Th2-biased immune responses to body migrating Ascaris larvae in primary infection are associated with pathology but not protection.

Helminth infections lead to an overdispersion of the parasites in humans as well as in animals. We asked whether early immune responses against migrating Ascaris larvae are responsible for the unequal distribution of worms in natural host populations and thus investigated a susceptible versus a resistant mouse strain. In mice, the roundworm larvae develop until the lung stage and thus early anti-Ascaris immune responses against the migrating larvae in the liver and lung can be deciphered. Our data show that susceptible C57BL/6 mice respond to Ascaris larval migration significantly stronger compared to resistant CBA mice and the anti-parasite reactivity is associated with pathology. Increased eosinophil recruitment was detected in the liver and lungs, but also in the spleen and peritoneal cavity of susceptible mice on day 8 post infection compared to resistant mice. In serum, eosinophil peroxidase levels were significantly higher only in the susceptible mice, indicating functional activity of the recruited eosinophils. This effect was associated with an increased IL-5/IL-13 production by innate lymphoid cells and CD4+ T cells and a pronounced type 2 macrophage polarization in the lungs of susceptible mice. Furthermore, a comparison of wildtype BALB/c and eosinophil-deficient dblGATA-1 BALB/c mice showed that eosinophils were not essential for the early control of migrating Ascaris larvae. In conclusion, in primary infection, a strong local and systemic type 2 immune response during hepato-tracheal helminth larval migration is associated with pathology rather than protection.

Ascaris Mouse Model Protocols: Advancing Research on Larval Ascariasis Biology.

Ascariasis, caused by both Ascaris lumbricoides and Ascaris suum, is the most prevalent parasitic disease worldwide, affecting both human and porcine populations. However, due to the difficulties of assessing the early events of infection in humans, most studies of human ascariasis have been restricted to the chronic intestinal phase. Therefore, the Ascaris mouse model has become a fundamental tool for investigating the immunobiology and pathogenesis of the early infection stage referred to as larval ascariasis because of the model's practicality and ability to replicate the natural processes involved. The Ascaris mouse model has been widely used to explore factors such as infection resistance/susceptibility, liver inflammation, lung immune-mediated pathology, and co-infections and, notably, as a pivotal element in preclinical vaccine trials. Exploring the immunobiology of larval ascariasis may offer new insights into disease development and provide a substantial understanding of key components that trigger a protective immune response. This article focuses on creating a comprehensive guide for conducting Ascaris experimental infections in the laboratory as a foundation for future research efforts. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Acquisition and embryonation of Ascaris suum eggs from adult females Alternate Protocol: Cleaning and purification of Ascaris suum from female A. suum uteri Basic Protocol 2: Preparation of Ascaris suum eggs and murine infection Basic Protocol 3: Measurement of larval burden and Ascaris-larva-induced pathogenesis Basic Protocol 4: In vitro hatching and purification of Ascaris L3 larvae Support Protocol: Preparation of crude antigen from Ascaris infectious stages Basic Protocol 5: Ultrastructure-expansion microscopy (U-ExM) of Ascaris suum larval stages.

Acute necrotising pancreatitis from ascariasis in a gravid patient.

Rarer causes of acute pancreatitis may be considered in certain settings, such as parasitism in endemic regions. This report describes a pregnant female (second trimester) in her 20s who presented with 3-day steady epigastric pain radiating to the back and passage of worm from the mouth. She was diagnosed with mild acute pancreatitis, given a significantly elevated serum lipase and absence of organ failures. Fecalysis showed Ascaris lumbricoides ova; hence, she was treated with mebendazole. Plain MR cholangiopancreatography showed an 842 mL necrotic pancreatic fluid collection and tubular flow void foci within the gallbladder and duodenum consistent with helminthiasis. The patient was managed conservatively in the absence of indications for drainage. The abdominal pain remarkably improved, and she underwent eventual vacuum-assisted delivery to a healthy term baby 4 months after the bout of acute pancreatitis.

Biliary Ascariasis in a Pediatric Patient in Lithuania: Case Report and Literature Review.

Hepatobiliary ascariasis is caused by the entry of the nematode A. lumbricoides from the duodenum into the biliary duct. We report a case of an Ascaris-induced extrahepatic biliary tract obstruction in a pediatric patient admitted to the hospital due to a wide spectrum of symptoms of biliary disease, which included abdominal pain in the upper abdominal quadrants, vomiting, and jaundice. Imaging tests-including ultrasound, magnetic resonance cholangiopancreatography (MRCP), and endoscopic retrograde cholangiopancreatography (ERCP)-were performed to confirm the diagnosis of biliary ascariasis. The tests did, in fact, demonstrate signs of this disease. Nevertheless, during the ERCP, only the remains of Ascaris parasites in the form of tissue fragments were extracted. We also aim to discuss the prevalence of ascariasis in children, the causes of migration of Ascaris parasites into the bile ducts, together with its clinical manifestations, as well as the diagnostic and treatment methods of this disease.

