Microbiome of lovebug (Plecia longiforceps) in Seoul, South Korea.

Lovebugs appeared in large numbers across a wide area in Seoul, South Korea, in June 2023. The sudden appearance of exotic insects not only discomforts people but also fosters anxiety, as their potential for pathogen transmission would be unknown. In this study, targeted next-generation sequencing (NGS) of the 16S rRNA gene V4 region was performed using iSeq 100 to screen for bacteria in lovebugs. Forty-one lovebugs (20 females and 21 males) collected in Seoul, Korea, were identified as Plecia longiforceps based on mitochondrial cytochrome oxidase subunit 1 sequencing data using PCR. We analyzed the microbiome of the lovebugs and detected 453 species of bacteria. Among all bacteria screened based on NGS, Rickettsia was detected in all samples with an average relative abundance of 80.40%, followed by Pandoraea and Ewingella. Diversity (alpha and beta) between females and males did not differ; however, only Tumebacillus showed a higher relative abundance in females. Sequencing analysis of Rickettsia using a gltA gene-specific primer by PCR showed that it had higher sequence similarity to the Rickettsia symbiont of arthropods than to the spotted fever group rickettsiae. Eleven samples in which Pandoraea was detected by iSeq 100 were confirmed by PCR and exhibited 100% sequence identity to Pandoraea oxalativorans strain DSM 23570. Consequently, the likelihood of pathogen transmission to humans is low. The applied method may play a crucial role in swiftly identifying bacterial species in the event of future outbreaks of exotic insects that may be harmful to humans.IMPORTANCELovebugs have recently emerged in large numbers in Seoul, causing major concern regarding potential health risks. By performing the next-generation sequencing of the 16S rRNA gene V4 region, we comprehensively examined the microbiome of these insects. We identified the presence of numerous bacteria, including Rickettsia and Pandoraea. Reassuringly, subsequent tests confirmed that these detected bacteria were not pathogenic. The present study addresses health concerns related to lovebugs and shows the accuracy and efficiency of our detection technique. Such methods prove invaluable for rapidly identifying bacterial species during potential outbreaks of unfamiliar insects, thereby ensuring public safety.

Metabarcoding of pathogenic parasites based on copro-DNA analysis of wild animals in South Korea.

Four species of dominant wild animals, namely, Prionailurus bengalensis euptilurus, Nyctereutes procyonoides koreensis, Hydropotes inermis argyropus, and Sus scrofa coreanus, are hosts of potential infectious agents, including helminths and protozoa. Therefore, it is necessary to analyze the infectious agents present in these wild animals to monitor and control the spread of pathogens. In the present study, fecal samples from 51 wild animals were collected from the mountains of Yangpyeong, Hoengseong, and Cheongyang in South Korea and metabarcoding of the V9 region of the 18S rRNA gene was performed to identify various parasite species that infect these wild animals. Genes from nematodes, such as Metastrongylus sp., Strongyloides spp., Ancylostoma sp., and Toxocara sp., were detected in the fecal samples from wild animals. In addition, platyhelminthes, including Spirometra sp., Echinostomatidae gen. sp., Alaria sp., Neodiplostomum sp., and Clonorchis sp., and protozoa, including Entamoeba sp., Blastocystis sp., Isospora sp., Tritrichomonas sp., Pentatrichomonas sp., and Cryptosporidium sp., were detected. In the present study, various parasites infecting wild animals were successfully identified using metabarcoding. Our technique may play a crucial role in monitoring parasites within wild animals, especially those causing zoonoses.

NF-κB p65 and TCF-4 interactions are associated with LPS-stimulated IL-6 secretion of macrophages.

Proinflammatory cytokine plays a central role in host defense and acute inflammatory responses. Both positive and negative correlations of NF-κB and Wnt/ß-catenin pathways have been reported depending on cell types in response to inflammatory stimuli for IL-6 cytokine production. Macrophages are vital to the regulation of immune responses and the development of inflammation, but the crosstalk between two pathways has not been elucidated so far in macrophages. We observed a positive cross-regulation between the NF-κB and Wnt/ß-catenin pathways for IL-6 production in human macrophages. To verify the functional validity of this interaction, LY294002 or PNU74654, representative blockers of each pathway, were treated. IL-6 secretion was reduced to the basal level by both inhibitor treatments, even when stimulated by LPS. We also found that NF-κB p65 migrated to the nucleus and interacted with the transcription factor TCF-4 in macrophages upon LPS stimulation.

