Asymptomatic Enteric Virus Infections and Association with the Gut Microbiome in Rural Residents of Northern Laos.

Viral gastrointestinal infections are an important public health concern, and the occurrence of asymptomatic enteric virus infections makes it difficult to prevent and control their spread. This study aimed to determine the prevalence of and factors associated with asymptomatic enteric virus infection in adults in northern Laos. Fecal samples were collected from apparently healthy participants who did not report diarrhea or high fever at the time of the survey in northern Laos, and enteric viruses were detected using polymerase chain reaction (PCR) and reverse transcription (RT)-PCR. Individual characteristics, including the gut microbiome, were compared between asymptomatic carriers and noncarriers of each enteric virus. Of the participants (N = 255), 12 (4.7%) were positive for norovirus genogroup I (GI), 8 (3.1%) for human adenovirus, and 1 (0.4%) for norovirus GII; prevalence tended to be higher in less-modernized villages. Gut microbial diversity (evaluated by the number of operational taxonomic units) was higher in asymptomatic carriers of norovirus GI or human adenovirus than in their noncarriers. Gut microbiome compositions differed significantly between asymptomatic carriers and noncarriers of norovirus GI or human adenovirus (permutational analysis of variance, P <0.05). These findings imply an association between asymptomatic enteric virus infection and modernization and/or the gut microbiome in northern Laos.

Transition to a market economy and chronic psychosocial stress in northern Laos: An exploratory study of urinary free cortisol in rural residents.

OBJECTIVES: The indigenous population in northern Laos has experienced a transition from self-sufficiency to a market-oriented economy, which may have brought about unprecedented chronic psychosocial stress. This study examined the association between the transition to a market economy and urinary free cortisol (UFC) concentration as a stress biomarker among rural residents of three villages with different degrees of integration into the market economy. METHODS: An interview survey and urine sample collection were conducted in August 2018 and March 2019. We measured the UFC concentration in spot urine samples collected in the morning from participants aged 20-60 years (n = 168) using an enzyme-linked immunosorbent assay. Multiple linear regression analysis was performed to examine differences in UFC concentrations among villages by sex, with time of arrival for the survey, age, and body mass index included as covariates. RESULTS: The UFC concentration was higher in men living in the village with the highest degree of integration into the market economy than in those in the two villages with a lower dependence on cash, possibly linking increased stress levels with a change in employment type. This trend was not observed in women. CONCLUSION: Socioeconomic changes incidental to a transition to a market economy may increase the stress levels of men in northern Laos.

Recovery of microbial DNA by agar-containing solution from extremely low-biomass specimens including skin.

Recovering a sufficient amount of microbial DNA from extremely low-biomass specimens, such as human skin, to investigate the community structure of the microbiome remains challenging. We developed a sampling solution containing agar to increase the abundance of recovered microbial DNA. Quantitative PCR targeting the 16S rRNA gene revealed a significant increase in the amount of microbial DNA recovered from the developed sampling solution compared with conventional solutions from extremely low-biomass skin sites such as the volar forearm and antecubital fossa. In addition, we confirmed that the developed sampling solution reduces the contamination rate of probable non-skin microbes compared to the conventional solutions, indicating that the enhanced recovery of microbial DNA was accompanied by a reduced relative abundance of contaminating microbes in the 16S rRNA gene amplicon sequencing data. In addition, agar was added to each step of the DNA extraction process, which improved the DNA extraction efficiency as a co-precipitant. Enzymatic lysis with agar yielded more microbial DNA than conventional kits, indicating that this method is effective for analyzing microbiomes of low-biomass specimens.

A comprehensive microbial analysis of pediatric patients with acute appendicitis.

