Search

Metagenomic next-generation sequencing (mNGS) is the process of sequencing all genetic material in a biological sample. The technique is growing in popularity with myriad applications including outbreak investigation, biosurveillance, and pathogen detection in clinical samples. However, mNGS programs are costly to build and maintain, and additional obstacles faced by low- and middle-income countries (LMICs) may further widen global inequities in mNGS capacity. Over the past two decades, several important infectious disease outbreaks have highlighted the importance of establishing widespread sequencing capacity to support rapid disease detection and containment at the source. Using lessons learned from the COVID-19 pandemic, LMICs can leverage current momentum to design and build sustainable mNGS programs, which would form part of a global surveillance network crucial to the elimination of infectious diseases.

The year 2019 witnessed the highest number of dengue cases ever reported globally. We analyzed epidemiological, serological, and phylogenomic data to investigate the drivers of the 2019 epidemic in Cambodia. Using epidemiological models fit to a 19-year national dataset, we identified an overall trend of declining annual force of infection (FOI) for dengue virus (DENV) in Cambodia, interspersed with FOI spikes corresponding to epidemic year caseloads that exceeded demographic predictions. We constructed time-resolved phylogenetic trees with 105 DENV genomes sequenced from the 2019 Cambodian epidemic, paired with historical Southeast Asian data, to document the first-recorded introduction of DENV-2 Cosmopolitan genotype into Cambodia. This introduction yielded highly localized transmission and decreased genomic diversity when compared to endemic DENV-1, supporting the hypothesis of epidemic invasion. Introduction of this genetically distinct lineage into a population with limited prior immunity—paired with a spike in FOI—was a key driver of the 2019 Cambodian epidemic.These studies were registered at clinicaltrials.gov under NCT04034264 and NCT03534245.

Mosquito-borne viruses are a growing global threat. Initial viral inoculation occurs in the skin via the mosquito ‘bite’, eliciting immune responses that shape the establishment of infection and pathogenesis. We aimed to cutaneous innate and adaptive immune responses to mosquito bites in individuals from endemic areas. In this single-arm, cross-sectional interventional study, we enrolled 30 healthy adult participants aged 18 to 45 years of age from Cambodia between October 2020 and January 2021. We performed 3-mm skin biopsies at baseline as well as 30 minutes, 4 hours, and 48 hours after a controlled feeding by uninfected Aedes aegypti mosquitos. The primary endpoints were measurement of changes in early and late innate responses in bitten vs unbitten skin by gene expression profiling, immunophenotyping, and cytokine profiling. Results revealed induction of neutrophil degranulation and recruitment of skin-resident dendritic cells and M2-macrophages in ‘bitten’ skin. As the immune reaction progressed over time, T cell priming and regulatory pathways were upregulated along with a shift to a Th2-driven response and CD8+ T cell activation. In accordance, participants’ bitten skin cells produced less pro-inflammatory cytokines when stimulated by Ae. aegypti salivary gland extract. No unexpected adverse events occurred, and one patient was lost to follow-up at Day 14. These results identify key immune genes, cell types, and pathways in the human response to mosquito bites that can be leveraged to develop novel therapeutics and vector-targeted vaccine candidates to arboviral diseases.Graphical AbstractPanel shows evolution of the human skin response to Aedes aegypti bites over time.

Significance Metagenomic pathogen sequencing offers an unbiased approach to characterizing febrile illness. In resource-scarce settings with high biodiversity, it is critical to identify disease-causing pathogens in order to understand burden and to prioritize efforts for control. Here, metagenomic next-generation sequencing (mNGS) characterization of the pathogen landscape in Cambodia revealed diverse vector-borne and zoonotic pathogens irrespective of age and gender as risk factors. Identification of key pathogens led to changes in national program surveillance. This study is a “real world” example of the use of mNGS surveillance of febrile individuals, executed in-country, to identify outbreaks of vector-borne, zoonotic, and other emerging pathogens in a resource-scarce setting.

