Disseminated ascariasis infection with encephalopathy: Molecular confirmation and phylogenetic insights

Abstract

Ascariasis remains a major parasitic infection in low-resource regions, with children at highest risk. We describe a rare and severe pediatric case from rural Colombia involving intestinal obstruction, peritonitis, cholecystitis, and encephalopathy. A recovered helminth underwent stereoscopic examination and molecular characterization through amplification of 18S-rRNA and COII genes, followed by long-read sequencing using Oxford Nanopore technology. Taxonomic analysis with Centrifuge and BLAST confirmed the specimen as Ascaris, showing high similarity to Ascaris suum, while COII phylogenetics clustered it with Ascaris lumbricoides. The sequence formed a monophyletic group with Ascaris suum, Ascaris lumbricoides, and Parascaris spp., suggesting potential hybridization or cryptic diversity within Ascaris. The child required surgical management, albendazole, and supportive care including ECMO, ultimately recovering without neurological sequelae. This case highlights the zoonotic potential and extremely rare neurological manifestations of Ascaris infection and demonstrates the diagnostic value and challenges of long-read sequencing in resolving complex taxonomic relationships.

Highlights

• •Ascariasis remains a significant pediatric health concern in low-resource settings.
• •Severe ascariasis in children can involve the central nervous system.
• •Manifestations from Ascaris lumbricoides and Ascaris suum can be indistinguishable.
• •Improved molecular methods are needed to enhance taxonomic precision.

Introduction

Ascariasis is among the most prevalent neglected tropical disease worldwide, affecting millions of individuals with a global prevalence estimated at 11 %. Transmission primarily occurs through ingesting food contaminated with embryonated eggs, via a fecal-oral route [1]. Children are particularly vulnerable to complications due to higher parasite burdens, which can lead to intestinal obstruction, extra-intestinal migration, and malnutrition [2]. After ingestion, larvae hatch from the eggs, penetrate the intestinal mucosa, migrate through the circulation to the lungs, and are then swallowed, maturing into adult worms in the small intestine. While extra-intestinal migration to various sites, including the hepato-biliary-pancreatic system, respiratory system, urinary bladder, and rarely the brain, has been documented, neurological involvement is rare [3]. Few cases of neurological complications related to Ascaris species have been reported, primarily involving Ascaris lumbricoides, with only one instance possibly caused by Ascaris suum [4], [5]. In this report, we describe the case of a 2-year-old with Ascaris superinfection presenting with intestinal obstruction, cholecystitis, peritonitis, and associated encephalopathy, in which Ascaris spp. was confirmed by molecular detection as the etiological agent.

Case report

A 2-year-old female toddler from Turbo, Antioquia, a tropical area located on the Atlantic coast of Colombia, presented with a three-week history of abdominal pain, intermittent diarrhea, malaise, and progressive clinical deterioration. Her past medical history was notable for mild malnutrition, an incomplete immunization scheme, a history of geophagia, and previous residence in a rural zone with close contact with farm animals.

After multiple visits to various local ambulatory healthcare centers, she was admitted to the Pediatric Intensive Care Unit (PICU). Exploratory laparotomy revealed intestinal obstruction due to the presence of adult ascarid worms and necrosis in the ileum-jejunal zone. Subsequently, she underwent small bowel resection and received anthelmintic therapy with Piperazine citrate. Although piperazine was administered, it is important to note that current first-line therapy for ascariasis consists of single-dose oral albendazole, mebendazole, or ivermectin, which have demonstrated high efficacy; however, these agents were not available in the rural, underserved setting where the patient initially received care. The patient developed multiorgan dysfunction syndrome (MODS) and experienced a 3-min cardiac arrest, responding well to resuscitation. She was then transferred to Bogota (the capital) for extracorporeal membrane oxygenation (ECMO). Subsequent exploratory abdominal surgery revealed abundant ascarids helminths in the abdominal cavity with associated peritonitis. Intestinal resection, removal of nematodes, cholecystectomy, and intra-abdominal lavage and drainage were performed.

