Use of a dual priming oligonucleotide system to detect multiple sexually transmitted pathogens in clinical specimens.

AIMS: To evaluate a new dual priming oligonucleotide (DPO)-based multiplex polymerase chain reaction (PCR) assay for detection of six sexually transmitted pathogens, including Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma urealyticum and Trichomonas vaginalis. METHODS AND RESULTS: Using 130 clinical specimens, the results obtained by the multiplex PCR, previously established in-house PCR and COBAS Amplicor PCR assays were compared. The specimens frequently contained multiple pathogens (34/130 specimens). The multiplex PCR assay had an overall sensitivity of 96% and specificity of 100% compared to the in-house PCR assay at >20 microg ml(-1) of DNA concentrations in samples and there was no cross-reaction with nonpathogenic Neisseria species that cause the majority of false-positive results with the COBAS Amplicor PCR assay. CONCLUSIONS: The DPO-based multiplex PCR assay detected the six sexually transmitted pathogens in clinical specimens with a high sensitivity and specificity, although its sensitivity was dependent on the DNA content of the samples. SIGNIFICANCE AND IMPACT OF THE STUDY: It is the first report about the new DPO-based technique to detect multiple sexually transmitted pathogens in a single assay, which has considerable potential to diagnose the infections accurately and rapidly.

Effective human herpesvirus 8 infection of human umbilical vein endothelial cells by cell-mediated transmission.

Cell-free transmission of human herpesvirus 8 (HHV-8) to human cells in vitro has been reported to be difficult, if not impossible. The present experiments were conducted with the idea that cell-cell contact may produce much more effective transmission, so-called cell-mediated transmission. Primary human umbilical vein endothelial cells (HUVECs) were cocultured with an HHV-8-infected lymphoma cell line, BCBL-1 cells. When a ratio of 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated BCBL-1 cells to HUVECs of 10:1 was used, more than 20% of HUVECs were found to express the HHV-8 latency-associated nuclear antigen (LANA) 48 h after the start of coculturing; this value increased to more than 30% after 72 h. HHV-8-encoded ORF26, K8, K8.1, K10, K11, ORF59, and ORF65 proteins were not detected in these HHV-8-infected HUVECs until 72 h. The HHV-8 antigens were not observed in HUVECs cocultured with TPA-treated BCBL-1 cells separated by a membrane. Thirty days after removal of the BCBL-1 cells from the cell-mediated transmission experiment, the HUVECs still expressed LANA and the HHV-8 genome was detected by PCR in these cells. Moreover, the ORF59 protein, a DNA replication-associated protein of HHV-8, was expressed in such HUVECs in the presence of TPA stimulation. These results indicated a far more effective transmission mechanism, cell-cell contact, suggesting the possibility that such a mechanism works in vivo.

[Clinical and fundamental studies of streptococcal toxic shock syndrome].

Streptococcal toxic shock syndrome(STSS) is known to progress rapidly into septic shock and multiple organ failure with frequently necrotizing fasciitis, and high mortality (approximately 40%). The diagnosis of STSS is confirmed based on the diagnosis criteria induced by the working group of the United State. Several extracellular products such as SPE A, B, and C having pyrogenic and superantigenic activity as well as SPE F, SPE G, H, J, SME Z, SME Z2, SSA are likely to involved in the pathogenesis of STSS. Recent studies have demonstrated an important role of certain cytokines, such as TNF-alpha, in laboratory animals. The exact role of such products in the pathogenesis of STSS, however, is currently unknown. The role of several virulence factors of group A streptococci in the pathogenesis of STSS was discussed in this review.

Clonotypes of Staphylococcus aureus isolated from continuous ambulatory peritoneal dialysis patients: what is the vector between nares and infection site?

Staphylococcus aureus is frequently isolated from patients with infections related to continuous ambulatory peritoneal dialysis (CAPD). In many cases, the organism is also isolated simultaneously from the anterior nares. To clarify the transmission trail of S. aureus, we used DNA analysis to identify clonotypes of clinical strains. The nares and exit sites of 32 CAPD patients were swabbed, and PD fluid samples were taken for pathogen culture. Genome DNA of S. aureus was digested with restriction enzyme Sma I for pulsed-field gel electrophoresis. We also asked the patients how they usually performed the PD procedure. S. aureus was isolated from 4 patients, including 3 who hosted two strains isolated separately from different sites. The DNA patterns of the strains isolated from these latter 3 patients were identical. However, the clonotypes from all 4 patients were different. Most of the patients did not wash their hands and wear masks while exchanging PD bags and caring for their exit sites. After the patients were disinfected and re-educated in proper procedures, S. aureus was not detected in any of them. These data suggest that no outbreak occurred in our hospital and that the vectors of endogenous infection were the patients themselves, probably their hands. A bacteriological study presents an efficient opportunity to re-educate patients in PD procedure.

