Salmonella enterica serovar Typhi in Japan, 2001-2006: emergence of high-level fluoroquinolone-resistant strains.

The phage types and antimicrobial susceptibilities of 226 isolates of Salmonella enterica serovar Typhi from imported cases in Japan between 2001 and 2006 were investigated. Most (93.8%) had travelled to Asian countries, particularly South East Asia. Twenty-one phage types were identified with E1 (30.5%), UVS (15.9%) and B1 (9.3%) being the most common. The frequency of multidrug-resistant strains reached 37.0% in 2006 with phage types E1 and E9 predominating. Almost half (48.2%) of the isolates were resistant to nalidixic acid and two isolates displayed high-level fluoroquinolone resistance. Three mutations, two in gyrA and one in parC, were identified in both isolates.

A possible animal model for critical colonisation.

OBJECTIVE: To develop a critical colonisation model in rats that will facilitate investigation of its pathophysiology and the development of new and effective diagnosis and treatment protocols. METHOD: Three groups of rats were given full-thickness dorsal wounds: a control group received phosphate-buffered saline; an experimental group was inoculated with Pseudomonas aeruginosa; an infection group with streptozotocin-induced diabetes was also inoculated with P. aeruginosa. All groups were assessed on a number of parameters at days 1, 3, 5 and 7 following wounding. Parameters included gross observations, histopathological observations, quantification of redness and swelling, serum C-reactive protein (CRP) measurement and tissue bacterial counts. RESULTS: Healing was delayed in the experimental group when compared with the control group, with no signs of inflammation. Although the numbers of bacteria were similar in the experimental and infection groups, polymorphonuclear neutrophil (PMN) infiltration was localised to granulation tissue in the experimental group, whereas it extended to muscular tissue in the experimental group. CRP levels remained low in the experimental group. CONCLUSION: These findings suggest that the inoculation of bacteria provides a possible model of critical colonisation in rats. We believe this will contribute to a better understanding of critical colonisation.

FcepsilonRI-mediated thymic stromal lymphopoietin production by interleukin-4-primed human mast cells.

A significant increase of mRNA expression of thymic stromal lymphopoietin (TSLP) has been reported in the bronchial mast cells (MCs) of asthmatic subjects; however, the mechanism underlying the upregulation of TSLP mRNA and protein remains unknown. FcepsilonRI-mediated activation of human MCs upregulated TSLP mRNA expression by 5.2+/-2.9-fold, while activation of the MCs using lipopolysaccharide and polyriboinosinic:polyribocytidylic acid failed to upregulate TSLP. Stimulation of MCs with interleukin (IL)-4 alone did not affect the TSLP mRNA expression, while pre-incubation of MCs with IL-4 for 48 h significantly enhanced the FcepsilonRI-mediated TSLP mRNA expression (by 53.7+/-15.9-fold; p<0.05) and the amount of TSLP in the cell pellets increased significantly from 23.4+/-4.3 pg mL(-1) to 121.5+/-3.7 pg mL(-1) (p<0.0001). However, the released TSLP was rapidly degraded by proteases that were released by MCs. We identified the population of cells expressing TSLP in the lungs of 16 asthmatic and 11 control subjects by immunohistochemistry. The percentage of TSLP-positive MCs in the total population of MCs was significantly increased in asthmatic airways (p<0.0001). Thus, MCs are able to store TSLP intracellularly and to produce TSLP following aggregation of FcepsilonRI in the presence of IL-4.

The eta isoform of protein kinase C is localized on rough endoplasmic reticulum.