Acute Ascaris infection impairs the effector functions of natural killer cells in single and Salmonella co-infected pigs.

Natural killer (NK) cells play a key role in defense against Salmonella infections during the early phase of infection. Our previous work showed that the excretory/secretory products of Ascaris suum repressed NK activity in vitro. Here, we asked if NK cell functionality was influenced in domestic pigs during coinfection with Ascaris and Salmonella enterica serotype Typhimurium. Ascaris coinfection completely abolished the IL-12 and IL-18 driven elevation of IFN-γ production seen in CD16 + CD8α + perforin + NK cells of Salmonella single-infected pigs. Furthermore, Ascaris coinfection prohibited the Salmonella-driven rise in NK perforin levels and CD107a surface expression. In line with impaired effector functions, NK cells from Ascaris-single and coinfected pigs displayed elevated expression of the inhibitory KLRA1 and NKG2A receptors genes, contrasting with the higher expression of the activating NKp46 and NKp30 receptors in NK cells during Salmonella single infection. These differences were accompanied by the highly significant upregulation of T-bet protein expression in NK cells from Ascaris-single and Ascaris/Salmonella coinfected pigs. Together, our data strongly indicate a profound repression of NK functionality by an Ascaris infection which may hinder infected individuals from adequately responding to a concurrent bacterial infection.

Evaluation of copromicroscopy, multiplex-qPCR and antibody serology for monitoring of human ascariasis in endemic settings.

BACKGROUND: The standard diagnosis of Ascaris lumbricoides and other soil-transmitted helminth (STH) infections relies on the detection of worm eggs by copromicroscopy. However, this method is dependent on worm patency and shows only limited accuracy in low-intensity infection settings. We aimed to decipher the diagnostic accuracy of different antibodies using various Ascaris antigens in reference to copromicroscopy and quantitative PCR (qPCR), four months after national STH preventative chemotherapy among school children in western Kenya. METHODOLOGY: STH infection status of 390 school children was evaluated via copromicroscopy (Kato-Katz and mini-FLOTAC) and qPCR. In parallel, Ascaris-specific antibody profiles against larval and adult worm lysates, and adult worm excretory-secretory (ES) products were determined by enzyme-linked immunosorbent assay. Antibody cross-reactivity was evaluated using the closely related zoonotic roundworm species Toxocara cati and Toxocara canis. The diagnostic accuracy of each antibody was evaluated using receiver operating curve analysis and the correspondent area under the curve (AUC). PRINCIPAL FINDINGS: Ascaris was the predominant helminth infection with an overall prevalence of 14.9% (58/390). The sensitivity of mini-FLOTAC and Kato-Katz for Ascaris diagnosis reached only 53.5% and 63.8%, respectively compared to qPCR. Although being more sensitive, qPCR values correlated with microscopic egg counts (R = -0.71, P<0.001), in contrast to antibody levels. Strikingly, IgG antibodies recognizing the ES products of adult Ascaris worms reliably diagnosed active Ascaris infection as determined by qPCR and microscopy, with IgG1 displaying the highest accuracy (AUC = 0.83, 95% CI: 0.75-0.91). CONCLUSION: IgG1 antibody responses against adult Ascaris-ES products hold a promising potential for complementing the standard fecal and molecular techniques employed for monitoring Ascaris infections. This is of particular importance in the context of deworming programs as the antibody diagnostic accuracy was independent of egg counts.

Serology survey of Ascaris suum and Trichinella spiralis in rural pigs in Southwestern Mexico.

BACKGROUND: Parasitic diseases of pigs are a public and veterinary health problem. Helminths influence pork production, whereas backyard pigs can transmit these parasites. OBJECTIVES: This work aimed to investigate the prevalence of antibodies against Ascaris suum and Trichinella spiralis in backyard pigs from Jamiltepec, Region de la Costa, Oaxaca, in Southwestern Mexico. METHODS: Six hundred sixty-four serum samples were obtained from backyard pigs from 23 rural villages distributed in 5 municipalities; samples were taken in a non-probabilistic manner with the owner's consent. The presence of serum antibodies against a total extract of A. suum adult worm was determined by ELISA. In contrast, antibodies to the excretion-secretion products of the T. spiralis muscle larva were determined by Western blot. RESULTS: The global seroprevalence for A. suum was 5.12% and 2.41% for T. spiralis; however, antibodies were only found in 8 villages and distributed in 3 municipalities. The highest frequency of positivity for Ascaris was found in the municipality of Santa Catarina Mechoacán (13.01%), whereas, in Santa María Huazalotitlán, the highest frequency of positivity for Trichinella was found (5.75%). In San Andrés, frequencies were 7.23% and 4.82%, respectively. No statistical differences were observed between populations. CONCLUSIONS: Our data suggest that helminth transmission is restricted by locality. However, further studies must be conducted to understand the factors limiting this transmission to promote pork meat production in parasite-free zones.