BACKGROUND: Pathogenesis of pediatric acute appendicitis (AA) is yet to be elucidated. Therefore, we performed a comprehensive microbial analysis of saliva, feces, and appendiceal lumen of AA patients using 16S ribosomal RNA (rRNA) gene amplicon sequencing to elucidate the pathogenesis of pediatric AA. METHODS: This study included 33 AA patients and 17 healthy controls (HCs) aged <15 y. Among the AA patients, 18 had simple appendicitis, and 15 had complicated appendicitis. Salivary and fecal samples were obtained from both groups. The contents of the appendiceal lumen were collected from the AA group. All samples were analyzed using 16S rRNA gene amplicon sequencing. RESULTS: The relative abundance of Fusobacterium was significantly higher in the saliva of AA patients as compared to that in HCs (P = 0.011). Bacteroides, Escherichia, Fusobacterium, Coprobacillus, and Flavonifractor were significantly increased in the feces of AA patients, as compared to that in HCs (P = 0.020, 0.010, 0.029, 0.031, and 0.002, respectively). In the appendiceal lumen, Bacteroides, Parvimonas, Fusobacterium, and Alloprevotella were the top bacterial genera with an average relative abundance >5% (16.0%, 9.1%, 7.9%, and 6.0%, respectively). CONCLUSIONS: The relative abundance of Fusobacterium was high in the appendiceal lumen of pediatric AA patients. Moreover, the relative abundance of Fusobacterium was significantly higher in the saliva and feces of pediatric AA patients than in those of healthy children. These results suggest that ectopic colonization of oral Fusobacterium in the appendix might play an important role in the pathogenesis of pediatric AA.

Impact of modernization on oxidative stress among indigenous populations in northern Laos.

OBJECTIVES: To explore the impact of modernization on oxidative stress during a momentous health transition process, we investigated differences in oxidative stress among the indigenous populations of villages in northern Laos with different levels of modernization. METHODS: We conducted a cross-sectional study of 380 adults in three villages with different levels of modernization. Three biomarkers related to oxidative stress were measured: urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) and 8-isoprostane concentrations (both measured by liquid chromatography-tandem mass spectrometry), and blood telomere length (measured with qPCR). We examined associations between village-level modernization and oxidative stress-related biomarkers in a multilevel analysis including a random effect and covariates. RESULTS: The geometric means of urinary 8-OHdG and 8-isoprostane concentrations were 2.92 and 0.700 µg/g creatinine, respectively, in our study population. Higher urinary 8-OHdG concentrations and shorter telomeres were observed in participants from the more modernized villages, whereas urinary 8-isoprostane concentrations did not differ significantly among villages. CONCLUSIONS: Our findings imply that modernization-induced changes in lifestyle may increase oxidative DNA damage. Baseline levels of oxidative lipid damage are expected to be high in the indigenous populations of northern Laos. Assessments of oxidative stress may provide valuable insights into the mechanisms of health transition in specific populations.

Determinants of oxidative stress among indigenous populations in Northern Laos: Trace element exposures and dietary patterns.

OBJECTIVES: To investigate determinants of oxidative stress in an indigenous population, we examined associations of trace element exposures and dietary patterns with three oxidative stress-related biomarkers among indigenous populations in Northern Laos. METHODS: This cross-sectional study included 341 adults from three villages with different levels of modernization. We used three oxidative stress-related biomarkers: urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) and 8-isoprostane concentrations, which were measured using liquid chromatography-tandem mass spectrometry, and blood telomere lengths, which were measured using a quantitative polymerase chain reaction method. We used multilevel analysis to examine associations of urinary arsenic, cadmium, and selenium concentrations, their interaction terms, and wild-plant-food scores (principal component scores calculated from food consumption frequencies) with oxidative stress-related biomarkers. RESULTS: Urinary arsenic and cadmium concentrations were positively associated with urinary 8-isoprostane concentrations. Urinary selenium concentrations were positively associated with urinary 8-OHdG concentrations. Interaction terms ([arsenic or cadmium] × selenium) showed negative associations with urinary 8-OHdG and 8-isoprostane concentrations, respectively. Urinary cadmium concentrations were negatively associated with telomere lengths. Wild-plant-food scores did not exhibit associations with oxidative stress-related biomarkers. CONCLUSION: Our findings imply that exposure to arsenic and cadmium is associated with greater oxidative lipid damage, whereas selenium may attenuate arsenic-induced oxidative DNA damage and cadmium-induced oxidative lipid damage. Cadmium exposure may accelerate telomere attrition. Trace element exposure may be a determinant of oxidative stress among indigenous populations in Northern Laos.