Association between Smoking Status and the Risk of Hip Fracture in Patients with Type 2 Diabetes: A Nationwide Population-Based Study.

BACKGRUOUND: Limited longitudinal evidence exists regarding the potential association between smoking status and hip fracture among individuals with type 2 diabetes. We investigated this association using large-scale, nationwide cohort data for the Korean population. METHODS: This nationwide cohort study included 1,414,635 adults aged 40 and older who received Korean National Health Insurance Service health examinations between 2009 and 2012. Subjects with type 2 diabetes were categorized according to their smoking status, amount smoked (pack-years), number of cigarettes smoked per day, and duration of smoking. The results are presented as hazard ratios (HRs) and 95% confidence intervals (CIs) for the associations between smoking status parameters and risk of hip fracture in multivariable Cox proportional hazard regression analysis. RESULTS: Compared with never-smokers, an increased adjusted HR (aHR) for hip fracture was observed in current smokers (1.681; 95% CI, 1.578 to 1.791), and a comparable aHR for hip fracture was found in former smokers (1.065; 95% CI, 0.999 to 1.136). For former smokers who had smoked 20 pack-years or more, the risk was slightly higher than that for never-smokers (aHR, 1.107; 95% CI, 1.024 to 1.196). The hip fracture risk of female former smokers was similar to that of female current smokers, but the hip fracture risk in male former smokers was similar to that of male never-smokers. CONCLUSION: Smoking is associated with an increased risk of hip fracture in patients with type 2 diabetes. Current smokers with diabetes should be encouraged to quit smoking because the risk of hip fracture is greatly reduced in former smokers.

Morphological studies of fly puparia (Diptera: Calliphoridae) in the soil from a Joseon Dynasty grave in Korea.

Archaeoentomological investigations were conducted on soil contents from a grave belonging to the Joseon Dynasty as part of the Urban Environment Maintenance Project (UEMP) in Cheongjin 12-16 dong (districts), Jongno-gu, Seoul, Korea, from December 01, 2008 to February 19, 2011. A total of 28 insect puparia with hard shells of the common green bottle fly Lucilia sericata were identified in the soil. Evidence suggested that the corpse was placed outside for some days instead of being buried immediately after death. This is the first report of fly puparia in soil samples from a tomb of the Joseon Dynasty during 16-17 AD in Korea. Our findings may help determine the timeframe of burial and offer archaeological insights into the funerary customs of the period.

Forsythia velutina Nakai extract: A promising therapeutic option for atopic dermatitis through multiple cell type modulation.

BACKGROUND: Atopic dermatitis (AD) is a complex condition characterized by impaired epithelial barriers and dysregulated immune cells. In this study, we demonstrated Forsythia velutina Nakai extract (FVE) simultaneously inhibits basophils, macrophages, keratinocytes, and T cells that are closely interrelated in AD development. METHODS: We analyzed the effect of FVE on nitric oxide and reactive oxygen species (ROS) production in macrophages, basophil degranulation, T cell activation, and tight junctions in damaged keratinocytes. Expression of cell-type-specific inflammatory mediators was analyzed, and the underlying signaling pathways for anti-inflammatory effects of FVE were investigated. The anti-inflammatory effects of FVE were validated using a DNCB-induced mouse model of AD. Anti-inflammatory activity of compounds isolated from FVE was validated in each immune cell type. RESULTS: FVE downregulated the expression of inflammatory mediators and ROS production in macrophages through TLR4 and NRF2 pathways modulation. It significantly reduced basophil degranulation and expression of type 2 (T2) and pro-inflammatory cytokines by perturbing FcεRI signaling. Forsythia velutina Nakai extract also robustly inhibited the expression of T2 cytokines in activated T cells. Furthermore, FVE upregulated the expression of tight junction molecules in damaged keratinocytes and downregulated leukocyte attractants, as well as IL-33, an inducer of T2 inflammation. In the AD mouse model, FVE showed superior improvement in inflammatory cell infiltration and skin structure integrity compared to dexamethasone. Dimatairesinol, a lignan dimer, was identified as the most potent anti-inflammatory FVE compound. CONCLUSION: Forsythia velutina Nakai extract and its constituent compounds demonstrate promising efficacy as a therapeutic option for prolonged AD treatment by independently inhibiting various cell types associated with AD and disrupting the deleterious link between them.