Understanding the regional pathogen landscape and surveillance of emerging pathogens is key to mitigating epidemics. Challenges lie in resource-scarce settings, where outbreaks are likely to emerge, but where laboratory diagnostics and bioinformatics capacity are limited. Using unbiased metagenomic next generation sequencing (mNGS), we identified a variety of vector-borne, zoonotic and emerging pathogens responsible for undifferentiated fevers in a peri-urban population in Cambodia. From March 2019 to October 2020, we enrolled 473 febrile patients aged 6 months to 65 years of age presenting to a large peri-urban hospital in Cambodia. We collected sera and prepared sequencing libraries from extracted pathogen RNA for unbiased metagenomic sequencing and subsequent bioinformatic analysis on the global cloud-based platform, IDseq. We employed multivariate Bayesian models to evaluate specific pathogen risk causing undifferentiated febrile illness. mNGS identified vector-borne pathogens as the largest clinical category with dengue virus (124/489) as the most abundant pathogen.Underappreciated zoonotic pathogens such as Plasmodium knowlesi, leptospirosis, and co-infecting HIV were also detected. Early detection of chikungunya virus presaged a larger national outbreak of more than 6,000 cases. Pathogen-agnostic mNGS investigation of febrile persons in resource-scarce Southeast Asia is feasible and revealing of a diverse pathogen landscape. Coordinated and ongoing unbiased mNGS pathogen surveillance can better identify the breadth of endemic, zoonotic or emerging pathogens and deployment of rapid public health response.Clinical Trial NumbersNCT04034264 and NCT03534245.Significance StatementPublic health authorities recently advocated for global expansion of sequencing capacity worldwide; however, the importance of genomics-based surveillance to detect emerging pathogens or variants in resource-limited settings is paramount, especially in a populous, biodiverse Southeast Asia. From 2019 to 2020, pathogen metagenomic next generation sequencing (mNGS) of febrile patients in Cambodia identified several vector-borne and zoonotic pathogens, both common and underappreciated, and resulted in a variety of actionable health interventions. Understanding these pathogen discoveries, and the attendant challenges of mNGS in these outbreak-prone settings, is critical for today’s global society and decision-makers in order to implement sequencing-based pathogen or variant detection.Significance StatementMetagenomic pathogen sequencing offers an unbiased approach to characterizing febrile illness. In resource-scarce settings with high biodiversity, it is critical to identify disease-causing pathogens in order to understand burden and to prioritize efforts for control. Here, mNGS characterization of the pathogen landscape in Cambodia revealed diverse vector-borne and zoonotic pathogens irrespective of age and gender as risk factors. Identification of key pathogens led to changes in national program surveillance. This study provides a recent ‘real world’ example for the use of mNGS surveillance in both identifying diverse microbial landscapes and detecting outbreaks of vector-borne, zoonotic, and other emerging pathogens in resource-scarce settings.ClassificationBiological Sciences; microbiology; medical sciences

Greater Mekong inhabitants are exposed to pathogens, zoonotic and otherwise, that may influence SARS-CoV-2 seroreactivity. A pre-pandemic (2005 to 2011) serosurvey of from 528 malaria-experienced Cambodians demonstrated higher-than-expected (up to 13.8 %) positivity of non-neutralizing IgG to SARS-CoV-2 spike and RBD antigens. These findings have implications for interpreting large-scale serosurveys., Article Summary Line: In the pre-COVID19 pandemic years of 2005 to 2011, malaria-experienced Cambodians from rural settings had higher-than-expected seroreactivity to SARS-CoV-2 spike and receptor binding domain proteins.

