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.

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.

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.

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.

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.

Combinational therapy with antibiotics and antibiotic-loaded adipose-derived stem cells reduce abscess formation in implant-related infection in rats.

Implant-related infection is difficult to treat without extended antibiotic courses. However, the long-term use of antibiotics has led to the development of multidrug- and methicillin-resistant Staphylococcus aureus. Thus, alternatives to conventional antibiotic therapy are needed. Recently, mesenchymal stem cells have been shown to have antimicrobial properties. This study aimed to evaluate the antimicrobial activity and therapeutic effect of local treatment with antibiotic-loaded adipose-derived stem cells (ADSCs) plus an antibiotic in a rat implant-associated infection model. Liquid chromatography/tandem mass spectrometry revealed that ADSCs cultured in the presence of ciprofloxacin for 24 h showed time-dependent antibiotic loading. Next, we studied the therapeutic effects of ADSCs and ciprofloxacin alone or in combination in an implant-related infection rat model. The therapeutic effects of ADSCs plus antibiotics, antibiotics, and ADSCs were compared with no treatment as a control. Rats treated with ADSCs plus ciprofloxacin had the lowest modified osteomyelitis scores, abscess formation, and bacterial burden on the implant among all groups (P < 0.05). Thus, local treatment with ADSCs plus an antibiotic has an antimicrobial effect in implant-related infection and decrease abscess formation. Thus, our findings indicate that local administration of ADSCs with antibiotics represents a novel treatment strategy for implant-associated osteomyelitis.

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.

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.

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.

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.

Human case of subcutaneous nodule because of a novel genetic variation of Dirofilaria sp.

A 75-year-old man presented with a 1-cm large elastic soft subcutaneous nodule on the left side of the umbilicus, which when excised showed presence of a helminthic form within the granulomatous lesions. Morphologically, the helminth was considered to be of the genus Dirofilaria, and the patient showed increased serum antibody titer against canine filaria. The partial DNA sequence of the mitochondrial 12S rRNA gene locus of this clinical isolate showed the highest nucleotide identity (89.6%) with Dirofilaria repens; however, the phylogenetic analysis addressed the haplotype and Dirofilaria ursi as outgroups of the clusters of D. repens and Dirofilaria immitis, which are the causal agents of most human dirofilariasis. As like bear filaria D. ursi, a wide variety of other carnivore-parasitizing filaria species have rarely been reported in humans. The newly detected genetic haplotype in this case may correspond to one of these species of Dirofilaria, though the genetic references are not available thus far.

Molecular characterization of highly pathogenic Eimeria species among beef cattle on Java Island, Indonesia.

Gastrointestinal parasites including Eimeria spp. are known to affect domestic animal productivity causing watery or lethal bloody diarrhea. However, there are few reports on the detailed distribution of bovine Eimeria spp. in cattle, particularly in developing tropical and sub-tropical areas. Using a total of 289 fecal samples collected from beef cattle on Java Island, one of the five main islands of Indonesia, fecal examinations by the Whitlock and sugar flotation methods and molecular surveys were conducted to reveal the prevalence of 6 Eimeria spp. As a result of morphological screening using Whitlock methods and sugar flotation, Eimeria spp. prevalences of 9.4% and 52.3% were confirmed, respectively. The prevalence was higher in younger cattle [under 1 year (63.9%), 1-2 years (75.0%) and more than in 2 year old cattle (42.3%)]. The prevalences of identified species were as follows: 10.4% for E. bovis, 2.8% for E. ellipsoidalis, 2.1% for E. alabamensis, 1.4% for E. zuernii, 1.1% for E. auburnensis, and 0.4% for E. cylindrica. Moreover, prevalences of 12.8% for Strongyloides papillosus, 7.3% for Trichuris globulosa, and 0.3% for Capillaria bovis were detected. Although the average number of oocysts per gram of feces was <100 among the positive samples, and cases of heavy infection were limited, the endemicity of these pathogenic Eimeria species among farms in Indonesia should be noted.

Possible pathogenicity of commensal Entamoeba hartmanni revealed by molecular screening of healthy school children in Indonesia.

