An autopsy case of granulomatous amebic encephalitis caused by Balamuthia mandrillaris involving prior amebic dermatitis.

An 82-year-old man, who was healthy and had worked as a farmer, experienced worsening neurological symptoms over a seven-month period, which eventually caused his death. Multiple fluctuating brain lesions were detected radiographically. Clinically, sarcoidosis was ranked high among the differential diagnoses because of the presence of skin lesions showing granulomatous inflammation, confirmed by biopsy. The patient's cerebrospinal fluid was also examined, but no definitive diagnosis was made while he was alive. An autopsy revealed multiple granulomatous amebic encephalitis lesions in the brain. Genetic and immunohistochemical analyses identified Balamuthia (B.) mandrillaris, a free-living ameba, which resides in soil and fresh water, as the causative organism. A retrospective examination revealed B. mandrillaris in the biopsied skin as well as cerebrospinal fluid, strongly suggesting that the ameba had spread into the brain percutaneously. Few studies have detailed the cutaneous pathology of B. mandrillaris infections. In general, granulomatous amebic encephalitis is extremely difficult to diagnose without autopsy, but the present case provides a clue that could allow similar cases to be diagnosed earlier; that is, the presence of skin lesions.

Antibacterial Activity in Iodine-coated Implants Under Conditions of Iodine Loss: Study in a Rat Model Plus In Vitro Analysis.

BACKGROUND: We developed iodine-coated titanium implants to suppress microbial activity and prevent periprosthetic joint infection (PJI); their efficacy was demonstrated in animal and in vitro models. The iodine content in iodine-coated implants naturally decreases in vivo. However, to our knowledge, the effect of reduced iodine content on the implant's antimicrobial activity has not been evaluated to date. QUESTIONS/PURPOSES: (1) How much does the iodine content on the implant surface decrease after 4 and 8 weeks in vivo in a rat model? (2) What effect does the reduced iodine content have on the antimicrobial effect of the implant against multiple bacteria in an in vitro model? METHODS: This experiment was performed in two parts: an in vivo experiment to determine attenuation of iodine levels over time in rats, and an in vitro experiment in which we sought to assess whether the reduced iodine content observed in the in vivo experiment was still sufficient to deliver antimicrobial activity against common pathogens seen in PJI. For the in vivo experiment, three types of titanium alloy washers were implanted in rats: untreated (Ti), surface-anodized to produce an oxide film (Ti-O), and with an iodine layer on the oxidation film (Ti-I). The attenuation of iodine levels in rats was measured over time using inductively coupled plasma-mass spectrometry. Herein, only the Ti-I washer was used, with five implanted in each rat that were removed after 4 or 8 weeks. For the 4- and 8-week models, two rats and 15 washers were used. For the in vitro study, to determine the antibacterial effect, three types of washers (Ti, Ti-O, and Ti-I) (nine washers in total) were implanted in each rat. Then, the washers were removed and the antibacterial effect of each washer was examined on multiple bacterial species using the spread plate method and fluorescence microscopy. For the spread plate method, six rats were used, and five rats were used for the observation using fluorescence microscopy; further, 4- and 8-week models were made for each method. Thus, a total of 22 rats and 198 washers were used. Live and dead bacteria in the biofilm were stained, and the biofilm coverage percentage for quantitative analysis was determined using fluorescence microscopy in a nonblinded manner. Ti-I was used as the experimental group, and Ti and Ti-O were used as control groups. The total number of rats and washers used throughout this study was 24 and 213, respectively. RESULTS: Iodine content in rats implanted with Ti-I samples decreased to 72% and 65% after the in vivo period of 4 and 8 weeks, respectively (p = 0.001 and p < 0.001, respectively). In the in vitro experiment, the Ti-I implants demonstrated a stronger antimicrobial activity than Ti and Ti-O implants in the 4- and 8-week models. Both the median number of bacterial colonies and the median biofilm coverage percentage with live bacteria on Ti-I were lower than those on Ti or Ti-O implants for each bacterial species in the 4- and 8-week models. There was no difference in the median biofilm coverage percentage of dead bacteria. In the 8-week model, the antibacterial activity using the spread plate method had median (interquartile range) numbers of bacteria on the Ti, Ti-O, and Ti-I implants of 112 (104 to 165) × 105, 147 (111 to 162) × 105, and 55 (37 to 67) × 105 of methicillin-sensitive Staphylococcus aureus (Ti-I versus Ti, p = 0.026; Ti-I versus Ti-O, p = 0.009); 71 (39 to 111) × 105, 50 (44 to 62) × 105, and 26 (9 to 31)× 105 CFU of methicillin-resistant S. aureus (Ti-I versus Ti, p = 0.026; Ti-I versus Ti-O, p = 0.034); and 77 (74 to 83) × 106, 111 (95 to 117) × 106, and 30 (21 to 45) × 106 CFU of Pseudomonas aeruginosa (Ti-I versus Ti, p = 0.004; Ti-I versus Ti-O, p = 0.009). Despite the decrease in the iodine content of Ti-I after 8 weeks, it demonstrated better antibacterial activity against all tested bacteria than the Ti and Ti-O implants. CONCLUSION: Iodine-coated implants retained their iodine content and antibacterial activity against methicillin-sensitive S. aureus, methicillin-resistant S. aureus, and P. aeruginosa for 8 weeks in vivo in rats. To evaluate the longer-lasting antibacterial efficacy, further research using larger infected animal PJI models with implants in the joints of both males and females is desirable. CLINICAL RELEVANCE: Iodine-coated titanium implants displayed an antibacterial activity for 8 weeks in rats in vivo. Although the findings in a rat model do not guarantee efficacy in humans, they represent an important step toward clinical application.

