Central Nervous System Infection by Free-Living Nematode Cephalobus cubaensis in a Human Host in Africa

In August 2022, a clergyman in his mid-seventies travelled from the Democratic Republic of Congo to Durban, South Africa to seek medical care, having developed otalgia and tinnitus eight months prior. He had been well previously, did not use tobacco or alcohol, and had no known history of skin, ear or eye problems. There had been no exposure to farm animals or pets. Initial symptoms were followed by a growing mass in the external auditory canal, swelling in the temporomandibular region, otorrhoea, hearing loss and right facial muscle weakness. Over the three months prior to admission he developed confusion, drowsiness, slurred speech, eating difficulties, urge incontinence and inability to walk. Examination showed mild generalised wasting, but no obvious pallor, no head and neck lymphadenopathy, and no skin lesions. A right-side lower motor neuron seventh nerve palsy was present. All other cranial nerves were intact. Otorhinolaryngological examination found trismus, a prominent firm swelling over the right temporomandibular area, and an exophytic fleshy mass in the ipsilateral external auditory canal, with a serosanguinous discharge (Video 1, supplementary information). Laboratory investigations revealed microcytic anaemia (haemoglobin of 8.8 g/dL) and mild eosinophilia of 0.53 x 10⁹/L. Computed tomography of the brain showed erosions of the right temporal bone with a soft tissue mass extending through the middle ear into the external auditory canal (Figure 1 A, B). There was also a minimally-enhancing 3.1 cm diameter hyperdense lesion with surrounding vasogenic oedema in the left parietal lobe (Figure 1 C, D). The differential diagnoses included temporal bone squamous cell carcinoma and lymphoma. Other malignancies or cholesteatoma were thought less likely. Infectious causes considered were necrotising otitis externa, tuberculosis, and fungal skull base osteomyelitis. Histopathology of tissue biopsies of the ear mass revealed an inflammatory background with granuloma formation, central microabscesses and associated nematode larvae, possibly Strongyloides stercoralis. Bone destruction by the inflammatory process was observed, without evidence of malignancy. Lumbar puncture revealed a normal opening pressure and clear colourless cerebrospinal fluid (CSF), with an elevated protein (0.83 g/L), normal glucose (3.9 mmol/L), lymphocyte count of 44/µL, erythrocyte count of 90/µL and no polymorphonuclear cells. No organisms were observed on Gram stain and bacterial culture was negative. C-reactive protein was 69 mg/L, and glycated haemoglobin 5.4 %. HIV-1/2 ELISA and HTLV-1 serology were negative. Cerebrospinal fluid, auditory canal fluid and fresh tissue from the biopsied ear mass were referred to the Parasitology Reference Laboratory (PRL) at the National Institute for Communicable Diseases (NICD) for further investigation. Empiric treatment included albendazole (400 mg bd) and intravenous methylprednisone (750 mg daily for three days), followed by dexamethasone (8 mg daily IVI). The level of consciousness declined. On day six of admission praziquantel (1500 mg tds) and oral ivermectin (18 mg daily) were added. Subsequently the patient developed respiratory failure and died after transfer to the Intensive Care Unit for ventilatory support. At autopsy, external examination showed right temporomandibular area swelling with necrotic material in the external auditory canal. Further dissection revealed extradural necrotic tissue involving the middle and inner ear with erosion of the petrous and squamous parts of the temporal bone extending externally, eroding the mastoid portion of temporal bone (Figure 2 A). Microscopic examination of the brain and meninges showed extensive necrosis, inflammation, and nematodes primarily affecting the left temporal lobe and to a lesser extent, the right temporal lobe, pons, and cerebellum. Meningoencephalitis, with perivascular inflammation comprising lymphocytes and macrophages was present. Adult female nematodes, larvae, and eggs surrounded by a granulomatous inflammatory response were observed within the brain parenchyma, some within blood vessels (Figure 2 B, C). The necrotic material from the right temporomandibular region and brain biopsies showed abundant nematodes. The spinal cord was not examined. The lungs showed bronchopneumonic changes. Nematodes were not seen in any other organs. The cause of death and final diagnosis was that of progressive parasitic meningoencephalitis with terminal bronchopneumonia.

We performed light microscopy on preparations of ear discharge fluid, CSF and ear tissue mass. Direct wet preparations were viewed under low-power magnification (100x). Giemsa-stained preparations were examined at low and higher (500x, 1000x) magnifications.