Efficacy of Single-Dose Albendazole and Albendazole Plus Ivermectin for Soil-Transmitted Helminth Infection in Children in the Peruvian Amazon.

In countries where soil-transmitted helminth (STH) infections are endemic, deworming programs are recommended to reduce morbidity; however, increasing levels of resistance to benzimidazoles are of concern. In an observational study in Peru, we studied the clinical efficacy of 400 mg of albendazole 20 days after treatment among children aged 2-11 years. Of 426 participants who provided samples, 52.3% were infected with a STH, 144 (33.8%) were positive for Ascaris (41.8% light, 50.8% moderate, and 7.4% heavy infections), 147 (34.5%) were positive for Trichuris (75.2% light, 22.5% moderate, and 2.3% heavy infections), and 1.1% were positive for hookworm species (100% light infections). Additional stool samples were examined at 20, 90, and 130 days after the initial treatment. At 20 days post-administration of albendazole, the cure rate (CR) of Ascaris infection was 80.1% (95% CI: 73.5-86.7), and the egg reduction rate (ERR) was 70.8% (95% CI: 57.8-88.7); the CR for Trichuris infection was 27.1% (95% CI: 20.0-34.3), and the ERR was 29.8% (95% CI: -1.40 to 57.5). Among participants with persistent or recurrent infections with Trichuris, the combined therapy of albendazole (400 mg) and ivermectin at 600 µg/dose increased overall CR for Trichuris infection to 75.2% (95% CI: 67.3-83.2%) with an ERR of 84.2% (95% CI: 61.3-93.8%). Albendazole administration alone for the control of STH was associated with high rates of treatment failure, especially for Trichuris. Combined single doses of albendazole and ivermectin was observed to have improved efficacy.

Soil-Transmitted Helminth Infections in Shanghai, China: Surveillance from 2014 to 2023.

Soil-transmitted helminthiasis remains a notable health problem in developing countries. In 1990 in Shanghai, a city in eastern China, 47% of the population was affected. Due to a series of comprehensive approaches, the prevalence decreased to 0.5% in 2009. We collected 10-year surveillance data to assess the epidemic situation of soil-transmitted helminth (STH) infections in Shanghai. Stool samples and questionnaires from participants were collected. The Kato-Katz technique was used to detect infections with Ascaris lumbricoides, hookworm (Ancylostoma duodenale and Necator americanus), and Trichuris trichiura. From 2014 to 2023, a total of 77,685 participants were screened for parasitic infections, and the overall prevalence of STH infections decreased from 0.1% to 0% in Shanghai. Of 77,685 participants, 25 (0.03%) were positive for intestinal helminths, with the most common parasite being A. lumbricoides (72.0%). Generally, elderly participants from rural areas with lower education levels were more likely to be infected with STHs. A total of 2,914 questionnaires were collected, and most respondents (72.6%) had good knowledge about the transmission routes of STHs. However, 12.3% of participants did not know the damage to health caused by STHs, and 19.3% had no idea of how to prevent infection. These data demonstrate the current state of STH infections in Shanghai. The results suggest that various comprehensive measures should be encouraged, continuously implemented, and strengthened accordingly so that STH elimination can be integrated into the Healthy China initiative by 2030.

Worldwide absence of canonical benzimidazole resistance-associated mutations within ß-tubulin genes from Ascaris.

