Localization and RNA Interference-Driven Inhibition of a Brugia malayi-Encoded Interleukin-5 Receptor Binding Protein.

A molecule we termed Brugia malayi IL-5 receptor (IL-5R) binding protein (BmIL5Rbp; also known as Bm8757) was identified from B. malayi filarial worms and found to inhibit human interleukin-5 (IL-5) binding to its human receptor competitively. After the expression and purification of a recombinant BmIL5Rbp and generation of BmIL5Rbp-specific rabbit antibody, we localized the molecule on B. malayi worms through immunohistochemistry and immunoelectron microscopy. RNA interference (RNAi) was used to inhibit BmIL5Rbp mRNA and protein production. BmIL5Rbp was shown to localize to the cuticle of Brugia malayi and to be released in its excretory/secretory products. RNAi inhibited BmIL5Rbp mRNA production by 33%, reduced the surface protein expression by ~50%, and suppressed the release of BmIL5Rbp in the excretory/secretory products. RNAi has been used successfully to knock down the mRNA and protein expression of BmIL5Rbp in the early larval stages of B. malayi and provided a proof of principle for the local inhibition of the human IL-5R. These findings provide evidence that a parasite-encoded IL-5R antagonist may locally inhibit a vital host innate immune activation of IL-5 on eosinophils.

Strongyloides stercoralis Infection in Solid Organ Transplant Patients Is Associated With Eosinophil Activation and Intestinal Inflammation: A Cross-sectional Study.

BACKGROUND: Strongyloidiasis can cause devastating morbidity and death in immunosuppressed patients. Identification of reliable biomarkers for strongyloidiasis in immunosuppressed patients is critical for the prevention of severe disease. METHODS: In this cross-sectional study of solid organ transplant (SOT) candidates and recipients, we quantified Strongyloides-specific IgG to the recombinant NIE-Strongyloides antigen and/or to a soluble extract of S. stercoralis somatic antigens ("crude antigen") using enzyme-linked immunosorbent assays (ELISAs). We also measured peripheral eosinophilia, 4 different eosinophil granule proteins, and intestinal fatty acid-binding protein (IFABP). RESULTS: We evaluated serum biomarkers in 149 individuals; 77 (52%) pre-SOT and 72 (48%) post-SOT. Four percent (6/149) tested positive by NIE ELISA and 9.6% (11/114) by crude antigen ELISA (overall seropositivity of 9.4% [14/149]). Seropositive patients had higher absolute eosinophil counts (AECs) than seronegative patients (P = .004). AEC was positively correlated to the levels of eosinophil granule proteins eosinophil cationic protein (ECP) and eosinophil peroxidase (EPO) (P < .05), while IFABP was positively related to the 2 other eosinophil granule proteins (major basic protein [MBP] and eosinophil-derived neurotoxin [EDN]; Spearman's r = 0.3090 and 0.3778, respectively; P < .05; multivariate analyses slopes = 0.70 and 2.83, respectively). CONCLUSIONS: This study suggests that, in SOT patients, strongyloidiasis triggers both eosinophilia and eosinophil activation, the latter being associated with intestinal inflammation. These data provide insight into the pathogenesis of S. stercoralis infection in the immunocompromised population at high risk of severe strongyloidiasis syndromes.

Schistosomiasis Induces Persistent DNA Methylation and Tuberculosis-Specific Immune Changes.

Epigenetic mechanisms, such as DNA methylation, determine immune cell phenotype. To understand the epigenetic alterations induced by helminth coinfections, we evaluated the longitudinal effect of ascariasis and schistosomiasis infection on CD4+ T cell DNA methylation and the downstream tuberculosis (TB)-specific and bacillus Calmette-Guérin-induced immune phenotype. All experiments were performed on human primary immune cells from a longitudinal cohort of recently TB-exposed children. Compared with age-matched uninfected controls, children with active Schistosoma haematobium and Ascaris lumbricoides infection had 751 differentially DNA-methylated genes, with 72% hypermethylated. Gene ontology pathway analysis identified inhibition of IFN-γ signaling, cellular proliferation, and the Th1 pathway. Targeted real-time quantitative PCR after methyl-specific endonuclease digestion confirmed DNA hypermethylation of the transcription factors BATF3, ID2, STAT5A, IRF5, PPARg, RUNX2, IRF4, and NFATC1 and cytokines or cytokine receptors IFNGR1, TNFS11, RELT (TNF receptor), IL12RB2, and IL12B (p < 0.001; Sidak-Bonferroni). Functional blockage of the IFN-γ signaling pathway was confirmed, with helminth-infected individuals having decreased upregulation of IFN-γ-inducible genes (Mann-Whitney p < 0.05). Hypomethylation of the IL-4 pathway and DNA hypermethylation of the Th1 pathway was confirmed by Ag-specific multidimensional flow cytometry demonstrating decreased TB-specific IFN-γ and TNF and increased IL-4 production by CD4+ T cells (Wilcoxon signed-rank p < 0.05). In S. haematobium-infected individuals, these DNA methylation and immune phenotypic changes persisted at least 6 mo after successful deworming. This work demonstrates that helminth infection induces DNA methylation and immune perturbations that inhibit TB-specific immune control and that the duration of these changes are helminth specific.