Molecular identification of Eimeria hestermani and Eimeria prionotemni from a red-necked wallaby (Macropodidae; Macropus rufogriseus) in Japan.

To date, more than 50 Eimeria spp. have been isolated from marsupials of the family Macropodidae. Although 18 species of Eimeria have been previously detected from multiple animal species belonging to the genus Macropus of the family, limited genetic analyses of the parasites are available, and their pathogenicity remains unclear. Here, we report the isolation of Eimeria spp. from a zoo specimen of red-necked wallaby (Macropodidae; Macropus rufogriseus). Specifically, two distinct types of Eimeria oocysts were recovered, one from the feces before treatment with an anthelmintic and the second from the intestinal contents after death of the animal. The oocysts obtained from the two sources were morphologically identified as E. hestermani and E. prionotemni, respectively. We successfully determined partial gene sequences from the two isolates, including segments of the 18S rRNA genes, and for the first time have used phylogenetic analyses of these sequences to assign the species to distinct clades. In combination with further genetic data, these results are expected to help elucidate the pathogenicity and host ranges of Eimeria spp. within the respective family and genus.

Occurrence and genetic identifications of porcine Entamoeba, E. suis and E. polecki, at Tangerang in West Java, Indonesia.

Entamoeba suis and E. polecki subtype (ST) 1 and ST3 recently have been inferred to be virulent in pigs. However, because relevant molecular epidemiological surveys have been limited, the prevalences of these species remain unknown and their pathogenicities are still controversial. We surveyed 196 fecal samples of pigs (118 of adults, 78 of piglets) at Tangerang in West Java, Indonesia, in 2017, employing PCR using porcine Entamoeba-specific primers. E. suis was the more frequently detected species, observed in 81.1% of samples, while E. polecki ST1 and ST3 were detected in 18.4% and 17.3% of samples, respectively; mixed infections (harboring 2-3 species or subtypes of Entamoeba) were confirmed in 29.3% of positive samples. Statistically significant differences in the positive rates were not seen between adult pigs and piglets, except for those of E. polecki ST3. The prevalences of Eimeria spp. and/or Cystoisospora suis (79.1%), strongyles (55.6%), and Strongyloides spp. (6.1%) were also observed morphologically in the samples. Further chronological or seasonal investigations of pigs and humans in these high-prevalence areas are needed to assess the virulence of the Entamoeba parasites, including the effects on pig productivity, and to evaluate the zoonotic impacts of these organisms.

Molecular phylogeny of Blastocystis isolates from wild rodents captured in Indonesia and Japan.