Laboratory testing on admission revealed cell blood count (CBC) with moderate leukocytosis (18.4 cel × 10⁹/L reference range: 5.5 – 15.5 × 10⁹/L), anemia (hemoglobin 9.3 g/dL reference range: 10.5 – 13.5 g/dL), and thrombocytopenia (51 ×10⁹/L. reference range: 150 – 450 10⁹/L), elevated procalcitonin (19.6 ng/mL reference range: <0.05 ng/mL), and C-Reactive Protein values (11.2 mg/dL reference range: < 1 mg/dL),), liver (alanine aminotransferase 307 U/L reference range: 5 – 45 U/L) and kidney dysfunction (blood urea 67.18 md/dL reference range: 5 – 18 mg/dL; and creatinine 1.2 mg/dL reference range: 0.2 – 0.5 mg/dL), low fibrinogen (91 mg/dL reference range: 200 – 400 mg/dL), elevated ferritin (5072 ng/mL reference range: 10 – 140 ng/mL)), hyponatremia (serum sodium127 mEq/L reference range: 135 – 145 mEq/L), and hypoalbuminemia (2.2 g/dL reference range: 3.5 – 5.2 g/dL), elevated lactic dehydrogenase (3037 U/L reference range: 160–370 U/L), negative HIV 1 and 2 antibodies, and normal levels of immunoglobulins G, A, M, and E.

Chest X-rays revealed bilateral alveolar opacities, more intense in the right lobe. Initial simple abdominal images showed no relevant findings, but later detection of roundworms in the biliary tree was observed on Magnetic Resonance Imaging (MRI). Broad-spectrum antibiotics were initiated, including linezolid, meropenem, and caspofungin, with initial negative blood, urine, and peritoneal cultures. Several nematodes were removed from the gastric tube during the first two days in the PICU.

On the second day of ECMO therapy, a simple and contrasted brain CT scan revealed multiple focal, hypertense, cortical and subcortical lesions located in the occipital and parieto-temporal areas), the etiology of which remained unclear, possibly related to embolic events or vasculitis secondary to Ascaris migration or ECMO. (Fig. 1) Albendazole therapy was continued for four weeks, along with corticosteroid therapy with sequential tapering doses. A brain MRI performed after 4 weeks of admission revealed residual brain lesions related to hemorrhages and suspected septic emboli with mild brain atrophy. (Fig. 2)

Fig. 1.

Head CT with contrast 1 A axial and 1B-1C coronal showing multiple focal hyperdense cortical and subcortical lesions in the occipital and parieto-temporal regions.

Fig. 2.

Brain MRI Fig. 2A T1 and Fig. 2B-2C T2 were performed four weeks after admission, showing residual lesions consistent with prior hemorrhages and suspected septic emboli, along with mild cerebral atrophy.

During her hospitalization, the patient developed multiple infectious complications, including infections with Pseudomonas aeruginosa, Enterococcus faecium, and Candida parapsilosis, requiring specific antibiotic therapy. However, she experienced progressive clinical recovery and was successfully removed from ECMO after 10 days. Two months post-admission, she was discharged home without evident neurological involvement.

Methods and results

One of the helminths retrieved from the gastric tube was sent for processing. Stereoscopic analysis to morphologically identify the parasite revealed three lips surrounding the mouth at the anterior end of the parasite, consistent with the genus Ascaris. (Fig. 3)

Fig. 3.

Photograph of the helminth isolated from the patient's intestines.

DNA extraction was conducted using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany), following the manufacturer’s instructions. Subsequently, a PCR assay targeting the small ribosomal subunit gene of 18S rRNA and the Cytochrome oxidase II unit were performed. To enhance amplicon size and ensure better taxonomic characterization, we employed a combination of previously reported primers: Nem_18S_F (5’-CGCGAATRGCTCATTACAACAGC-3’) and 1289-R (5’- ACTAAGAACGGCCATGCACC-3’) [6], [7]. The concentration of DNA and amplicons was quantified using the Qubit 4 Fluorometer, with DNA integrity evaluated through 1.5 % agarose gel electrophoresis.