Parasitic infections represent a significant health threat among recent immigrants in Chicago.

Parasitic infections are likely under-recognized among immigrant populations in the USA. We conducted a cross-sectional study to evaluate if such infections have health impacts among recent immigrants in Chicago and to identify predictive factors for parasitic infections. A total of 133 recent immigrants were enrolled, filling out a standardized medical questionnaire and providing blood and stool samples. Appriximately 12% of subjects (15/125) who provided a blood or stool sample for testing were found to have evidence of current or prior infection with a pathogenic parasite, of which Toxocara spp. (8 subjects, 6.4%) and Strongyloides stercoralis (5 subjects, 4%) were most commonly identified. Parasitic infection was more likely among subjects who had immigrated within the previous 2 years and those with a self-reported history of worms in the stool. The most useful surrogate markers identified for parasitic infections were an elevated immunoglobulin E level (seen in 46.7% (7/15) of subjects with parasitic infections and 20% (22/110) of uninfected individuals, p = 0.04) and the presence of Blastocystis hominis cysts on Ova & Parasite exam (detected in 38.5% (5/13) of subjects with parasitic infections who provided a stool sample and 5.1% (5/98) of uninfected subjects, p = 0.002). Our study found that parasitic infections may be common in recent US immigrants, which highlights an important health disparity among a vulnerable population that merits further study. Additionally, clinical risk factors, symptoms, and laboratory findings traditionally thought to be associated with parasites were commonly found but not predictive of infection in this study population.

Multiplexed Recombinase Polymerase Amplification Assay To Detect Intestinal Protozoa.

This work describes a proof-of-concept multiplex recombinase polymerase amplification (RPA) assay with lateral flow readout that is capable of simultaneously detecting and differentiating DNA from any of the diarrhea-causing protozoa Giardia, Cryptosporidium, and Entamoeba. Together, these parasites contribute significantly to the global burden of diarrheal illness. Differential diagnosis of these parasites is traditionally accomplished via stool microscopy. However, microscopy is insensitive and can miss up to half of all cases. DNA-based diagnostics such as polymerase chain reaction (PCR) are far more sensitive; however, they rely on expensive thermal cycling equipment, limiting their availability to centralized reference laboratories. Isothermal DNA amplification platforms, such as the RPA platform used in this study, alleviate the need for thermal cycling equipment and have the potential to broaden access to more sensitive diagnostics. Until now, multiplex RPA assays have not been developed that are capable of simultaneously detecting and differentiating infections caused by different pathogens. We developed a multiplex RPA assay to detect the presence of DNA from Giardia, Cryptosporidium, and Entamoeba. The multiplex assay was characterized using synthetic DNA, where the limits-of-detection were calculated to be 403, 425, and 368 gene copies per reaction of the synthetic Giardia, Cryptosporidium, and Entamoeba targets, respectively (roughly 1.5 orders of magnitude higher than for the same targets in a singleplex RPA assay). The multiplex assay was also characterized using DNA extracted from live parasites spiked into stool samples where the limits-of-detection were calculated to be 444, 6, and 9 parasites per reaction for Giardia, Cryptosporidium, and Entamoeba parasites, respectively. This proof-of-concept assay may be reconfigured to detect a wide variety of targets by re-designing the primer and probe sequences.

Molecular detection of intestinal parasites in a rural community of Colombia: A one health approach to explore potential environmental-zoonotic transmission.