BACKGROUND: The giant roundworm Ascaris is an intestinal nematode, causing ascariasis by infecting humans and pigs worldwide. Recent estimates suggest that Ascaris infects over half a billion people, with chronic infections leading to reduced growth and cognitive ability. Ascariasis affects innumerable pigs worldwide and is known to reduce production yields via decreased growth and condemnation of livers. The predominant anthelminthic drugs used to treat ascariasis are the benzimidazoles. Benzimidazoles interact with ß-tubulins and block their function, and several benzimidazole resistance-associated mutations have been described in the ß-tubulins of ruminant nematodes. Recent research on ascarids has shown that these canonical benzimidazole resistance-associated mutations are likely not present in the ß-tubulins of Ascaris, Ascaridia or Parascaris, even in phenotypically resistant populations. METHODS: To further determine the putative absence of key ß-tubulin polymorphisms, we screened two ß-tubulin isotypes of Ascaris, highly expressed in adult worms. Using adult and egg samples of Ascaris obtained from pigs and humans worldwide, we performed deep amplicon sequencing to look for canonical resistance-associated mutations in Ascaris ß-tubulins. Subsequently, we examined these data in closer detail to study the population dynamics of Ascaris and genetic diversity within the two isotypes and tested whether genotypes appeared to partition across human and pig hosts. RESULTS: In the 187 isolates, 69 genotypes were found, made up of eight haplotypes of ß-tubulin isotype A and 20 haplotypes of isotype B. Single nucleotide polymorphisms were seen at 14 and 37 positions for ß-tubulin isotype A and isotype B, respectively. No evidence of any canonical benzimidazole resistance-associated mutations was found in either human- or pig-derived Ascaris isolates. There was, however, a difference in the genetic diversity of each isotype and distribution of ß-tubulin genotypes between human- and pig-derived Ascaris. Statistical tests of population differentiation show significant differences (p < 0.001) between pig- and human-derived worms; however, more diversity was seen between worms from different populations than worms from different hosts. CONCLUSIONS: Our work suggests an absence of canonical ß-tubulin mutations within Ascaris, but alternative modes of anthelminthic resistance may emerge necessitating continued genetic scrutiny alongside monitoring of drug efficacy.

Epidemiology of soil-transmitted helminthiasis among school-aged children in pastoralist communities of Kenya: A cross-sectional study.

INTRODUCTION: Soil-transmitted helminthiasis (STH) are a major public health problem in Sub-Saharan Africa. In Kenya, the National School Based Deworming Program (NSBDP) was launched in 2012 with a goal of reducing STH prevalence in school-aged children (SAC) to <1%, however monitoring and evaluation results have consistently showed > 20% prevalence in Narok County. We conducted a study to investigate factors associated with STH infections among SAC in Narok County. METHODOLOGY: A cross-sectional study was conducted among 514 SAC from five schools in Trans Mara West sub-county, Narok County. The sub-county was selected because it had participating schools within the NSBDP with a high prevalence of STH infection. Participants were selected using systematic random sampling. Stool samples collected from participants were examined for STH eggs using Kato-Katz technique. An open data kit questionnaire was used to collect socio-demographics, household, and STH knowledge information from 139 of the 514 SAC. Descriptive statistics was used to summarize the data, prevalence and mean intensity of infections were calculated, and logistic regression used to determine factors associated with STH infections. RESULTS: The overall prevalence of any STH infection was 24.6% (95%CI: 21.1-28.6). Trichuris trichiura 14.4% (95%CI: 11.7-17.8), Ascaris lumbricoides 12.5% (95%CI: 9.9-15.7) and hookworm 0%. From multivariable analysis the only factors significantly associated with increased risk of STH infection were, children attending Karda and Nkarano schools with aOR = 5.29 (95%CI: 1.45-19.24); p = 0.011 and aOR = 4.53 (95%CI: 1.29-15.97); p = 0.019 respectively. For A. lumbricoides, children attending Nkarano School were associated with a significant risk of infection with aOR = 7.81 (95%CI: 1.81-33.63); p = 0.006. CONCLUSIONS: Despite the ongoing work of NSBDP, the STH prevalence is still ≥ 20% in Trans Mara West sub-county Narok County, among SAC. This underscores the need for continued annual MDA. Additionally, if possible, drug combinations may effectively manage T. trichiura, the region's most common helminth. The study found a correlation between children attending specific schools and STH infection risk, suggesting the importance of health education and improved water, sanitation, and hygiene practices holistically both in schools and associated catchment areas that can act as STH reservoirs to alleviate the burden of STH.

Ascaris suum infection in juvenile pigs elicits a local Th2 response in a setting of ongoing Th1 expansion.