Human Gut Microbiota and Its Metabolites Impact Immune Responses in COVID-19 and Its Complications.

BACKGROUND & AIMS: We investigate interrelationships between gut microbes, metabolites, and cytokines that characterize COVID-19 and its complications, and we validate the results with follow-up, the Japanese 4D (Disease, Drug, Diet, Daily Life) microbiome cohort, and non-Japanese data sets. METHODS: We performed shotgun metagenomic sequencing and metabolomics on stools and cytokine measurements on plasma from 112 hospitalized patients with SARS-CoV-2 infection and 112 non-COVID-19 control individuals matched by important confounders. RESULTS: Multiple correlations were found between COVID-19-related microbes (eg, oral microbes and short-chain fatty acid producers) and gut metabolites (eg, branched-chain and aromatic amino acids, short-chain fatty acids, carbohydrates, neurotransmitters, and vitamin B6). Both were also linked to inflammatory cytokine dynamics (eg, interferon γ, interferon λ3, interleukin 6, CXCL-9, and CXCL-10). Such interrelationships were detected highly in severe disease and pneumonia; moderately in the high D-dimer level, kidney dysfunction, and liver dysfunction groups; but rarely in the diarrhea group. We confirmed concordances of altered metabolites (eg, branched-chain amino acids, spermidine, putrescine, and vitamin B6) in COVID-19 with their corresponding microbial functional genes. Results in microbial and metabolomic alterations with severe disease from the cross-sectional data set were partly concordant with those from the follow-up data set. Microbial signatures for COVID-19 were distinct from diabetes, inflammatory bowel disease, and proton-pump inhibitors but overlapping for rheumatoid arthritis. Random forest classifier models using microbiomes can highly predict COVID-19 and severe disease. The microbial signatures for COVID-19 showed moderate concordance between Hong Kong and Japan. CONCLUSIONS: Multiomics analysis revealed multiple gut microbe-metabolite-cytokine interrelationships in COVID-19 and COVID-19related complications but few in gastrointestinal complications, suggesting microbiota-mediated immune responses distinct between the organ sites. Our results underscore the existence of a gut-lung axis in COVID-19.

Rat trade and leptospirosis: Molecular epidemiology of Leptospira species in rats exported from Cambodia to Vietnam.

Rats are an important maintenance host of Leptospira spp., the causative agents of leptospirosis, a zoonotic disease with worldwide distribution. Rats are traded as food in the Mekong Delta, where Cambodia exports tons of rats to Vietnam. Handling wild rats is a potential health risk, but the information on Leptospira spp. carried by rats traded in the region remains limited. In this study, we investigated the carriage of Leptospira spp. in rats exported from Cambodia to Vietnam using bacterial culture, nested PCR and DNA sequencing. Isolates were then assessed using serological analysis and whole-genome sequencing (WGS), and Leptospira DNA detected in rat kidney tissues was also analysed by multilocus sequence typing (MLST). Seventy-two rats (2 Bandicota indica, 57 Rattus argentiventer, 11 R. losea, 1 R. norvegicus and 1 R. rattus) were subjected to bacterial culture, and three L. borgpetersenii serogroup Javanica isolates were obtained from R. argentiventer (5.3%). WGS revealed that although Cambodian isolates were genetically related to L. borgpetersenii serogroup Javanica strains widely distributed in East and South-East Asian countries, they formed a different cluster from other strains. In addition to the three L. borgpetersenii sequences, the flaB sequence of L. interrogans was detected in 18 R. argentiventer and 7 R. losea kidney tissue samples (38.9%) using nested PCR followed by DNA sequencing. The L. interrogans flaB-positive samples were further analysed by MLST, revealing that seven housekeeping genes (glmU, pntA, sucA, tpiA, pfkB, mreA and caiB) contained novel sequences with distinct lineages from other sequence types. This study revealed a high prevalence of Leptospira spp. among rats exported from Cambodia to Vietnam, indicating a potential risk to people engaging in rat trade and demonstrating that a fastidious L. interrogans strain circulates among Cambodian rats.

