Yosaburo Oikawa

In vivo and ex vivo laser confocal microscopy findings in patients with early-stage acanthamoeba keratitis.

Objective: This study included in vivo and ex vivo investigations of patients with early-stage Acanthamoeba keratitis by using new-generation laser confocal microscopy (Heidelberg Retina Tomograph 2 Rostock Cornea Module [HRT 2-RCM]). Methods: Three patients (2 men and 1 woman; mean age, 22.0 years) with early-stage Acanthamoeba keratitis diagnosed by direct examination (Parker ink-potassium hydroxide stain), culture from corneal epithelial scrapings, or both methods were enrolled in this study. All patients were examined by slit-lamp biomicroscopy. The area of the affected cornea was examined by HRT 2-RCM. Selected images of in vivo corneal layers and ex vivo cultured microorganisms were evaluated qualitatively for shape and degree of light reflection of the corneal structural changes or Acanthamoeba cysts. In addition, cultured Acanthamoeba were examined ex vivo by HRT 2-RCM. Results: In vivo laser confocal microscopy showed highly reflective round-shaped, high-contrast Acanthamoeba cysts (10-20 μm in diameter) in the corneal epithelium in all cases, leading to rapid confirmation of the clinical diagnosis. In all culture samples of Acanthamoeba, ex vivo laser confocal microscopy showed highly reflective round- or stellate-shaped high-contrast particles (10-20 μm in diameter). Conclusions: In vivo laser confocal microscopy enables rapid and noninvasive diagnosis of early-stage Acanthamoeba keratitis with high resolution. In addition, ex vivo laser confocal images of Acanthamoeba cysts may be helpful when similar structures are identified and have to be interpreted under in vivo conditions.

Paragonimus ohirai : immunobiochemical characterization on the tegumental glycocalyx of excysted juvenile recognized by a monoclonal antibody

The tegumental glycocalyx of excysted juvenile (EJ) of Paragonimus ohirai was immunobiochemically characterized using a monoclonal antibody (MS-Mab). HPLC gel filtration showed that the antigens detected by two-site ELISA had a molecular weight of greater than or equal to 2 x 10(6) Da (dextran marker). On reduced SDS-PAGE, the glycocalyx antigen retained in the stacking gel was cleaved into several much smaller antigens after pronase treatment. The antigenic activity of the glycocalyx was stable in two-site ELISA to heat and acid treatments, but sensitive to alkali, periodate, base/borohydride, and pronase treatments. Precipitin formation in immunodouble diffusion between MS-Mab and EJ crude antigen was inhibited only by two monosaccharides: galactose and N-acetylgalactosamine. The purified glycocalyx bound strongly to PNA lectin, fairly well to RCA120 lectin, and slightly to SBA lectin, but not to Con A, WGA, UEA-1, DBA, or LFA lectins. Exo-beta-galactosidase treatment increased SBA binding, whereas it decreased PNA binding. PNA was observed to strongly bind to the body surface of living EJ. The antigenic activity of the glycocalyx was remarkably lost by incubation with exo-beta-galactosidase and O-glycanase. The glycocalyx was reactive with sera of P. ohirai-infected rats, and its reactivity was remarkably reduced by O-glycanase treatment. The ELISA level was higher in sera at an early stage of infection than in a late one. These studies show that the EJ tegumental glycocalyx is antigenic in infection, a marked, high molecular weight glycoprotein containing antigenic O-linked sugars, and that the sugar epitope is at the nonreducing terminal of the O-linked sugars and is composed of galactose and N-acetylgalactosamine.

Bowman’s layer encystment in cases of persistent Acanthamoeba keratitis

Background The purpose of this study was to report Acanthamoeba encystment in Bowman’s layer in Japanese cases of persistent Acanthamoeba keratitis (AK). Methods Laser confocal microscopic images of the cornea were obtained in vivo from 18 consecutive eyes from 17 confirmed AK patients. Retrospectively, 14 cases treated over 4 months were categorized as a nonpersistent group and three cases that required prolonged therapy for more than 6 months were categorized as a persistent group. Clinical outcomes based on final best-corrected visual acuity were retrospectively analyzed, and selected confocal images were evaluated qualitatively for abnormal findings. Results The final best-corrected visual acuity was significantly lower (P < 0.01) for patients in the persistent group compared with that in the nonpersistent group. At the initial visit, in vivo confocal microscopy demonstrated Acanthamoeba cysts exclusively in the epithelial layer in both the nonpersistent group (80%) and the persistent group (100%). At a subsequent follow-up visit, numerous Acanthamoeba cysts were observed in the epithelial cell layer and in Bowman’s layer in all patients with persistent AK, but Acanthamoeba cysts were undetectable in all cases with nonpersistent AK tested. Conclusion Invasion of cysts into Bowman’s layer was characteristically observed in patients with persistence of AK. This finding suggests that invasion of Acanthamoeba cysts into Bowman’s layer may be a useful predictor for a persistent clinical course.

