Kenji Yagita

Molecular Characterization of Cryptosporidium Isolates Obtained from Human and Bovine Infections in Japan

Abstract. Cryptosporidium oocysts, morphologically identified as Cryptosporidium parvum, were isolated from 22 human and 14 bovine cases in Japan, and were genotyped by means of a PCR/RFLP analysis of the poly-threonine gene. DNA profiles of human isolates gave three distinct genotypes, namely an anthroponotic genotype 1, zoonotic genotype 2 and a new genotype. Isolates from bovine samples gave zoonotic genotype 2. The unusual genotype of Cryptosporidium was isolated from the feces of three immunologically healthy adults, and was further characterized by the sequence analysis of the 18S rRNA gene. The third genotype was identified as Cryptosporidium meleagridis, demonstrating that C. meleagridis, which occurs worldwide, has the potential to infect humans regardless of their immunological condition.

The Largest Outbreak of Legionellosis in Japan Associated with Spa Baths

In July 2002, a large outbreak of legionellosis occurred in a bathhouse with spa facilities in Miyazaki Prefecture. Two hundred-ninety-five patients (including suspected cases) that had pneumonia and/or symptoms of fever, cough and so forth were reported; 37% of them were hospitalized and seven people died. In environmental investigations, Legionella pneumophila serogroups (SGs) land 8, L. dumoffii, L. londiniensis, some other Legionella species and many kinds of amoeba were isolated from 55 samples of bathtub water, tank water, filters and so forth in the spa facilities. The dominant isolates from the bathtab waters belonged to L. londiniensis, L. dumoffii and L. pneumophila SG1, and their maximum concentrations were 1.5 x 10(6), 5.2 x 10(5) and 1.6 x 10(5) cfu/100 mL, respectively. L. pneumophila SG1 strains isolated from bathtub water, tank water, filters and sputa of patients showed a indistinguishable DNA fingerprint pattern by pulsed-field gel electrophoresis (PFGE), confirming that the source of infection was the spa water. Our study indicate that spas may be a significant health hazard if hygienic management fails.

Assessment of Real-Time Polymerase Chain Reaction Detection of Acanthamoeba and Prognosis Determinants of Acanthamoeba Keratitis

OBJECTIVE To evaluate the diagnostic value of real-time polymerase chain reaction (PCR) for detecting Acanthamoeba in eyes diagnosed with Acanthamoeba keratitis (AK) by conventional tests. In addition, to determine the preoperative prognosis-determining factors in eyes with AK. DESIGN Retrospective, cross-sectional study. PARTICIPANTS A total of 104 eyes of 103 patients who were diagnosed with AK or with bacterial or bacteria-associated keratitis (BK) by conventional tests. METHODS Twenty-nine eyes with AK and 75 eyes with BK were evaluated for Acanthamoeba and bacterial DNA by real-time PCR. The Acanthamoeba copy numbers, bacterial load, and clinical parameters in the patients with AK were assessed for those significantly associated with poor outcome, that is, final visual acuity of <20/50 or requiring keratoplasty, by logistic regression analysis. MAIN OUTCOME MEASURES Acanthamoeba DNA copy number, bacterial DNA copy number, and odds ratio (OR) for poor prognosis. RESULTS The detection of amoebic DNA was 50 times more sensitive by real-time PCR than by conventional cyst counting. The Acanthamoeba copy numbers at the first visit (mean: 4.7×10(5)±3.2×10(5) copies) were significantly correlated with the AK stage, and both were significant risk factors for a poor outcome. The Acanthamoeba DNA copy numbers at the first visit and AK stage had a significantly high risk for poor outcome (OR of Acanthamoeba DNA copy per logarithm of copy numbers: 3.48, 95% confidence interval [CI], 1.04-111.63, P<0.05; OR of AK stage: 2.8 per stage increase, 95% CI, 1.07-7.30, P<0.05, after adjustment of age). In the AK cases with poor outcome, the amoebic DNA was not reduced by more than 90% after 1 month of treatment. The weak amoebic reduction was significantly associated with advanced AK stages or previous use of steroids. Bacterial 16S rDNA was detected in 53.6% of the eyes with AK, but it was not associated with any risk for refractoriness. CONCLUSIONS Real-time PCR was effective in detecting and managing AK. The Acanthamoeba copy number and AK stage at the first visit were significantly associated with poor outcome. FINANCIAL DISCLOSURE(S) The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Multisite Performance Evaluation of an Enzyme-Linked Immunosorbent Assay for Detection of Giardia, Cryptosporidium, and Entamoeba histolytica Antigens in Human Stool

ABSTRACT A novel fecal antigen detection assay for fresh and frozen human samples that detects but does not differentiate Giardia spp, Cryptosporidium spp, and Entamoeba histolytica, the Tri-Combo parasite screen, was compared to three established enzyme-linked immunosorbent assays (ELISAs) at three international sites. It exhibited 97.9% sensitivity and 97.0% specificity, with positive and negative predictive values of 93.4% and 99.1%, respectively. The Tri-Combo test proved a reliable means to limit the use of individual parasite ELISAs to positive samples.

Heterogeneity in cyst morphology within isolates of Acanthamoeba from keratitis patients in Thailand.

