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Koki Aoki

Publications and source records attributed to Koki Aoki.

17 recordsLinked to original sources

Anti-adenoviral effect of anti-HIV agents in vitro in serotypes inducing keratoconjunctivitis.

BACKGROUND: Around one million people are affected by adenoviral keratoconjunctivitis a year in Japan, and it is recognized as one of the major pathogens of ophthalmological nosocomial infection worldwide. Although cidofovir can be used systemically for immunocompromised patients with disseminated adenoviral infection, no specific anti-adenoviral agent has been established for the treatment of adenoviral infection. We evaluated the anti-adenoviral effect of anti-HIV (human immunodeficiency virus) agents in this study. METHODS: Five anti-HIV agents (zalcitabine, stavudine, nevirapine, indinavir and amprenavir) were subjected to in vitro evaluation. A549 cells were used for viral cell culture, and adenovirus serotypes 3, 4, 8, 19 and 37 were used. After calculating CC(50) (50% cytotoxic concentration) of each agent by MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) method, we cultured adenovirus with the agents for seven days and quantitatively measured extracted adenoviral DNA by real-time PCR. RESULTS: Among the anti-HIV drugs, zalcitabine and stavudine, both nucleoside reverse transcriptase inhibitors, showed significant anti-adenoviral activity. In contrast, nevirapine, a non-nucleoside reverse transcriptase inhibitor, and indinavir and amprenavir, which are both protease inhibitors, were ineffective against adenovirus. CONCLUSIONS: These results indicate that zalcitabine and stavudine are possible candidates for the local and systemic treatment of adenoviral infection, and the anti-adenoviral effect might depend on the pharmacological properties of anti-HIV agents. The chemical properties on the clinical safety for systemic and local application need to be determined in order to for these drugs to be accepted for the treatment of adenovirus in clinical settings.

Adenovirus Infections, Human↗

Quantitative detection and rapid identification of human adenoviruses.

We have established a method of quantitative detection and rapid identification of human adenoviruses (hAdVs). Using LightCycler PCR with a primer set, we were able to amplify 554 bp of the hexon gene from each of 51 prototype strains of hAdVs. The sensitivity of LightCycler PCR was 10 copies of hAdV DNA/reaction. When LightCycler PCR was performed using a set of primers, hAdV was positive for 74.4% (99 of 133) of conjunctivitis patients and for 27.3% (81 of 297) of respiratory infection patients. We also attempted to measure hAdV in the potentially contaminated eye drops used by patients, detecting 5.4 x 10(2) to 1.6 x 10(6) copies/ml of hAdV. We determined the 350-bp nucleotide sequence of the amplified hexon gene and compared it with the sequences of the 51 prototype strains. Phylogenetic analysis based on 350 bp of the hexon gene identified 99 positive conjunctival swabs as 24 cases of AdV type 3 (AdV-3), 14 cases of AdV-4, 1 case of AdV-8, 19 cases of AdV-19a, and 41 cases of AdV-37. The 81 sequences from pharyngeal or nasal mucus swabs were identified as 29 cases of AdV-2, 18 cases of AdV-1, 18 cases of AdV-5, 12 cases of AdV-4, 2 cases of AdV-37, 1 case of AdV-3, and 1 case of AdV-6. LightCycler PCR followed by phylogenetic analysis provides an effective tool for the rapid identification of hAdVs and for studying molecular epidemiology.

Adenovirus Infections, Human↗

Molecular epidemiology of adenoviral conjunctivitis in Hanoi, Vietnam.

PURPOSE: To investigate the serotypes of adenovirus causing conjunctivitis in Hanoi, Vietnam. DESIGN: Clinical diagnosis of adenoviral conjunctivitis and laboratory-based experimental study. METHODS: We collected 21 conjunctival swabs from 21 different patients with a clinical presentation compatible with adenoviral conjunctivitis, in Hanoi, Vietnam. Immunochromatography and real-time polymerase chain reaction (PCR) were performed to detect human adenovirus (HAdV). The sequence of PCR products was analyzed to determine the serotype of HAdV. RESULTS: Of 21 samples, HAdV DNA was detected in 14 samples (66.7%) by real-time PCR. The serotype analysis showed HAdV-8 in 11 samples (78.6%), HAdV-3 in two samples (14.3%), and HAdV-37 in one sample (7.1%). Of 11 HAdV-8 samples, one sample (9.1%) was prototype, and the other 10 samples (90.9%) had identical nucleotide sequence and were identified as a variant of HAdV-8. CONCLUSIONS: HAdV-8 was found to be the predominant serotype in Hanoi, Vietnam. Most of the HAdV-8 samples were a variant of HAdV-8.