AIMS: Protozoan and helminth parasitic infections pose significant public health challenges, especially in developing countries with rural populations marked by suboptimal hygiene practices and socio-economic constraints. The parasites are the etiological agents of these infections and have a notably elevated global prevalence. Therefore, this study focuses on estimating the frequency and transmission dynamics of several parasitic species, including Blastocystis, Giardia, Cryptosporidium spp., Entamoeba histolytica, Ascaris lumbricoides, Trichuris trichiura, Taenia spp. and hookworms, within a rural community in southwest Colombia with a particular emphasis on the One Health framework, considering environmental and zoonotic transmission potentials. METHODS AND RESULTS: This study involved the analysis of 125 samples, encompassing human participants (n = 99), their domestic pets (dogs) (n = 24) and water sources (n = 2). Parasite detection was carried out utilizing a combination of microscopy and molecular techniques. Furthermore, the characterization of Blastocystis subtypes (STs) was achieved through Oxford Nanopore sequencing of the rRNA-18S gene. The investigation also entailed the examination of potential associations between intestinal parasitism and various sociodemographic factors. Results revealed a high frequency of parasitic infections when employing molecular methods, with Blastocystis (n = 109/87%), Giardia (n = 20/16%), Ancylostoma duodenale (n = 28/22%), Ancylostoma ceylanicum (n = 7/5.6%), E. histolytica (n = 6/4.8%), Cryptosporidium spp. (n = 12/9.6%) and even Taenia (n = 1/0.8%) detected. Cryptosporidium spp. was also identified in water samples. Coinfections were prevalent, with 57% (n = 70) of samples exhibiting single-parasite infections and 43% (n = 53) showing various degrees of polyparasitism, emphasizing the complexity of transmission dynamics. Blastocystis subtyping, conducted via Oxford Nanopore sequencing, revealed a diversity of subtypes and coexistence patterns, with ST2 being the most prevalent. CONCLUSIONS: This research underscores the importance of using molecular techniques for frequency estimation, particularly emphasizing the relevance of zoonotic transmission in parasitic infections. It highlights the significance of the One Health approach in comprehending the circulation of parasites among animals, humans and environmental sources, thereby directly impacting public health and epidemiological surveillance.

Environmental Detection of Parasites in the Marginalized Paiute Reservations Compared to a Nearby Area.

The amounts of parasite DNA in soil samples from different playgrounds and other public areas can help identify areas of possible microbe transmission and give indications of the possible occurrence of parasite infection in nearby communities. We collected 207 soil samples from parks in Paiute indigenous tribal areas in southwestern Utah and from the higher income city of St. George, Utah, and tested them for 11 parasites that can cause human disease. Molecular tests revealed an elevated odds ratio (OR) of 3.072 (range, 1.114-8.065) for detecting the helminth Trichuris trichiura and an elevated OR of 3.036 (range, 1.101-7.966) for any protozoa (not including Acanthamoeba) in the tribal land playgrounds compared with St. George parks. These findings support previous studies showing that areas in lower socioeconomic communities, especially marginalized communities, tend to have more parasites in the soil, which may lead to higher disease prevalence rates.

Comparison of multi-parallel quantitative real-time PCRs targeting different DNA regions and detecting soil-transmitted helminths in stool.

BACKGROUND: Soil-transmitted helminths infect an estimated 18% of the world's population, causing a significant health burden. Microscopy has been the primary tool for diagnosing eggs from fecal samples, but its sensitivity drops in low-prevalence settings. Quantitative real-time polymerase chain reaction (qPCR) is slowly increasing in research and clinical settings. However, there is still no consensus on preferred qPCR targets. METHODS: We aimed to compare soil-transmitted helminth (STH) DNA detection methods by testing naïve stool samples spiked with known quantities of STH eggs and larvae. DNA extracts from spiked samples were tested using independent quantitative realtime PCR (qPCR) assays targeting ribosomal or putative non-protein coding satellite sequences. RESULTS: For Trichuris trichiura, there was a strong correlation between egg/larvae counts and qPCR results using either qPCR method (0.86 and 0.87, respectively). Strong correlations also existed for A. lumbricoides (0.60 and 0.63, respectively), but weaker correlations were found for Ancylostoma duodenale (0.41 for both assays) and Strongyloides stercoralis (0.48 and 0.65, respectively). No correlation for Necator americanus was observed when testing with either qPCR assay. Both assays had fair-to-moderate agreement across targets when using field-collected stool samples (0.28-0.45, for all STHs), except for S. stercoralis (0.12) with slight agreement. CONCLUSIONS: There is a strong correlation between qPCR results and egg/larvae counts. Our study confirms that qPCR is an effective diagnostic tool, even with low-intensity infections, regardless of the DNA-based diagnostic marker used. However, the moderate agreement between the two different qPCR assays when testing field samples highlights the need to understand the role of these targets in the genome so that the parasite burden can be quantified more accurately and consistently by qPCR.