Effects of "isolating hemodialysis" on prevention of methicillin-resistant Staphylococcus aureus cross-infection in a hemodialysis unit.

AIM: To evaluate the effects of a contact isolation program against methicillin-resistant Staphylococcus aureus (MRSA) cross-infection among patients in a hemodialysis unit. CLINICAL SETTING AND METHODS: In all patients maintaining hemodialysis therapy were tested for MRSA infection and who had MRSA infection, not only inpatients but also outpatients were separated into a designated area (isolating hemodialysis). Clinically isolated MRSA strains were clonotyped with coagulase typing, staphylococcal enterotoxin typing and restriction enzyme analysis of plasmid DNA. RESULTS: The frequency of patients with MRSA infection was 4.5% before starting this protocol and was reduced to 2.9% two and a half years later. At this time, MRSA was isolated from the 8 patients. These 8 clinical strains were differentiated into 6 clonotypes and 3 strains showed the same patterns. Two of 3 were isolated from inpatients and the other was from a patient with community onset MRSA colitis. In this case, most MRSA infections were independent under prophylaxis control and cross-infection was observed only once between hospitalized patients who stayed in a same ward. CONCLUSION: This "isolating hemodialysis" should be useful to prevent cross-infection among patients in end-stage renal disease in a dialysis unit.

Synergistic bactericidal effects of acrinol and tetracycline against Pseudomonas aeruginosa.

Combined treatment of acrinol (Ac) and tetracycline hydrochloride (Tc) against Pseudomonas aeruginosa strains isolated from clinical specimens synergistically increased the bactericidal effect. The minimum bactericidal concentration (MBC) of Ac against P. aeruginosa strain no. 985 was 200 microg/ml, while the MBC of Ac against strains no. 47 and no. 783 was above 800 microg/ml for each. The MBC of Tc was above 400 microg/ml against each of the tested strains. However, simultaneous treatment with 25 microg/ml Ac and 200 microg/ml Tc against P. aeruginosa strain no. 985 decreased the viable cell number from 107 cfu/ml to <10 cfu/ml within 24 h, while a higher concentration of Tc (400 microg/ml) with Ac (25 microg/ml) reduced the viable cell number to <10 cfu/ml within 8 h. A similar synergistic bactericidal effect of Ac and Tc was observed in strains no. 47 and no. 783 by treatment with 200 microg/ml Ac and 200 microg/ml or 400 microg/ml Tc. The degree of bactericidal effect against P. aeruginosa was proportional to the concentration of Tc under the condition of a constant concentration of Ac. Furthermore, Ac-treated cells of strain no. 47 were killed by a following Tc treatment, but cells pretreated with Tc did not show such a sensitivity to Ac. To induce the synergistic effect of Ac and Tc, Ac must be applied to P. aeruginosa before or at the same time as Tc.

Streptococcus peroris sp. nov. and Streptococcus infantis sp. nov., new members of the Streptococcus mitis group, isolated from human clinical specimens.

Taxonomic studies were performed on eight strains of alpha-haemolytic streptococci that showed very low DNA-DNA hybridization similarity values with all established members of the mitis group of the genus Streptococcus. These strains were isolated from the tooth surface and pharynx of humans. 16S rRNA gene sequence analysis showed that these strains belonged to the mitis group, but that they fell into two new branches. DNA-DNA hybridization demonstrated two new similarity groups. From the results of the present study, the names Streptococcus peroris sp. nov. and Streptococcus infantis sp. nov. are proposed for these new groups. The type strains are O-66T (= GTC 848T = JCM 10158T) and O-122T (= GTC 849T = JCM 10157T), respectively.

[Report of surveillance on streptococcal toxic shock syndrome in Japan and presentation of the criteria].

