[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.

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.

Hepatobiliary Ascariasis in a Piglet.

PURPOSE: Ascariasis caused by the helminth Ascaris suum is the most common parasitosis of swine worldwide and it may involve all age categories of pigs. The present study reports an unusual localization of A. suum worms in the biliary system of a piglet slaughtered for human consumption. METHODS: The liver was subjected to ultrasound scan and pathological examination. The isolated worms were morphologically examined and the DNA was extracted for the molecular identification of the species involved. RESULTS: A total of 43 preadult nematodes were found within the gallbladder and the bile ducts. Parasites were morphologically identified as belonging to the genus Ascaris and molecularly as A. suum. At gross examination, the liver was moderately enlarged, with the bile ducts severely dilated. A chronic inflammatory infiltrate was noted, often centered around ectatic bile ducts (up to 5 mm in diameter), lined by hyperplastic epithelium and filled with sections of nematodes. The worm sections showed smooth cuticle, coelomyarian musculature, and an intestinal tract lined by columnar, uninucleated cells within a pseudocoelom. The ex vivo ultrasonographic examination of the liver allowed the visualization of several nematodes in the bile duct lumen and could be suggested for in vivo diagnosis. Unfortunately, the absence of the intestine did not allow to define the pathogenesis of the infection. CONCLUSION: Although, given the unusual nature of this finding, it is difficult to identify predisposing factors for this A. suum localization, it suggests that ascariasis should be considered in the differential diagnosis of pigs with hepatobiliary disease.

What was the main factor in successful control of ascariasis in Korea?

In the l950s, under the legacy of traditional agriculture, Ascaris lumbricoides, spread epidemically in the war-bitten society of Korea. Consensus on the parasite control was drafted in the Parasite Disease Prevention Act, which passed a parliamentary agreement in 1966, and established safe disposal of feces and mass chemotherapy as control strategies. Biannual stool examinations and treating infected schoolchildren were basic scheme of the control activity through which revenue could be secured for organized business. In the 27 years following 1969, a maximum of 16 million stool examinations had been done every year. Cellophane thick smear enabled the task. The infection declined remarkably in the 1970s when industrialization and green revolution proceeded. A population study of A. lumbricoides in the late 1970s helped us better understand its epidemiology. The data also settled down the understandable protest of teachers against the repeated stool examinations. In the 9 years following 1987, the target population was gradually reduced when the egg positive rate was below 0.1%. An article in the Korean Law, stipulating obligatory stool examinations, was made optional. Although the long-term Korean effort of Ascaris control was a success, the effect of mass chemotherapy was not as succinct in terms of lowering reinfection. In the period of control, Korean agricultural technology changed, and the economy grew and supplied sanitary facilities by which the vicious cycle was disconnected. Reduction of morbidity was a benefit of mass chemotherapy, which is the only control method feasible in economically difficult countries. The most important hurdle of parasite control in the 1960s was poverty of general population and limited financial resources in Korea but the society formed a consensus on the priority of intestinal helminthiasis control during the ordeal period. The national consensus in the 1960s was the critical milestone for Ascaris control in Korea. Under the social agreement, application of timely technical and research advancements in parasitology achieved the success of ascariasis elimination. The successful experience of ascariasis elimination in Korea can be a benchmark for countries where neglected tropical diseases are endemically recycled.

Biliary ascariasis misidentified as a biliary stent in a patient undergoing liver resection.

Ascaris lumbricoides causes one of the most common soil-transmitted helminthiases globally. The worms mostly infect the human small intestine and elicit negligible or nonspecific symptoms, but there are reports of extraintestinal ectopic ascariasis. We describe a rare case of biliary ascariasis mistaken for biliary stent in a 72-year-old female patient with a history of liver resection. She visited our outpatient clinic complaining of right upper quadrant pain and fever for the past week. She had previously undergone left lateral sectionectomy for recurrent biliary and intrahepatic duct stones 2 years ago. Besides mildly elevated gamma-glutamyl transferase levels, her liver function tests were normal. Magnetic resonance cholangiopancreatography revealed a linear filling defect closely resembling an internal stent from the common bile duct to the right intrahepatic bile duct. A live female A. lumbricoides adult worm was removed by endoscopic retrograde cholangiopancreatography (ERCP). Despite a significant decrease of the ascariasis prevalence in Korea, cases of biliary ascariasis are still occasionally reported. In this study, a additional case of biliary ascariasis, which was radiologically misdiagnosed as the biliary stent, was described in a hepatic resection patient by the worm recovery with ERCP in Korea.