Investigating the microbiome of house dust mites in South Korea.

Understanding the house dust mites (HDMs) microbiome is crucial due to its potential effects on the development of allergic diseases. In 1998, our laboratory collected Dermatophagoides farinae and D. pteronyssinus from beds in a Korean household and began cultivating these HDMs. Our laboratory has been actively investigating several topics about HDMs in recent years, including the bacterial and fungal microbiome and their interactions, as well as the impact of the HDM microbiome on airway inflammation. To study the D. farinae microbiome, we employed high-throughput sequencing of the 16S rDNA amplicons. The results revealed that the two most abundant bacteria were Enterococcus faecalis and Bartonella spp. In contrast, we found almost no bacteria in D. pteronyssinus. By inoculating bacteria to HDMs, we found that D. farinae is more susceptible to bacteria than D. pteronyssinus. This susceptibility was associated with the presence of certain fungal species in D. pteronyssinus. Additionally, we have recently made efforts to produce HDMs with reduced levels of symbiotic bacteria. We believe that standardizing and controlling the microbiome in HDMs are crucial steps for the future development and improvement of allergic immunotherapies.

Intestinal parasitic infections among children aged 12-59 months in Nyamasheke District, Rwanda.

Intestinal parasitic infections are a public health burden and a major cause of illness in developing countries. The diseases lead to various health threats, including growth retardation and mental health-related disorders, especially in children. We assessed the risk factors for intestinal parasitic infections among children aged 12-59 months residing in Nyamasheke District, Rwanda. A cross-sectional descriptive study was conducted using secondary data from 1,048 children aged 12-59 months whose stool samples were examined for the presence of intestinal parasites and whose results were registered in the laboratory information system in 2020. The prevalence of intestinal parasites in children aged 12-59 months was 53.2%. The dominant parasites were Ascaris lumbricoides (13.1%), followed by Giardia lamblia (10.9%), Entamoeba histolytica (7.9%), Trichuris trichiura (6.5%), hookworms (1.7%), and Taenia species (1.4%). A significant association was observed between intestinal parasites and the literacy of mothers or children's caregivers (odds ratio (OR)=5.09, P<0.001). Children from farming households were 2.8-fold more likely to contract intestinal parasitic infections than those from nonfarming households (OR=2.8, P<0.001). A significant association was also observed between intestinal parasites and food safety (OR=4.9, P<0.001). Intestinal parasitic infections were significantly associated with hand hygiene practices after using the toilet and washing fresh fruits before eating (P<0.001). The information gathered will help public health providers and partners develop control plans in highly endemic areas in Rwanda.

Gut microbiota differences induced by Toxoplasma gondii seropositivity in stray cats in South Korea.

T. gondii is a highly prevalent parasite worldwide, with cats serving as its final host. However, few studies have investigated the impact of T. gondii infection on cat gut microbiota. Therefore, this study examined the influence of T. gondii infection on the gut microbiota of stray cats and identified potential pathogens in their feces. This study examined T. gondii infection through blood of stray cats and the influence of microbiota in their feces using 16S rRNA gene amplicon sequencing. The results revealed significant differences in gut microbiota composition and diversity between the T. gondii seropositive and seronegative groups. Seropositive samples displayed a lower number of operational taxonomic units and reduced Shannon index than the seronegative samples. The seropositive and seronegative groups exhibited enrichment of taxa, including Escherichia and Enterobacteriaceae and Collinsella, Bifidobacterium, and Roseburia, respectively. Furthermore, potential pathogen species, including Campylobacter, Escherichia, and Streptococcus, were identified in the fecal samples. These findings suggest that T. gondii infection significantly impacts gut microbiota composition and diversity in stray cats. Additionally, an increased potential pathogen load, represented by Escherichia spp., was observed. These results underscore the importance of monitoring the prevalence of zoonotic pathogens in stray cats, as they can serve as reservoirs for zoonotic diseases.