Background We established the first prospective cohort to understand how infection with dengue virus is influenced by vector-specific determinants such as humoral immunity to Aedes aegypti salivary proteins. Methods Children aged 2–9 years were enrolled in the PAGODAS (Pediatric Assessment Group of Dengue and Aedes Saliva) cohort with informed consent by their guardians. Children were followed semi-annually for antibodies to dengue and to proteins in Ae. aegypti salivary gland homogenate using enzyme-linked immunosorbent assays and dengue-specific neutralization titers. Children presented with fever at any time for dengue testing. Results From 13 July to 30 August 2018, we enrolled 771 children. At baseline, 22% (173/770) had evidence of neutralizing antibodies to 1 or more dengue serotypes. By April 2020, 51 children had symptomatic dengue while 148 dengue-naive children had inapparent dengue defined by neutralization assays. In a multivariate model, individuals with higher antibodies to Ae. aegypti salivary proteins were 1.5 times more likely to have dengue infection (hazard ratio [HR], 1.47 [95% confidence interval {CI}, 1.05–2.06]; P = .02), particularly individuals with inapparent dengue (HR, 1.64 [95% CI, 1.12–2.41]; P = .01). Conclusions High levels of seropositivity to Ae. aegypti salivary proteins are associated with future development of dengue infection, primarily inapparent, in dengue-naive Cambodian children. Clinical Trials Registration NCT03534245

Rapid production and publication of pathogen genome sequences during emerging disease outbreaks provide crucial public health information. In resource-limited settings, especially near an outbreak epicenter, conventional deep sequencing or bioinformatics are often challenging. Here we successfully used metagenomic next generation sequencing on an iSeq100 Illumina platform paired with an open-source bioinformatics pipeline to quickly characterize Cambodia’s first case of COVID-2019.

BackgroundMetagenomic next generation sequencing (mNGS) has enabled the rapid, unbiased detection and identification of microbes without pathogen-specific reagents, culturing, or a priori knowledge of the microbial landscape. mNGS data analysis requires a series of computationally intensive processing steps to accurately determine the microbial composition of a sample. Existing mNGS data analysis tools typically require bioinformatics expertise and access to local server-class hardware resources. For many research laboratories, this presents an obstacle, especially in resource limited environments.FindingsWe present IDseq, an open source cloud-based metagenomics pipeline and service for global pathogen detection and monitoring (https://idseq.net). The IDseq Portal accepts raw mNGS data, performs host and quality filtration steps, then executes an assembly-based alignment pipeline which results in the assignment of reads and contigs to taxonomic categories. The taxonomic relative abundances are reported and visualized in an easy-to-use web application to facilitate data interpretation and hypothesis generation. Furthermore, IDseq supports environmental background model generation and automatic internal spike-in control recognition, providing statistics which are critical for data interpretation. IDseq was designed with the specific intent of detecting novel pathogens. Here, we benchmark novel virus detection capability using both synthetically evolved viral sequences, and real-world samples, including IDseq analysis of a nasopharyngeal swab sample acquired and processed locally in Cambodia from a tourist from Wuhan, China, infected with the recently emergent SARS-CoV-2.ConclusionThe IDseq Portal reduces the barrier to entry for mNGS data analysis and enables bench scientists, clinicians, and bioinformaticians to gain insight from mNGS datasets for both known and novel pathogens.

Nematode infection upregulates interleukin-4 (IL-4) and IL-13 and induces STAT6-dependent changes in gut function that promote worm clearance. IL-4 and IL-13 activate the type 2 IL-4 receptor (IL-4R), which contains the IL-13Rα1 and IL-4Rα chains. We used mice deficient in IL-13Rα1 (IL-13Rα1 −/− ) to examine the contribution of IL-13 acting at the type 2 IL-4R to immune and functional responses to primary (Hb1) and secondary (Hb2) infections with the gastrointestinal nematode parasite Heligmosomoides bakeri . There were differences between strains in the IL-4 and IL-13 expression responses to Hb1 but not Hb2 infection. Following Hb2 infection, deficient mice had impaired worm expulsion and higher worm fecundity despite normal production of Th2-derived cytokines. The upregulation of IL-25 and IL-13Rα2 in Hb1- and Hb2-infected wild-type (WT) mice was absent in IL-13Rα1 −/− mice. Goblet cell numbers and resistin-like molecule beta (RELM-β) expression were attenuated significantly in IL-13Rα1 −/− mice following Hb2 infections. IL-13Rα1 contributes to the development of alternatively activated macrophages, but the type 1 IL-4R is also important. Hb1 infection had no effects on smooth muscle function or epithelial permeability in either strain, while the enhanced mucosal permeability and changes in smooth muscle function and morphology observed in response to Hb2 infection in WT mice were absent in IL-13Rα1 −/− mice. Notably, the contribution of claudin-2, which has been linked to IL-13, does not mediate the increased mucosal permeability following Hb2 infection. These results show that activation of IL-13Rα1 is critical for key aspects of the immune and functional responses to Hb2 infection that facilitate expulsion.