Parasitic Contamination of Soil in the Southern United States.

Parasites are generally associated with lower income countries in tropical and subtropical areas. Still, they are also prevalent in low-income communities in the southern United States. Studies characterizing the epidemiology of parasites in the United States are limited, resulting in little comprehensive understanding of the problem. This study investigated the environmental contamination of parasites in the southern United States by determining each parasite's contamination rate and burden in five low-income communities. A total of 499 soil samples of approximately 50 g were collected from public parks and private residences in Alabama, Louisiana, Mississippi, South Carolina, and Texas. A technique using parasite floatation, filtration, and bead-beating was applied to dirt samples to concentrate and extract parasite DNA from samples and detected via multiparallel quantitative polymerase chain reaction (qPCR). qPCR detected total sample contamination of Blastocystis spp. (19.03%), Toxocara cati (6.01%), Toxocara canis (3.61%), Strongyloides stercoralis (2.00%), Trichuris trichiura (1.80%), Ancylostoma duodenale (1.42%), Giardia intestinalis (1.40%), Cryptosporidium spp. (1.01%), Entamoeba histolytica (0.20%), and Necator americanus (0.20%). The remaining samples had no parasitic contamination. Overall parasite contamination rates varied significantly between communities: western Mississippi (46.88%), southwestern Alabama (39.62%), northeastern Louisiana (27.93%), southwestern South Carolina (27.93%), and south Texas (6.93%) (P <0.0001). T. cati DNA burdens were more significant in communities with higher poverty rates, including northeastern Louisiana (50.57%) and western Mississippi (49.60%) compared with southwestern Alabama (30.05%) and southwestern South Carolina (25.01%) (P = 0.0011). This study demonstrates the environmental contamination of parasites and their relationship with high poverty rates in communities in the southern United States.

Evaluation of a novel Tc-24 recombinant antigen ELISA for serologic testing for Trypanosoma cruzi in dogs.

Chagas disease is a parasitic infection caused by Trypanosoma cruzi. Diagnosis of chronic Chagas disease in dogs relies on limited serological test options. This study used a new Tc-24 recombinant antigen ELISA on an archival set of 70 dog serum samples from multi-dog kennel environments in Texas subjected to three existing Chagas serological tests. Tc-24 ELISA produced a quantitative result and could detect anti-T. cruzi antibodies in dogs with high sensitivity and specificity. Comparing individual tests to Tc-24 ELISA resulted in strong associations and correlations, which suggest that Tc-24 ELISA is a reliable and accurate diagnostic tool for dogs with a single test.

Detection of soil-transmitted helminths and Schistosoma spp. by nucleic acid amplification test: Results of the first 5 years of the only international external quality assessment scheme.

BACKGROUND: Infections with soil-transmitted helminths (STH) and schistosomiasis (SCH) result in a significant global health burden, particularly in rural communities in low and middle-income countries. While microscopy remains the primary diagnostic method for STH and SCH in resource-limited settings, nucleic acid amplification tests (NAATs) are gaining prominence as tools for evaluation of public health control programs in endemic countries, and individual diagnosis in high-income countries. Despite the high sensitivity and specificity of NAATs, previous research has highlighted inter-laboratory variations, both in technical and clinical performance, justifying the need for continuous proficiency testing. METHODOLOGY: Results from 5 rounds over a 5-year period of the so far only longitudinal international Helminth External Molecular Quality Assessment Scheme (HEMQAS), coordinated by the Dutch Foundation for Quality Assessment in Medical Laboratories (SKML), were examined in order to (i) assess the diagnostic proficiency of laboratories in detecting helminths in stool and (ii) identify potential factors contributing to variations in performance. OUTCOME AND CONCLUSIONS: Thirty-six laboratories, from 18 countries and 5 continents, participated in HEMQAS. The overall diagnostic performances were satisfying, with remarkably low numbers (<2%) of false-positive results. False-negative results were more often reported for stool (15%) than for DNA (5%) samples. False-negative results varied largely between targets (the highest number (29%) for Trichuris trichiura). Twenty-five laboratories provided a sufficient number of results for a robust comparison between participating laboratories, which confirmed substantial inter-laboratory variability in quantitative NAAT results (Cq-values). This variability likely arises from differences in pre-treatment, DNA isolation and DNA-target amplification procedures. This study emphasizes the complexity of molecular diagnosis for STH and SCH, highlighting the critical role of proper stool preparation and DNA isolation methods. The results underscore the necessity for laboratory professionals and public health decision-makers to recognize these complexities and continuously undertake external quality assessment schemes to ensure accurate and reliable performance in molecular diagnosis.