Impact of modernization on urinary concentrations of arsenic, cadmium, lead, and selenium in rural residents of Northern Laos.

OBJECTIVES: The impacts of modernization on toxic heavy metal exposure and essential trace element intake in indigenous populations of subsistence societies are unknown. We assessed urinary trace element concentrations in rural residents of Northern Laos and examined associations with levels of modernization. METHODS: A cross-sectional study was conducted comprising 380 residents of three villages in Northern Laos with different levels of modernization. We surveyed general characteristics and measured the weight and height of 341 participants. Arsenic, cadmium, lead, and selenium concentrations were measured in spot urine samples by inductively coupled plasma mass spectrometry. We examined associations between urinary trace element concentrations and variables related to modernization (village, roofing material, possessions index [total number of possessions], and body mass index [BMI]) using multilevel analyses with household as a random effect, after adjusting for sex, age, and smoking status. RESULTS: Urinary concentrations of arsenic and cadmium were high, while those of lead and selenium were low in comparison to previous reports of populations in non-contaminated regions or without excess/deficiency. We observed associations between urinary trace element concentrations and village-level modernization: lead and selenium concentrations were higher in more modernized villages and cadmium concentration was highest in the least modernized village. Urinary arsenic concentration was not predicted by the modernization level of a village, although we observed significant differences among villages. In addition, urinary selenium concentration was higher in participants inhabiting more modernized houses. CONCLUSION: Modernization of villages may impact toxic heavy metal exposure and selenium intake in rural residents of Northern Laos.

The search for aliens within us: a review of evidence and theory regarding the foetal microbiome.

The microbiome is believed to be established during the birthing process through exposure to the maternal microbiome and immediate external environment. The absence of a microbiome prior to birth is based on the sterile womb hypothesis, which was formulated at the beginning of the 20th century and is supported primarily by the culture-based approach in microbiological studies.Findings of bacterial presence in products of fertilization such as the placenta, amniotic fluid, foetal membranes, and umbilical cord blood in studies using next-generation DNA sequencing technologies began to challenge the sterile nature of the intrauterine environment during gestation. These studies have been mainly criticized by their approach to contamination and inconclusive evidence of viability. The implications of bacterial presence in utero are far reaching in medicine and basic sciences. If commensal bacteria exist in the foetus, antibiotic therapies in pregnancy particularly for asymptomatic cases will need to be re-evaluated. Experimental studies utilizing gnotobiology may also be impacted by a realignment of theory.This review of existing literature aims to provide insight into the existence of bacteria in utero, specifically the foetal microbiome through analysis of experimental evidence and theoretical concepts, and to suggest approaches that may further provide clarity into this inquiry.

Dysregulation of the Intestinal Microbiome in Patients With Haploinsufficiency of A20.

INTRODUCTION: Haploinsufficiency of A20 (HA20) is a form of inborn errors of immunity (IEI). IEIs are genetically occurring diseases, some of which cause intestinal dysbiosis. Due to the dysregulation of regulatory T cells (Tregs) observed in patients with HA20, gut dysbiosis was associated with Tregs in intestinal lamina propria. METHODS: Stool samples were obtained from 16 patients with HA20 and 15 of their family members. Infant samples and/or samples with recent antibiotics use were excluded; hence, 26 samples from 13 patients and 13 family members were analyzed. The 16S sequencing process was conducted to assess the microbial composition of samples. Combined with clinical information, the relationship between the microbiome and the disease activity was statistically analyzed. RESULTS: The composition of gut microbiota in patients with HA20 was disturbed compared with that in healthy family members. Age, disease severity, and use of immunosuppressants corresponded to dysbiosis. However, other explanatory factors, such as abdominal symptoms and probiotic treatment, were not associated. The overall composition at the phylum level was stable, but some genera were significantly increased or decreased. Furthermore, among the seven operational taxonomic units (OTUs) that increased, two OTUs, Streptococcus mutans and Lactobacillus salivarius, considerably increased in patients with autoantibodies than those without autoantibodies. DISCUSSION: Detailed interaction on intestinal epithelium remains unknown; the relationship between the disease and stool composition change helps us understand the mechanism of an immunological reaction to microorganisms.