The eta isoform of protein kinase C, isolated from a cDNA library of mouse skin, has unique tissue and cellular distributions. It is predominantly expressed in epithelia of the skin, digestive tract, and respiratory tract in close association with epithelial differentiation. We report here that this isoform is localized on the rough endoplasmic reticulum in transiently expressing COS1 cells and constitutively expressing keratinocytes. By the use of polyclonal antibodies raised against peptides of the diverse D1 and D2/D3 regions, we found that immunofluorescent signals were strongest in the cytoplasm around the nucleus and became weaker toward the peripheral cytoplasm. Under immunoelectron microscopic examination, electron-dense signals were located on the rough endoplasmic reticulum and on the outer nuclear membrane which is continuous with the endoplasmic reticulum membrane. However, no signals were detected in the nucleus, inner nuclear membrane, smooth endoplasmic reticulum, Golgi apparatus, mitochondria, or plasma membrane. Treatment of the cells in situ with detergents suggested association of the isoform of protein kinase C with intracellular structures. By immunoblotting, a distinct single band with an M(r) of 80,000 was detected in whole-cell lysate and in rough microsomal and crude nuclear fractions, all of which contain outer nuclear membrane and/or rough endoplasmic reticulum. We further demonstrated the absence of a nuclear localization signal in the pseudosubstrate sequence. The present observation is not consistent with the report of Greif et al. (H. Greif, J. Ben-Chaim, T. Shimon, E. Bechor, H. Eldar, and E. Livneh, Mol. Cell. Biol. 12:1304-1311, 1992).

Differentiation and morphogenesis in pellet cultures of developing rat retinal cells.

We previously developed a reaggregate cell culture system (pellet cultures) in which retinal neuroepithelial cells proliferate and give rise to rod photoreceptor cells (rods) in vitro (Watanabe and Raff, 1990, Neuron 4:461-467). In the present study, we analyzed cell differentiation and morphogenesis in pellet cultures by using both cell-type-specific markers with immunofluorescence and electron microscopy. We demonstrated that, in addition to rods, the other major retinal cell types, including amacrine cells, bipolar cells, Müller cells, and ganglion cells were all present in the pellets, where most were able to develop from dividing precursor cells in vitro. The different cell types in the pellets became organized into two distinct structures: dark rosettes and pale rosettes. The cellular composition of these structures indicated that the dark rosettes correspond to the outer nuclear layer and the pale rosettes to the inner nuclear layer of the normal retina. Ultrastructural studies have indicated that the thin layer of neuronal processes surrounding the dark rosettes correspond to the outer plexiform layer, and the central region of the pale rosettes correspond to the inner plexiform layer of the normal retina. Other features of normal retinal development also occurred in the pellets, including programmed cell death and the formation of inner and outer rod cell segments and synapses. Thus, pellet cultures provide a convenient way to study different aspects of retinal development where one can control the size and the cellular composition of the initial reaggregate.

Defective degranulation and calcium mobilization of bone-marrow derived mast cells from Xid and Btk-deficient mice.

Regulation of adhesion and degranulation of mast cells plays an important role in allergy and inflammation. We investigated a possible role of Bruton's tyrosine kinase (Btk) in the regulation of adhesion and degranulation by using bone marrow-derived mast cells from X-linked immunodeficiency (Xid) and Btk-deficient mice. Cross-linking of the high affinity IgE receptor (Fc epsilonRI) and steel factor (SLF) induced indistinguishable adhesive responses of mast cells to fibronectin in kinetics, and these adhesive responses were comparable among wild type, Xid, and Btk-deficient mast cells. Cross-linking of Fc epsilonRI, but not SLF triggered degranulation of bone marrow-derived mast cells. However, Fc epsilonRI-induced degranulation was impaired in Xid and Btk-deficient mast cells. Calcium influx induced by Fc epsilonRI cross-linking and SLF were also reduced in Xid and Btk-deficient mast cells. Degranulation and calcium influx were reduced more severely in Btk-deficient than in Xid mast cells. Consistently, cross-linking Fc epsilonRI and SLF augmented Btk kinase activities transiently. Inositol triphosphate (IP3) production was also severely reduced in Btk-deficient mast cells, indicating Btk play a critical role of Fc epsilonRI-induced IP3 production. The differential sensitivity of wortmannin on calcium influx in wild type and Xid mast cells suggested that the activation of phosphatidylinositol 3 kinase (PI 3-kinase) was required in calcium influx. Furthermore, abnormal secretory granules with translucent contents and variable in size were observed both in Xid and Btk-deficient mast cells. Our study demonstrated a critical role of Btk in regulating intracellular calcium and granule exocytosis.