Obesity is associated with a chronic low-grade inflammation characterized by increased levels of proinflammatory cytokines that are implicated in disrupted metabolic homeostasis. Parasitic nematode infection induces a polarized Th2 cytokine response and has been explored to treat autoimmune diseases. We investigated the effects of nematode infection against obesity and the associated metabolic dysfunction. Infection of RIP2-Opa1KO mice or C57BL/6 mice fed a high-fat diet (HFD) with Nippostrongylus brasiliensis decreased weight gain and was associated with improved glucose metabolism. Infection of obese mice fed the HFD reduced body weight and adipose tissue mass, ameliorated hepatic steatosis associated with a decreased expression of key lipogenic enzymes/mediators, and improved glucose metabolism, accompanied by changes in the profile of metabolic hormones. The infection resulted in a phenotypic change in adipose tissue macrophages that was characterized by upregulation of alternative activation markers. Interleukin-13 (IL-13) activation of the STAT6 signaling pathway was required for the infection-induced attenuation of steatosis but not for improved glucose metabolism, whereas weight loss was attributed to both IL-13/STAT6-dependent and -independent mechanisms. Parasitic nematode infection has both preventive and therapeutic effects against the development of obesity and associated features of metabolic dysfunction in mice.

SerpinB2, a member of the serine protease inhibitor family, is expressed by macrophages and is significantly upregulated by inflammation. Recent studies implicated a role for SerpinB2 in the control of Th1 and Th2 immune responses, but the mechanisms of these effects are unknown. In this study, we used mice deficient in SerpinB2 (SerpinB2−/−) to investigate its role in the host response to the enteric nematode, Heligmosomoides bakeri. Nematode infection induced a STAT6-dependent increase in intestinal SerpinB2 expression. The H. bakeri–induced upregulation of IL-4 and IL-13 expression was attenuated in SerpinB2−/− mice coincident with an impaired worm clearance. In addition, lack of SerpinB2 in mice resulted in a loss of the H. bakeri–induced smooth muscle hypercontractility and a significant delay in infection-induced increase in mucosal permeability. Th2 immunity is generally linked to a CCL2-mediated increase in the infiltration of macrophages that develop into the alternatively activated phenotype (M2). In H. bakeri–infected SerpinB2−/− mice, there was an impaired infiltration and alternative activation of macrophages accompanied by a decrease in the intestinal CCL2 expression. Studies in macrophages isolated from SerpinB2−/− mice showed a reduced CCL2 expression, but normal M2 development, in response to stimulation of Th2 cytokines. These data demonstrate that the immune regulation of SerpinB2 expression plays a critical role in the development of Th2-mediated protective immunity against nematode infection by a mechanism involving CCL2 production and macrophage infiltration.