Neurofilament light chain and cardiac MIBG uptake as predictors for phenoconversion in isolated REM sleep behavior disorder.

BACKGROUND: Isolated rapid-eye-movement (REM) sleep behavior disorder (iRBD) is considered as a prodromal stage of either multiple system atrophy (MSA) or Lewy body disease (LBD; Parkinson's disease and dementia with Lewy bodies). However, current knowledge is limited in predicting and differentiating the type of future phenoconversion in iRBD patients. We investigated the role of plasma neurofilament light chain (NfL) and cardiac metaiodobenzylguanidine (MIBG) uptake as predictors for phenoconversion. METHODS: Forty patients with iRBD were enrolled between April 2018 and October 2019 and prospectively followed every 3 months to determine phenoconversion to either MSA or LBD. Plasma NfL levels were measured at enrollment. Cardiac MIBG uptake and striatal dopamine transporter uptake were assessed at baseline. RESULTS: Patients were followed for a median of 2.92 years. Four patients converted to MSA and 7 to LBD. Plasma NfL level at baseline was significantly higher in future MSA-converters (median 23.2 pg/mL) when compared with the rest of the samples (median 14.1 pg/mL, p = 0.003). NfL level above 21.3 pg/mL predicted phenoconversion to MSA with the sensitivity of 100% and specificity of 94.3%. Baseline MIBG heart-to-mediastinum ratio of LBD-converters (median 1.10) was significantly lower when compared with the rest (median 2.00, p < 0.001). Heart-to-mediastinum ratio below 1.545 predicted phenoconversion to LBD with the sensitivity of 100% and specificity of 92.9%. CONCLUSIONS: Plasma NfL and cardiac MIBG uptake may be useful biomarkers in predicting phenoconversion of iRBD. Elevated plasma NfL levels may suggest imminent phenoconversion to MSA, whereas low cardiac MIBG uptake suggests phenoconversion to LBD.

Importance of Balanced Attention Toward Coronavirus Disease 2019 and Neglected Tropical Diseases.

Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, has been spreading since 2019, causing a worldwide pandemic. Amid the COVID-19 pandemic, tuberculosis, AIDS, and malaria have adversely affected the quality of life of patients and killed millions of people. In addition, COVID-19 continues to impede the delivery of health services, including those for the control of neglected tropical diseases (NTDs). Furthermore, NTDs have been reported as possible co-pathogens among patients infected with COVID-19. However, studies regarding parasitic co-infection among these patients have been limited. This review aimed to explore and describe the cases and reports of parasitic infections in the backdrop of COVID-19 to provide comprehensive knowledge regarding this aspect. We reviewed seven cases of patients who had parasitic co-infection and tested positive for COVID-19, and summarized the literature on the importance of controlling parasitic diseases. In addition, we identified recommendations for the control of parasitic diseases under possible difficulties, such as declining funding for parasitic diseases in 2020. This review highlights the growing burden of NTDs under COVID-19 that may be caused by the deficiency of healthcare infrastructure and human resources as the main reasons. Clinicians should remain vigilant for possible co-infections with parasites in COVID-19 patients, while policymakers are urged to reinforce a balanced and long-term health strategy that addresses both NTDs and COVID-19.

Production of Dermatophagoides farinae Having Low Bacterial Content Using Ampicillin.