Blastocystis sp. is a common intestinal protist found worldwide in a variety of animals, including humans. Currently, 17 subtypes (STs) of Blastocystis isolates from mammalian and avian host species have been reported based on the small subunit ribosomal RNA gene (SSU rDNA). Among these, human Blastocystis were only identified among STs 1-9. Except ST9, all other STs comprised isolates from humans and other animal species. Entire sequence data of the SSU rDNA of nine Blastocystis isolates from laboratory rats or guinea pigs previously showed ST4, whereas Blastocystis isolates from wild rodents have not been addressed genetically. In this study, Blastocystis infection in wild rodents was surveyed in Indonesia and Japan, and 11 and 12 rodent Blastocystis parasites were obtained from Rattus exulans and R. novercious, respectively. All new Blastocystis isolates from wild rodents were identified as ST4 based on the SSU rDNA sequences. The best tree inferred with the entire sequences of the SSU rDNA of all ST4 isolates including 17 data registered in GenBank clearly showed monophyletic ST4A and ST4B clades. Although ST4 isolates from laboratory rats were separated into these two clades, all Blastocystis isolates from wild rodents in the present study were positioned into the clade ST4A and further separated into two sub-clusters within the clade ST4A according to the location of the host species. Considering the fact that laboratory rats were susceptible to both ST4A and ST4B, separation of the monophyletic sub-clusters of Blastocystis isolates from Indonesian Polynesian rats and Japanese brown rats may indicate the presence of geographical variations rather than a host-specific separation. In either way, the robust host preference to rodent species of ST4 Blastocystis was also confirmed.

Impact of HIV Infection and Anti-Retroviral Therapy on the Immune Profile of and Microbial Translocation in HIV-Infected Children in Vietnam.

CD4⁺ T-lymphocyte destruction, microbial translocation, and systemic immune activation are the main mechanisms of the pathogenesis of human immunodeficiency virus type 1 (HIV) infection. To investigate the impact of HIV infection and antiretroviral therapy (ART) on the immune profile of and microbial translocation in HIV-infected children, 60 HIV vertically infected children (31 without ART: HIV(+) and 29 with ART: ART(+)) and 20 HIV-uninfected children (HIV(-)) aged 2-12 years were recruited in Vietnam, and their blood samples were immunologically and bacteriologically analyzed. Among the HIV(+) children, the total CD4⁺-cell and their subset (type 1 helper T-cell (Th1)/Th2/Th17) counts were inversely correlated with age (all p < 0.05), whereas regulatory T-cell (Treg) counts and CD4/CD8 ratios had become lower, and the CD38⁺HLA (human leukocyte antigen)-DR⁺CD8⁺- (activated CD8⁺) cell percentage and plasma soluble CD14 (sCD14, a monocyte activation marker) levels had become higher than those of HIV(-) children by the age of 2 years; the CD4/CD8 ratio was inversely correlated with the plasma HIV RNA load and CD8⁺-cell activation status. Among the ART(+) children, the total CD4⁺-cell and Th2/Th17/Treg-subset counts and the CD4/CD8 ratio gradually increased, with estimated ART periods of normalization being 4.8-8.3 years, whereas Th1 counts and the CD8⁺-cell activation status normalized within 1 year of ART initiation. sCD14 levels remained high even after ART initiation. The detection frequency of bacterial 16S/23S ribosomal DNA/RNA in blood did not differ between HIV-infected and -uninfected children. Thus, in children, HIV infection caused a rapid decrease in Treg counts and the early activation of CD8⁺ cells and monocytes, and ART induced rapid Th1 recovery and early CD8⁺-cell activation normalization but had little effect on monocyte activation. The CD4/CD8 ratio could therefore be an additional marker for ART monitoring.

First molecular identification of Entamoeba polecki in a piglet in Japan and implications for aggravation of ileitis by coinfection with Lawsonia intracellularis.

Parasitic Entamoeba spp. are found in many vertebrate species including humans, as well as many livestock including pigs. In pigs, three Entamoeba spp., E. suis, and E. polecki and E. histolytica as zoonotic species, have been identified, but their pathogenicity has not been fully characterized. Here, we report the bacteriological, virological, and histopathological examination of three piglets with chronic diarrhea. Two animals appeared to be additionally infected with Lawsonia intracellularis, which caused a characteristic proliferative ileitis. In the piglet infected with Entamoeba spp., the trophozoites (approximately 10-15 µm with one nucleus in their cytoplasm) invaded into the lamina propria and the disease was worsened by the formation of ulcers and pseudomembranes. Genetic analysis identified the parasite as E. polecki (99.5% identity). Although E. polecki in humans or animals might be less pathogenic in the case of a single infection, coinfections with other pathogens including L. intracellularis may increase the severity of the disease.