Ocellar system of the insect: comparison of dorsal ocellus and lateral ocellus.

The ocellar receptor and neuropil in the dorsal ocellar system of the cockroach and the lateral ocellar system of the swallowtail butterfly larva have been compared. In the dorsal ocellus of the cockroach more than 10,000 retinular cells each with a rhabdomere are piled up one upon another without a specific pattern in the ocellar capsule. Retinular axons synapse exclusively with branches of four thick second order neurons in the lower region of the capsule. Dark granule-containing neurons, originating in the brain, also synapse with the second order neurons in the ocellar neuropil. The ocellar nerve is comprised of four thick second order processes (ca. 6-16 micron) and 20-40 thin processes (ca. 1-5 micron), which include retinular axons, collaterals of thick processes, possible third order processes and unidentified processes. Synapses occur against these processes in the ocellar nerve and in the extreme proximal region of the ocellar neuropil. Six lateral ocelli occur on both sides of head in the butterfly larva. Each ocellus contains seven retinular cells, which are grouped into three distal cells and four proximal cells, or four distal cells, two peripheral cells and one central cell by the position of their rhabdomeres. Retinular axons extend to the brain, and enter the distal optic neuropil. There are six neuropils, each of which contains the seven axons from one ocellus and a dozen or so second order processes. Of seven retinular axons, four occur peripherally and the remaining three occur centrally in the neuropil. All axons synapse with fine branches of interneurons. Second order processes together with at least three central retinular axons extend to the second optic neuropil, but the peripheral axons could not be followed there. The highly organized receptors and the neuropil in the lateral ocellar system suggest a complexly integrated function. An ocellar nerve and proximal ocellar neuropil appear like an extension of the CNS, and direct connections of retinular axons with interneurons in the CNS appear characteristic of the dorsal ocellus. This difference may be related to the distribution of the two ocellar systems: the dorsal ocelli co-exist with the compound eyes of the adult, whereas the lateral ocelli are the only photoreceptor organs of the larval insect.

Oral administration of fluoroquinolones in the treatment of typhoid fever and paratyphoid fever in Japan.

OBJECTIVE: To study the adverse reactions and therapeutic effects of fluoroquinolones to investigate whether they can be used for the treatment of patients with typhoid fever and paratyphoid fever. METHODS: The adverse reactions and therapeutic effects of fluoroquinolones were studied retrospectively in patients with typhoid fever and paratyphoid fever. PATIENTS: 58 patients (54 Japanese) with typhoid fever, 42 patients (41 Japanese) with paratyphoid fever, and 1 Japanese patient with both typhoid fever and paratyphoid fever, who were admitted in hospitals in Tokyo, Kawasaki, Yokohama, Kyoto, and Osaka from 1995 to 1998 and treated with fluoroquinolones. RESULTS: Almost 80% of the patients were treated with tosufloxacin (TFLX) and the remaining 20 % were treated with norfloxacin, ciprofloxacin, levofloxacin, or sparofloxacin. Side effects (nausea, urticaria, aphthous stomatitis) and elevation of serum amylase were found in 3.6% and 8.3 % of patients treated with TFLX, respectively, but these adverse reactions disappeared in all of these cases either with or without a change in the drug used. No adverse reactions were found in patients treated with the other fluoroquinolones. The clinical and bacteriological effects of these drugs were adequate. CONCLUSION: Though further studies still need to be performed on the fluoroquinolones other than TFLX, we can preliminarily conclude that fluoroquinolones are safe drugs and they can be recommended for the initial therapy of patients with typhoid fever and paratyphoid fever.

The effect of anti-VLA-4 monoclonal antibody on eosinophil accumulation and leukotriene production in nasal mucosa.