Ascaris spp. undergo extensive migration within the body before establishing patent infections in the small intestinal tract of humans and pigs. However, whether larval migration is critical for inducing efficient type 2 responses remains poorly understood. Therefore, we investigated systemic versus local adaptive immune responses along the hepato-tracheal migration of Ascaris suum during primary, single infections in conventionally raised pigs. Neither the initial invasion of gut tissue nor migration through the liver resulted in discernable Th2 cell responses. In contrast, lung-stage larvae elicited a Th2-biased pulmonary response, which declined after the larvae had left the lungs. In the small intestine, we observed an accumulation of Th2 cells upon the arrival of fourth-stage larvae (L4) to the small intestinal lumen. In parallel, we noticed robust and increasing Th1 responses in circulation, migration-affected organs, and draining lymph nodes. Phenotypic analysis of CD4+ T cells specifically recognizing A. suum antigens in the circulation and lung tissue of infected pigs confirmed that the majority of Ascaris-specific T cells produced IL-4 (Th2) and, to a much lesser extent, IL-4/IFN-g (Th2/1 hybrids) or IFN-g alone (Th1). These data demonstrate that lung-stage but not the early liver-stage larvae lead to a locally restricted Th2 response. Significant Th2 cell accumulation in the small intestine occurs only when L4 complete the body migration. In addition, Th2 immunity seems to be hampered by the concurrent, nonspecific Th1 bias in growing pigs. Together, the late onset of Th2 immunity at the site of infection and the Th1-biased systemic immunity likely enable the establishment of intestinal infections by sufficiently large L4 stages and pre-adult worms, some of which resist expulsion mechanisms.

Anthelmintic Activity of Saussurea costus (Falc.) Lipsch. Against Ascaridia galli, a Pathogenic Nematode in Poultry: In Vitro and In Vivo Studies.

AIM OF THE STUDY: The growing resistance of helminth parasites to currently available commercial anthelmintic drugs, combined with apprehensions regarding detrimental chemical residues in livestock products, has sparked an interest in exploring medicinal plants as an alternative strategy for treating helminthiasis. As a result, this study was designed to investigate the anthelmintic activity of crude methanolic extracts (CME) of Saussurea costus root on Ascaridia.galli, a pathogenic nematode of poultry. MATERIALS AND METHODS: In vitro, the anthelmintic effect of Saussurea costus root was evaluated in comparison to commercial anthelmintic, levamisole on the adult nematode parasites, A.galli using worm motility inhibition (WMI) test. The CME of S.costus was also evaluated for in vivo anthelmintic activity in chickens experimentally infected with Ascaridia galli. For the in vivo study, one hundred-day-old chickens were orally infected with embryonated eggs of A. galli worms. The efficacy of the plant extract as an anthelmintic was assessed through two tests: faecal egg count reduction (FECR) test and worm count reduction (WCR) test. The study investigated three distinct doses of plant extract under in vivo setup: 500 mg kg-1 body weight (bw), 1000 mg kg-1 bw, and 2000 mg kg-1 bw. RESULTS: In vitro, all the tested concentrations of S.costus (25 mg/ml, 50 mg/ml, and 100 mg/ml) showed a significant (P < 0.001) anthelmintic effects on live adult A. galli worms in terms of inhibition of worm motility at different hours post-treatment. At the highest concentration of the extract, we observed worm motility inhibition of 100% at 24 h post-exposure. On day 14 post-treatment, all birds were slaughtered, and adult A. galli worms were subsequently retrieved from their small intestines. Birds treated with CME extract of S. costus root exhibited a significant (P < 0.001) reduction in faecal egg count. However, the administration of the extract at the dosage of 500 mg kg-1bw to the birds did not reveal any significant (P > 0.05) differences in the worm count compared to the negative control group. The CME of S. costus at a dose of 2000 mg kg-1bw showed the highest anthelmintic activity by inducing 83.10% FECR and 76.47% WCR. CONCLUSION: In conclusion, the root extract of S. costus has a promising anthelmintic activity on A. galli as demonstrated by the results of the present experiment.

[Cytokine measurement using a multiplex technique with spectral flow cytometry: application in the study of the immunobiology of ascariasis]. / Medición de citoquinas mediante una técnica multiplex por citometría espectral: aplicación en el estudio de la inmunobiología de la ascariasis.