Background: Symbiotic bacteria in house dust mites pose a risk of immunological side effects in the clinical use of immunotherapeutic agents. In this study, we investigated the duration for which the bacterial concentration in Dermatophagoides farinae could be kept low with antibiotic treatment, and whether the allergenic properties of the mite changed under ampicillin treatment. Methods: D. farinae was cultivated in the presence of ampicillin powder in an autoclaved medium for 6 weeks. After subsequent subcultures without ampicillin, the mites were harvested, and the extract was prepared. The amounts of bacteria, lipopolysaccharides (LPS), and two major allergens (Der f 1 and Der f 2) were measured. Human bronchial epithelial cells and mice were treated with the D. farinae extract to assess the allergic airway inflammation. Results: The number of bacteria and level of LPS were reduced by 150-fold and 33-fold, respectively, at least 18 weeks after ampicillin treatment. The concentration of Der f 1 and Der f 2 remained unchanged by ampicillin treatment. The secretion of interleukin (IL)-6 and IL-8 from the human airway epithelial cells decreased when treated with the extract of ampicillin-treated D. farinae compared with that of ampicillin-untreated D. farinae. A mouse asthma model was developed using ampicillin-treated D. farinae. We observed that the level of lung function, airway inflammation, and serum-specific immunoglobulin were not different for the mouse asthma model developed using ampicillin-treated D. farinae than the model developed using ampicillin-untreated D. farinae. Conclusions: We showed that bacterial content in D. farinae was reduced by ampicillin treatment, which was sufficient to induce allergic sensitization and an immune response. This method will be used to develop more controlled allergy immunotherapeutic agents.

Ampicillin treated German cockroach extract leads to reduced inflammation in human lung cells and a mouse model of Asthma.

Cockroaches can cause allergic sensitization in humans via contact with their feces or frass. Antibiotics can affect concentration of major allergen and total bacteria production in German cockroaches (Blattella germanica). This study examined the ability of antibiotic-treated German cockroaches to induce allergic airway inflammation and the effect of antibiotics on their lipopolysaccharide and Bla g1, 2, and 5 expression levels. Specifically, we measured the ability of German cockroach extract (with or without prior antibiotic exposure) to induce allergic inflammation in human bronchial epithelial cells and a mouse model of asthma. Bacterial 16S rRNA and lipopolysaccharide levels were lower in ampicillin-treated cockroaches than in the control group. The Bla g1, Bla g2, and Bla g5 expression in ampicillin-treated cockroaches decreased at both the protein and RNA levels. In human bronchial epithelial cell lines BEAS-2B exposed to the ampicillin-treated extract, expression levels of interleukin-6 and interleukin-8 were lower than that in the control group. The total cell count and eosinophil count in bronchoalveolar lavage fluid was also lower in mice exposed to the ampicillin-treated extract than in those exposed to normal cockroach extract. Mouse lung histopathology showed reduced immune cell infiltration and mucus production in the ampicillin group. Our results showed that ampicillin treatment reduced the symbiont bacterial population and major allergen levels in German cockroaches, leading to reduced airway inflammation in mice. These results can facilitate the preparation of protein extracts for immunotherapy or diagnostics applications.

The microbiota in feces of domestic pigeons in Seoul, Korea.

In Korea, feral pigeons pose significant public health risks because they carry various zoonotic pathogens. Human population density is a significant factor in zoonotic disease events. Seoul is one of the largest cities by population density among developed countries and where most of the homeless population in Korea exists. We designed this study to compare the microbiota of pigeon feces by regional characteristics and the presence of homeless individuals. Therefore, this study used 16S rRNA amplicon sequencing to detect possible pathogenic microbes and assess the current risk of zoonosis in Seoul, South Korea. Pigeon fecal samples (n = 144) obtained from 19 public sites (86 and 58 fecal samples from regions in and outside Seoul, respectively) were examined. Potentially pathogenic bacteria were also detected in the fecal samples; Campylobacter spp. was found in 19 samples from 13 regions, Listeriaceae was found in seven samples, and Chlamydia spp. was found in three samples from two regions. Principal coordinates analysis and permutational multivariate analysis of variance revealed a significant difference in bacterial composition between the regions in Seoul (n = 86) and outside Seoul (n = 58) and between the regions with (n = 81) and without (n = 63) homeless individuals. Overall, this study identified various potentially pathogenic microorganisms in pigeon feces at public sites in South Korea. Moreover, this study demonstrates that the microbial composition was influenced by regional characteristics and homelessness. Taken together, this study provides important information for public health strategic planning and disease control.

Metabarcoding of bacteria and parasites in the gut of Apodemus agrarius.