Geographic Distribution of Human Infections with Zoonotic Ancylostoma ceylanicum and Anthropophilic Hookworms in Ecuador: A Retrospective Analysis of Archived Stool Samples.

Zoonotic human infections with Ancylostoma ceylanicum have recently been reported in the Americas. We used archived human stool samples to study the geographic distribution of human infections with A. ceylanicum and anthropophilic hookworms in different geoclimatic regions (coastal, Andean, and Amazon) of Ecuador. We analyzed retrospectively archived human stool samples from five studies previously screened for hookworm infection by microscopy, of which four included hookworm-positive samples only and one involved hookworm-negative samples to increase geographic distribution of sampling. Stools were analyzed using multi-parallel quantitative polymerase chain reaction (qPCR) assays to detect Necator americanus, Ancylostoma duodenale, A. ceylanicum, Ascaris lumbricoides, Trichuris trichiura, and Strongyloides stercoralis. Sequencing was done for the A. ceylanicum cox1 gene. A total of 132 samples were analyzed, of which 69 (52.3%) were from hookworm-positive and 63 (47.7%) from hookworm-negative individuals by microscopy. Overall, 82.6% of microscopy-positive samples and 33.3% of microscopy-negative samples were positive for hookworm by qPCR. Of microscopy-positive samples, 36.2% were A. ceylanicum, 37.7% A. duodenale, and 33.3% N. americanus, whereas equivalent proportions for microscopy-negative samples were 1.6%, 31.7%, and 1.6%, respectively. Ancylostoma duodenale was the most widely dispersed geographically, followed by N. americanus. Ancylostoma ceylanicum was least dispersed but was detected in coastal and Amazon regions. In conclusion, human infections with A. ceylanicum, A. duodenale, and N. americanus were detected in different geoclimatic regions of Ecuador. Additional studies are required to further define the epidemiology of human A. ceylanicum infections, but the potentially widespread presence of this helminth in human populations in Ecuador has implications for hookworm control strategies.

Performance of a stool-based quantitative PCR assay for the diagnosis of tuberculosis in adolescents and adults: a multinational, prospective diagnostic accuracy study.

BACKGROUND: Despite increasing availability of rapid molecular tests for the diagnosis of tuberculosis in high-burden settings, many people with tuberculosis are undiagnosed. Reliance on sputum as the primary specimen for tuberculosis diagnostics contributes to this diagnostic gap. We evaluated the diagnostic accuracy and additive yield of a novel stool quantitative PCR (qPCR) assay for the diagnosis of tuberculosis in three countries in Africa with high tuberculosis burdens. METHODS: We undertook a prospective diagnostic accuracy study in Eswatini, Mozambique, and Tanzania from Sept 21, 2020, to Feb 2, 2023, to compare the diagnostic accuracy for tuberculosis of a novel stool qPCR test with the current diagnostic standard for Mycobacterium tuberculosis DNA detection from sputum and stool, Xpert-MTB/RIF Ultra (Xpert Ultra). Sputum, stool, and urine samples were provided by a cohort of participants, aged 10 years or older, diagnosed with tuberculosis. Participants with tuberculosis (cases) were enrolled within 72 h of treatment initiation for tuberculosis diagnosed clinically or following laboratory confirmation. Participants without tuberculosis (controls) consisted of household contacts of the cases who did not develop tuberculosis during a 6-month follow-up. The performance was compared with a robust composite microbiological reference standard (CMRS). FINDINGS: The cohort of adolescents and adults (n=408) included 268 participants with confirmed or clinical tuberculosis (cases), 147 (55%) of whom were living with HIV, and 140 participants (controls) without tuberculosis. The sensitivity of the novel stool qPCR was 93·7% (95% CI 87·4-97·4) compared with participants with detectable growth on M tuberculosis culture, and 88·1% (81·3-93·0) compared with sputum Xpert Ultra. The stool qPCR had an equivalent sensitivity as sputum Xpert Ultra (94·8%, 89·1-98·1) compared with culture. Compared with the CMRS, the sensitivity of the stool qPCR was higher than the current standard for tuberculosis diagnostics on stool, Xpert Ultra (80·4%, 73·4-86·2 vs 73·5%, 66·0-80·1; p=0·025 on paired comparison). The qPCR also identified 17-21% additional tuberculosis cases compared to sputum Xpert Ultra or sputum culture. In controls without tuberculosis, the specificity of the stool qPCR was 96·9% (92·2-99·1). INTERPRETATION: In this study, a novel qPCR for the diagnosis of tuberculosis from stool specimens had a higher accuracy in adolescents and adults than the current diagnostic PCR gold standard on stool, Xpert-MTB/RIF Ultra, and equivalent sensitivity to Xpert-MTB/RIF Ultra on sputum. FUNDING: National Institutes of Health (NIH) Allergy and Infectious Diseases, and NIH Fogarty International Center.