Utilizing a conditional (intestinal-specific) knockout (cKO) mouse model, we have recently shown that the sodium-dependent multivitamin transporter (SMVT) ( SLC5A6) is the only biotin uptake system that operates in the gut and that its deletion leads to biotin deficiency. Unexpectedly, we also observed that all SMVT-cKO mice develop chronic active inflammation, especially in the cecum. Our aim here was to examine the role of SMVT in the maintenance of intestinal mucosal integrity [permeability and expression of tight junction (TJ) proteins]. Our results showed that knocking out the mouse intestinal SMVT is associated with a significant increase in gut permeability and with changes in the level of expression of TJ proteins. To determine whether these changes are related to the state of biotin deficiency that develops in SMVT-cKO mice, we induced (by dietary means) biotin deficiency in wild-type mice and examined its effect on the above-mentioned parameters. The results showed that dietary-induced biotin deficiency leads to a similar development of chronic active inflammation in the cecum with an increase in the level of expression of proinflammatory cytokines, as well as an increase in intestinal permeability and changes in the level of expression of TJ proteins. We also examined the effect of chronic biotin deficiency on permeability and expression of TJ proteins in confluent intestinal epithelial Caco-2 monolayers but observed no changes in these parameters. These results show that the intestinal SMVT plays an important role in the maintenance of normal mucosal integrity, most likely via its role in providing biotin to different cells of the gut mucosa.

In this study, nonhuman primates (NHPs) exposed to lethal doses of total body irradiation (TBI) within the gastrointestinal (GI) acute radiation syndrome range, sparing ∼5% of bone marrow (TBI-BM5), were used to evaluate the mechanisms involved in development of the chronic GI syndrome. TBI increased mucosal permeability in the jejunum (12-14 Gy) and proximal colon (13-14 Gy). TBI-BM5 also impaired mucosal barrier function at doses ranging from 10-12.5 Gy in both small intestine and colon. Timed necropsies of NHPs at 6-180 days after 10 Gy TBI-BM5 showed that changes in small intestine preceded those in the colon. Chronic GI syndrome in NHPs is characterized by continued weight loss and intermittent GI syndrome symptoms. There was a long-lasting decrease in jejunal glucose absorption coincident with reduced expression of the sodium-linked glucose transporter. The small intestine and colon showed a modest upregulation of several different pro-inflammatory mediators such as NOS-2. The persistent inflammation in the post-TBI-BM5 period was associated with a long-lasting impairment of mucosal restitution and a reduced expression of intestinal and serum levels of alkaline phosphatase (ALP). Mucosal healing in the postirradiation period is dependent on sparing of stem cell crypts and maturation of crypt cells into appropriate phenotypes. At 30 days after 10 Gy TBI-BM5, there was a significant downregulation in the gene and protein expression of the stem cell marker Lgr5 but no change in the gene expression of enterocyte or enteroendocrine lineage markers. These data indicate that even a threshold dose of 10 Gy TBI-BM5 induces a persistent impairment of both mucosal barrier function and restitution in the GI tract and that ALP may serve as a biomarker for these events. These findings have important therapeutic implications for the design of medical countermeasures.

Protective immunity against enteric parasitic nematodes is dependent on IL-4, IL-13 activation of their exclusive transcription factor STAT6. The precise pathways by which enteric parasitic nematodes are recognized by the host is unclear, but elimination of this important interaction in developed nations is thought to contribute to the dysregulated immune responses that are a characteristic of autoimmune diseases. Nematode-derived products are involved in evading host defenses to promote their life cycle leading to modulation of host immune responses. Host protective immunity has adapted to enteric parasitic nematode infection by elaboration of mucins, increasing intraluminal fluid to control access to the surface epithelium, increasing cell turnover to maintain an effective barrier to their invasion, initiating immune responses through activation of resident immune cells, and recruitment of additional immune cells to release immune mediators that help orchestrate these responses. Both the immune and functional outcomes depend largely on IL-4/IL-13 signaling through STAT6, with a dominant role for IL-13 working through the type 2 IL-4 receptor (IL-4R). The recent observation that enteric nematode infection prevents the onset of a number of experimental models of IBD, diabetes, and several extraintestinal autoimmune diseases including multiple sclerosis has generated considerable interest in the identification of worm/egg products involved in the generation and maintenance of Th2 cytokines that may mediate the beneficial effects of nematode infection in autoimmune and inflammatory pathologies.

