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BACKGROUND: Pinworm, Enterobius vermicularis, is a common parasitic illness, particularly among children in Palestine. On rare occasions, it can cause serious consequences such as acute appendicitis, which can still present a challenging diagnosis especially in children. CASE PRESENTATION: This report describes two cases (a 9 year old male and a 11 year old female, both Palestinian children from Hebron district) of acute appendicitis referred to Queen Alia Hebron Governmental Hospital in Palestine. The clinical examination revealed a lower abdominal pain, and they were diagnosed with appendicitis. The pathological examination after appendectomy showed the presence of eggs and pinworms. Anthelminthic medication was provided, and they were followed up for 6 weeks after the operation without any incidents. CONCLUSION: These cases highlight the importance of considering Enterobius vermicularis infestation in children with abdominal pain, as the parasites can mimic appendicitis. Prompt recognition and cautious laparoscopic appendectomy are crucial to prevent unnecessary surgery and peritoneal contamination, with the infestation being easily treatable with anthelmintic medication.
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Antihelmínticos , Apendicectomía , Apendicitis , Enterobiasis , Enterobius , Humanos , Apendicitis/diagnóstico , Apendicitis/parasitología , Apendicitis/cirugía , Enterobiasis/diagnóstico , Enterobiasis/tratamiento farmacológico , Niño , Masculino , Femenino , Diagnóstico Diferencial , Animales , Enterobius/aislamiento & purificación , Antihelmínticos/uso terapéutico , Dolor Abdominal/etiología , Árabes , Medio OrienteRESUMEN
OBJECTIVE: To investigate the prevalence of Enterobius vermicularis infections among children in China from 2016 to 2021, so as to provide insights into enterobiasis control and formulation of the enterobiasis control strategy among children. METHODS: Publications pertaining to the prevalence of E. vermicularis infections among children were retrieved from Wanfang Data, CNKI, VIP and PubMed databases published from January 2016 to June 2023. Eligible publications were screened according to inclusion and exclusion criteria, and the publication bias was evaluated using the assessment tool for prevalence studies proposed by the Joanna Briggs Institute Evidence-Based Practice Resources in Australia. The study period, study areas, study subjects, sample size and number of infections were extracted from publications, and a pooled analysis was performed using a meta-analysis. A meta-regression analysis was performed with the prevalence of E. vermicularis infections as an independent variable, and sample size, source of samples, study area, study method, geographical area and province as dependent variables to identify the source of the study heterogeneity. RESULTS: A total of 66 studies were included, covering 23 provinces (municipalities, autonomous regions) in China, and with the investigations conducted between 2016 and 2021. Meta-analysis showed that the pooled prevalence of E. vermicularis infections was 4.5% [95% confidence interval (CI): (3.1%, 6.0%)] among children in China from 2016 to 2021, and the annual prevalence was 4.1% [95% CI: (2.2%, 6.5%)], 4.2% [95% CI: (2.4%, 6.6%)], 4.2% [95% CI: (2.2%, 6.8%)], 3.2% [95% CI: (1.5%, 5.4%)], 2.3% [95% CI: (0.9%, 4.3%)] and 1.1% [95% CI: (0.4%, 2.1%)] from 2016 to 2021. The pooled prevalence of E. vermicularis infections was 4.9% [95% CI: (3.4%, 6.8%)] in studies with a sample size of < 5 000 cases, which was higher than that in studies with a sample size of 5 000 cases and higher [2.1%, 95% CI: (0.2%, 3.6%)], and the pooled prevalence of E. vermicularis infections was 5.2% [95% CI: (2.9%, 8.2%)] among subjects from schools, which was higher than that among subjects from communities [4.2%, 95% CI: (2.7%, 6.0%)]. The pooled prevalence of E. vermicularis infections was 4.4% [95% CI: (2.8%, 6.2%)] among children included in comprehensive surveillance, which was higher than that among children included in specific surveillance [4.8%, 95% CI: (2.6%, 7.7%)], and the pooled prevalence of E. vermicularis infections was 5.7% [95% CI: (3.8%, 7.8%)] among children included in county-level surveys, which was higher than that among children included in city-[4.8%, 95% CI: (2.3%, 8.0%)] and province-level surveys [1.8%, 95% CI: (0.3%, 4.7%)]. In addition, the pooled prevalence of E. vermicularis infections was higher among children in southern China [11.3%, 95% CI: (7.5%, 15.7%)] than that in central China [5.2%, 95% CI: (2.8%, 8.2%)], eastern China [5.2%, 95% CI: (2.8%, 8.2%)] and southwestern China [2.6%, 95% CI: (1.4%, 4.1%)]. Meta-regression analysis identified geographical area and survey province as factors affecting the study heterogeneity. CONCLUSIONS: Currently, the overall prevalence of E. vermicularis infections is moderate among children in China, and the prevalence varies greatly in regions, with a high prevalence rate in southern China and presence of small-scale clusters. Enterobiasis surveillance and health education pertaining to enterobiasis control are required with adaptations to local circumstance to reduce the prevalence of E. vermicularis infections among children.