The influences of low protein diet on the intestinal microbiota of mice.

Recent research suggests that protein deficiency symptoms are influenced by the intestinal microbiota. We investigated the influence of low protein diet on composition of the intestinal microbiota through animal experiments. Specific pathogen-free (SPF) mice were fed one of four diets (3, 6, 9, or 12% protein) for 4 weeks (n = 5 per diet). Mice fed the 3% protein diet showed protein deficiency symptoms such as weight loss and low level of blood urea nitrogen concentration in their serum. The intestinal microbiota of mice in the 3% and 12% protein diet groups at day 0, 7, 14, 21 and 28 were investigated by 16S rRNA gene sequencing, which revealed differences in the microbiota. In the 3% protein diet group, a greater abundance of urease producing bacterial species was detected across the duration of the study. In the 12% diet protein group, increases of abundance of Streptococcaceae and Clostridiales families was detected. These results suggest that protein deficiency may be associated with shifts in intestinal microbiota.

Detection and phylogenetic analysis of phlebovirus, including severe fever with thrombocytopenia syndrome virus, in ticks collected from Tokyo, Japan.

Severe fever with thrombocytopenia syndrome (SFTS) was detected for the first time in China in 2011. Since then, human cases have been reported in endemic regions, including Japan. To investigate the presence of tick-borne pathogens in Tokyo, 551 ticks (266 samples) were collected from October 2015 to October 2016. Although the SFTS virus was not detected by RT-PCR, a novel phlebovirus was detected in one sample. In a phylogenetic analysis based on the partial nucleotide sequences of the L and S segments of the virus, the virus clustered with Lesvos virus (Greece), Yongjia tick virus, and Dabieshan tick virus (China). Further studies involving virus isolation are required to characterize this novel phlebovirus and to expand the epidemiological knowledge of related pathogens.

Transition of the intestinal microbiota of cats with age.

The transition of intestinal microbiota with age has been well described in humans. However, the age-related changes in intestinal microbiota of cats have not been well studied. In the present study, we investigated the composition of intestinal microbiota of cats in 5 different age groups (pre-weanling, weanling, young, aged, senile) with a culture-based method. For lactobacilli and bifidobacteria, we also quantified with molecular-based method, real-time PCR. The results suggested that the composition of the feline intestinal microbiota changes with age, while the changes were different from those of humans and dogs. Bifidobacteria which are predominant in human intestine or lactobacilli which are predominant in dog intestine, did not appear to be important in cat intestines. Enterococci, instead, seem to be major lactic acid producing bacteria in cats. We also identified lactobacilli and bifidobacteria at the species level based on 16S rRNA gene sequences and found that the species composition of Lactobacillus also changed with age.

Transition of the intestinal microbiota of dogs with age.

Although it is established that the composition of the human intestinal microbiota changes with age, transition of the intestinal microbiota of animals with age has not been well studied. In the present study, we collected fresh fecal samples from dogs of 5 different age groups (pre-weanling, weanling, young, aged, senile) and analyzed the compositions of their intestinal microbiota with a culture-based method. The results suggested that the composition of the canine intestinal microbiota also changes with age. Among intestinal bacteria predominant in dog intestines, lactobacilli appeared to change with age. Both the number and the prevalence of lactobacilli tended to decrease when dogs became older. Bifidobacteria, on the other hand, was not predominant in the intestine of the dogs. We also identified lactobacilli at the species level based on 16S rRNA gene sequences and found that the species composition of Lactobacillus also changed with age. It was further suggested that bacteria species beneficial to host animals may differ depending on the host species.