A survey was made on the situation of Group A Streptococcal toxic shock syndrome (STSS) based on questionnaires. The survey was divided into two parts. The first survey was done by sending out an outline of the STSS inquiring if any STSS cases were observed by mail to university hospitals, residence training hospital and other major hospitals totaling 2512 institutes. The second survey was subsequently done to the institutes that had STSS cases asking for the clinical course, data and sampling of the bacteria. The diagnosis of STSS was confirmed based on the diagnostic criteria induced by the working group of the United States. We have found 97 cases of STSS which 48.5% had fatal outcomes. There was no significant sex difference in the onset or the mortality rate. It occurred more in the older population, and occurred through out Japan but was not found to be epidemic. The first case was backed in 1978 and it began to increase since 1993, reaching its peak in 1994 and now decreasing in number. Most of the isolated Group A streptococcus were of type M1 and M3. We have modified the United States diagnostic criteria creating a new Japanese criteria, which includes the symptoms of the central nervous system in the term MOF. The aim for the Japanese criteria is to search for the etiology of the disease. The Japanese criteria requires that the disease progresses rapidly and that the patient be free from any conditions that might suppress the immunal system.

Purification and partial characterization of a novel human platelet aggregation factor in the extracellular products of Streptococcus mitis, strain Nm-65.

A human blood platelet aggregation factor was purified from the extracellular products (ECP) of Streptococcus mitis, strain Nm-65 by sequential chromatography on DEAE-Sepharose CL-6B, hydroxyapatite and Superdex 75 columns. The purified factor (S. mitis-derived human platelet aggregation factor, Sm-hPAF) gave a single band with a molecular weight of 66 kDa on SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Sm-hPAF showed a peak absorption at 278 nm and an isoelectric point of around 8.5. Chemical analyses revealed that Sm-hPAF contained no sugars and that its first 15 amino-terminal amino acid residues were H-DEQGNRPVETENIAR. Platelet aggregation activity of Sm-hPAF was abolished by heating at 45 degrees C for 10 min. Platelet aggregation by Sm-hPAF was accompanied by a release of prostaglandin E2 (PGE2) in a dose-dependent manner. The platelet aggregation was not inhibited by either prostaglandin E1 (PGE1) or Gly-Arg-Gly-Asp-Ser (GRGDS), that inhibit the platelet aggregation induced by collagen. Twenty (77%) platelet rich-plasma (PRP) specimens derived from 26 healthy volunteers were aggregated by Sm-hPAF, but the remaining 6 (23%) were not reactive. A preliminary study suggested the presence of an inhibitory factor against Sm-hPAF in the plasma from a non-reactive donor.

Induction of lymphocytes cytotoxic to oral epithelial cells by Streptococcus mitis superantigen.

The preparation of a superantigenic fraction F-2 from the culture supernatant of Streptococcus mitis 108, a fresh isolate from human tooth surfaces, was reported previously. Now, to determine the possible pathogenic role of the superantigen in oral mucosal diseases, we examined the cytotoxic effects of human peripheral blood T-cells activated with F-2 on human oral epithelial cells. T-cells activated with F-2 were cytotoxic to the human squamous carcinoma HO-1-N-1 cells derived from the oral mucosa, similar to those activated with Staphylococcus aureus enterotoxin B (SEB). This cytotoxic effect was increased in a dose-dependent manner by the addition of the respective stimulant, F-2 or SEB, to the cytotoxic assay system. F-2 endowed mainly CD8+ T-cells with cytotoxic activity. Pretreatment with human interferon gamma increased the sensitivity of the HO-1-N-1 cells to the cytotoxic effects of F-2-activated T-cells. The F-2-activated T-cells were also cytotoxic to human keratinocytes derived from gingiva. There was no correlation between the degree of cytotoxicity and the levels of tumor necrosis factor alpha in co-cultures of F-2-activated T-cells and HO-1-N-1 cells. A double-chamber plate experiment revealed no cytotoxic effects when the F-2-activated T-cells were separated from the HO-1-N-1 cells. Supernatants of the co-cultures of target and effector cells were not cytotoxic to HO-1-N-1 cells. These findings suggest that the cytotoxic effects of the F-2-activated T-cells on HO-1-N-1 cells were mediated not by soluble factors but by the direct interaction between the activated T-cells and the target cells. The cytotoxicity of the F-2-activated T-cells against HO-1-N-1 cells was markedly inhibited by monoclonal antibodies (MAbs) against CD11a and CD54, but was only slightly inhibited by MAbs against human leukocyte antigen (HLA)-DR and CD2. Thus, the interaction between lymphocyte-function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) was crucial for the F-2-dependent T-cell-mediated cytotoxicity against oral epithelial cells, while HLA-DR and CD2 molecules are not necessarily involved in the cytotoxicity observed.