OBJECTIVE: To quantify the production of Th1/Th2/Th17 cytokines induced by Ascaris lumbricoides antigens in peripheral blood mononuclear cells (PBMCs) using a multiplex technique. METHODS: PBMCs were cultured from individuals with mild A. lumbricoides infection (n = 20) and uninfected individuals (n = 21) and stimulated with A. lumbricoides extract (ExtAscaris), a mix of anti-CD2/CD3/CD28 (CDmix) as a positive control, and only medium (negative control). Cytokines in the supernatants were measured using the BD™ Cytometric Bead Array Human Th1/Th2/Th17 kit, to identify IFN-γ, TNF, IL-10, IL-6, IL-4, IL-2, and IL-17A. Readings were taken on a spectral cytometer (Northern Lights, Cytek, USA), and analysis was performed using R software with packages "tidyverse," "beadplexr," "flowCore," and "arsenal." Cytokine concentrations were calculated using a 5-parameter logistic curve. The t-test was used to compare cases and controls, and statistical significance was set at p < 0.05. The study was approved by the Ethics Committee of the University of Cartagena and the participants provided informed consent. This study was financially supported by the Colombian Sistema General de Regalías under the BPIN2020000100405 - BPIN2020000100364. RESULTS: Efficient fluorescence intensity extraction for each cytokine was achieved using detection channel R8 and the "mclust" clustering model (Figure 1). No significant differences were found in the levels of the seven cytokines between cases and controls (Figure 2). Although the IFN-γ response to ExtAscaris was higher in cases than in controls (252.5 ng/mL vs. 173.1 ng/mL), the difference was not significant. IL-17A (detection limit: 18.9 pg/mL) was more detectable in cases than controls (5 cases, 23% vs. 2 controls, 9.5%). IL-4 was only detected in the supernatants from CDmix-stimulated cultures but not with the Ascaris extract (Figure 2). CONCLUSIONS: The multiplex technique using spectral flow cytometry combined with open-source software analysis proved applicable for quantifying cytokines induced by A. lumbricoides antigens in PBMCs. However, a more sensitive method is needed to evaluate IL-4 response in the context of ascariasis. The results did not reveal significant differences in cytokine production between cases and controls for the evaluated stimuli.

OBJETIVOS: Cuantificar la producción de citoquinas Th1/Th2/Th17, inducida por antígenos de Ascaris lumbricoides en PBMCs, utilizando una técnica de multiplex. MÉTODOS: Se realizaron cultivos de PBMCs de personas con infección leve por A. lumbricoides (n = 20), y no infectadas (n = 21), y se estimularon con extracto de A. lumbricoides (ExtAscaris), un mix de anti-CD2/CD3/CD28 (CDmix), como control positivo, y solo medio (control negativo). Las citoquinas en los sobrenadantes, se midieron usando el estuche BD™ Cytometric Bead Array Human Th1/Th2/Th17, para identificar IFN-γ, TNF, IL-10, IL-6, IL-4, IL-2 e IL-17A. La lectura se realizó en un citómetro espectral (Northern Lights, Cytek, USA), y el análisis en software R, usando los paquetes tidyverse, beadplexr, flowCore y arsenal. Se calculó la concentración de citoquinas mediante ajuste de curva logística de cinco parámetros. Se empleó la prueba t para comparar casos y controles y una p < 0,05, se consideró como significativa. Se contó con autorización del Comité de Ética de la Universidad de Cartagena para hacer la investigación y con el consentimiento informado por parte de los participantes. Este trabajo fue financiado por el Sistema General de Regalías de Colombia, bajo el BPIN2020000100405 - BPIN2020000100364. RESULTADOS: Al utilizar el canal de detección R8 para identificar las citoquinas y el modelo de agrupamiento mclust, se extrajo eficientemente la intensidad de fluorescencia para cada citoquina (Figura 1). No se encontraron diferencias significativas en los niveles de las siete citoquinas entre casos y controles (Figura 2). Aunque la respuesta de IFN-, γ hacia ExtAscaris fue más alta en los casos de controles (252,5 ng/mL vs 173,1 ng/mL), la diferencia no fue significativa. La IL-17A (límite de detección: 18,9 pg/mL) fue más detectable en casos que en controles (cinco casos, 23% vs dos controles, 9,5%). La IL-4 solo se detectó en los sobrenadantes de cultivos estimulados con CDmix, pero no con el extracto de Ascaris (Figura 2). CONCLUSIONES: La técnica multiplex por citometría espectral, combinada con el análisis en software de licencia libre, se mostró aplicable para cuantificar citoquinas inducidas por antígenos de A. lumbricoides en PBMCs. Sin embargo, se requiere de un método más sensible para evaluar la respuesta de IL-4 en el contexto de la ascariasis. Los resultados no revelaron diferencias significativas en la producción de citoquinas entre casos y controles para los estímulos evaluados.

[Characterization of peripheral blood cellular populations in relation to Ascaris Lumbricoides infection in rural areas of Bolívar]. / Caracterización de poblaciones celulares en sangre periférica, con relación a la infección por Ascaris lumbricoides en zonas rurales de Bolívar.