Sequencing libraries were constructed from the obtained amplicons and subjected to long-read sequencing on the Oxford Nanopore MinION platform using the MinKNOW 23.04.5 application. Raw Fast5 files underwent base-calling and demultiplexing using Guppy, followed by quality and length filtration to eliminate potential chimeric and low-quality sequences.

For bioinformatics analyses, a custom database was constructed using 18S-rRNA and COII sequences from nematodes and trematodes available in NCBI. Taxonomic assignment was performed via Centrifuge, utilizing the custom database and considering reads with a minimum length of 800 bp. The results were visualized using Pavian [8] and further validated through BLAST [9].

Reads were aligned using Minimap2 (version 2.24) to a selected reference sequence of 18S-rRNA for Ascaris suum (GenBank: U94367.1). A Variant Call Format (VCF) file was generated from the mapped sequences using Samtools (v 1.17) to identify differences compared to the reference sequence. The consensus sequence was then generated using Bedtools (version 2.25.0), serving as the basis for subsequent analyses.

A phylogenetic reconstruction was conducted to assess the evolutionary relationships between the obtained Ascaris sequence and other Ascaridomorpha groups. Representative sequences for Asacaridodea, Cosmocercoidea, Heterakoidea, and Seuratoidea species were retrieved from NCBI. Multiple sequence alignment was performed using MAFFT v7.407, followed by the construction of a maximum likelihood (ML) tree using IQ-TREE multicore v1.6.12. The phylogenetic tree was visualized using Interactive Tree Of Life (iTOL) v5. (Fig. 4)

Fig. 4.

Phylogenetic Reconstruction of Ascaridomorpha. This figure depicts the phylogenetic relationships of the consensus sequence produced in this study and other Ascaridomorpha species. Nodes supported by a bootstrap value greater than 75 are indicated.

Taxonomic assignment of reads obtained through long-read sequencing using Oxford Nanopore technology was conducted using Centrifuge against a custom database comprising 18S-rRNA gene sequences of nematodes and trematodes from NCBI, revealing clustering with Ascaris suum a parasite responsible for infections in pigs. The high phylogenetic proximity to the parasite Ascaris lumbricoides, coupled with low genetic and morphological divergence, could potentially explain the infection of this parasite in humans and its subsequent erratic migration to the brain [10], [11]. The bioinformatics findings support the observations made through stereoscopic analysis.

Phylogenetic analysis revealed the evolutionary relationships of the generated consensus sequence with other reported Ascaridomorpha. (Fig. 4) The Ascaris sequence reported here falls within the superfamily Asacaridodea, forming a monophyletic group with Ascaris (A. suum and A. lumbricoides) and Parascaris species (bootstrap = 89). Additionally, our sequence demonstrates phylogenetic distinction from other Ascaridoidea genera, such as Toxocara, Anisakis, and Contracaecum. (Fig. 4) However, when observing the results of COII, the sequence clustered with A. lumbricoides and not with A. suum. These results posed a taxonomic conflict, leading to identification only to genus-level (Ascaris spp.).

Discussion

Ascaris infection is among the most common helminthic infections worldwide. Humans are the definitive hosts for Ascaris lumbricoides, while Ascaris suum typically infects swine; however, Ascaris suum may also infect humans [12]. Originally thought to be the same species, Ascaris lumbricoides and Ascaris suum are now recognized as two different but closely related species of ascarid helminths, which for years have been at the center of a taxonomic debate [1]. Overtime, a number of different hypotheses have offered different perspectives on the relationship between A. lumbricoides and A. suum, shedding light on their possible evolutionary history and conspecificity [11]. Possible evolutionary scenarios include the possibility that these two species could have originated from a common ancestor through a speciation event occurring before humans domesticated pigs or that A. lumbricoides in humans might have directly evolved from A. suum, the pig-infecting species, with A. suum persisting as an ancestral species subsequent to the formation of A. lumbricoides [11]. Conversely, A. suum may have directly evolved from A. lumbricoides, with A. lumbricoides as the persistent ancestor and A. suum as the newly derived species [11].