In vivo imaging of radial keratoneuritis in patients with Acanthamoeba keratitis by anterior-segment optical coherence tomography.

PURPOSE: To investigate in vivo corneal changes of radial keratoneuritis in early-stage Acanthamoeba keratitis (AK) using anterior-segment optical coherence tomography (AS-OCT). DESIGN: Single-center, prospective clinical study. PARTICIPANTS: Four eyes (4 patients with a mean age of 28.5 years) with early-stage AK showing radial keratoneuritis were included in this study. Definitive diagnosis was made by confirmation of AK cysts using in vivo confocal microscopy and culture. METHODS: Anterior-segment OCT examination was performed on the initial visit and at follow-up visits paying special attention to radial keratoneuritis. MAIN OUTCOME MEASURES: Selected AS-OCT images of the cornea were evaluated qualitatively for the shape and degree of light reflection of abnormal neurons. RESULTS: With the use of AS-OCT, we successfully obtained high-resolution images of putative radial keratoneuritis in all patients as highly reflective bands or lines in the corneal stroma. The depth and width of the highly reflective bands/lines varied from case to case (anterior stroma to mid-stroma, from 20 to 200 µm). Some lines ran obliquely from the deep peripheral stroma toward the anterior stroma, and some were located at different depths (subepithelial and mid-stroma) and ran relatively parallel to the corneal layers. After appropriate treatment, radial keratoneuritis was resolved by both slit-lamp biomicroscopy and AS-OCT in all patients. CONCLUSIONS: High-resolution Fourier-domain AS-OCT provides novel and detailed visual information of radial keratoneuritis in patients with early-stage AK. Visualization of radial keratoneuritis by AS-OCT may be a useful adjunct to the diagnosis and follow-up of early-stage AK.

Molecular characterization of various trichomonad species isolated from humans and related mammals in Indonesia.

Trichomonad species inhabit a variety of vertebrate hosts; however, their potential zoonotic transmission has not been clearly addressed, especially with regard to human infection. Twenty-one strains of trichomonads isolated from humans (5 isolates), pigs (6 isolates), rodents (6 isolates), a water buffalo (1 isolate), a cow (1 isolate), a goat (1 isolate), and a dog (1 isolate) were collected in Indonesia and molecularly characterized. The DNA sequences of the partial 18S small subunit ribosomal RNA (rRNA) gene or 5.8S rRNA gene locus with its flanking regions (internal transcribed spacer region, ITS1 and ITS2) were identified in various trichomonads; Simplicimonas sp., Hexamastix mitis, and Hypotrichomonas sp. from rodents, and Tetratrichomonas sp. and Trichomonas sp. from pigs. All of these species were not detected in humans, whereas Pentatrichomonas hominis was identified in humans, pigs, the dog, the water buffalo, the cow, and the goat. Even when using the high-resolution gene locus of the ITS regions, all P. hominis strains were genetically identical; thus zoonotic transmission between humans and these closely related mammals may be occurring in the area investigated. The detection of Simplicimonas sp. in rodents (Rattus exulans) and P. hominis in water buffalo in this study revealed newly recognized host adaptations and suggested the existence of remaining unrevealed ranges of hosts in the trichomonad species.

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. Bowman's 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.

Giardiasis in a patient undergoing chemotherapy for retinoblastoma and acute myelogenous leukemia.

Giardiasis is a common cause of diarrhea in undeveloped countries, but is very rare in developed countries. A patient with acute myelogenous leukemia and retinoblastoma presented with a high fever and severe watery diarrhea during induction chemotherapy. On microscopy, cysts were seen in her stool, suggesting Giardia intestinalis, which was confirmed on polymerase chain reaction (PCR). G. intestinalis was also detected in the patient's asymptomatic parents, who may have transmitted it to the patient. Giardiasis should be tested for in patients with severe and persistent diarrhea during chemotherapy, when other etiologies have been excluded. PCR used to amplify the DNA of G. intestinalis is rapid and sensitive.