An unusual presentation of diabetic ketoacidosis associated with ascariasis and fungal esophagitis: A case report.

RATIONALE: Gastrointestinal symptoms are most commonly observed in patients with diabetic ketoacidosis (DKA), which usually resolves completely with therapy. However, gastrointestinal symptoms may persist after DKA is resolved. PATIENT CONCERNS: A 22-year-old female was admitted to hospital for epigastric pain and persistent vomiting. The results of laboratory examination showed fungal esophagitis complicated by DKA. DIAGNOSIS: The patient was diagnosed with DKA associated with ascariasis and fungal esophagitis. OUTCOME: The patient was discharged after treatment. LESSONS: In this case, despite the correction of metabolic acidosis, persistent nausea, vomiting and dysphagia can be a sign of esophagitis in patients with type 1 diabetes. Therefore, physicians should be aware of fungal infections associated with type 1 diabetes.

Case Report: Laparoscopic Management of Acute Appendicitis Resulting from Ascaris lumbricoides.

Appendicitis is one of the most common conditions that leads to an acute abdomen. Surgical management is the gold standard for therapy. The main cause of appendicitis is an obstruction in the appendicular lumen. Intestinal ascariasis infection is undoubtedly one of the least frequent. We present a female patient with acute appendicitis resulting from Ascaris lumbricoides as an incidental intraoperative diagnosis.

[Ascaris lumbricoides-induced pancreatic lesion]. / Porazhenie podzheludochnoi zhelezy Ascaris lumbricoides.

Ascaris lumbricoides is nematode localized in human small bowel in most cases. This nematode is capable for migration. Pancreatitis is one of the most common surgical diseases. One of the causes of this pancreatitis may be migration of Ascaris lumbricoides from small bowel through the major duodenal papilla to the pancreatic ducts. This migration is provided by complex of morphological and functional disorders of digestive system associated with this helminthiasis, as well as certain risk factors. These are previous cholecystectomy, sphincterotomy, living in endemic areas, massive Ascaris lumbricoides invasion, pregnancy. Damage of the pancreas develops due to direct mechanical effect of nematodes and allergic reactions, papillitis and functional disorders of digestive tract. Pancreatitis is usually mild, but severe course of disease is also possible. A typical symptom of pancreatitis caused by Ascaris lumbricoides is helminths in vomit and/or feces.

Development of allele-specific PCR methodology (AS-PCR) to screening A. lumbricoides and A. suum.

Ascaris lumbricoides and Ascaris suum are described as helminths that infect humans and pigs, respectively. It is estimated that infection by A. lumbricoides affects about 447 million individuals living in tropical regions of developing countries. However, there is an increasing number of cases of human ascariasis in countries with no recent history of autochthonous infection by A. lumbricoides. In these places, pigs have been incriminated as the main source of human infection. Conventional parasitological diagnosis does not allow species-specific identification, and the real epidemiological scenario of human and swine ascariasis is still uncertain. Therefore, this work presents the application of a species-specific molecular diagnosis, based on the allele-specific PCR methodology (AS-PCR), using the Internal Transcript Space 1 (ITS-1) of the ribosomal DNA, as a target for differentiating between the two species, using DNA obtained from eggs. To validate the methodology, stool samples positive for Ascaris spp, were obtained from 68 humans from seven Brazilian states and from six pigs from the state of Minas Gerais. All samples obtained from humans were genotyped as A. lumbricoides and all samples obtained from swine were genotyped as A. suum. These results are in agreement with the literature, which demonstrates that in most endemic regions, transmission cycles are separate. Therefore, the execution of this work allowed the availability of a useful methodology for the differential diagnosis of the species, which may contribute to the characterization of the real epidemiological profile of human and swine ascariasis, and to the implementation of future control strategies.

Longitudinal assessment of the exposure to Ascaris lumbricoides through copromicroscopy and serology in school children from Jimma Town, Ethiopia.