Summary We isolated Acanthamoebae from the first two keratitis patients identified in Thailand in 1988 and 1990. The patients developed decreased vision, severe photophobia, severe eye pain and foreign body sensation after minor corneal trauma. The lesions included generalized superficial punctate keratitis, stromal corneal ulcer with keratic precipitate and uveitis in one case, and corneal ulcer with abscess in the other. Both cases were diagnosed by isolation of characteristic trophozoites and cysts of Acanthamoeba from corneal tissue by non‐nutrient agar culture method. Based on cyst morphology, A. castellanii and A. polyphaga were detected in one case, and A. castellanii and A. triangularis in the other. Restriction fragment length polymorphism analysis of mitochondrial DNA (mtDNA‐RFLP) revealed that each patient harboured a single parasite population. One shared mtDNA‐RFLP with an authentic strain of A. castellanii, and the other gave a new unique pattern. Thus species identification of Acanthamoeba based on cyst morphology per se can be arbitrary, and mtDNA‐RFLP may be more appropriate for accurate species/strain differentiation amongst morphologically heterogeneous populations of Acanthamoebae.

Clustering of Acanthamoeba isolates from human eye infections by means of mitochondrial DNA digestion patterns.

Abstract A total of 90 Acanthamoeba isolates from human eye infections from 15 countries were clustered into distinct genotypes according to their mtDNA restriction fragment patterns. Closely related digestion phenotypes (sequence difference 0.1–1.5%) were integrated into a single genotype, whereas phenotypes with greater than 4.76% difference were considered distinct. Approximately 80% of the human isolates studied fell into 7 of 22 genotypes, indicating that virulence may be associated with specific clusters of cladistic groups of Acanthamoeba. This technique is useful for large-scale surveying of this particular pathogen.

High‐temperature adapted primitive Protochlamydia found in Acanthamoeba isolated from a hot spring can grow in immortalized human epithelial HEp‐2 cells

To elucidate how ancient pathogenic chlamydiae could overcome temperature barriers to adapt to human cells, we characterized a primitive chlamydia found in HS-T3 amoebae (Acanthamoeba) isolated from a hot spring. Phylogenetic analysis revealed the primitive species to be Protochlamydia. In situ hybridization staining showed broad distribution into the amoebal cytoplasm, which was supported by transmission electron microscopic analysis showing typical chlamydial features, with inclusion bodies including both elementary and reticular bodies. Interestingly, although most amoebae isolated from natural environments show reduced growth at 37°C, the HS-T3 amoebae harbouring the Protochlamydia grew well at body temperature. Although infection with Protochlamydia did not confer temperature tolerance to the C3 amoebae, the number of infectious progenies rapidly increased at 37°C with amoebal lysis. In immortalized human epithelial HEp-2 cells, fluorescence microscopic study revealed atypical inclusion of the Protochlamydia, and quantitative real-time polymerase chain reaction analyses also showed an increase in 16S ribosomal RNA DNA amounts. Together, these results showed that the Protochlamydia found in HS-T3 amoebae isolated from a hot spring successfully adapted to immortalized human HEp-2 cells at 37°C, providing further information on the evolution of ancient Protochlamydia to the present pathogenic chlamydiae.

Depletion of Cryptosporidium parvum Oocysts from Contaminated Sewage by Using Freshwater Benthic Pearl Clams (Hyriopsis schlegeli)

ABSTRACT The freshwater benthic pearl clam, Hyriopsis schlegeli, was experimentally exposed to Cryptosporidium parvum oocysts, and it was verified that the oocysts were eliminated predominantly via the fecal route, retaining their ability to infect cultured cells (HCT-8). The total fecal oocyst elimination rate was more than 90% within 5 days after exposure to the oocysts. H. schlegeli was able to survive in the final settling pond of a sewage plant for long periods, as confirmed by its pearl production. In the light of these findings, the clam was placed in the final settling pond in a trial to test its long-term efficacy in depleting oocysts contaminating the pond water. The number of clams placed was set to ensure a theoretical oocyst removal rate of around 50%, and the turbidity and the density of feed microbes in the overflow trough water of the pond were about 35% and 40 to 60% lower, respectively, than in the control water throughout the year. It was found that the clam feces containing oocysts were sufficiently heavy for them to settle to the bottom of the pond, despite the upward water flow. From these results, we concluded that efficient depletion of oocysts in the sewage water of small or midscale sewage treatment plants can be achieved by appropriate placement of H. schlegeli clams.

Development and evaluation of a reverse transcription-loop-mediated isothermal amplification assay for rapid and high-sensitive detection of Cryptosporidium in water samples

We describe a novel assay for simple, rapid and high-sensitive detection of Cryptosporidium oocysts in water samples using a reverse transcription-loop-mediated isothermal amplification (RT-LAMP). The assay is based on the detection of 18S rRNA specific for Cryptosporidium oocysts. The detection limit of the developed RT-LAMP assay was as low as 6 x 10(-3) oocysts/test tube, which theoretically enables us to detect a Cryptosporidium oocyst and perform duplicated tests even if water samples contain only one oocyst. The developed RT-LAMP assay could more sensitively detect Cryptosporidium oocysts in real water samples than the conventional assay based on microscopic observation.

Restriction-fragment-length polymorphism and variation in electrophoretic karyotype inNaegleria fowleri from Japan

Strains ofNaegleria fowleri isolated in Japan from two different places were found to differ in electro-phoretic karyotype and restriction-fragment-length polymorphism (RFLP) both from each other and from strains isolated on other continents. Using the Wagmer parsimony method on the RFLP, we found that the Japanese isolates are most closely related to the Australian isolates and most distinct from the European isolates.