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.

A case of amebic colitis and liver abscesses that occurred after treatment of coronavirus disease 2019 with dexamethasone.

Entamoeba histolytica infections, which can be asymptomatic, are endemic to developing countries; traveling to such countries is a risk factor for contracting these infections. A 65-year-old Japanese man was hospitalized for coronavirus disease 2019 (COVID-19)-associated respiratory distress, and was treated with remdesivir, dexamethasone, and oxygen supplementation. Although his respiratory condition improved and the oxygen support was discontinued, he developed a fever, severe abdominal pain, and diarrhea on day 13 of hospitalization. Fifteen years ago, he was hospitalized for diarrhea of an unknown origin in Suzhou, China, and had a history of passing loose stools for 1 year. Contrast-enhanced abdominal and pelvic computed tomography revealed liver abscesses in both lobes and intestinal edema from the ascending colon to the descending colon. The abscesses were suspected to be amebic based on the characteristics of the drained abscess fluid. The patient was treated with cefotaxime and metronidazole, and his temperature declined and abdominal pain improved. A culture analysis of abscess fluid yielded negative findings; however, polymerase chain reaction analyses of abscess and stool samples were positive for Entamoeba histolytica. We speculated that the patient was infected with Entamoeba histolytica while in China, and that the corticosteroid usage for COVID-19 had exacerbated the infection. Clinicians should be aware that corticosteroid treatments can lead to recurrent invasive amebiasis in asymptomatic amebic carriers.

Intestinal parasitic infections and risk factors for infection in Kenyan children with and without HIV infection.

It has been reported that HIV infection is not a risk factor for Entamoeba species infection but is for Giardia intestinalis assemblage B in children living in Western Kenya. This study aimed to investigate the prevalence of and the risk factors for Entamoeba spp. and G. intestinalis infection in children living in Nairobi, Kenya. This cross-sectional study included 87 children with HIV [HIV(+)] and 85 without HIV [HIV(-)]. Stool and blood samples were collected for the detection of the parasites by PCR and immunological analyses using flow cytometry. Sociobehavioral and hygienic data were collected using questionnaires and analyzed statistically. The prevalence of Entamoeba spp. infection was significantly lower in the HIV(+) than in the HIV(-) children (63.2% vs. 78.8%, P = 0.024), whereas the prevalence of G. intestinalis infection was not (27.6% vs. 32.9%, P = 0.445). "Not boiling drinking water" (adjusted odds ratio [aOR]: 3.8, P = 0.044) and "helping in nursery care" (aOR: 2.8, P = 0.009) were related to G. intestinalis assemblage B infection, and "CD4/CD8 ratio ≥1" was related to Entamoeba spp. infection (aOR: 3.3, P = 0.005). In stratified regression analyses, HIV infection was negatively associated with G. intestinalis assemblage B infection in females (aOR: 0.3, P = 0.022), but positively associated in males (aOR 3.8, P = 0.04). These results suggest that G. intestinalis assemblage B infection is related to hygienic conditions, while Entamoeba spp. infection is an indicator of better immunological status, and that the role of HIV infection in Giardia infection may differ between Kenyan boys and girls.

Antibacterial effect and biological reaction of calcium phosphate cement impregnated with iodine for use in bone defects.

Calcium phosphate cement (CPC) is often used to repair bone defects that occur after bone tumor and fracture treatment. To address bone defect cases with a high infection risk, developing CPCs with a longlasting wide-spectrum antibacterial effect is critical. Povidone-iodine has a wide antibacterial spectrum. Though there have been some reports of CPC containing antibiotics, no report of CPC with iodine has been described. In this study, the antibacterial effect and biological reaction of CPC impregnated with iodine was investigated. Iodine release from CPC and bone cement with various iodine contents (2.5, 5, and 20%) was evaluated, and 5 %-iodine CPC retained more iodine than the other CPCs after one week. Antibacterial activity against Staphylococcus aureus and Escherichia coli was also investigated, showing that 5 %-iodine had an antibacterial effect for up to eight weeks. Cytocompatibility was assessed, and 5 %-iodine CPC showed the same amount of fibroblast colony formation as control samples. CPCs with varying iodine contents (0, 5, and 20%) were then inserted into lateral femora of Japanese white rabbits for histological analysis. Osteoconductivity was evaluated using scanning electron microscopy, and hematoxylin-eosin staining. Consecutive bone formation was observed around all CPCs at eight weeks. These results indicate that CPC impregnated with iodine exhibits antimicrobial activity and cytocompatibility, and therefore, it may be effective for bone defect cases with high infection risk.