Adjuvant activity of diesel exhaust particulates (DEP) in production of anti-IgE and anti-IgG1 antibodies to mite allergen in mice.

The present study indicates that diesel exhaust particulates (DEP) and pyrene contained in DEP have an adjuvant activity on IgE and IgG1 antibody productions in mice immunized intranasally with a mite allergen. The effect of pyrene on IgE and IgG1 antibody productions in mice was investigated to clarify the relation between mite allergy and adjuvancy of the chemical compounds in DEP. Der f II, one of the major allergens of house dust mite (Dermatophagoides farinae), was used as a mite allergen. Mice were grouped, and immunized with 5 micrograms of Der f II alone, 5 micrograms of Der f II plus 200 micrograms of pyrene and 5 micrograms of Der f II plus 100 micrograms of DEP intranasally seven times at two week intervals. The separate groups of mice were also immunized with 10 micrograms of Der f II plus the same dose of adjuvants in the same way. The IgE antibody responses to Der f II in mice immunized with Der f II plus pyrene or Der f II plus DEP were markedly enhanced compared with those immunized Der f II alone. The anti-Der f II IgE antibody production increased with increasing the dose of Der f II from 5 micrograms to 10 micrograms in mice immunized with Der f II plus the same dose of adjuvants. The IgG1 antibody responses to Der f II in mice immunized with 10 micrograms of Der f II plus 200 micrograms of pyrene or 10 micrograms of Der f II plus 100 micrograms of DEP were extremely higher than those immunized with 10 micrograms of Der f II alone. In addition, when the peritoneal macrophages obtained from normal mice were incubated with pyrene or DEP in vitro, an enhanced interleukin-1 alpha production of the macrophages was observed. When the spleen lymphocytes obtained from the mice immunized with 10 micrograms of Der f II plus 100 micrograms of DEP or 10 micrograms Der f II plus 200 micrograms of pyrene were stimulated with 10 micrograms of Der f II in vitro, an enhanced IL-4 production of the lymphocytes was also observed compared with those immunized with Der f II alone. These results suggest that the adjuvancy of DEP and pyrene on the production of IgE and IgG1 antibodies to Der f II may be one of the factors responsible for an incidence of asthma caused by house dust mite.

Adjuvant activities of pyrene, anthracene, fluoranthene and benzo(a)pyrene in production of anti-IgE antibody to Japanese cedar pollen allergen in mice.

We have previously demonstrated that pyrene in diesel-exhaust particles (DEP) has an adjuvant activity on immunoglobulin E (IgE) antibody production in mice immunized with Japanese cedar pollen allergen (JCPA) or ovalbumin (OA) intraperitoneally. The present study is concerned with the adjuvant activity in IgE antibody production against JCPA of pyrene or DEP inoculated intranasally in mice. We show that anthracene, fluoranthene and benzo(a)pyrene in DEP have the ability to enhance anti-JCPA IgE antibody production in mice by intranasal immunization. Mice were grouped, immunized with 10 micrograms of JCPA plus 400 micrograms of pyrene, 10 micrograms of JCPA plus 100 micrograms of DEP, 10 micrograms of JCPA plus 2 mg of aluminum hydroxide and 10 micrograms of JCPA alone intranasally 7 times at 2 week intervals. Mice were also grouped, and immunized with JCPA (10 micrograms) plus 40 micrograms of anthracene, JCPA (10 micrograms) plus 400 micrograms of fluoranthene, JCPA (10 micrograms) plus 40 micrograms of benzo(a)pyrene, and JCPA (10 micrograms) plus 400 micrograms of pyrene and JCPA (10 micrograms) alone. We found that the IgE antibody responses to JCPA in mice immunized with JCPA plus pyrene, JCPA plus DEP or JCPA plus the three chemical organic compounds mentioned above were significantly enhanced compared with those immunized with JCPA alone. In addition, when the intraperitoneal macrophages obtained from the normal mice (unimmunized mice) were incubated with pyrene, anthracene, fluoranthene or benzo(a)pyrene in vitro, an enhanced chemiluminescence (CI) response and interleukin-1 alpha (IL-1 alpha) production of the macrophages was observed in each instance. These results suggest that in the production of IgE antibody to JCPA the adjuvancy of polycyclic aromatic hydrocarbons (PAHs) in DEP may be important in an attack of Japanese cedar pollinosis.