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Enterobiasis , Enterobius , Enterobiasis/epidemiología , Enterobiasis/parasitología , China/epidemiología , Humanos , Niño , Prevalencia , Enterobius/fisiología , Enterobius/aislamiento & purificación , Animales , Preescolar , AdolescenteRESUMEN
BACKGROUND: Enterobius vermicularis (E. vermicularis), also referred to as pinworm, is a widespread human intestinal parasite which predominantly occurs in young children, making their caretakers a population at risk for the transmission of this helminth. It can occasionally affect extraintestinal organs and tissues, including the female genital tract. Infestation can be asymptomatic or manifest as different kinds of gynaecological disorders, such as pelvic inflammation mimicking tumours, abnormal uterine bleeding, or vaginitis. Diagnosis is made by identifying ova in the sample collected from the perineal skin using a transparent adhesive tape or microscopic examination of resected tissue. Mebendazole is the first-line medication and should also be administered to all household members. CASE PRESENTATION: We present a case of a patient who had undergone surgery for invasive cervical cancer with an accidental finding of E. vermicularis eggs in the cervix. CONCLUSIONS: Although not very common, infestation with E. vermicularis should be considered in differential diagnoses of various gynaecological disorders accompanied by histological findings of granulomatous inflammation.
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Enterobiasis , Enterobius , Neoplasias del Cuello Uterino , Humanos , Femenino , Enterobiasis/diagnóstico , Enterobiasis/complicaciones , Neoplasias del Cuello Uterino/diagnóstico , Neoplasias del Cuello Uterino/cirugía , Enterobius/aislamiento & purificación , Animales , Mebendazol/uso terapéutico , Cuello del Útero/parasitología , Cuello del Útero/patología , Diagnóstico Diferencial , Persona de Mediana Edad , AdultoRESUMEN
The German cockroach Blattella germanica is commonly infected with the pinworm Blatticola blattae. To determine the effect of pinworm infection on cockroach survival, we artificially altered the pinworm infection status of cockroaches and determined the number of survival days under no-feeding conditions, with and without opportunities for fecal feeding. Four lines of the German cockroach (Wn, Wp, Nn, and Np groups) were used in the experiment. Wp and Np were pinworm-infected lines. Wn and Nn were pinworm-free lines. The 50% survival days of cockroaches in the absence of opportunities for fecal feeding were not significantly different in Wp (3.45 days) vs Wn (3.27), and in Np (4.60) vs Nn (4.48). In contrast, in the presence of fecal feeding, the 50% survival times for the pinworm-infected Wp (4.04) and Np (6.65) were significantly longer than those for the pinworm-free Wn (2.77) and Nn (5.46). The number of survival days without feeding was significantly higher in the pinworm-infected group given the opportunity to eat feces than in the non-infected group. These results suggest that pinworm infection of cockroaches during starvation, in association with fecal feeding, may be associated with longer survival.