Environmental detection of parasites in the marginalized Paiute reservations compared to a nearby area.

The amounts of parasite DNA in soil samples from different playgrounds and other public areas can help identify areas of possible microbe transmission as well as giving indications of possible occurrence of parasite infection in nearby communities. We collected 207 soil samples from parks located on Paiute indigenous tribal areas in southwest Utah and from higher income city of St. George, Utah, and tested them for the presence of 11 parasites that can cause human disease. Molecular tests revealed elevated odds ratio of detecting the helminth Trichuris trichiura 3.072 (1.114 to 8.065) and any protozoa (not including Acanthamoeba ) 3.036 (1.101 to 7.966) in the tribal land playgrounds compared to St. George parks. These findings support previous studies showing that areas in lower socioeconomic communities, especially marginalized communities, tend to have higher presence of parasites in the soil that may lead to higher rates of disease prevalence.

Optimizing DNA Extraction from Pediatric Stool for Diagnosis of Tuberculosis and Use in Next-Generation Sequencing Applications.

The WHO has endorsed the use of stool samples for diagnosis of tuberculosis (TB) in children, and targeted next-generation sequencing (tNGS) of stool has been shown to support diagnosis and provide information about drug susceptibility (DS). Optimizing extraction of DNA from stool for sequencing is critical to ensure high diagnostic sensitivity and accurate DS information. Human stool samples were spiked with various concentrations of Mycobacterium bovis bacillus Calmette-Guérin (BCG), and DNA was extracted from the samples using four different DNA extraction kits. Each sample was subjected to quantitative PCR for identifying Mycobacterium tuberculosis complex bacteria and underwent further analysis to assess the overall DNA yield, fragment length, and purity. This same process was performed with 10 pediatric participants diagnosed with pulmonary TB, and the samples underwent tNGS. The FastDNA spin kit for soil showed the best results on model samples spiked with known quantities of BCG, compared to the other extraction methods evaluated. For clinical samples, the FastDNA and PowerFecal Pro DNA (PowerFecal) kits both showed an increase in the overall DNA quantity, M. tuberculosis-specific DNA quantity, and successful targeted sequencing when testing was performed on stool samples, compared to the two other kits. Three samples extracted via PowerFecal and three samples extracted via FastDNA (from different patients) provided successful sequencing data, with an average depth of coverage of the rpoB region for FastDNA of 298 (range, 107 to 550) and for PowerFecal of 310 (range, 182 to 474), results that were comparable to one another (P = 0.946). The PowerFecal Pro and FastDNA spin kits were superior for extracting DNA from pediatric stool samples for tNGS. IMPORTANCE This is the first study to compare Mycobacterium tuberculosis DNA extraction techniques from pediatric stool samples for use with sequencing technologies. It provides an important starting point for other researchers to isolate quality DNA for this purpose to further the field and to continue to optimize protocols and approaches.

Microbiome Alterations Driven by Trypanosoma cruzi Infection in Two Disjunctive Murine Models.