Parasitic enteric nematodes induce a type 2 immune response characterized by increased production of Th2 cytokines, IL-4 and IL-13, and recruitment of alternatively activated macrophages (M2) to the site of infection. Nematode infection is associated with changes in epithelial permeability and inhibition of sodium-linked glucose absorption, but the role of M2 in these effects is unknown. Clodronate-containing liposomes were administered prior to and during nematode infection to deplete macrophages and prevent the development of M2 in response to infection with Nippostrongylus brasiliensis. The inhibition of epithelial glucose absorption that is associated with nematode infection involved a macrophage-dependent reduction in SGLT1 activity, with no change in receptor expression, and a macrophage-independent down-regulation of GLUT2 expression. The reduced transport of glucose into the enterocyte is compensated partially by an up-regulation of the constitutive GLUT1 transporter consistent with stress-induced activation of HIF-1α. Thus, nematode infection results in a "lean" epithelial phenotype that features decreased SGLT1 activity, decreased expression of GLUT2 and an emergent dependence on GLUT1 for glucose uptake into the enterocyte. Macrophages do not play a role in enteric nematode infection-induced changes in epithelial barrier function. There is a greater contribution, however, of paracellular absorption of glucose to supply the energy demands of host resistance. These data provide further evidence of the ability of macrophages to alter glucose metabolism of neighboring cells.

IL-33 is a recently identified cytokine member of the IL-1 family. The biological activities of IL-33 are associated with promotion of Th2 and inhibition of Th1/Th17 immune responses. Exogenous IL-33 induces a typical “type 2” immune response in the gastrointestinal tract, yet the underlying mechanisms remain to be fully elucidated. In addition, the role of IL-33 in the regulation of gastrointestinal function is not known. The present study investigated IL-33-dependent intestinal immunity and function in mice. Exogenous IL-33 induced a polarized type 2 cytokine response in the intestine that was entirely MyD88 dependent but STAT6 and IL-13 independent. Mice injected with recombinant IL-33 exhibited intestinal smooth muscle hypercontractility, decreased epithelial responses to acetylcholine and glucose, and increased mucosal permeability. IL-33 effects on intestinal epithelial function were STAT6 dependent, and both IL-4 and IL-13 appeared to play a role. The effects on smooth muscle function, however, were attributable to both STAT6-dependent and -independent mechanisms. In addition, IL-13 induction of insulin-like growth factor-1 was implicated in IL-33-induced smooth muscle hypertrophy. Finally, alternative activation of macrophages induced by IL-33 revealed a novel pathway that is IL-4, IL-13, and STAT6 independent. Thus manipulating IL-33 or related signaling pathways represents a potential therapeutic strategy for treating inflammatory diseases associated with dysregulated intestinal function.

Type 2 immunity is essential for host protection against nematode infection but is detrimental in allergic inflammation or asthma. There is a major research focus on the effector molecules and specific cell types involved in the initiation of type 2 immunity. Recent work has implicated an important role of epithelial-derived cytokines, IL-25 and IL-33, acting on innate immune cells that are believed to be the initial sources of type 2 cytokines IL-4/IL-5/IL-13. The identities of the cell types that mediate the effects of IL-25/IL-33, however, remain to be fully elucidated. In the present study, we demonstrate that macrophages as IL-25/IL-33-responsive cells play an important role in inducing type 2 immunity using both in vitro and in vivo approaches. Macrophages produced type 2 cytokines IL-5 and IL-13 in response to the stimulation of IL-25/IL-33 in vitro, or were the IL-13-producing cells in mice administrated with exogenous IL-33 or infected with Heligmosomoides bakeri. In addition, IL-33 induced alternative activation of macrophages primarily through autocrine IL-13 activating the IL-4Rα-STAT6 pathway. Moreover, depletion of macrophages attenuated the IL-25/IL-33-induced type 2 immunity in mice, while adoptive transfer of IL-33-activated macrophages into mice with a chronic Heligmosomoides bakeri infection induced worm expulsion accompanied by a potent type 2 protective immune response. Thus, macrophages represent a unique population of the innate immune cells pivotal to type 2 immunity and a potential therapeutic target in controlling type 2 immunity-mediated inflammatory pathologies.