Adenovirus Infections, Human↗

Tolerability and efficacy of N-chlorotaurine in epidemic keratoconjunctivitis--a double-blind, randomized, phase-2 clinical trial.

The aim of this study was to assess the tolerability and efficacy of N-chlorotaurine (NCT), an endogenous antimicrobial agent, in epidemic keratoconjunctivitis. In a prospective double-blind, randomized phase 2b study, the infected eyes were treated for 7 days with eye drops containing 1% aqueous solution of N-chlorotaurine (33 subjects) or gentamicin (27 subjects, control group). Adenovirus types 3, 4, 8, 19, and 37 were detected in 39 subjects (65%), enteroviruses in 8 (13.3%), and staphylococci in 5 (8.3%). Subjective and objective symptoms were scaled and added to a subjective and objective score, respectively, on day 1 (baseline), day 4, and day 8. Analyzing the whole study population, the subjective score on day 8 was lower in the NCT group (P = 0.016), whereas there were no differences in the objective score. However, in severe infections caused by adenovirus type 8 (n = 20) both the subjective and objective score were lower in the NCT group on day 4 (P = 0.003 and 0.015, respectively), which was also true for the subjective score on day 8 (P = 0.004) in this subgroup. The frequency of subepithelial infiltrates was similar in both groups. N-chlorotaurine was well-tolerated, shortened the duration of illness, and seems to be a useful causative therapeutic approach in severe epidemic keratoconjunctivitis.

Adenoviridae↗

Five new genome types of adenovirus type 37 caused epidemic keratoconjunctivitis in Sapporo, Japan, for more than 10 years.

Human adenovirus type 37 (HAdV-37) is a major cause of epidemic keratoconjunctivitis and has recently been the largest causative agent of keratoconjunctivitis in Japan. To investigate the genetic characteristics of HAdV-37 strains isolated in Sapporo, we analyzed the genome types and genetic relationships of 51 strains isolated there from 1990 through 2001. By using DNA restriction analysis, eight genome types (HAdV-37/D1, HAdV-37/D3, and HAdV-37/D6 to HAdV-37/D11) were identified, including five new ones. The restriction fragments of these genome types shared more than 95% identity with those of the prototype strain. By DNA sequence analysis, five and three single nucleotide substitutions, respectively, were found in partial sequences of the hexon and fiber genes. The combinations of mutations resulted in four hexon and fiber types (hx1 to hx4 and f1 to f4) and six hexon/fiber pairs (hx1/f1, hx2/f1, hx1/f2, hx1/f3, hx3/f4, and hx4/f4). The six pairs correlated well with certain genome types. In all three epidemics of keratoconjunctivitis to strike Sapporo in the past 12 years, specific genome types and fiber types were usually isolated: in the first epidemic, HAdV-37/D1 (f1) and HAdV-37/D3 (f1); in the second, HAdV-37/D6 (f2) and HAdV-37/D8 (f3); and in the third, HAdV-37/D10 (f4) and HAdV-37/D11 (f4). We conclude that mutations in the adenovirus genome occurred chronologically and that certain mutations were correlated with the epidemics of adenoviral keratoconjunctivitis.

Adenovirus Infections, Human↗

Sensitive and rapid detection of herpes simplex virus and varicella-zoster virus DNA by loop-mediated isothermal amplification.

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method in which reagents react rapidly and efficiently, with a high specificity, under isothermal conditions. We used a LAMP assay for the detection of herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), and varicella-zoster virus (VZV). The virus specificities of primers were confirmed by using 50 HSV-1, 50 HSV-2, and 8 VZV strains. The assay was performed for 45 min at 65 degrees C. The LAMP assay had a 10-fold higher sensitivity than a PCR assay. An analysis of nucleotide sequence variations in the target and primer regions used for the LAMP assay indicated that 3 of 50 HSV-1 strains had single nucleotide polymorphisms. No HSV-2 or VZV strains had nucleotide polymorphisms. Regardless of the sequence variation, there were no differences in sensitivity with the HSV-1-specific LAMP assay. To evaluate the application of the LAMP assay for clinical diagnosis, we tested clinical samples from 40 genital herpes patients and 20 ocular herpes patients. With the LAMP assay, 41 samples with DNA extraction and 26 direct samples without DNA extraction were identified as positive for HSV-1 or HSV-2, although 37 samples with DNA extraction and just one without DNA extraction were positive by a PCR assay. Thus, the LAMP assay was less influenced than the PCR assay by the presence of inhibitory substances in clinical samples. These observations indicate that the LAMP assay is very useful for the diagnosis of HSV-1, HSV-2, and VZV infections.