Combined adipose-derived mesenchymal stem cell and antibiotic therapy can effectively treat periprosthetic joint infection in rats.

Periprosthetic joint infection (PJI) is characterized by biofilm infection, which is difficult to alleviate while preserving implant integrity. Furthermore, long-term antibiotic therapy may increase the prevalence of drug-resistant bacterial strains, necessitating a non-antibacterial approach. Adipose-derived stem cells (ADSCs) exert antibacterial effects; however, their efficacy in PJI remains unclear. This study investigates the efficacy of combined intravenous ADSCs and antibiotic therapy in comparison to antibiotic monotherapy in a methicillin-sensitive Staphylococcus aureus (MSSA)-infected PJI rat model. The rats were randomly assigned and equally divided into 3 groups: no-treatment group, antibiotic group, ADSCs with antibiotic group. The ADSCs with antibiotic group exhibited the fastest recovery from weight loss, with lower bacterial counts (p = 0.013 vs. no-treatment group; p = 0.024 vs. antibiotic group) and less bone density loss around the implants (p = 0.015 vs. no-treatment group; p = 0.025 vs. antibiotic group). The modified Rissing score was used to evaluate localized infection on postoperative day 14 and was the lowest in the ADSCs with antibiotic group; however, no significant difference was observed between the antibiotic group and ADSCs with antibiotic group (p < 0.001 vs. no-treatment group; p = 0.359 vs. antibiotic group). Histological analysis revealed a clear, thin, and continuous bony envelope, a homogeneous bone marrow, and a defined, normal interface in the ADSCs with antibiotic group. Moreover, the expression of cathelicidin expression was significantly higher (p = 0.002 vs. no-treatment group; p = 0.049 vs. antibiotic group), whereas that of tumor necrosis factor (TNF)-α and interleukin(IL)-6 was lower in the ADSCs with antibiotic group than in the no-treatment group (TNF-α, p = 0.010 vs. no-treatment group; IL-6, p = 0.010 vs. no-treatment group). Thus, the combined intravenous ADSCs and antibiotic therapy induced a stronger antibacterial effect than antibiotic monotherapy in a MSSA-infected PJI rat model. This strong antibacterial effect may be related to the increased cathelicidin expression and decreased inflammatory cytokine expression at the site of infection.

In vitro assessment of anticryptosporidial efficacy and cytotoxicity of adenosine analogues using a SYBR Green real-time PCR method.

OBJECTIVES: The aims of this study were to provide a cost-effective and valuable method for evaluating drug efficacy against Cryptosporidium parvum using a quantitative SYBR Green real-time PCR (qPCR) and to assess the efficacy of adenosine analogues as drug templates. METHODS: C. parvum HNJ-1 strain growing in human ileocaecal adenocarcinoma cells was employed as an in vitro culture system. To normalize the DNA extraction efficiency, a specific plasmid was added to each sample before DNA purification; the genomic DNA of infected cells was quantified by qPCR using specific primers to confirm drug efficacy and cytotoxicity. To determine the mechanism of action, enzymatic inhibition analyses were conducted using C. parvum S-adenosyl-l-homocysteine hydrolase (CpSAHH) recombinant protein. RESULTS: The dose-dependent growth inhibition of C. parvum was confirmed; 50% effective concentrations of neplanocin A (NPA) and 2-fluoroadenosine (2FA) were 139 µM and 0.842 µM, respectively. Cytotoxicity evaluation showed that the 50% growth inhibition concentration of 2FA was 1.18 µM; NPA did not exhibit any cytotoxicity up to 200 µM. The screening system revealed the specific but marginal efficacy of NPA and showed 2FA to be cytotoxic. Recombinant CpSAHH inhibition analyses showed that NPA competitively inhibited CpSAHH activity (K(i )= 0.395 µM), whereas 2FA did not. CONCLUSIONS: This novel qPCR system confirmed not only drug efficacy against C. parvum but also cytotoxicity to host cells. Moreover, since the SYBR Green method is cost effective, it could therefore be used in a wide variety of clinical and research-oriented applications of Cryptosporidium analysis.