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Heces , Animales , Heces/parasitología , Blattellidae/parasitología , Análisis de Supervivencia , Enterobius/aislamiento & purificación , Cucarachas/parasitología , Conducta AlimentariaRESUMEN
BACKGROUND: The aim of this study was to determine and compare the miRNA profile in the immune response with the parasite in pediatric patients with acute appendicitis caused by Enterobius vermicularis and in pediatric patients with enterobiasis. METHODS: A total of 30 tissue samples, which were operated with the diagnosis of pediatric acute appendicitis in the last 10 y and Enterobius vermicularis was detected by histopathological findings, were analyzed. In addition, blood samples were taken from 30 pediatric patients diagnosed with enterobiasis for this study. The miRNAs that activate T and B cells were evaluated by a quantitative real-time PCR, statistically calculated within ΔΔCt values, and fold changes were evaluated by Welch's T test, in which p<0.5 was considered to be significant. RESULTS: It was found that 48 out of 136 (35.3%) miRNAs differed between the pediatric patient and healthy control groups. It was determined that 22 (57.9%) of the different miRNAs were T cell activating miRNAs and 26 (68.4%) were B cell activating miRNAs. While there was a significant difference in miRNA values activating T cells in two patient groups (p<0.01), there was no significant difference in miRNA values activating B cells (p>0.01). CONCLUSIONS: In the study, although Enterobius vermicularis was the causative agent in both patient groups, it was revealed that the immune response of patients with acute appendicitis was more affected than enterobiasis patients.
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Apendicitis , Enterobiasis , Enterobius , MicroARNs , Humanos , Apendicitis/parasitología , Apendicitis/inmunología , Niño , Masculino , Femenino , Animales , Preescolar , Adolescente , Linfocitos B/inmunología , Enfermedad Aguda , Linfocitos T/inmunologíaRESUMEN
Larvae of European rose chafer Cetonia aurata (Linnaeus, 1758) and cockchafer Melolontha sp. beetles were collected in Hungary for parasitological study. Intestinal examination revealed the presence of three well-known thelastomatid nematodes belonging to the genus Cephalobellus Cobb, 1920. We report for the first-time Cephalobellus cuspidatum (Rudolphi, 1814) Leibersperger, 1960, C. osmodermae Leibersperger, 1960, and C. potosiae Leibersperger, 1960 in Hungary, all found in scarab beetle larvae. Due to incomplete original descriptions, a comprehensive redescription with detailed morphological data is presented. Additionally, an identification key for closely related Cephalobellus, Thelastoma and Severianoia species infesting scarab beetles worldwide is provided. Newly generated 18S and 28S rDNA gene sequences of C. osmodermae place it as one of the early branches within Thelastomatidae.
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Escarabajos , Nematodos , Animales , Enterobius , Hungría , Especificidad de la EspecieRESUMEN
Cockroaches have been identified as mechanical carriers of pathogens capable of infesting humans. The primary objective of this research was to detect and separate medically significant parasites found within the collected cockroaches. A total of 185 cockroaches were captured. In the laboratory, cockroaches underwent a series of steps. Initially, they were washed in a saturated salt solution and rinsed with 70% alcohol, dried, and subsequently dissected to examine endoparasites. In this study, a transport rate of 29.729% was documented, and four types of helminths were identified, which included Ascaris lumbricoides (47.27%), Enterobius vermicularis (30.91%), Trichuris spp. (7.27%), and Hymenolepis nana (14.55%). The parasites were more frequently found on the external surface of cockroaches (38/69.09%), compared to the internal surface (17/30.91%). Cockroaches captured in toilets were found to carry a higher percentage of parasites (31/16.75%) compared to those from kitchens (11/5.94%) and houses (13/7.02%). These findings suggest that cockroaches can serve as carriers for intestinal parasites and potentially act as mechanical agents in the dissemination of these parasites.
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Cucarachas , Parásitos , Humanos , Animales , Ascaris lumbricoides , Enterobius , EtanolRESUMEN
Enterobius vermicularis is the most common helminthic infection in the United States, infecting an estimated 40 million persons. A rare complication of E. vermicularis infestation is appendicitis. The diagnosis must be considered during pathologic examination to ensure appropriate treatment with anti-helminthic therapy.
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Apendicitis , Apéndice , Enterobiasis , Animales , Humanos , Enterobius , Apendicitis/diagnóstico , Apendicitis/complicaciones , Apendicectomía , Enterobiasis/complicaciones , Enterobiasis/diagnóstico , Enterobiasis/tratamiento farmacológicoRESUMEN
Myelitis is a rare inflammatory myelopathy, and known associated etiologies only account for a small number of causes. A significant percentage of cases have an unknown etiology and are considered idiopathic. With 64% to 68% of cases fitting into the idiopathic category, helminth infections, and specifically pinworm parainfections, should be considered in cases that would otherwise be classified as idiopathic. This case report outlines a pediatric patient diagnosed with myelitis given her progressive weakness, fussiness, refusal to bear weight as well as magnetic resonance imaging (MRI) demonstrating T2-hyperintense signal and/or T1 gadolinium enhancement, and/or positive cerebrospinal fluid (CSF) inflammatory markers. This patient had a negative evaluation for typical known etiologies for myelitis including no signs of multiple sclerosis and neuromyelitis optica spectrum disorder on brain MRI, oligoclonal banding and aquaporin-4 autoantibodies, and no evidence of bacterial or viral meningitis given normal cell counts and cultures in CSF. She was found to have a pinworm infection, suggesting a parasitic parainfectious etiology of her myelitis. This case outlines the first case noting the correlation between myelitis and pinworm infection in a pediatric patient.