Alterations caused by Trypanosoma cruzi in the composition of gut microbiome may play a vital role in the host-parasite interactions that shapes physiology and immune responses against infection. Thus, a better understanding of this parasite-host-microbiome interaction may yield relevant information in the comprehension of the pathophysiology of the disease and the development of new prophylactic and therapeutic alternatives. Therefore, we implemented a murine model with two mice strains (BALB/c and C57BL/6) to evaluate the impact of Trypanosoma cruzi (Tulahuen strain) infection on the gut microbiome utilizing cytokine profiling and shotgun metagenomics. Higher parasite burdens were observed in cardiac and intestinal tissues, including changes in anti-inflammatory (interleukin-4 [IL-4] and IL-10) and proinflammatory (gamma interferon, tumor necrosis factor alpha, and IL-6) cytokines. Bacterial species such as Bacteroides thetaiotaomicron, Faecalibaculum rodentium, and Lactobacillus johnsonii showed a decrease in relative abundance, while Akkermansia muciniphila and Staphylococcus xylosus increased. Likewise, as infection progressed, there was a decrease in gene abundances related to metabolic processes such as lipid synthesis (including short-chain fatty acids) and amino acid synthesis (including branched-chain amino acids). High-quality metagenomic assembled genomes of L. johnsonii and A. muciniphila among other species were reconstructed, confirming, functional changes associated with metabolic pathways that are directly affected by the loss of abundance of specific bacterial taxa. IMPORTANCE Chagas disease (CD) is caused by the protozoan Trypanosoma cruzi, presenting acute and chronic phases where cardiomyopathy, megaesophagus, and/or megacolon stand out. During the course of its life cycle, the parasite has an important gastrointestinal tract transit that leads to severe forms of CD. The intestinal microbiome plays an essential role in the immunological, physiological, and metabolic homeostasis of the host. Therefore, parasite-host-intestinal microbiome interactions may provide information on certain biological and pathophysiological aspects related to CD. The present study proposes a comprehensive evaluation of the potential effects of this interaction based on metagenomic and immunological data from two mice models with different genetic, immunological, and microbiome backgrounds. Our findings suggest that there are alterations in the immune and microbiome profiles that affect several metabolic pathways that can potentially promote the infection's establishment, progression, and persistence. In addition, this information may prove essential in the research of new prophylactic and therapeutic alternatives for CD.

Epidemiology of giardiasis and assemblages A and B and effects on diarrhea and growth trajectories during the first 8 years of life: Analysis of a birth cohort in a rural district in tropical Ecuador.

BACKGROUND: There are limited longitudinal data on the acquisition of Giardia lamblia infections in childhood using molecular assays to detect and type assemblages, and measure effects of infections on diarrhea risk and childhood growth. METHODS: We analysed stool samples from a surveillance sample within a birth cohort in a rural district in tropical Ecuador. The cohort was followed to 8 years of age for the presence of G. lamblia in stools by quantitative PCR and A and B assemblages by Taqman assay or Sanger sequencing. We explored risk factors associated with infection using generalized estimating equations applied to longitudinal binary outcomes, and longitudinal panel data analysis to estimate effects of infection on diarrhea and growth trajectories. RESULTS: 2,812 stool samples collected between 1 month and 8 years of age from 498 children were analyzed and showed high rates of infection: 79.7% were infected at least once with peak prevalence (53.9%) at 5 years. Assemblage B was accounted for 56.8% of genotyped infections. Risk factors for infection included male sex (P = 0.001), daycare attendance (P<0.001), having a household latrine (P = 0.04), childhood (P<0.001) and maternal soil-transmitted helminth (P = 0.029) infections, and exposures to donkeys (age interaction P = 0.034). G. lamblia was associated with increased risk of diarrhea (per episode, RR 1.03, 95% CI 1.01-1.06, P = 0.011) during the first 3 years of life and a transient impairment of weight (age interaction P = 0.017) and height-for-age (age interaction P = 0.025) trajectories between 1 and 4 years of age. There was no increased risk of either assemblage being associated with outcomes. CONCLUSION: Our data show a relatively high edemicity of G. lamblia transmission during childhood in coastal Ecuador, and evidence that infection is associated with a transiently increased risk of diarrhea during the first 3 years of life and impairment of weight and height between 1 and 4 years.

Screening, prevention, and treatment for hyperinfection syndrome and disseminated infections caused by Strongyloides stercoralis.

PURPOSE OF REVIEW: This review discusses the latest approaches to the diagnosis and treatment of patients with strongyloidiasis, with an emphasis on infection in the immunocompromised host and the risk for disseminated strongyloidiasis. RECENT FINDINGS: The differences in acute, chronic, accelerated autoinfection, and disseminated disease in Strongyloides stercoralis infection are explored with particular emphasis on early diagnosis, treatment, and prevention. The goals of treatment are investigated for the different infection states. Predisposing risks for dissemination are delineated, and the roles played for newer diagnostics in the identification of at-risk individuals are detailed. SUMMARY: The use of newer diagnostic tests and broader screening of immunocompromised patients from Strongyloides-endemic areas is of paramount importance, particularly if prevention of life-threatening dissemination is the goal.