BACKGROUND: Although parasites are still endemic in developing areas, residents in those regions seem not to be affected by the presence of intestinal protozoans. This study aimed to investigate whether pathogenic and commensal protozoans are the causal agents of diarrhea via a school-based cross-sectional survey conducted in Indonesia, in September 2016. RESULTS: Molecular screening for intestinal protozoans in collected 144 stool samples from healthy students (age range 7-15 years) was carried out. The prevalence of protozoan parasites was as follows: Giardia intestinalis (56.3%), Entamoeba histolytica (0%), E. dispar (6.9%), E. moshkovskii (0%), E. hartmanni (31.3%), and E. coli (44.4%). Observational evaluation of stool conditions using the Bristol stool chart confirmed the loose stool rate (33.3-90.9%) in each age group. Logistic regression analysis of protozoan infection or colonization for loose stool (mild to severe diarrhea) as an outcome revealed no significant findings in examined protozoans including pathogenic G. intestinalis infection [adjusted odds ratio (AOR) 0.78, 95% confidence interval (CI) 0.36-1.67], except in E. hartmanni colonization (AOR 2.81, 95% CI 1.1-3.7, P = 0.026). CONCLUSIONS: The molecular survey of intestinal protozoans targeting healthy population with their stool form evaluation could address the pathogenicity of those parasites appropriately. In comparatively higher-age children at least 7 years of age or greater in the endemic area, G. intestinalis could regard commensal, while E. hartmanni seems to possess a certain pathogenicity as a causal agent of mild diarrhea.

Three monophyletic clusters in Retortamonas species isolated from vertebrates.

Retortamonas spp. has been reported as an intestinal parasite among various host organisms, including humans; however, its intra-genus molecular diversity has not yet been elucidated. Haplotypes of the 18S small subunit ribosomal RNA locus (1836-1899 bp) of Retortamonas spp. from humans (n = 8), pigs (n = 6), dogs (n = 1), goats (n = 16), water buffalos (n = 23), cattle (n = 7), rats (n = 3), and chickens (n = 5) were analyzed with references isolated from non-human mammals, amphibians, and insects. Phylogenetic and network analyses revealed a statistically supported three cluster formation among the vertebrate-isolated haplotypes, while insect-isolated haplotypes were independently clustered with Chilomastix. In the clade of vertebrate isolates, assemblage A (amphibian genotype), which included the amphibian references, was addressed as an out-group of the other clusters. Assemblage B (mammalian and chicken genotype) included most haplotypes from various mammals including humans with the haplotypes isolated from a chicken. Human isolates were all classified into this assemblage, thus assemblage B might correspond to R. intestinalis. Assemblage C (bovine genotype), which included specific haplotypes from water buffalos and cattle, was addressed as a sister lineage of assemblage B. Among the diversified haplotypes of assemblage B, a specific haplotype, which was identified from multiple host mammals (humans, dogs, pigs, cattle, water buffalos, elks, goats, and rats), indicates the potential zoonotic transmission of the Retortamonas among them. The genotyping classification of retortamonads could contribute to a better understanding of its molecular epidemiology, especially among humans and related host 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.

Adenovirus-prime and baculovirus-boost heterologous immunization achieves sterile protection against malaria sporozoite challenge in a murine model.

With the increasing prevalence of artemisinin-resistant malaria parasites, a highly efficacious and durable vaccine for malaria is urgently required. We have developed an experimental virus-vectored vaccine platform based on an envelope-modified baculovirus dual-expression system (emBDES). Here, we show a conceptually new vaccine platform based on an adenovirus-prime/emBDES-boost heterologous immunization regimen expressing the Plasmodium falciparum circumsporozoite protein (PfCSP). A human adenovirus 5-prime/emBDES-boost heterologous immunization regimen consistently achieved higher sterile protection against transgenic P. berghei sporozoites expressing PfCSP after a mosquito-bite challenge than reverse-ordered or homologous immunization. This high protective efficacy was also achieved with a chimpanzee adenovirus 63-prime/emBDES-boost heterologous immunization regimen against an intravenous sporozoite challenge. Thus, we show that the adenovirus-prime/emBDES-boost heterologous immunization regimen confers sterile protection against sporozoite challenge by two individual routes, providing a promising new malaria vaccine platform for future clinical use.

Extra-gastrointestinal anisakidosis caused by Pseudoterranova azarasi manifesting as strangulated inguinal hernia.

The incidence of anisakidosis continues to increase worldwide due to increased consumption of undercooked fish. Although anisakidosis can be categorized into four clinical phenotypes (gastric, intestinal, ectopic, and allergic), ectopic forms of the condition are much less common than gastric or intestinal forms when caused by Pseudoterranova spp. We report the case of a 5-year-old patient who presented with a rapidly growing left inguinal mass and was subsequently diagnosed with extra-gastrointestinal anisakidosis caused by Pseudoterranova azarasi.