BACKGROUND: We previously demonstrated that serology holds promise as an alternative diagnostic tool to copromicroscopy to monitor and evaluate deworming programs targeting soil-transmitted helminths (STHs). Here we explored the dynamics of anti-Ascaris antibodies (Ab) and evaluated the Ab-isotype of choice to assess the longitudinal exposure to Ascaris in Ethiopian school children. METHODOLOGY: Between October 2018 and February 2020, stool and blood samples were collected every four months from school children (4 to 6 years of age). Stool samples were analyzed by duplicate Kato-Katz to assess the presence and intensity of any STH infection. Plasma Ab-responses against the total extract of Ascaris suum lung third stage larvae were measured through in-house Ab-ELISA's for seven different Ab-isotypes. PRINCIPAL FINDINGS: At baseline, 42.4% of the 66 children were excreting eggs of any STH, Trichuris (37.9%) being the most prevalent. The cumulative prevalence (proportion of children tested that positive at least once over the entire study period) was 56.1% for Trichuris and 31.8% for Ascaris. For Ascaris, re-infections were frequently observed, whereas for Trichuris, children often remained excreting eggs following drug administration. When measuring anti-Ascaris Ab-levels, the cumulative seroprevalence was generally higher (IgG4: 60.6%; IgG1: 50.0%; IgE: 36.4%). The individual anti-Ascaris IgG4 levels at baseline were positively associated with the fecal egg counts averaged over the study period, the rate of egg-appearance and the number of positive test results. There was no apparent cross-reactivity between the anti-Ascaris IgG4 Ab-ELISA and Trichuris. CONCLUSIONS/SIGNIFICANCE: We demonstrate that the children are exposed to STH before the age of four and that the exposure to Ascaris is underestimated when measured with copromicroscopy. Compared to other Ab-isotypes, IgG4 is the Ab-isotype of choice to measure Ascaris exposure in STH endemic settings. Finally, the results also highlight that measuring anti-Ascaris IgG4 levels holds promise as a tool to identify individuals at higher risk for continued exposure to this STH.

Ascaris lumbricoides and the surgical complications: our experience from Medical College Hospital.

BACKGROUND: Intestinal ascariasis is considered as state subject in our valley of Kashmir. The aim of our study was to analyse the age, clinical presentation, diagnosis, surgical complications, and management of Ascaris lumbricoides in children and adults. METHODS: Total of 312 patients above 2 years of age with definite clinical and radiological diagnosis of intestinal ascariasis and associated complications were included in our study. All our patients received anthelminthic drug (Albenzadole 400 mg stat) after termination of acute phase and attached to our Out-Patient Department for follow-up. Each patient was given second dose of antihelminthic drug at second follow-up visit. RESULTS: Total of 312 subjects included 131 (41.99%) males and 181 (58.01%) females. The highest number of patients was in age group of 6-10 years (46.47%). Colicky abdomen pain was the most common presentation and present in 80.12% patients followed by vomiting (64.1%). Palpable worm bolus was a cardinal sign present in 46.47% of our subjects. Two-hundred sixty-five (84.94%) patients responded uneventfully and were relieved of colic and obstruction after conservative non-operative treatment. Twenty (6.41%) patients underwent enterotomy and evacuation of worms, 2 (0.64%) subjects underwent resection anastomosis, and 8 (2.56%) patients had laparotomy and milking of worms into colon. None of patient expired during the study period. CONCLUSION: Ascaris lumbricoides is common cause of acute abdomen in our valley attributed to poor hygiene and low socioeconomic background. In patients of high clinical suspicion of worm obstruction, prompt investigations should be advised to reach a definitive diagnosis and prevent the development of complications. Significant efforts must be channelised at political and society levels for the prevention of this disease. Mass deworming programs should be adopted to overcome this menace.

Food handlers: an important reservoir of protozoans and helminth parasites of public health importance / Manipuladores de alimentos: um importante reservatório de protozoários e helmintos parasitas de importância para a saúde pública

Food handlers plays a primary role in the transmission of pathogenically important protozoans and helminth parasites. This study was aimed to evaluate the prevalence of intestinal pathogenic protozoans and helminth parasites among food handlers in and around University of Malakand, Lower Dir, Pakistan. Stool samples were collected from 642 food handlers (all of male) in a cross-sectional study from January to November, 2017. Wet Mount Techniques and concentration methods by using salt and formol–ether solutions. Three hundred and eighty four cases (59.8%) were found infected with one more parasites. Most of the individuals were found infected with helminth (47.6%) as compared to intestinal protozoans (0.93%). Seventy two cases (11.2%) of the cases presented mixed infection with both intestinal protozoan and helminth parasites. The order of prevalence for intestinal helminth was Ancylostoma duodenale (n = 258, 40.1%), followed by Taeniasa ginata (n=96, 14.9%) Ascaris lumbricoides (n = 54, 8.40%) and Trichuris trichura (n=30, 4.60%). For intestinal protozoa, Entamoeba histolytica/dispar (n = 36, 5.64%) was the only protozoan detected. Mono-parasitism was higher than poly-parasitism. Family size income and education level were the factors significantly (P<0.05) associated in the parasites prevalence. Current research showed that IPIs are primarily the foodborne pathogens still an important public health problem in Pakistan. Effective control programs on parasitic diseases transfer and their associated factors are recommended.

