for antibodies to H. pylori by Western blot analysis and inhibition ELISA. Conclusions. The urine-based ELISA (URINELISA H. pylori Antibody) is very accurate and should be useful as an alternative to serum-based ELISAs for screening of H. pylori infection.

α-1,4 glucosidic bonds of cyclodextrins (CyDs) are barely capable of being hydrolyzed and only slightly absorbed in passage through the stomach and small intestine; however they are fennented by colonic microflora into small saccharides and thus absorbed in the large intestine. This kind of biodegradable property of CyDs is particularly useful as a colon-targeting carrier [1], and thus CyD conjugates may serve as a source of site-specific delivery of drugs to colon Therefore, we prepared six conjugates of the antiinflammatory drug, 4-biphenylylacetic acid (BPAA) coupled to α-, s- and γ-CyDs through an ester or amide linkage (Fig. 1), and investigated the in-vitro and in-vivo release behavior of BPAA from the prodrugs, oral absorption behavior, and antiinflammatory effect in rat model to evaluate them as a colon-specific dmg delivery system.

Biphenylyl acetic acid was selectively conjugated to one of the primary hydroxyl groups of β-cyclodextrin through an ester- or amide-linkage, and the physicochemical properties (aqueous solubility and hydrolysis) were investigated. Aqueous solubility of the conjugates was lower than those of either drug or parent β-cyclodextrin. The amide conjugate was stable in aqueous solution and in rat biological fluids and gastrointestinal contents. The ester conjugate was hydrolysed to β-cyclodextrin and biphenylyl acetic acid at moderate rates resulting in a V-shaped rate-pH profile in aqueous solution. The ester conjugate released the drug preferentially when incubated with the contents of caecum or colon, whereas no appreciable drug release was observed on incubation with contents of stomach or intestine, nor on incubation with intestinal or liver homogenates, nor on incubation with rat blood. The present results suggest that the ester-type drug conjugate of β-cyclodextrin may serve as a colon-targeting prodrug.

Biphenylyl acetic acid (BPAA) was conjugated with one of primary hydroxyl groups of α-, β-, and γ-cyclodextrins (α-, β- and γ-CyDs) through an ester-linkage. The serum levels of BPAA after oral administration of the α- and γ-CyD conjugates to rats were significantly delayed, accompanying a marked increase in the serum levels, whereas the β-CyD conjugate resulted in little increase to the serum levels. The in vitro drug release behavior also suggests that the ester-type drug conjugates of α- and γ-CyD serve as colon-targeting prodrugs providing a delayed drug action.