BACKGROUND: The striped field mouse Apodemus agrarius is a wild rodent commonly found in fields in Korea. It is a known carrier of various pathogens. Amplicon-based next-generation sequencing (NGS) targeting the 16S ribosomal RNA (rRNA) gene is the most common technique used to analyze the bacterial microbiome. Although many bacterial microbiome analyses have been attempted using feces of wild animals, only a few studies have used NGS to screen for parasites. This study aimed to rapidly detect bacterial, fungal and parasitic pathogens in the guts of A. agrarius using NGS-based metabarcoding analysis. METHODS: We conducted 18S/16S rDNA-targeted high-throughput sequencing on cecal samples collected from A. agrarius (n = 48) trapped in May and October 2017. Taxa of protozoa, fungi, helminths and bacteria in the cecal content were then identified. RESULTS: Among the protozoa identified, the most prevalent was Tritrichomonas sp., found in all of the cecal samples, followed by Monocercomonas sp. (95.8% prevalence; in 46/48 samples) and Giardia sp. (75% prevalence; in 36/48 samples). For helminths, Heligmosomoides sp. was the most common, found in 85.4% (41/48) of samples, followed by Hymenolepis sp. (10.4%; 5/48) and Syphacia sp. (25%; 12/48). The 16S rRNA gene analysis showed that the microbial composition of the cecal samples changed by season (P = 0.005), with the linear discriminant analysis effect size showing that in the spring Escherichia coli and Lactobacillus murinus were more abundant and Helicobacter rodentium was less abundant. Helicobacter japonicus was more abundant and Prevotella_uc was less abundant in males. The microbial composition changed based on the Heligmosomoides sp. infection status (P = 0.019); specifically, Lactobacillus gasseri and Lactobacillus intestinalis were more abundant in the Heligmosomoides sp.-positive group than in the Heligmosomoides sp.-negative group. CONCLUSIONS: This study demonstrated that bacterial abundance changed based on the season and specific parasitic infection status of the trapped mice. These results highlight the advantages of NGS technology in monitoring zoonotic disease reservoirs.

Periprosthetic Fracture around a Cemented Stem in Total Hip Arthroplasty.

The increase in the number of primary total hip arthroplasties that will be performed over the next several decades will lead to an increase in the incidence of periprosthetic fractures around the femoral stem. A search of targeted articles was conducted using on-line databases of PubMed (National Library of Medicine) and articles were obtained from January 2008 to November 2021. Reliable prediction of treatment can be achieved using the Vancouver classification; internal fixation is indicated in fractures involving a stable implant and revision arthroplasty is indicated in those with unstable prostheses. To the best of our knowledge, relatively fewer studies regarding periprosthetic proximal femur fractures of cemented stems have been reported. The focus of this review is on the risk factors and strategies for treatment of these fractures for periprosthetic femoral fractures around a cemented hip arthroplasty.

Accuracy and efficiency of automatic tooth segmentation in digital dental models using deep learning.

This study evaluates the accuracy and efficiency of automatic tooth segmentation in digital dental models using deep learning. We developed a dynamic graph convolutional neural network (DGCNN)-based algorithm for automatic tooth segmentation and classification using 516 digital dental models. We segmented 30 digital dental models using three methods for comparison: (1) automatic tooth segmentation (AS) using the DGCNN-based algorithm from LaonSetup software, (2) landmark-based tooth segmentation (LS) using OrthoAnalyzer software, and (3) tooth designation and segmentation (DS) using Autolign software. We evaluated the segmentation success rate, mesiodistal (MD) width, clinical crown height (CCH), and segmentation time. For the AS, LS, and DS, the tooth segmentation success rates were 97.26%, 97.14%, and 87.86%, respectively (p < 0.001, post-hoc; AS, LS > DS), the means of MD widths were 8.51, 8.28, and 8.63 mm, respectively (p < 0.001, post hoc; DS > AS > LS), the means of CCHs were 7.58, 7.65, and 7.52 mm, respectively (p < 0.001, post-hoc; LS > DS, AS), and the means of segmentation times were 57.73, 424.17, and 150.73 s, respectively (p < 0.001, post-hoc; AS < DS < LS). Automatic tooth segmentation of a digital dental model using deep learning showed high segmentation success rate, accuracy, and efficiency; thus, it can be used for orthodontic diagnosis and appliance fabrication.

Parasites and blood-meal hosts of the tsetse fly in Tanzania: a metagenomics study.