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Enterobiasis , Mielitis Transversa , Mielitis , Neuromielitis Óptica , Femenino , Animales , Humanos , Niño , Mielitis Transversa/diagnóstico por imagen , Mielitis Transversa/etiología , Enterobius , Enterobiasis/complicaciones , Medios de Contraste , Gadolinio , Mielitis/complicaciones , Mielitis/diagnóstico por imagen , Imagen por Resonancia Magnética/métodos , Autoanticuerpos/líquido cefalorraquídeo , Acuaporina 4RESUMEN
Neotropical primates (Platyrrhines) are commonly parasitized by pinworm nematodes of the genus Trypanoxyuris Vevers, 1923. The taxonomic identity of Trypanoxyuris sampled in night monkeys (Aotus Iliger) has been rather controversial. Two species have been described, namely T. microon (Linstow, 1907) and T. interlabiata (Sandosham, 1950). The latter was synonymized with T. microon considering that the observed morphological differences corresponded to different developmental stages of the nematode rather than to differences between both species. Here, we used an integrative taxonomy approach, based on morphological and molecular data along with host identity, in order to assess the validity of both species. Our results evidenced that these different morphotypes correspond to different and reciprocally monophyletic groups; thus, we propose the resurrection of T. interlabiata. We redescribe both pinworm species using specimens sampled in Aotus monkeys from Colombia and discuss the advantages of combining molecular and morphological data to uncover pinworm diversity, and to understand the potential forces determining the diversification process in pinworms from platyrrhine primates.
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Enterobius , Oxyuroidea , Animales , Aotidae , Especificidad de la Especie , Oxyuroidea/genética , Oxyuroidea/anatomía & histología , PrimatesRESUMEN
Enterobius vermicularis, an intestinal helminth, is transmitted through the ingestion of eggs found in food, water, dust, or other fomites, including infected individuals. This review aimed to examine the frequency and distribution of E. vermicularis infections in Brazil between 1991 and 2022. The conducted bibliographic survey revealed that the frequency of E. vermicularis infections in Brazil ranged from 0.1 to 26.1%, depending on factors such as population ethnicity, individual age group, geographic area, time frame, and diagnostic method. However, these findings were based on a limited number of publications, suggesting that the actual prevalence rates of E. vermicularis infection may still be unknown and potentially underestimated.
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Enterobiasis , Helmintiasis , Parasitosis Intestinales , Humanos , Animales , Enterobius , Brasil/epidemiología , Helmintiasis/epidemiología , Parasitosis Intestinales/diagnóstico , Parasitosis Intestinales/epidemiología , Prevalencia , Enterobiasis/diagnóstico , Enterobiasis/epidemiologíaRESUMEN
Phthorimaea absoluta (Meyrick) (= Tuta absoluta) (Lepidoptera: Gelechiidae), is the most damaging insect pest threatening the production of tomato and other solanaceous vegetables in many countries. In this study, we predicted the risk of establishment and number of generations for P. absoluta in the current and future climatic conditions under two Shared Socioeconomic Pathways (SSP2-4.5 and SSP5-8.5) of the years 2050 and 2070 using insect life cycle modelling (ILCYM) software. We used a temperature-dependent phenology model to project three risk indices viz., establishment risk index (ERI), generation index (GI), and activity index (AI) based on temperature data. The model projected large suitable areas for P. absoluta establishment in the Southern hemisphere under current and future climatic scenarios, compared to the Northern part. However, the risk of P. absoluta is expected to increase in Europe, USA, Southern Africa, and some parts of Asia in the future. Under current conditions, P. absoluta can complete between 6 and 16 generations per year in suitable areas. However, an increase in GI between 1 and 3 per year is projected for most parts of the world in the future, with an increase in AI between 1 and 4. Our results provide information on the risk of establishment of P. absoluta which could guide decision-makers to develop control strategies adapted for specific agro-ecological zones.