Recent research has prompted a reassessment of Ascaris transmission dynamics, particularly in regions where both Ascaris lumbricoides and Ascaris suum are endemic. Molecular studies have distinguished between the two species, challenging previous assumptions. Evidence indicates significant cross-species transmission between humans and pigs in certain areas, leading to the recognition of A. suum as a zoonotic agent [13], [14], [15]. In this case, the patient is from a rural area endemic for A. lumbricoides, dominated by agricultural settlements, and reports having had close contact with farm animals, including pigs.

Our findings provide valuable insights into the taxonomic and phylogenetic relationships of the helminth identified in this study. Using long-read sequencing and the Centrifuge classifier against a curated 18S-rRNA database, we were unable to confidently reach a taxonomic assignment to a species level. Interestingly, COII-based phylogenetic analysis revealed a closer relationship to A. lumbricoides than A. suum, suggesting the potential presence of cryptic diversity or hybridization events within Ascaris spp. These findings support the hypothesis that the helminth belongs to the Ascaris genus while highlighting the complexities in distinguishing between A. suum and A. lumbricoides. Further comparative genomic analyses could elucidate the evolutionary dynamics and host associations within this group, providing a deeper understanding of their zoonotic and ecological significance.

Cases of ascariasis caused by A. lumbricoides and A. suum can be clinically indistinguishable [12]. Patients may be asymptomatic, but common symptoms include abdominal pain and distension, along with nonspecific gastrointestinal symptoms such as diarrhea, vomiting, and anorexia. Gastrointestinal complications include bowel obstruction and biliary-pancreatic involvement, such as pancreatitis, cholangitis, and cholecystitis. Extraintestinal manifestations are rare and usually occur in cases of superinfection, leading to respiratory and, more rarely, neurological involvement [12], [13], [14], [15], [16].

Neurological manifestations of ascariasis are extremely rare, with only a few reports in the medical literature [4], [17]. In a study conducted by Tatter and Hopkins, two cases involving intestinal A. lumbricoides accompanied by cerebral manifestations were reported. The first case involved a 53-year-old Syrian woman with intestinal A. lumbricoides who experienced a generalized seizure and exhibited a cerebral lesion that deteriorated over several days but resolved after treatment. The second case involved a 2-year-old girl who presented with fever and progressive irritability. Unfortunately, she passed away due to a brain abscess, despite having passed an adult A. lumbricoides in her stool five days earlier [4].

Subsequently, Inatomi et al. reported a case involving a 35-year-old patient presenting with encephalopathy characterized by drowsiness, quadriparesis, eosinophilia, elevated serum IgE levels, and MRI findings showing multiple cerebral cortical and white matter lesions. The diagnosis of A. suum infection was established through a multiple-dot enzyme-linked immunosorbent assay (ELISA) involving 12 different parasite antigens. The assay exhibited strong reactivity to A. suum antigen. Additionally, a double diffusion test revealed prominent precipitin bands against A. suum antigen and faint bands against A. lumbricoides, while no reaction was observed against antigens from Toxocara canis or Trichinella spiralis. These findings strongly suggested A. suum as the likely causative agent, although molecular confirmation was not performed at the time [17].

The marked elevation of procalcitonin observed in this case may be attributable to several mechanisms, including tissue migration facilitating translocation of gut flora, bacterial invasion through ulcerated mucosa, or subsequent transient bacteremia and sepsis [18], [19], [20]. Furthermore, previous studies have suggested that Ascaris infection can increase host susceptibility to bacterial pathogens [20], thereby heightening the risk of systemic infection—a phenomenon that may explain the recurrent infections observed during the patient’s hospitalization. These findings highlight the need for further investigation into the potential utility of procalcitonin as a biomarker for systemic complications associated with Ascaris infection.

