Jpn. J. Infect. Dis., 73, 44–50, 2020 Original Article Molecular Identification of the Etiological Agent of Human Gnathostomiasis in an Endemic Area of Mexico Sylvia Paz Díaz-Camacho1, Jesús Ricardo Parra-Unda2, Julián Ríos-Sicairos2, and Francisco Delgado-Vargas2* 1Research Unit in Environment and Health, Autonomous University of Occident, Sinaloa; and 2Public Health Research Unit "Dra. Kaethe Willms", School of Chemical and Biological Sciences, Autonomous University of Sinaloa, University city, Culiacan, Sinaloa, Mexico SUMMARY: Human gnathostomiasis, which is endemic in Mexico, is a worldwide health concern. It is mainly caused by the consumption of raw or insufficiently cooked fish containing the advanced third-stage larvae (AL3A) of Gnathostoma species. The diagnosis of gnathostomiasis is based on epidemiological surveys and immunological diagnostic tests. When a larva is recovered, the species can be identified by molecular techniques. Polymerase chain reaction (PCR) amplification of the second internal transcription spacer (ITS-2) is useful to identify nematode species, including Gnathostoma species. This study aims to develop a duplex-PCR amplification method of the ITS-2 region to differentiate between the Gnathostoma binucleatum and G. turgidum parasites that coexist in the same endemic area, as well as to identify the Gnathostoma larvae recovered from the biopsies of two gnathostomiasis patients from Sinaloa, Mexico. The duplex PCR established based on the ITS- 2 sequence showed that the length of the amplicons was 321 bp for G. binucleatum and 226 bp for G. turgidum. The amplicons from the AL3A of both patients were 321 bp. Furthermore, the length and composition of these amplicons were identical to those deposited in GenBank as G. binucleatum (accession No. JF919679), corroborating our previous morphological finding that G. binucleatum is the etiological agent for human gnathostomiasis in the endemic area of Sinaloa, Mexico. Polymerase chain reaction (PCR) amplification INTRODUCTION of ribosomal DNA (rDNA) has been employed for Gnathostomiasis is a food-borne zoonotic systematics and phylogeny studies of eukaryotic nematodiasis in Mexico. It is mainly caused by the organisms and diagnosis of several parasitoses such consumption of raw or insufficiently cooked fish that as toxocariasis, taeniasis, and schistosomiasis (6). In contains the advanced third-stage larvae (AL3A) of nematodes, the second internal transcription spacer (ITS- different Gnathostoma species. Gnathostomiasis induces 2) is a marker for species identification, including those the larva migrans syndrome that can affect different of the genus Gnathostoma (7–9). The main etiological tissues (e.g., skin, eyes, viscera, and the central nervous agent of human gnathostomiasis are Gnathostoma system) and is endemic to countries such as Thailand, spinigerum in Asia and G. binucleatum in the Americas Japan, Mexico, and Ecuador (1,2). Globalization has (7,10). The species G. binucleatum, G. turgidum, and G. also increased the number of cases among international lamothei have been identified in Mexico, but the latter travelers and migrants (2,3). The definitive diagnosis two were found only in animals (Fig. 1) (11). Sinaloa of human gnathostomiasis has been achieved in only State is located in northwest Mexico, where human a small number of cases, as it depends on the recovery gnathostomiasis is endemic (Fig. 1). In Sinaloa, the and identification of the Gnathostoma larva, which is AL3A of G. binucleatum has been identified in different difficult due to its unpredictable migratory behavior species of fish, birds, and reptiles, whereas adult worms within the infected human. Thus, the diagnosis of and AL3A of G. turgidum have only been isolated from gnathostomiasis is based on epidemiological surveys, opossums (Didelphis virginiana) (12,13). clinical signs and symptoms, and immunological This study aims to develop a duplex PCR diagnostic tests. Nevertheless, these evaluations cannot amplification method of the ITS-2 region to differentiate discriminate among Gnathostoma species associated between the G. binucleatum and G. turgidum parasites with the infection and they cannot discern whether the and to identify the Gnathostoma larvae obtained from infection is active (4,5). the biopsies of two patients with gnathostomiasis. Received May 20, 2019. Accepted September 2, 2019. MATERIALS AND METHODS J-STAGE Advance Publication September 30, 2019. DOI: 10.7883/yoken.JJID.2019.180 Clinical data and recovery of AL3A in cases * Corresponding author: Mailing address: Public Health of cutaneous gnathostomiasis: The two cutaneous Research Unit "Dra. Kaethe Willms", School of Chemical gnathostomiasis cases from Sinaloa, Mexico included and Biological Sciences, Autonomous University of in this study were treated at the Public Health Research Sinaloa, University city, Culiacan, Sinaloa 80010, Mexico. Unit "Dra. Kaethe Willms" (PHRU), School of Chemical Tel: +52-667-7520460, Fax: +52-667-7520460, E-mail: and Biological Sciences, Autonomous University of [email