OBJECTIVE: To compare the relative frequencies of immune cell populations in the peripheral blood according to A. lumbricoides infection status. METHODS: Peripheral blood samples were collected from participants infected (n = 35) and uninfected with A. lumbricoides (n=27) residing in different rural municipalities of Bolívar. Infection was diagnosed using two coprological examinations and the Kato-Katz technique. Immunophenotyping was performed using two panels of markers and staining in fresh blood. The flow cytometry reading was performed on a spectral cytometer (Northern Lights, Cytek, USA). The populations identified in the first panel (Figure 1) were T lymphocytes (CD45+ CD3+), CD4+ or CD8+, B lymphocytes (CD45+ SSClow CD3- CD19+), neutrophils (CD45+ SSChi CD3- CD16+), and eosinophils (CD45+ SSChi CD3- CD16low). Monocytes were identified in another panel (Figure 2): classical (CD14++ CD16 -), intermediate (CD14++ CD16+), and non-classical (CD14+ CD16++). Dendritic cells, including CD123 + + CD303 + (plasmacytoid), HLA-DR + + CD1c + (myeloid CD1c +), and CD14-CD141 + + (myeloid CD141 +), were also identified. The study received approval from the Ethics Committee of the University of Cartagena, and participants provided informed consent. Funding was provided by the Colombian Sistema General de Regalías under BPIN2020000100405 - BPIN2020000100364. RESULTS: No significant differences were observed in age [mean cases: 35.69 (SD: 17.7) vs. controls: 37.04 (SD: 15.6) years] or sex (cases: 62.9% vs. controls: 74.1%) (Table 1). All infections were mild, with a median of 96 eggs (IQR, 48-216). A marginally significant difference was observed only in the percentage of neutrophils (45.37% in cases vs. 54.79% in controls, p=0.041) (Figure 3). Although the frequency of eosinophils was higher in the cases (8.1% vs. 6%), this difference was not significant (p=0.138) (Figure 3). No significant differences were observed in the populations of monocytes or dendritic cells between cases and controls (Figure 4). CONCLUSION: Mild A. lumbricoides infection appears to affect the number of neutrophils in peripheral blood. The low infection intensity in the studied samples may explain the lack of a significant impact on other cellular populations.

OBJETIVO: Comparar las frecuencias relativas de poblaciones de células inmunes en sangre periférica de acuerdo con el estado de infección por A. lumbricoides. MÉTODOS: Se recolectaron muestras de sangre periférica de participantes infectados (n=35) y no infectados con A. lumbricoides (n=27), residentes en distintos municipios rurales de Bolívar. La infección se diagnosticó por dos métodos coprológicos y la técnica de Kato-Katz. El inmunofenotipo se determinó con dos baterías de marcadores y tinciones en sangre fresca. La lectura fue realizada en un citómetro espectral (Northern Lights, Cytek, USA). Las poblaciones identificadas en la primera batería (Figura 1) fueron linfocitos T (CD45+ CD3+) CD4+ o CD8+, linfocitos B (CD45+ SSClow CD3- CD19+), neutrófilos (CD45+ SSChi CD3- CD16+), y eosinófilos (CD45+ SSChi CD3- CD16low). Los monocitos se identificaron en otra batería (Figura 2): clásicos (CD14++ CD16­), intermedios (CD14++ CD16+), y no clásicos (CD14+ CD16++). También se identificaron células dendríticas, tales como: CD123++ CD303+ (plasmocitoides), HLA-DR++ CD1c+ (mieloides CD1c+), y CD14- CD141++ (mieloides CD141+). El estudio recibió la aprobación del Comité de Ética de la Universidad de Cartagena, y los participantes otorgaron su consentimiento informado. La financiación fue proporcionada por el Sistema General de Regalías de Colombia, bajo el BPIN2020000100405 - BPIN2020000100364. RESULTADOS: No se observaron diferencias significativas en edad [media = casos: 35,69 (DE: 17,7) vs controles: 37,04 (DE: 15,6 años] o sexo (casos: 62,9% vs. controles: 74,1%). Todas las infecciones fueron leves con una mediana de huevos de 96 (RIC: 48 - 216). Solo se encontró diferencia significativa marginal en el porcentaje de neutrófilos (45,37% en los casos vs 54,79% en controles, p=0,041). Si bien la frecuencia de eosinófilos fue más alta en los casos (8,1% vs. 6%), esta diferencia no alcanzó la significancia (p=0,138). No se observaron diferencias significativas en las poblaciones de monocitos o células dendríticas entre casos y controles (Figura 4). CONCLUSIÓN: La infección leve por A. lumbricoides parece afectar el número de neutrófilos en sangre periférica. Es posible que por la baja intensidad de la infección en la muestra estudiada, no se detecte un impacto importante de la misma sobre el resto de las poblaciones celulares. Palabras claves: Helmintos; Ascaris lumbricoides; Citometría de flujo; Inmunofenotipado; Neutrófilos.