Animals↗

[Clinical and virological studies of nosocomial conjunctivitis infection caused by adenovirus type 37 variant].

PURPOSE: To study the clinical features and virological analysis of the nosocomial adenoviral conjunctivitis cases occurring in the ophthalmology ward of Fukushima Medical University Hospital. MATERIALS AND METHODS: We studied the symptoms and clinical course of 61 patients who had adenoviral conjunctivitis caused by nosocomial infections in our hospital. We attempted to detect the adenovirus antigen, analyze the viral DNA, and isolate the virus from conjunctival swabs. RESULTS: The clinical symptoms of adenoviral conjunctivitis were mainly conjunctival hyperemia, discharge and conjunctival follicles. Adenoviral conjunctivitis patients who had undergone ophthalmic surgery had conjunctivitis in the operated eye. The sensitivity of Adeno-check was 78.9% in the in-patients. Adenovirus type 37 variant was detected by molecular analysis and viral isolation. CONCLUSIONS: Adenoviral conjunctivitis can often lead to outbreaks of nosocomial infection in the ophthalmic ward and sometimes requires makes necessary restriction of hospitalization and closing of the ward. Therefore, patients need to be observed carefully. The virological analysis of specimens from conjunctival swabs detected pathogens and provided useful information concerning adenoviral conjunctivitis.

Adenoviruses, Human↗

The cotton rat model for adenovirus ocular infection: antiviral activity of cidofovir.

To determine the antiviral effects of compounds against ocular adenovirus (AdV) infection, we established an animal model of AdV infection in cotton rat eyes. Cotton rat eyes were inoculated intrastromally and topically with four AdV serotypes 4, 5, 8, and 37, and treated topically with 1% HPMPC (cidofovir) eye drops twice a day. The infected corneas were extracted and homogenized, and virus titers in the cornea specimens were determined by a plaque assay. The virus titer in AdV type 5-inoculated eyes peaked on days 0 through 3 after inoculation and virus shedding was detected for 18.0+/-2.8 days. AdV 5 antigen in the infected corneas was demonstrated in the corneal epithelial cells by immunofluorescence stain. However, for AdV serotypes 4, 8, and 37, no evidence of continued virus replication in cotton rat eyes was noted. Specimens from cidofovir-treated eyes infected with AdV 5 demonstrated a statistically significant reduction in the mean virus titer (days 3-15) (P=0.028) and virus shedding duration (P=0.0014), as compared with those of the control group.

Adenoviridae Infections↗

Molecular diagnosis of human adenoviruses d and e by a phylogeny-based classification method using a partial hexon sequence.

Human adenoviruses (HAdVs) are the major causes of a variety of acute illnesses. Virus isolation and neutralization tests are usually done to identify the causative virus, but these tests are labor-intensive and time-consuming, and standardized antisera are in limited supply. This study investigated a rapid and reliable method of virus identification based on PCR and phylogenetic analysis. The phylogenetic tree constructed by neighbor joining on the basis of the newly determined partial hexon sequences from 33 prototypes of HAdV-D and -E, along with 11 available prototypes of HAdV-A to -C and -F from GenBank, allowed HAdVs to be grouped into six distinct clusters. These clusters correspond closely to the six newly designated species, HAdV-A to -F. The partial hexon sequences of 57 isolates from patients with acute conjunctivitis obtained over 20 years plus those of 44 prototype strains were analyzed. Each isolate formed a monophyletic cluster along with its respective prototype strain, allowing serotype identification. Partial-hexon-based classification appears to be an effective tool for studying the molecular epidemiology of HAdVs.

Adenovirus Infections, Human↗

New genome type of adenovirus serotype 4 caused nosocomial infections associated with epidemic conjunctivitis in Japan.