Eosinophil derived leukotrienes and platelet activating factor are known to cause nasal swelling. Toxic proteins induce nasal hyperreactivity to non-specific stimuli including histamine. Recent studies strongly suggest that very late activation antigen-4 (VLA-4) on the eosinophil surface plays a prominent role in the recruitment of eosinophils from blood vessels and eosinophil locomotion in inflammatory tissues. To test this hypothesis, we examined the effect of a monoclonal antibody (mAb) to VLA-4 on eosinophil accumulation in nasal mucosa after antigen challenge in a guinea pig model. Here we have demonstrated that the mAb depressed the eosinophil accumulation in nasal mucosa. In addition, we have shown that this mAb also inhibited eosinophil activation and leukotriene production. Our results raise a possibility that eosinophils might be activated during the journey from bloodstream to inflammatory tissues by the adhesion to endothelial cells and fibronectin. VLA-4 might act as a signalling as well as an adhesion receptor on eosinophils.

A case of Penicillium marneffei infection in an AIDS patient: the first case in Japan.

A 38-year-old Japanese AIDS patient developed papular lesions which rapidly increased in number, eroded and crusted, and spread over not only skin but also the mucosal surface. High fever, sore throat, malaise and hepatosplenomegaly were also noted, and he died despite 2 months of intensive treatment. An autopsy revealed numerous histiocytes infected with Penicillium marneffei in the lymph nodes, liver, spleen, bone marrow, skin, and mucosal surface of the oral cavity to the pharynx. This case is thought to be the first Japanese case of penicilliosis marneffei.

[Effect of sparfloxacin on fecal microflora and its fecal concentration in patients with bacillary dysentery].

The effect of sparfloxacin (SPFX) on fecal microflora and its fecal concentrations were examined in four patients and one carrier with bacillary dysentery. SPEX was administered to five cases with a daily dose of 200 mg, once a day, for five days after breakfast. The results were as follows: Escherichia coli, a main bacterium in Enterobacteriaceae, was not detected during the drug administration, except one from whose feces mucoid type of E. coli was found all the time, and the colony forming units (CFUs) were not recovered to the initial level at the seventh day after the medication. Enterococcus, found in all of the cases before, was eliminated or decreased during and the CFUs showed slow recovery after the medication. Total CFUs of aerobes were decreased during and not returned to the initial level at the seventh day after the medication. Total CFUs of anaerobes were kept invariable, although variation of the CFUs with the species was seen. The peak levels of the fecal concentrations of SPEX were 177 to 535 micrograms/g on the third to the fifth day of the medication. The fecal concentrations were 1.3 to 4.4 micrograms/g at the seventh day after the medication in all of the cases. The clinical efficacy was excellent in three cases, poor in one evaluated for the other bacteria than Shigella, and unknown in one. No adverse reaction was observed.

[Gastrointestinal diseases associated with HIV infection].

A clinical studies were carried out on gastrointestinal diseases associated with HIV infection. During the 6 years between January 1993 and December 1998, 71 HIV infected cases visited to Yokohama Municipal Citizen's hospital, and 26 of them developed gastrointestinal complications during the course of their illness. They consisted of 24 males and 2 females, with the mean age of 44.7 years and the medial value of 42.5 years. Of the 26 patients, 21 were Japanese, and the remaining 5 were Southeast Asian. The mean CD4 count was 143/microliter and the medial value was 32/microliter at the time of development of complications. Gastrointestinal complications were esophageal candidiasis in 6 patients, cytomegalovirus (CMV) gastritis and gastric Kaposi's sarcoma in 1 patient each, amebiasis in 8 patients, infectious colitis in 11 patients, and asymptomatic pathogen carriers in 3 patients. Esophageal and gastric complications were common in patients with low count of CD4, and endoscopy was useful for diagnosis. Amebiasis developed even in patients with normal CD4 and was common in males with experience in homosexual contact. It seems that homosexual contact acquire not only HIV infection but also Entamoeba histolytica through sexual contact. Protozoan and acid-fast bacteria were detected at high rate in patients with infectious colitis and asymptomatic pathogen carriers. Besides food-born infections, imported infections were seen in foreign and Japanese patients who had traveled abroad. The gastrointestinal diseases associated with HIV infections for the most part were opportunistic infections or tumors but imported, food-born, and sexually transmitted infections were also observed. It seems necessary to take into consideration of varying background of patients in the treatment of gastrointestinal diseases associated with HIV infections.