Molecular investigations employing the ribosomal internal transcribed spacer (ITS) region and the mitochondrial cytochrome c oxidase subunit-1 (COX-1) gene have been used to distinguish between A. lumbricoides and A. suum. In this study, we adopted a Next-Generation Sequencing (NGS) methodology utilizing the 18S rRNA gene (18S rDNA) to enhance taxonomic precision and to accurately classify the parasite, ultimately being unable to confirm its identification at a species level. This study highlights the genomic complexity governing species-level variations leading to nuclear-mitochondrial discordance, reigniting the single/separate species debate and / or potential hybridization instances. Further whole genome sequencing studies are needed to provide genome-wide resolution to address this longstanding taxonomic conundrum.

Conclusions

This case of superinfection with encephalopathy represents a rare clinical manifestation of helminthic infection. Regardless of the specific species involved, it underscores the potential for zoonotic transmission of these pathogens. Additionally, it highlights the importance of raising physician awareness about extra-intestinal ascariasis and improving recognition of the rare but significant neurological complications associated with Ascaris infections in the most vulnerable pediatric population.

Ethics Approval and Consent

This case report was conducted in accordance with institutional and ethical standards. Written informed consent was obtained from the patient for publication of this case report and any accompanying images. Patient anonymity has been preserved.

Authorship and Contributions

All authors certify that they meet the authorship criteria of the International Committee of Medical Journal Editors (ICMJE). Each author has made substantial contributions to the conception and design of the work, acquisition of data, analysis or interpretation of data, drafting and/or critical revision of the manuscript, and has approved the final version for submission.

Originality

The authors confirm that this manuscript is original, has not been published previously, and is not under consideration for publication elsewhere, in whole or in part.

Copyright and Publishing Agreement

The authors agree to the terms of the Elsevier Journal Publishing Agreement. Copyright will be transferred to Elsevier upon acceptance of the manuscript, or the article will be published under the selected open access license, in accordance with the journal’s policies.

Author Approval

All authors have reviewed and approved the final manuscript and agree to its submission to IDCases.

Funding

The costs of personal, sequencing, and bioinformatic analyses were funded by by Vicerrectoría de Investigación e Innovación from Universidad del Rosario, Colombia. This project was also supported by internal departmental funds from the Department of Pathology, Molecular and Cell-Based Medicine at the Icahn School of Medicine at Mount Sinai.

CRediT authorship contribution statement

Santiago Correa: Visualization, Data curation, Conceptualization. Ramirez Juan: Writing – review & editing, Writing – original draft, Methodology, Investigation, Data curation, Conceptualization. Suarez Jose: Writing – review & editing, Writing – original draft, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Ruth Garcia-Redondo: Writing – review & editing, Writing – original draft. Paniz-Mondolfi Alberto Enrique: Writing – review & editing, Writing – original draft, Visualization, Methodology, Investigation, Data curation, Conceptualization. Alvarez-Olmos Martha: Writing – review & editing, Writing – original draft, Methodology, Formal analysis, Data curation, Conceptualization. Patiño Luz: Methodology, Investigation, Data curation, Conceptualization. Mauricio Sarta-Mantilla: Writing – original draft, Data curation, Conceptualization. Carolina Hernandez: Writing – review & editing, Writing – original draft, Methodology, Investigation, Conceptualization. Ramirez Angie: Writing – review & editing, Writing – original draft, Methodology, Formal analysis.

Declaration of Generative AI and AI-assisted technologies in the writing process

During the preparation of this work the author(s) used ChatGPT, a language model trained by OpenAI, to providing helpful insights and corrections during the process of improving the drafting and readability. After using this tool/service, the author(s) reviewed and edited the content as needed and take(s) full responsibility for the content of the publication.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.