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Lepidópteros , Mariposas Nocturnas , Solanum lycopersicum , Animales , Enterobius , Cambio Climático , LarvaRESUMEN
Background: Human infection with Enterobius vermicularis occurs worldwide. The most common clinical manifestation of a pinworm infection is an itchy anal region. This parasite is incidentally found in appendicitis. This study aims to characterize and genotype this parasite from different samples inferred by mt-DNA. Methods: Forty appendectomies for acute clinical appendicitis, 40 positive scotch-tape samples, and 10 adult females worm isolated from patients. Genetic differentiation, haplotype differences, and isolates population structure were analyzed based on the cytochrome c oxidase subunit I (cox1) gene. Results: It has been demonstrated that all isolations in the appendectomies specimens are similar, and the genetic difference divergence is seen in adult worm specimens. The neutral indices of the samples did not show a significant difference and show that there is no intra-specific and population distribution diversity. Conclusion: Our results show different haplotypes in the B type of E. vermicularis population and add new information about genotyping of these parasites in Iran. In comparison with other studies, intra-specific variation of this parasite from Iran was observed.
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Apendicitis , Enterobiasis , Adulto , Animales , Femenino , Humanos , Enterobius/genética , Irán/epidemiología , Enterobiasis/epidemiología , Enterobiasis/parasitología , Haplotipos , Enfermedad AgudaRESUMEN
OBJECTIVE: To investigate the prevalence and influencing factors of soil-transmitted nematode infections in Ningbo City from 2016 to 2021, so as to provide insights into the development of targeted control measures against soil-transmitted nematodiasis. METHODS: Permanent residents at ages of 3 years and older were sampled in Ningbo City using a multi-stage sampling method each year during the period from 2016 to 2021. Soil-transmitted nematode eggs were detected in stool samples using a modified Kato-Katz thick smear method (two slides for each stool sample), and Enterobius vermicularis eggs were additionally identified among children at ages of 3 to 12 years using the adhesive cellophane-tape perianal swab method. The time- and regions-specific prevalence of soil-transmitted nematode infections was calculated, and the factors affecting hookworm infections were identified using a multivariate logistic regression model. RESULTS: A total of 11 573 person-times were detected for soil-transmitted nematode infections in Ningbo City from 2016 to 2021, and 296 egg-positives were detected, with a mean prevalence rate of 2.56% [95% confidential interval (CI): (2.28%, 2.87%)]. Hookworm was the predominant species of soil-transmitted nematode among egg-positives in Ningbo City (98.31%, 291/296), and there was a significant difference in the prevalence of hook-worm infections among years (χ2 = 190.27, P < 0.01). The highest prevalence of hook-worm infections was observed in Ninghai County (4.06%), and there was a region-specific prevalence rate of hookworm infection in Ningbo City (χ2 = 148.43, P < 0.01). Multivariate logistic regression analysis showed that elderly residents at ages of over 60 years [odds ratio (OR)= 1.94, 95% CI: (1.07, 3.54), P < 0.05], males [OR = 2.19, 95% CI: (1.72, 2.80), P < 0.01], farmers [OR = 6.94, 95% CI: (3.37, 14.29), P < 0.01] and residents with a low education level [illiteracy or semi-illiterate: OR = 3.82, 95% CI: (1.56, 9.35), P < 0.05; primary school: OR = 2.70, 95% CI: (1.11, 6.59), P < 0.05] were at a higher risk for hookworm infections. CONCLUSIONS: The overall prevalence of soil-transmitted nematode infections was low among residents in Ningbo City from 2016 to 2021. The surveillance and health education for human hookworm disease remain to be reinforced among male farmers with a low education level at ages of over 60 years in Ninghai County.