Samples were planted on blood and malt extract agar plates with lawns of E. coli (ATCC 25922) and incubated at 30°C. Antibiotics were added to inhibit a Proteus sp. bacterial contamination. Minimum Essential Medium (MEM) (ThermoFisher, Johannesburg, SA) with and without gentamicin (0.5 µg/ml), streptomycin (0.1 µg/ml) and ampicillin (50 µg/ml) (Merck, Johannesburg, SA) was also inoculated. Cultures were monitored microscopically.

DNA was extracted from all samples except ear tissue, using the QIAamp DNA mini kit (Qiagen, Hilden, Germany). Strongyloides stercoralis PCR targeting a non-coding repeat region was initially performed, [1] followed by nested PCR using primers targeting the large nuclear subunit ribosomal RNA gene of cephalob nematodes [4]. (Table, supplementary information).

Amplicons from the cephalob PCR were purified using the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany), and submitted for Sanger sequencing (Inqaba Biotec, Pretoria, South Africa). Raw data from all samples were edited and cleaned, to generate a consensus sequence (BioEdit, Informer Technologies, Los Angeles, USA).

The ear mass biopsy sample was prepared using the NEBNext Microbiome DNA Enrichment Kit (New England Biolabs, Massachusetts, USA) to remove human DNA. Original and cultured samples were subjected to whole genome sequencing. Paired-end libraries (2 ×150 bp) were prepared using the Illumina DNA Prep kit (Illumina, San Diego, USA), followed by sequencing on the Illumina NextSeq 2000 platform (Illumina, San Diego, USA). Raw sequence reads were processed with quality checks and removal of low quality and adapter regions by Trim galore v0.6.2 (Babraham Bioinformatics, Cambridge, UK), followed by removal of human host reads using Bowtie2 v2.4.2. [5]. Remaining reads were used for de novo metagenome assembly with MetaSPAdes v3.14.1. [6].The genome completeness of the targeted nematode was evaluated using benchmarking universal single-copy orthologs (BUSCO) [7].

The 18S and 28S rRNA sequences of species from the same nematode clade were accessed from the National Center for Biotechnology Information (NCBI). The rRNA sequences of the sample were extracted from the de novo metagenome assembly results using BAsic Rapid Ribosomal RNA Predictor version 0.9 (https://github.com/tseemann/barrnap). The 18S rRNA sequence alignment (1759 bp) was used for maximum likelihood phylogenetic tree construction with RAxMLv8.2.12, [8] with 1000 bootstrap and GTRGAMMAX model. The partial sequence of the 28S rRNA sequence alignment (1080 bp) was used for maximum likelihood phylogenetic tree construction using IQ-TREE v.2 [9] with 1000 bootstrap and GTR+I+G model.

All tissue samples showed the presence of nematodes. Serous fluid (negative for beta-2-transferrin, therefore excluding CSF) from the auditory canal demonstrated nematode adults, larvae and ova on microscopy (Figure 3). Rhabditiform morphology of the larvae initially suggested Strongyloides or Halicephalobus species. Adult nematodes measured 70 to 270 μm by 10 to 20 μm, showing a smooth cuticle, a rhabditiform oesophagus and a female genital tract; however, absence of a dorsiflexed ovary and double-bulbed oesophagus, and the measured size of all stages, indicated a different free-living nematode species than first thought.

Motile larvae and ova were observed microscopically on the blood agar plate after three days’ incubation (Video 2, supplementary information). There was overgrowth of Proteus sp., which potentially led to failure of subsequent culture attempts.

The following supporting information can be downloaded at the website of this paper posted on Preprints.org. Figure S1. Maximum likelihood 28S rRNA phylogenetic tree showing Cephalobus cubaensis’ relationship to related species with bootstrap support above 70% (GenBank accession numbers in parentheses). The isolate sample in this study is in black text. The scale bar represents the inferred substitutions per nucleotide position. Outgroup comprises Cervidellus alutus and Acrobeles complexus (green text). Table S1. Primer sequences for nested PCR targeting the large nuclear subunit ribosomal RNA gene of cephalob nematodes. Video S1. Otoscopy of the right external auditory canal, showing the exudative inflammatory tissue mass obstructing the canal. Video S2. External auditory canal exudate cultured on blood agar with E. coli lawn, showing an adult gravid female nematode moving on the surface.