protected] Sinaloa. Gnathostomiasis was diagnosed by applying a 44 Molecular Identification of Gnathostoma Larvae Recovered from Human Patients pre-coded clinical-epidemiological survey, examining an edematous lesion on the right thigh, causing a hot and the skin lesions present at the time of the medical indurated reddish plaque associated with itching, pain, consultation, and performing an indirect ELISA test (14) and burning sensations. The patient became ill one year to detect IgG antibodies against a crude antigenic extract prior to the consultation and showed skin inflammation of G. binucleatum AL3A. and itching. Subsequently, the larva migrated to the Case report 1: A 31-year-old woman presented with posterior part of the thigh. A medical doctor prescribed ). In Mexico, the highest number of human gnathostomiasis cases ). In Mexico, has been identified as the etiological agent. has been identified G. binucleatum in Mexico, adapted from Pérez-Álvarez, et al. (11). Map shows the states where the species have been the states where species have et al. (11). Map shows adapted from Pérez-Álvarez, in Mexico, Gnathostoma ), as well those states with cases of human gnathostomiasis ( □ ) and non-identified ( ) and non-identified ◆ ▲ ● have been registered in Sinaloa and Nayarit (shadowed), the only states where in Sinaloa and Nayarit (shadowed), been registered have identified ( identified Fig. 1. (Color online) Geographic distribution of the genus Fig. 1. (Color online) Geographic distribution 45 Chlorpheniramine, Diclofenac, and Dexamethasone finger (Fig. 3). Because the lesion was very superficial, at this stage. The lesion disappeared but it reoccurred the pustule was opened with a needle and a live one month later, registering larval migration in the Gnathostoma sp. AL3A was recovered. Interestingly, the same area. Laboratory analysis revealed only a 5% patient reported that she did not consciously consume of eosinophils, negative reactive protein C, negative raw fish. rheumatoid factor, and a high globular sedimentation The two recovered Gnathostoma AL3A were placed rate (24 mm/h). Moreover, the gnathostomiasis ELISA in isotonic saline solution (NaCl 0.85% p/v), observed test results were positive (1:800). The patient was then with a stereoscopic microscope (4X), separated from treated with 200 mg Albendazole tablets (6 tabs/day for the remaining tissue of the patients, fixed in absolute 15 days) to induce the migration of the parasite to the ethanol, and stored at 4°C until the extraction of superficial skin layers. Seven days post-treatment, the genomic DNA. patient exhibited a papule 10 cm in diameter. Dr. Rafael Purification of DNA from Gnathostoma AL3A Castro-Velázquez of the Dermatologic Center of Sinaloa and adult worms: Each Gnathostoma AL3A was performed an excisional biopsy (1.8×0.5×1.0 cm) placed in Eppendorf tubes with alkaline lysis buffer (5 which was analyzed in the PHRU, and a live AL3A of M NaCl, 1 M Tris, pH 8, 0.5 M EDTA, pH 8, and 10% the Gnathostoma sp. was recovered (Fig. 2). This patient SDS) and 500 μg/mL of proteinase K (Promega Co., has been eating raw fish 3-4 times a month since she Madison, WI, USA). Tubes were heated at 55°C for 30 was 10 years old. min and parasites were macerated with a sterile pistil. Case report 2: A 54-year-old woman with a body A volume of phenol equilibrated with 0.5 M Tris-HCl weight of 63 kg presented with an erythematous (pH 8.0) was added to each tube, gently mixed for 3 and hyperthermic plaque of approximately 13 cm in min, and centrifuged (5,000×g, 10 min). The aqueous diameter on her left forearm, which was associated phase was recovered into a new tube, and the extraction with an itching sensation. Moreover, the edema-causing process was repeated with the residue. The aqueous larvae migrated in a period of two or three days to phases were combined and two ethanol volumes at room different parts of the forearm. The woman informed that temperature were added. The samples were then mixed she was initially treated with antibiotics and Prednisone; and centrifuged (5,000×g, 15 min). The pellet was however, her discomfort persisted. The patient went recovered, washed with 70% ethanol, and centrifuged to the PHRU and was positive for the gnathostomiasis (5,000×g, 5 min). The ethanol solution was removed ELISA test (1:800). She was initially treated with 6 mg and the DNA was suspended in TE buffer (10 mM Tris- tablets of Ivermectin (200 μg/kg body weight/day for HCl, 1 mM EDTA, pH 8.0). The genomic DNA was three days). Her discomfort disappeared for four months, analyzed by electrophoresis on 2% agarose gels migrated but they were observed again on one of her hands, for 1 h at 100 V and stained with ethidium bromide. The which showed inflammation and redness. The ELISA DNA was stored at 4°C until use (15). test resulted positive and treatment was changed to 200 Amplification of Gnathostoma ITS-2 by PCR- mg Albendazole tablets (10 mg/kg body weight/day for Fig. 3 14 days). The discomfort disappeared again and 12 days post-treatment, the patient exhibited a pustule with a brownFig.