Brugia pahangi : production of a monoclonal antibody reactive with the surface of infective larvae

Monoclonal antibodies against infective third-stage larvae (L3) of Brugia pahangi were generated from mice immunized with L3 antigens. The monoclonal antibodies were L3 stage-specific or stage-nonspecific. A BpG1 monoclonal antibody (IgG1 subclass) showing L3 stage-specificity was examined in detail. BpG1 recognized the surface of B. pahangi L3 and also reacted with the surface of Brugia malayi L3 but not with the surface of filarial worms of other genera, such as Acanthocheilonema viteae and Litomosoides carinii. BpG1 promoted cellular adhesion to the surface of B. pahangi L3. BpG1 bound on living L3 was shed but the shedding rate was relatively slow. The surface antigen recognized by BpG1 had a molecular weight of 58 kDa. It was stable to heat and periodate treatments but sensitive to trypsin digestion and was released from living L3 by SDS but not by Triton X-100 or CTAB. Preincubation of L3 with BpG1 significantly reduced the recovery rate of worms compared with the preincubation with a monoclonal antibody (IgG1 subclass) against the inner tissues of B. pahangi L3 or control supernatant of P3U1 myeloma cells. This result suggests that the antigen containing the BpG1 epitope may be one of the targets of a protective immune response against Brugia infection.

Genetic Characterization of Clinical Acanthamoeba Isolates from Japan using Nuclear and Mitochondrial Small Subunit Ribosomal RNA

Because of an increased number of Acanthamoeba keratitis (AK) along with associated disease burdens, medical professionals have become more aware of this pathogen in recent years. In this study, by analyzing both the nuclear 18S small subunit ribosomal RNA (18S rRNA) and mitochondrial 16S rRNA gene loci, 27 clinical Acanthamoeba strains that caused AK in Japan were classified into 3 genotypes, T3 (3 strains), T4 (23 strains), and T5 (one strain). Most haplotypes were identical to the reference haplotypes reported from all over the world, and thus no specificity of the haplotype distribution in Japan was found. The T4 sub-genotype analysis using the 16S rRNA gene locus also revealed a clear sub-conformation within the T4 cluster, and lead to the recognition of a new sub-genotype T4i, in addition to the previously reported sub-genotypes T4a-T4h. Furthermore, 9 out of 23 strains in the T4 genotype were identified to a specific haplotype (AF479533), which seems to be a causal haplotype of AK. While heterozygous nuclear haplotypes were observed from 2 strains, the mitochondrial haplotypes were homozygous as T4 genotype in the both strains, and suggested a possibility of nuclear hybridization (mating reproduction) between different strains in Acanthamoeba. The nuclear 18S rRNA gene and mitochondrial 16S rRNA gene loci of Acanthamoeba spp. possess different unique characteristics usable for the genotyping analyses, and those specific features could contribute to the establishment of molecular taxonomy for the species complex of Acanthamoeba.

Observation of Live Ticks (Haemaphysalis flava) by Scanning Electron Microscopy under High Vacuum Pressure

Scanning electron microscopes (SEM), which image sample surfaces by scanning with an electron beam, are widely used for steric observations of resting samples in basic and applied biology. Various conventional methods exist for SEM sample preparation. However, conventional SEM is not a good tool to observe living organisms because of the associated exposure to high vacuum pressure and electron beam radiation. Here we attempted SEM observations of live ticks. During 1.5×10−3 Pa vacuum pressure and electron beam irradiation with accelerated voltages (2–5 kV), many ticks remained alive and moved their legs. After 30-min observation, we removed the ticks from the SEM stage; they could walk actively under atmospheric pressure. When we tested 20 ticks (8 female adults and 12 nymphs), they survived for two days after SEM observation. These results indicate the resistance of ticks against SEM observation. Our second survival test showed that the electron beam, not vacuum conditions, results in tick death. Moreover, we describe the reaction of their legs to electron beam exposure. These findings open the new possibility of SEM observation of living organisms and showed the resistance of living ticks to vacuum condition in SEM. These data also indicate, for the first time, the usefulness of tick as a model system for biology under extreme condition.

The localization of allergens of Paragonimus westermani by pleural exudates from patients

The allergens of the lung fluke Paragonimus westermani were localized by indirect immunostaining in adult fluke sections using pleural exudates from 3 patients with P. westermani. Immunostaining performed by using pleural exudate with the highest level of specific IgE revealed that the P. westermani major allergen (or allergens) was located in the gut epithelium and luminal contents and that minor allergens were in the tegument and parenchyma. The antigens recognized by specific IgG were located at various sites including those recognized by specific IgE. Paragonimus westermani-specific IgE cross-reacted with only the gut of 2 other Paragonimus species, Paragonimus miyazakii and Paragonimus ohirai. The major allergen in the gut also was recognized by the other 2 pleural exudates. These results indicate that the substance present in and secreted from the gut is not only a major allergen but is also a common allergen among Paragonimus species.