BACKGROUND: Tsetse flies can transmit various Trypanosoma spp. that cause trypanosomiasis in humans, wild animals, and domestic animals. Amplicon deep sequencing of the 12S ribosomal RNA (rRNA) gene can be used to detect mammalian tsetse hosts, and the 18S rRNA gene can be used to detect all associated eukaryotic pathogens, including Trypanosoma spp. METHODS: Tsetse flies were collected from the Serengeti National Park (n = 48), Maswa Game Reserve (n = 42), and Tarangire National Park (n = 49) in Tanzania in 2012-13. Amplicon deep sequencing targeting mammal-specific 12S rRNA and 18S rRNA genes was performed to screen the blood-feeding sources of tsetse flies and eukaryotic parasites in tsetse flies, respectively. RESULTS: 12S rRNA gene deep sequencing revealed that various mammals were blood-feeding sources of the tsetse flies, including humans, common warthogs, African buffalos, mice, giraffes, African elephants, waterbucks, and lions. Genes of humans were less frequently detected in Serengeti (P = 0.0024), whereas African buffaloes were detected more frequently as a blood-feeding source (P = 0.0010). 18S rRNA gene deep sequencing showed that six tsetse samples harbored the Trypanosoma gene, which was identified as Trypanosoma godfreyi and Trypanosoma simiae in subsequent ITS1 gene sequencing. CONCLUSIONS: Through amplicon deep sequencing targeting the 12S rRNA and 18S rRNA genes, various mammalian animals were identified as blood-meal sources, and two Trypanosoma species were detected in tsetse flies collected from the Maswa Game Reserve, Serengeti National Park, and Tarangire National Park in Tanzania. This study illustrates the patterns of parasitism of tsetse fly, wild animals targeted by the fly, and Trypanosoma spp. carried by the fly in Tanzania. It may provide essential data for formulating better strategies to control African trypanosomes.

Diagnosis of Balamuthia mandrillaris Encephalitis by Thymine-Adenine Cloning Using Universal Eukaryotic Primers.

BACKGROUND: Identifying the causal pathogen of encephalitis remains a clinical challenge. A 50-year-old man without a history of neurological disease was referred to our department for the evaluation of an intracranial lesion observed on brain magnetic resonance imaging (MRI) scans, and the pathology results suggested protozoal infection. We identified the species responsible for encephalitis using thymine-adenine (TA) cloning, suitable for routine clinical practice. METHODS: We extracted DNA from a paraffin-embedded brain biopsy sample and performed TA cloning using two universal eukaryotic primers targeting the V4-5 and V9 regions of the 18S rRNA gene. The recombinant plasmids were extracted, and the inserted amplicons were identified by Sanger sequencing and a homology search of sequences in the National Center for Biotechnology Information Basic Local Alignment Search Tool. RESULTS: The infection was confirmed to be caused by the free-living amoeba Balamuthia mandrillaris. Two of 41 colonies recombinant with 18S V4-5 primers and 35 of 63 colonies recombinant with the 18S V9 primer contained B. mandrillaris genes; all other colonies contained human genes. Pathogen-specific PCR ruled out Entamoeba histolytica, Naegleria fowleri, Acanthamoeba spp., and Toxoplasma gondii infections. CONCLUSIONS: This is the first report of B. mandrillaris-induced encephalitis in Korea based on molecular identification. TA cloning with the 18S rRNA gene is a feasible and affordable diagnostic tool for the detection of infectious agents of unknown etiology.

Microbiome of Haemaphysalis longicornis Tick in Korea.

Ticks can transmit pathogenic bacteria, protozoa, and viruses to humans and animals. In this study, we investigated the microbiomes of Haemaphysalis longicornis according to sex and life stages. The Shannon index was significantly higher for nymphs than adult ticks. Principal coordinates analysis showed that the microbiome composition of female adult and male adult ticks were different. Notably, Coxiella-like bacterium (AB001519), known as a tick symbiont, was found in all nymphs and female adult ticks, but only one out of 4 male adult ticks had Coxiella-like bacterium (AB001519). In addition, Rickettsia rickettsii, Coxiella burnetii, and Anaplasma bovis were detected in this study.