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Nematodos , Infecciones por Nematodos , Anciano , Animales , Niño , Humanos , Masculino , China/epidemiología , Enterobius , Heces , Infecciones por Nematodos/epidemiología , Prevalencia , Suelo , Femenino , Preescolar , Persona de Mediana EdadRESUMEN
The tomato pinworm, Tuta absoluta, or Phthorimaea absouta, is native to South America, but quickly spread to other regions of world, including Europe, Africa, and Asia, devastating to global tomato production. However, a lack of high-quality genome resources makes it difficult to understand its high invasiveness and ecological adaptation. Here, we sequenced the genome of the tomato pinworm using Nanopore platforms, yielding a genome assembly of 564.5 Mb with contig N50 of 3.33 Mb. BUSCO analysis demonstrated that this genome assembly has a high-level completeness of 98.0% gene coverage. In total, 310 Mb are repeating sequences accounting for 54.8% of genome assembly, and 21,979 protein-coding genes are annotated. Next, we used the Hi-C technique to anchor 295 contigs to 29 chromosomes, yielding a chromosome-level genome assembly with a scaffold N50 of 20.7 Mb. In sum, the high-quality genome assembly of the tomato pinworm is a useful gene resource that contributes to a better understanding of the biological characteristics of its invasiveness and will help in developing an efficient control policy.
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Enterobius , Solanum lycopersicum , Animales , Enterobius/genética , Solanum lycopersicum/genética , Anotación de Secuencia Molecular , Genoma , Cromosomas , FilogeniaRESUMEN
OBJECTIVE: Enterobius vermicularis is a common gastrointestinal parasite. Acute appendicitis is one of the most common clinical conditions requiring surgical intervention. The coexistence of enterobiasis and acute appendicitis has attracted the attention of researchers for a long time. In this article, it is aimed to present the characteristics of enterobiasis cases detected in appendectomy specimens in a tertiary healthcare institution. In addition, it is aimed to reveal the differences by comparing the cases diagnosed with acute appendicitis without enterobiasis. METHODS: All cases operated for acute appendicitis between January 1, 2008 and December 31, 2021 were retrospectively evaluated through the hospital registry system. The cases with histopathologically determined E. vermicularis were reviewed and a study group was formed. The control group consisted of patients diagnosed with acute appendicitis for reasons other than enterobiasis, matched with the study group in terms of age and gender. Complete blood count, C-reactive protein (CRP) ultrasonography evaluations were compared between the two groups. RESULTS: During the study period, 3.650 appendectomy materials were examined; E. vermicularis was detected in 1.32% (n=48) cases, and acute appendicitis was present in 33.33% (n=16) of the cases. Lymphoid hyperplasia was the most common histopathological change in the study group (n=25; 52.08%). When compared to the control group, leukocyte, neutrophil, CRP, ultrasonographic appendix diameter values were found to be significantly higher in the control group, and the percentage of eosinophils in the study group (p<0.0001; p<0.0001; p<0.0001; p<0.0001; p=0.001; respectively). CONCLUSION: Enterobiasis is a common parasite especially in children. Rarely, it can be seen in patients with acute appendicitis. Histopathological evaluation is necessary to ensure a safe surgical procedure and to complete the treatment with anthelmintic drugs. Enterobiasis should be kept in mind in cases when the acute phase response is lower than expected.
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Apendicitis , Apéndice , Enterobiasis , Niño , Animales , Humanos , Enterobiasis/diagnóstico , Enterobiasis/epidemiología , Enterobiasis/complicaciones , Apendicitis/cirugía , Apendicitis/parasitología , Estudios Retrospectivos , Apéndice/parasitología , Apendicectomía , Enterobius , Proteína C-Reactiva , Enfermedad AgudaRESUMEN
This study aimed to evaluate the positive rates for Enterobius vermicularis eggs among preschool children in Yeosu-si, Jeollanam-do, the Republic of Korea (Korea) over a period of 5 years (2017-2021). Perianal swab samples, obtained using cellotape, from 10,392 preschool children in 26 districts were examined microscopically for E. vermicularis eggs. The test results were notified through the local health center, and the families of children who tested positive were advised to provide them anthelmintics treatment. The annual positive rates were 5.0%, 5.2%, 4.4%, 2.2%, and 1.0% in 2017, 2018, 2019, 2020, and 2021, respectively. The overall positive rate was higher in boys than in girls (P< 0.05), and children aged 5-7 years were at a higher risk of being infected than those aged 0-4 years (P< 0.05). Although the rates of infection by E. vermicularis in the survey area, Yeosu-si, were still in the 1% range , the results of this study suggest that they can be significantly reduced through continuous intervention centered around the test-treatment strategy.