Phylogenetic characterization of Isospora jaracimrmani oocysts from a veiled chameleon (family Chamaeleonidae; Chamaeleo calyptratus) reared at a zoo in Ishikawa, Japan.

Oocysts of Isospora sp. were detected in the feces of a veiled chameleon (family Chamaeleonidae; Chamaeleo calyptratus) kept at a zoo in Ishikawa, Japan. Phylogenetic analysis placed the sequence in the cluster of Isospora spp. isolated from reptiles. Based on a comparison of morphological data of ten previously reported Isospora species from the Chamaeleonidae family, this isolate was morphologically similar to I. jaracimrmani, which has been considered to be a virulent species. This case study suggests the possibility that species of Isospora might not always cause disease because the animal that shed these oocysts showed no symptoms for more than two months.

Extremely diversified haplotypes observed among assemblage B population of Giardia intestinalis in Kenya.

In molecular epidemiological studies of Giardia intestinalis, an pathogenic intestinal flagellate, due to the presence of allelic sequence heterogeneity (ASH) on the tetraploid genome, the image of haplotype diversity in the field remains uncertain. Here we employed the nine assemblage B positive stool samples, which had previously reported from Kenyan children, for the clonal sequence analysis of multiple gene loci (glutamate dehydrogenase (GDH), triosephosphate isomerase (TPI), and beta-giardin (BG)). The diversified unique assemblage B haplotypes as GDH (n = 67), TPI (n = 84), and BG (n = 62), and the assemblage A haplotypes as GDH (n = 7), TPI (n = 14), and BG (n = 15), which were hidden in the previous direct-sequence results, were detected. Among the assemblage B haplotypes, Bayesian phylogeny revealed multiple statistically significant clusters (9, 7, and 7 clusters for GDH, TPI, and BG, respectively). A part of the clusters (2 for GDH and 1 for BG), which included >4 haplotypes from an individual sample, indicated the presence of co-transmission with multiple strains sharing a recent ancestor. Locus-dependent discrepancies, such as different compositions of derived samples in clusters and different genotyping results for the assemblages, were also observed and considered to be the traces of both intra- and inter-assemblage genetic recombination respectively. Our clonal sequence analysis for giardial population, which applied firstly in Kenya, could reveal the higher rates of ASH far beyond the levels reported in other areas and address the complex population structure. The clonal analysis is indispensable for the molecular field study of G. intestinalis.

Detections of gastrointestinal parasites, including Giardia intestinalis and Cryptosporidium spp., in cattle of Banten province, Indonesia.

Gastrointestinal parasites can induce low productivity in livestock by causing acute or chronic enteritis. Veterinarians make great efforts to design rational and effective hygienic protocols for both the prevention and treatment of diarrhea. Although prevalences can vary depending on the examined areas or the ages of the hosts, and the methods used for detections, it is helpful to accumulate data across many areas to evaluate parasitic distribution. A coprological survey in cattle was conducted in Tangerang, Banten Province of Indonesia, in order to determine the prevalence of the parasites, including those of diarrhea-associated diseases. Furthermore, the risk of transmission of Giardia intestinalis and Cryptosporidium spp. to human was genetically analyzed. Gastrointestinal parasites were detected in 87 of 109 cattle samples, including 85 carrying Eimeria spp., 36 carrying Fasciola gigantica, 35 carrying Strongyloides spp., 33 carrying Paramphistomum spp., and 15 carrying Capillaria spp. Giardia intestinalis and Cryptosporidium spp., parasites with zoonotic potential, were detected in 9 and 1 cattle samples, respectively. Molecular analyses identified the G. intestinalis isolate as a member of Assemblage E, which has been recently detected in humans in another country. These results may be helpful in understanding the hygienic risk affecting the livestock productivity and zoonotic potential of cattle in Indonesia.