Detection of Francisella tularensis and analysis of bacterial growth in ticks in Japan.

Francisella tularensis is distributed in the Northern hemisphere and it is the bacterial agent responsible for tularaemia, a zoonotic disease. We collected 4 527 samples of DNA from ticks in Japan, which were then analysed by real‐time PCR and nested PCR. Francisella DNA was detected by real‐time PCR in 2·15% (45/2 093) of Ixodes ovatus, 0·66% (14/2 107) of I. persulcatus, 8·22% (6/73) of I. monospinosus and 0·72% (1/138) of Haemaphysalis flava specimens. Finally, Francisella DNA was detected by nested PCR in 42 and five samples I. ovatus and I. persulcatus, respectively, which were positive according to real‐time PCR. Phylogenetic analysis showed that the sequence from I. ovatus and I. persulcatus were clustered with F. tularensis type B strains distributed in Eurasia. Microinjected live F. tularensis persisted in ticks, whereas heat‐killed F. tularensis decreased. Microinjected F. tularensis hlyD mutant decreased in ticks significantly compared to parent strain, thereby suggesting that HlyD in F. tularensis contributes to the adaptation or survive of bacterial infection in ticks.

In vivo laser confocal microscopy findings of radial keratoneuritis in patients with early stage Acanthamoeba keratitis.

OBJECTIVE To investigate in vivo corneal changes of keratoneuritis in early stage Acanthamoeba keratitis (AK) using in vivo laser confocal microscopy. DESIGN Single-center, prospective, clinical study. PARTICIPANTS Thirteen eyes (12 patients; 5 men and 7 women; mean age ± standard deviation, 22.3 ± 4.2 years) with keratoneuritis resulting from early stage AK were included in this study. TESTING In vivo laser confocal microscopy was performed, paying special attention to keratoneuritis. MAIN OUTCOME MEASURES Selected confocal images of corneal layers were evaluated qualitatively for shape and degree of light reflection of abnormal cells and deposits. RESULTS In all patients, Acanthamoeba cysts were observed clearly in the basal epithelial cell layer as highly reflective round particles with a diameter of 10 to 20 μm. Bowmans layer infiltration of Acanthamoeba cysts was observed in only 1 case, and no cases showed stromal or nerve infiltration of Acanthamoeba cysts. In the stroma, all cases showed highly reflective activated keratocytes forming a honeycomb pattern; these changes were significant around the keratoneuritis. Infiltration of inflammatory cells, possibly polymorphonuclear cells, was observed along with keratocyte bodies in all cases. Numerous highly reflective spindle-shaped materials were observed around the keratoneuritis. Most notably, highly reflective patchy lesions were observed around the keratoneuritis in 11 cases (84.6%). Inflammatory cells also were observed in the endothelial cell layer in 4 cases (30.8%). CONCLUSIONS In vivo laser confocal microscopy identified consistent corneal abnormalities around keratoneuritis in early stage AK patients, of which highly reflective patchy lesions may be characteristic of keratoneuritis. Further morphologic studies of corneas with early stage AK in a larger number of patients may elucidate the clinical significance of radial keratoneuritis and may help us to understand the interaction between Acanthamoeba organisms and host corneal cells or nerves.

Ex vivo laser confocal microscopy findings of cultured Acanthamoeba trophozoites

Purpose The purpose of the current study was to investigate ex vivo laser confocal microscopic findings of cultured Acanthamoeba trophozoites obtained from Acanthamoeba keratitis patients. Methods Eight cultured samples of Acanthamoeba trophozoites from eight eyes of seven patients (mean age, 26.9 years; age range, 18–52 years) were used. Seven samples were from corneal scrapings of Acanthamoeba keratitis patients and one sample was from the solution in a soft contact lens case. Ex vivo laser confocal microscopy was performed to qualitatively evaluate the shape and degree of light reflection of the living Acanthamoeba trophozoites. Results Ex vivo laser confocal microscopy demonstrated highly reflective, high-contrast Acanthamoeba trophozoites with no walls (mean size, 25.4 μm; range, 17.1–58.5 μm). The shapes of the trophozoites were highly pleomorphic, and some showed characteristic acanthopodia by laser confocal microscopy. Conclusion Ex vivo laser confocal microscopy was effective in demonstrating cultured Acanthamoeba trophozoites of various shapes and sizes. The observations of the current study may be helpful when similar structures are identified under in vivo conditions.