Assay for the simultaneous detection of Raillietina spp. (R. echinobothrida, R. tetragona, and R. cesticillus) and Ascaridia galli infection in chickens using duplex loop-mediated isothermal amplification integrated with a lateral flow dipstick assay.

Raillietina species and Ascaridia galli are two of the significant intestinal parasites that affect chickens in a free-range system production. They destroy the intestinal mucosa layer, leading to several clinical symptoms such as weight loss, a slowed growth rate, and economic value loss. Thus, the objective of this study was to develop an assay for simultaneously detecting Raillietina spp. (R. echinobothrida, R. tetragona, and R. cesticillus) and A. galli in a single reaction using duplex loop-mediated isothermal amplification (dLAMP) coupled with a lateral flow dipstick (LFD) assay. The analytical specificity of the dLAMP-LFD assay showed a high specific amplification of Raillietina spp. and A. galli without non-target amplification. Regarding the analytical sensitivity, this approach was capable of simultaneously detecting concentrations as low as 5 pg/µL of mixed-targets. To evaluate the efficiency of the dLAMP assay, 30 faecal samples of chickens were verified and compared through microscopic examination. The dLAMP-LFD assay and microscopic examination results showed kappa values of Raillietina spp. and A. galli with moderate (K= 0.615) to high (K= 1) agreements, respectively, while the McNemar's test indicated that the efficiency between assays was not significantly different. Therefore, the developed dLAMP-LFD assay can be used as an alternative screening method to the existing classical method for epidemiological investigation, epidemic control, and farm management, as well as for addressing poultry health problems.

Autochthonous Ascariasis, Mississippi, USA.

We describe a case of a 2-year-old child who expelled a single adult female Ascaris lumbricoides worm. The patient is from a rural county in Mississippi, USA, with no reported travel outside of the United States. The caregivers in the home practice good sanitation. Exposure to domestic pigs is the likely source of infection.

Management of paediatric soil-transmitted helminthiasis in a non-endemic area: experience in a reference international health unit.

Soil-transmitted helminth (STH) infections inflict disability worldwide, especially in the poorest communities. Current therapeutic options against STHs show limited efficacy, particularly against Trichuris trichiura. The empirical management of patients coming from high-prevalence areas has been suggested for non-endemic areas. This study aimed to describe the management of STH infections in a non-endemic setting using an individualised approach. We performed a retrospective, descriptive study of all patients up to 16 years of age with STH infections attended at an international health unit in a non-endemic area (2014-2018), including all T. trichiura, Necator americanus, Ancylostoma duodenale, and Ascaris lumbricoides infections diagnosed using a formol-ether concentration technique and direct visualisation. Patients were treated according to current international guidelines. Sixty-one stool samples from 48 patients testing positive for STHs were collected, with 96% (46/48) reporting a previous long-term stay in endemic areas. Cure rates with 3-day benzimidazole regimens were 72% for T. trichiura, 40% for hookworms, and 83% for A. lumbricoides. The results were not influenced by any reinfection risk due to the study being performed in a non-endemic area. Patients coming from STH-endemic areas should be evaluated with appropriate diagnostic tools and followed up until cure control results. Cure rates in our cohort were moderate to low, similar to those published in studies in endemic areas. The efficacy of current treatment options is insufficient to recommend a specific empirical approach in high-income countries' healthcare systems.

Host gastric corpus microenvironment facilitates Ascaris suum larval hatching and infection in a murine model.

Ascariasis (roundworm) is the most common parasitic helminth infection globally and can lead to significant morbidity in children including chronic lung disease. Children become infected with Ascaris spp. via oral ingestion of eggs. It has long been assumed that Ascaris egg hatching and larval translocation across the gastrointestinal mucosa to initiate infection occurs in the small intestine. Here, we show that A. suum larvae hatched in the host stomach in a murine model. Larvae utilize acidic mammalian chitinase (AMCase; acid chitinase; Chia) from chief cells and acid pumped by parietal cells to emerge from eggs on the surface of gastric epithelium. Furthermore, antagonizing AMCase and gastric acid in the stomach decreases parasitic burden in the liver and lungs and attenuates lung disease. Given Ascaris eggs are chitin-coated, the gastric corpus would logically be the most likely organ for egg hatching, though this is the first study directly evincing the essential role of the host gastric corpus microenvironment. These findings point towards potential novel mechanisms for therapeutic targets to prevent ascariasis and identify a new biomedical significance of AMCase in mammals.