[A case of leptospirosis infected in Borneo Island, Malaysia].

We report a case of leptospirosis infected in Sabah, Borneo island, Malaysia. The case is 25-year-old male who had participated in the EcoChallenge Sabah 2000 Expedition Race, a multisport event held during August 20 to September 3, 2000 at various sites in Sabah in Malaysian Borneo. He developed a high fever and headache on September 7, and he was admitted to our hospital on September 9. On admission he also had conjunctivitis and myalgias. Laboratory findings on admission revealed leukocytosis with left shift, slightly elevated transaminase levels, high CRP levels and proteinuria. Plasmodium spp. were negative on blood smears, and no bacteria were isolated from blood and feces cultures. We performed the laboratory tests for leptospirosis, based on the information about the probable leptospirosis outbreak among athletes who participated in the EcoChallenge Race, however both Leptospira antigens and antibodies were negative at that time. We diagnosed leptospirosis clinically because he manifested persistent symptoms, and minocycline 100 mg b.i.d. was administered intravenously resulting in excellent efficacy. Serum antibody tests by microscopic agglutination test (MAT) at convalescent stage revealed significant increased antibodies against Leptospira interrogans serovar hebdomadis, and the diagnosis of leptospirosis was confirmed. Infectious diseases have been global and it is important to have information concerning worldwide infectious disease situations as much as possible for accurate diagnosis.

[A case of AIDS complicated with isosporiosis as initial manifestation].

We reported a Thailander patient with isosporiasis accompanied by AIDS. The 34-year-old male, who had been living in Japan for six years, was admitted to our hospital complaining of diarrhea and body weight loss. After admission the patient developed acute renal failure due to dehydration resulting from diarrhea. Laboratory findings on admission showed neither significant microorganisms nor parasite ova in the stool. The patients was given both an intestinal drug and an antibacterial agent, but is vain. Frequent stool examinations disclosed Isospora oocyst in the feaces. Co-trimoxazole was administered resulting in remarkable diarrheal improvement. Renal function was also improved without dialysis. Although isosporiasis is rarely found in Japan, it is stressed that in the AIDS patient with persistent diarrhea who may develop a parasitic disease as did in this case, frequent fecal examinations should be conducted.

[Evaluation of an HIV screening assay kit for the combined detection of anti-HIV-1/2 antibodies and HIV p24 antigen].

We have evaluated a new HIV screening assay kit (Genscreen HIV Ag-Ab) for the HIV antigen-antibody combined test by comparing with two HIV antigen-antibody combined assay kits (VIDAS HIV DUO, Enzygnost HIV integral). Genscreen HIV Ag-Ab is a microwell plate enzyme immunoassay for the detection of HIV infection, based on the detection of anti-HIV-1/2 antibodies and HIV p24 antigen in human serum or plasma. In this study, 90 samples of HIV-1 antibody positive sera and 670 samples of HIV negative sera were examined. The sensitivity was 100% and the specificity was 99.7%. All of HIV-1 group M sera (subtypes A to G and B/D), HIV-1 group O sera and HIV-2 sera in worldwide HIV performance panel-302 were positive with Genscreen HIV Ag-Ab. Ten commercially available HIV-1 seroconversion panels were tested to evaluate sensitivity of three HIV antigen-antibody combined assay kits. Genscreen HIV Ag-Ab detected infection at the same bleeds as VIDAS HIV DUO in 8 of 10 seroconversion panels and 1 to 2 bleeds earlier than Enzygnost HIV integral in 5 of 10 seroconversion panels. However, VIDAS HIV DUO indicated false negative on 5th bleed in panel BB (PRA952). The result of the specimen was positive on 3rd bleed, equivocal on 4th bleed, negative on 5th bleed and again positive on 6th bleed. All of these specimens were positive by Genscreen HIV Ag-Ab. Therefore, Genscreen HIV Ag-Ab that shorten the window period is a useful and reliable for HIV screening test, especially in case of primary infection.