Os manipuladores de alimentos desempenham um papel fundamental na transmissão de protozoários e helmintos parasitas patogenicamente importantes. Este estudo teve como objetivo avaliar a prevalência de protozoários patogênicos intestinais e helmintos parasitas entre manipuladores de alimentos na Universidade de Malakand, Lower Dir, Paquistão. Amostras de fezes foram coletadas de 642 manipuladores de alimentos (todos do sexo masculino) em um estudo transversal de janeiro a novembro de 2017. Técnicas de montagem úmida e métodos de concentração usando soluções de sal e formol-éter. Trezentos e oitenta e quatro casos (59,8%) foram encontrados infectados com mais um parasita. A maioria dos indivíduos foi encontrada infectada por helmintos (47,6%) em comparação com protozoários intestinais (0,93%). Setenta e dois casos (11,2%) dos casos apresentavam infecção mista com protozoários intestinais e helmintos parasitas. A ordem de prevalência de helmintos intestinais foi Ancylostoma duodenale (n = 258, 40,1%), seguido por Taeniasa ginata (n = 96, 14,9%) Ascaris lumbricoides (n = 54, 8,40%) e Trichuris trichura (n = 30, 4,60 %). Para protozoários intestinais, Entamoeba histolytica / dispar (n = 36, 5,64%) foi o único protozoário detectado. Monoparasitismo foi maior do que poliparasitismo. A renda familiar e o nível de escolaridade foram os fatores significativamente (P <0,05) associados na prevalência de parasitos. A pesquisa atual mostrou que os IPIs são principalmente os patógenos de origem alimentar, ainda um importante problema de saúde pública no Paquistão. Programas eficazes de controle da transferência de doenças parasitárias e seus fatores associados são recomendados.

Ascariasis in a 75-year-old man with small bowel volvulus: a case report.

BACKGROUND: Ascaris lumbricoides and Ascaris suum are the most common soil-transmitted helminths of humans and pigs, respectively. The zoonotic potential of A. suum has been a matter of debate for decades. This study was aimed to present a case of human ascariasis caused by A. suum in southern Italy. CASE PRESENTATION: A 75-year-old man presented to the department of surgery in Avellino (southern Italy) complaining of abdominal pain and vomiting. Physical examination revealed bloating and abdominal tenderness. A computed tomography scan showed air-fluid levels and small bowel distension. During exploratory laparotomy a small bowel volvulus with mesenteritis was evident and surprisingly an intraluminal worm was detected. The worm was removed with a small enterotomy and identified as an adult female of A. suum based on morphological and molecular analysis. Faecal examination revealed the presence of unfertilized Ascaris eggs with an intensity of 16 eggs per gram (EPG) of faeces. The patient was treated with mebendanzole 100 mg twice a day for 3 days. The post-operative course was regular with re-alimentation after 3 days and discharge after 12 days. CONCLUSIONS: This report shows as A. suum can function as a relevant agent of human zoonosis. Therefore, in patients with bowel obstruction with no evident aetiology a helminthic infestation should be considered for an accurate diagnosis, especially in patients living in rural areas.

Live ascaris in urinary bladder: a case report.

INTRODUCTION: Ascaris in urinary bladder is an extremely rare phenomenon. It may occur after fistula formation between urinary and gastrointestinal tract or by retrograde migration of adult worm, and is associated with complications. CASE PRESENTATION: A 47-year-old Amhara woman from rural northwest Ethiopia presented with a complaint of difficulty to fully evacuate her bladder of 1 year duration. Ultrasonography showed thickened bladder wall with echo debris. There were also thickened bowel and fluid-filled loops of intestine adjacent to urinary bladder. On cystoscopy examination, there was live ascaris swimming inside the bladder. Enterovesical fistula was entertained and explorative laparotomy performed. Findings confirmed presence of iliovesical fistula. The fistula was divided and the continuity of the intestine restored. The inflammatory mass was subjected to histopathology study and turned out to be benign inflammatory reaction. She was also given antihelminthics. Postoperatively, her course was uneventful, and she was discharged cured. CONCLUSION: Though it is extremely rare to have urinary symptoms from ascariasis, it is important to have a high index of suspicion for all possibilities.