Peripheral blood stem cell transplant-related Plasmodium falciparum infection in a patient with sickle cell disease.

BACKGROUND: Although transmission of Plasmodium falciparum (Pf) infection during red blood cell (RBC) transfusion from an infected donor has been well documented, malaria parasites are not known to infect hematopoietic stem cells. We report a case of Pf infection in a patient 11 days after peripheral blood stem cell transplant for sickle cell disease. STUDY DESIGN AND METHODS: Malaria parasites were detected in thick blood smears by Giemsa staining. Pf HRP2 antigen was measured by enzyme-linked immunosorbent assay on whole blood and plasma. Pf DNA was detected in whole blood and stem cell retention samples by real-time polymerase chain reaction using Pf species-specific primers and probes. Genotyping of eight Pf microsatellites was performed on genomic DNA extracted from whole blood. RESULTS: Pf was not detected by molecular, serologic, or parasitologic means in samples from the recipient until Day 11 posttransplant, coincident with the onset of symptoms. In contrast, Pf antigen was retrospectively detected in stored plasma collected 3 months before transplant from the asymptomatic donor. Pf DNA was detected in whole blood from both the donor and the recipient after transplant, and genotyping confirmed shared markers between donor and recipient Pf strains. Lookback analysis of RBC donors was negative for Pf infection. CONCLUSIONS: These findings are consistent with transmission by the stem cell product and have profound implications with respect to the screening of potential stem cell donors and recipients from malaria-endemic regions.

Association of Midgut Bacteria and Their Metabolic Pathways with Zika Infection and Insecticide Resistance in Colombian Aedes aegypti Populations.

INTRODUCTION: Aedes aegypti is the vector of several arboviruses such as dengue, Zika, and chikungunya. In 2015-16, Zika virus (ZIKV) had an outbreak in South America associated with prenatal microcephaly and Guillain-Barré syndrome. This mosquito's viral transmission is influenced by microbiota abundance and diversity and its interactions with the vector. The conditions of cocirculation of these three arboviruses, failure in vector control due to insecticide resistance, limitations in dengue management during the COVID-19 pandemic, and lack of effective treatment or vaccines make it necessary to identify changes in mosquito midgut bacterial composition and predict its functions through the infection. Its study is fundamental because it generates knowledge for surveillance of transmission and the risk of outbreaks of these diseases at the local level. METHODS: Midgut bacterial compositions of females of Colombian Ae. aegypti populations were analyzed using DADA2 Pipeline, and their functions were predicted with PICRUSt2 analysis. These analyses were done under the condition of natural ZIKV infection and resistance to lambda-cyhalothrin, alone and in combination. One-step RT-PCR determined the percentage of ZIKV-infected females. We also measured the susceptibility to the pyrethroid lambda-cyhalothrin and evaluated the presence of the V1016I mutation in the sodium channel gene. RESULTS: We found high ZIKV infection rates in Ae. aegypti females from Colombian rural municipalities with deficient water supply, such as Honda with 63.6%. In the face of natural infection with an arbovirus such as Zika, the diversity between an infective and non-infective form was significantly different. Bacteria associated with a state of infection with ZIKV and lambda-cyhalothrin resistance were detected, such as the genus Bacteroides, which was related to functions of pathogenicity, antimicrobial resistance, and bioremediation of insecticides. We hypothesize that it is a vehicle for virus entry, as it is in human intestinal infections. On the other hand, Bello, the only mosquito population classified as susceptible to lambda-cyhalothrin, was associated with bacteria related to mucin degradation functions in the intestine, belonging to the Lachnospiraceae family, with the genus Dorea being increased in ZIKV-infected females. The Serratia genus presented significantly decreased functions related to phenazine production, potentially associated with infection control, and control mechanism functions for host defense and quorum sensing. Additionally, Pseudomonas was the genus principally associated with functions of the degradation of insecticides related to tryptophan metabolism, ABC transporters with a two-component system, efflux pumps, and alginate synthesis. CONCLUSIONS: Microbiota composition may be modulated by ZIKV infection and insecticide resistance in Ae. aegypti Colombian populations. The condition of resistance to lambda-cyhalothrin could be inducing a phenome of dysbiosis in field Ae. aegypti affecting the transmission of arboviruses.