[HIV-1 (human immunodeficiency virus type 1) mutant strain associated with zidovudine resistance detected from a Japanese patient with primary HIV-1 infection].

Sequences of HIV-1 pol gene were determined by direct sequencing from a Japanese patient with primary HIV-1 infection. The patient did not receive antiretroviral therapies. However we observed a HIV-1 mutant strain associated with zidovudine (ZDV) resistance. The patient had both the codon 70 and the codon 215 amino acid substitutions in the RT region. Our data indicated that the patient was infected with a HIV-1 mutant strain associated with ZDV resistance and this is the first report in Japan.

[A case of imported paratyphoid fever associated with Giardia lamblia and Isospora belli infection].

We report a case of imported paratyphoid fever associated with Giardia lamblia and Isospora belli infection. The patient was a 23-year-old Japanese female with complaints of high grade fever and diarrhea after 10 days traveling to Nepal. Salmonella Paratyphi A was isolated from the blood and fecal cultures on admission and Ciprofloxacin of 200 mg tid was administered for 14 days. Fecal examination revealed cysts of G. lamblia at the same time and metronidazole of 250 mg tid for 7 days was effective for their eradication. During the follow-up studies oocysts of I. belli were found and cotrimoxazole of 960 mg bid for 10 days was effective for their eradication. Fecal examinations on parasites which is rare in Japan such as I. belli are recommended to the cases returning from tropical areas.

[Basic and clinical studies of fleroxacin on infectious enteritis. Research Group of AM-833 on infectious enteritis].

A clinical study was conducted on fleroxacin (FLRX) in 143 patients and carriers with infectious enteritis (shigellosis, Salmonella enteritis, Campylobacter enteritis, pathogenic Escherichia coli enteritis, Vibrio parahaemolyticus enteritis, cholera, multiple bacterial infections, pathogen-negative enteritis). Furthermore, its antibacterial activity against clinical isolates, fecal concentration and effect on fecal microflora were conducted. FLRX was administered orally in doses of 200 mg once a day (200 mg group) or 300 mg once a day (300 mg group) for 3 days to cholera, for 7 days to Salmonella enteritis and for 5 days to the other infectious enteritis. The clinical efficacy rates were 100% in both the 200 mg and 300 mg groups. The bacteriological efficacy rates were 100% against Shigella spp., Salmonella spp., pathogenic E. coli, V. parahaemolyticus and V. cholerae O1, and 63.6% against Campylobacter spp. in the 200 mg group. The rates of the 300 mg group were 93.3% against Shigella spp., and 100% against Campylobacter spp. and pathogenic E. coli. As adverse effects, skin rash was observed in 1 case each in both groups (1.1%, 2.1%). Abnormal laboratory findings consisted of 1 case of increased eosinophils and 1 case of elevated GOT and GPT levels in the 200 mg group (2.8%), and 1 case of elevated GPT in the 300 mg group (2.9%). The clinical usefulness rates were 92.9% and 93.3% in the 200 mg and 300 mg groups, respectively. Antibacterial activity was somewhat inferior to that fo ciprofloxacin and equal to or better than that of norfloxacin, demonstrating MIC90 values against Shigella spp., Salmonella spp., pathogenic E. coli, V. parahaemolyticus and Campylobacter spp. of 0.1, 0.2, 0.1, 0.2 and 0.78 micrograms/ml, respectively. Peak fecal concentrations of the drug were 49.0 micrograms/g and 274.4 micrograms/g in the 200 mg group, and 43.3 micrograms/g and below the detection limit (5.0 micrograms/g) in the 300 mg group. With respect to fecal microflora (4 cases), a decrease in Enterobacteriaceae was observed in 3 cases during dosing. But this change showed a tendency to recover after completion of dosing. No effects were observed on anaerobic bacteria.