Since the patient continued to suffer from severe painful cutaneous swellings and hypereosinophilia, a third round of ivermectin (12 mg/d/3 d) was GDC-0068 chemical structure administered. After this last treatment, the patient quickly became asymptomatic. No cutaneous swellings reappeared and the eosinophil count rapidly normalized. The patient has remained asymptomatic to the present day, 2 years later. Since neither the multiple serological nor microscopy tests
performed were conclusive, and because the morphological analysis of the larval fragment suggested myiasis (Figure 2), immunodiagnostic tests for hypodermosis were performed using retrospective and tracking sera from the patient. Three consecutive serum samples were sent to the Lugo Veterinary School Laboratory. Anti-Hypoderma antibodies were sought by indirect ELISA using a crude extract obtained from the first instars of Hypoderma lineatum,
as described by Panadero et al.13 Different dilutions of the antigen, sera, and immunoconjugate were tested following a previously described protocol.14 The specificity of the procedure was assessed by testing three human sera positive for Gnathostoma. High titers of anti-Hypoderma antibodies were detected during the course of disease (OD 4.359 on November 24, 2006), at 3 months post-infection (p.i.) (on November 24, 2006), and after the treatment (OD 3.977 at 7 months p.i. and 4.044 at 15 months p.i.). These high levels of antibodies against H lineatum antigens confirmed the diagnosis of an infestation by oestrid larvae. Genomic DNA was extracted from the larval parasite tissues Gefitinib using CT99021 the Quantum Prep AquaPure Genomic DNA Kit (BioRad, Hercules, CA, USA). The hypervariable
sequence of the cytochrome oxidase I (cox1) gene coding for the region from the external loop 4 (E4) to the carboxy-terminal (COOH) of the protein (688 bp) was amplified by PCR as previously described.15 The PCR products were detected on 1.6% agarose-Tris-acetate-EDTA (TAE) gel, purified using Ultrafree–DA columns (Amicon, Billerica, MA, USA), and then directly sequenced in an ABI-PRISM 377 sequencer using the Taq DyeDeoxyTerminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). The mitochondrial fragments were sequenced in both directions. The sequences were aligned using the ClustalX program and examined by eye. Pairwise comparison of the sequences obtained showed them to be identical to the H sinense cox1 sequence available in the GenBank™ database (Accession number: AY350769). This is the first report of human infestation diagnosis caused by H sinense larvae in Europe, in a patient returning from India. It is very likely that the infestation resulted from contact with infested cattle or yaks in the region—which is endemic for hypodermosis—where the patient had been traveling.