Efficacy of gentian violet in the eradication of methicillin-resistant Staphylococcus aureus from skin lesions.

The efficacy of gentian violet (Gv) in eradicating methicillin-resistant Staphylococcus aureus (MRSA) in decubitus ulcers was investigated. Decubitus ulcers (a total of 18 cases) were scrubbed with Gv aqueous solution 0.1% and ointment containing Gv 0.1% was applied daily. MRSA was not detected in these lesions for 3-34 days (average, 10.5 +/- 2.5 days) after the application of Gv ointment. Before this trial, all patients were treated with povidone-iodine and antibiotics; however, those treatments were not effective in eradicating MRSA from skin lesions. Skin irritation and other systemic side effects caused by Gv were not observed. Our data suggest that Gv is a useful agent for treatment of the decubitus ulcers infected with MRSA.

Alveolar fibrosis and capillary alteration in experimental pulmonary silicosis in rats.

To analyze the evolution of fibrotic and vascular changes in pulmonary silicosis, ultrastructural and immunohistochemical studies were made of the lungs of rats given a single intratracheal injection of silica particles. Early lesions were characterized by accumulations of macrophages and neutrophils in alveolar lumina and interstitium and by damage to alveolar capillaries and epithelial cells. The intraluminal masses of inflammatory cells developed into granulomas and became associated with myofibroblasts that migrated from the interstitium through the damaged epithelial lining. Type II epithelial cells and bronchiolar cuboidal cells proliferated rapidly to line the intraluminal granulomas, incorporating them into the interstitium. This process mediated the transition from intraalveolar fibrosis to interstitial fibrosis. Vascular damage was repaired by proliferation and migration of endothelial cells. Some endothelial cells in alveolar capillaries expressed Factor VIII-related antigen at 2 wk after silica infusion. In normal animals, this feature was present in peribronchiolar but not in alveolar capillaries. Two patterns of endothelial cell migration were shown by staining for proliferating-cell nuclear antigen. The first pattern was characterized by endothelial cells that extended their cytoplasm over preexisting, denuded basement membranes and replaced necrotic cells in alveolar capillaries. At 4 mo after injury, some of these cells had developed fenestrations. The second pattern consisted of budlike sproutings that developed only in peribronchiolar connective tissue. These observations indicate that peribronchiolar vessels are sources for renewal of alveolar capillary endothelium as well as for neovascularization.

Immunopathological activities of extracellular products of Streptococcus mitis, particularly a superantigenic fraction.

Previously, we prepared extracellular products, fractions F-1 and F-2 of Streptococcus mitis 108, an isolate from the tooth surface of an infant, and showed that F-1 exhibited inflammatory cytokine-inducing activities. In the present study, we present evidence that fraction F-2 induced human T-cell proliferation in the presence of irradiated human peripheral blood mononuclear cells and selectively activated T cells bearing V beta 2 and V beta 5.1 in the T-cell receptor. F-1, on the other hand, stimulated human gingival fibroblasts to support the T-cell proliferation in the same way as human gamma interferon or Prevotella intermedia lipopolysaccharide (LPS). Fraction F-1 also primed gingival fibroblasts to support the production of interleukin-2 and gamma interferon by the T cells upon stimulation with F-2. Human gingival fibroblasts stimulated with fraction F-1, like those stimulated by P. intermedia LPS and human gamma interferon, exhibited human leukocyte antigen (HLA)-DR mRNA expression and cell surface HLA-DR molecules as detected by enzyme-linked immunosorbent assay. An anti-HLA-DR monoclonal antibody inhibited T-cell proliferation in response to F-2, probably through inactivating the accessory function of HLA-DR-bearing fibroblasts. T cells activated with F-2 in the presence of irradiated peripheral blood mononuclear cells exhibited definite cytotoxic effects against fibroblasts and squamous carcinoma cells originating from human oral tissues. These findings are strongly suggestive of an association of extracellular products of viridans streptococci with pathogenesis of oral mucosal diseases, particularly those disorders in gingiva which are accompanied by heavy infiltration of T cells.