Molecular Identification of Hypoderma sinense Cutaneous Myiasis in a Child from a Cattle-Raising Pastoral Region of China: A Case Report

Technical Summary: Molecular Identification of Hypoderma sinense Cutaneous Myiasis in a Child

Problem Statement
Cutaneous myiasis, an infestation by dipterous larvae, often mimics common pediatric skin infections such as furunculosis (boils), leading to delayed recognition and misdiagnosis. While the genus Hypoderma (botflies) primarily parasitizes livestock like cattle and yaks, human infestation is accidental and rare. Specifically, human cases caused by Hypoderma sinense (H. sinense) are scarcely documented in medical literature. Traditional morphological identification of larvae is frequently hindered by the specimen's developmental stage, incomplete structures, or a lack of specialized entomological expertise. Consequently, distinguishing H. sinense from other Hypoderma species (e.g., H. bovis, H. lineatum) in human cases remains a diagnostic challenge, limiting the understanding of this parasite's zoonotic potential and host range.

Methodology
The study presents a case report of a 3-year-2-month-old boy from a cattle-raising pastoral region in Aba Tibetan and Qiang Autonomous Prefecture, Sichuan Province, China. The diagnostic workflow integrated clinical, imaging, and advanced molecular techniques:

1. Clinical and Laboratory Evaluation: The patient presented with painful, treatment-resistant subcutaneous nodules. Laboratory analysis revealed marked eosinophilia (10.03 × 10⁹/L) and elevated total IgE (740.35 IU/mL), suggesting a parasitic etiology over bacterial infection.
2. Imaging: High-frequency ultrasonography (7–15 MHz linear probe) was employed to visualize the lesions. This non-invasive technique detected linear hyperechoic structures within the subcutaneous tissue and muscle layers, consistent with larval presence.
3. Extraction: Leveraging the ultrasound findings and a pre-existing skin opening (punctum), the medical team performed a minimally invasive extraction. Under local infiltration of 2% lidocaine, the larva was induced to protrude and removed intact using vascular forceps, avoiding wide incisions.
4. Molecular Identification:
   • DNA Extraction: Genomic DNA was extracted from the larval tissue using the CTAB method.
   • Sequencing: Whole-genome shotgun libraries were prepared and sequenced on the DNBSEQ-T7 platform (PE150 reads).
   • Bioinformatics Pipeline: Raw reads were aligned to the human reference genome (hg19) using BWA-MEM to subtract host sequences. Unmapped reads were assembled de novo using MEGAHIT.
   • Taxonomic Assignment: Assembled contigs were screened against a local invertebrate COI (cytochrome c oxidase subunit I) barcode database (COInr) using BLASTn. Candidate mitochondrial contigs were further validated against the NCBI nucleotide database.

Key Results

• Clinical Outcome: The patient's symptoms resolved following the extraction and subsequent treatment with albendazole and prophylactic antibiotics (cefaclor).
• Genomic Assembly: The bioinformatics pipeline generated a 16,888-bp mitochondrial contig (k141_17052) containing a full-length COI barcode region.
• Species Confirmation: BLASTn analysis of the COI region demonstrated high similarity to the complete mitochondrial genome of Hypoderma sinense. The mapping of non-human reads to this contig showed complete coverage and uniform high depth, confirming the larva's identity as H. sinense at the species level.
• Imaging Correlation: Ultrasonography successfully identified the linear structures of the larvae prior to extraction, validating its utility in guiding conservative management.

Significance and Claims
The paper claims that this case represents a molecularly confirmed record of accidental H. sinense cutaneous myiasis in a human child from a pastoral environment in China. The authors emphasize several key contributions:

1. Diagnostic Advancement: The study demonstrates that a combination of ultrasonography and minimally invasive extraction can effectively manage cutaneous myiasis while reducing tissue trauma, particularly when a pre-existing punctum is present.
2. Molecular Precision: By utilizing whole-genome shotgun sequencing and mitochondrial contig analysis, the authors successfully identified the species as H. sinense, overcoming the limitations of morphological identification. This confirms that H. sinense, typically a livestock parasite, can accidentally infest humans.
3. Epidemiological Insight: The case highlights the importance of considering myiasis in children with treatment-resistant subcutaneous nodules and relevant livestock exposure. It suggests that molecular identification is crucial for accurate surveillance of rare zoonotic parasites at the animal-environment-human interface.
4. Modest Interpretation: The authors explicitly state that this case should be interpreted as a rare accidental infestation rather than evidence of common human infection or human-to-human transmission. They acknowledge the limitation of not having environmental or livestock samples to trace the exact source of infestation but assert that the molecular confirmation provides a verifiable record of this parasite in a human host.