Human adenovirus type 4 is one of the major serotypes isolated from patients with adenoviral conjunctivitis. In 2001 we encountered nosocomial infections with epidemic conjunctivitis in the ophthalmology ward of one hospital in Sapporo, which is in the northern part of Japan. Adenoviruses were isolated from the patients with this nosocomial infection and identified as adenovirus type 4 (AdV-4) by a neutralization test with serotype-specific antiserum. When the cleavage patterns of the isolates were compared with the full viral genome with BamHI and SmaI, the cleavage patterns of the isolates were shown to be different from those of AdV-4p and other previously known AdV-4 variants. The nucleotide sequences of the fiber gene of the isolates showed the highest homologies (94.3%) with AdV-4 among the nucleotide sequences available from GenBank and formed a monophyletic cluster along with the prototype strain of AdV-4. The isolates, however, were located in a different lineage from those of AdV-4p and the AdV-4 variant from the sporadic infections. We conclude that the nosocomial infection that appeared in 2001 was caused by a new genome type of AdV-4, which was designated AdV-4c.

Adenoviridae↗

[Antiviral effect of sulfated sialyl lipid against a clinical strain of adenovirus].

PURPOSE: Currently, there is no antiviral drug for adenovirus(AdV). We have reported that sulfated sialyl lipid(NMSO) 3, a NMSO, has an antiviral effect against AdV prototype strains. We evaluated the antiviral inhibitory effect and the mechanism of NMSO 3 against AdV strains from patients with conjunctivitis in vitro. METHODS: Viruses used for the experiment were clinically isolated AdV type 3(AdV 3), AdV type 4(AdV 4), type 8(ADV 8), AdV type 19(AdV 19), and type 37(AdV 37). We examined three antiviral agents, i.e., NMSO 3, cidofovir(HPMPC), and zalcitabine(ddC). 50% effective concentration(EC50), 50% cytotoxic concentration(CC50), and selectivity index(SI) of compounds were determined for AdV infection in HEp-2 cells using 3-(4,5-dimetyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) methods. We also evaluated the anti-AdV activity of NMSO 3 when it was added during the stage of virus adsorption. RESULTS: NMSO 3, HPMPC, and ddC showed an inhibitory effect against all five AdV clinical strains. The EC50 values of NMSO3 were lower than those of HPMPC and ddC. NMSO 3 exhibited minimal cytotoxicity. NMSO 3 inhibited AdV infection only when it was added during the stage of virus adsorption. CONCLUSIONS: NMSO 3 inhibited the replication of all clinical AdV serotypes tested. NMSO 3 was the most potent and selective anti-AdV compound. The mechanism of anti-AdV activity by NMSO 3 was inhibition of viral adsorption to cells.

Adenoviridae↗

Adenovirus detected by polymerase chain reaction in multidose eyedrop bottles used by patients with adenoviral keratoconjunctivitis.

PURPOSE: We investigated the potential of a multidose eyedrop bottle used by patients with adenoviral keratoconjunctivitis as a source for spreading infection. DESIGN: Prospective consecutive case series. METHODS: The contents of multidose eyedrop bottles given to patients with adenoviral conjunctivitis and in use for 1 week were analyzed by polymerase chain reaction for adenovirus after as long as 9 weeks of preservation at room temperature. RESULTS: Of 26 patients with adenoviral keratoconjunctivitis, the eyedrop bottles of 19 patients (73%) were positive for adenovirus. The maximum detection interval was 9 weeks. Significantly higher prevalences of intrafamilial infection (P =.0098) and of corneal subepithelial opacity (P =.046) were observed among cases with adenoviral contamination than among cases without contamination. CONCLUSIONS: Multidose bottles used by patients with adenoviral keratoconjunctivitis are a possible vector for viral transmission for as long as 9 weeks.

Adenovirus Infections, Human↗

A twenty-one year surveillance of adenoviral conjunctivitis in Sapporo, Japan.

We have studied the clinicoetiological, serological, and molecular epidemiological features of adenoviral conjunctivitis under the auspices of the nationwide surveillance project in Sapporo, located in northern Japan. We were able to analyze the results of 1,454 cases of adenoviral, herpetic, and chlamydial conjunctivitis. We found that adenonovirus serotypes 8, 19, and 37 caused severe conjunctivitis without systemic symptoms, whereas serotypes Ad3, Ad7, and Ad11 caused mild conjunctivitis with systemic involvement. Ad4 showed a broad range of symptoms, from PCF to EKC. Adenoviral conjunctivitis had seasonal outbreaks in the summer in association with adequate temperature and humidity in Sapporo. Neutralization antibodies against Ad8, Ad19, and Ad37 were detected in fewer than 20% of cases, and so the incidence of epidemics with these serotypes may rise in the coming years. The main genome types of adenovirus in Sapporo were Ad4a, Ad8e, Ad19a, Ad19b, and Ad37p, a, and b.

Adenovirus Infections, Human↗