Case Report: Morphologic and Genetic Identification of Cerebral Sparganosis

Abstract.

A 50-year-old Chinese woman with a history of weakness and paroxysmal seizures of the left limb presented to our hospital with a ten-day history of neck pain. Imaging showed that there was a mass in the frontal lobe of her brain. On resection of the mass, a motile worm was identified. Morphological observation and molecular analysis of the mitochondrial COX1 and 28S rRNA genes of the worm extracted from the brain identified the causative agent as Spirometra mansoni. Homology search of the polymerase chain reaction (PCR)-amplified products from the case was conducted against gene fragments from local wild frogs. High homology was found between them, showing her likely exposure was frog consumption.

INTRODUCTION

Human sparganosis is a rare parasitic disease caused by the plerocercoid metaxestodes of Spirometra mansoni and related diphyllobothriidean cestodes. The infections have a worldwide distribution, but are most often seen in Asian countries including China.¹ Humans mostly serve as an intermediate host and are infected with larval worms. In the present study, we reported a female with brain sparganosis diagnosed using morphological observation and molecular sequencing on archived worm. We also highlight the clinical presentations and imaging, morphological, molecular characteristics of the case.

CASE REPORT

A 50-year-old female, a retired farmer without any organic diseases or psychosis, had a 1-year history of weakness and paroxysmal seizures (without impairment of consciousness) of the left limb. The patient denied eating raw meat, drinking unboiled water, or applying raw flesh on conjunctiva, mucosa, and open wounds. However, local consumption of frogs was common. Systemic migratory subcutaneous nodules have been seen at different parts of her body. She presented to our hospital with a 10-day history of neck pain. Magnetic resonance image of the brain revealed a frontoparietal lesion (1.5 × 0.9 cm) surrounded by parenchymal edema in the frontal lobe of her brain, and there was pneumocephalus in the right frontal and parietal area (Figure 1). However, after analyzing the blood routine and biochemical indexes, we found that levels of tumor markers were all normal. No eosinophilia was observed, and IgG against Clonorchis sinensis, Echinococcosis, and Schistosoma japonicum were all negative. The biochemical parameters were also unremarkable. The brain mass whose characteristics were unknown resulted in severe clinical manifestations, so complete surgical excision was deemed necessary for diagnosis and management. After separation of dural mater adherence, a yellow mass with a distinct margin was found 1.5 cm under the cortex of the precentral gyrus. Histological examination showed it consisted of many gliocytes. However, it was not a glioma, as lymphocyte infiltration showed that it was an inflammatory lesion. An 8-cm mobile worm was found during resection of the mass. The light microscope morphology of the carmine-stained worm (Figure 2) suggested it is a larva of Spirometra, owing to its transverse fold and depression at the scolex. For further determination of species, DNA was extracted using the DNA minikit for blood/tissue (QIAGEN, Hilden, Germany). PCR amplification was performed with primers specific for COX 1 (NC_011037.1) and 28S rRNA (KY552837.1) gene: cox1-F (5′-CGGCTTTTTTTGATCCTTTGGGTGG-3′), cox1-R (5′-GTATCATATGAACAACCTAATTTAC-3′), 28S-F (5′-CACCGAAGC CTGCGGTA-3′), and 28S-R (5′-GAAGGTCGACCTGGTGAA-3′) for the worm. Ultimately, the sequence of the patient returned COX1 (100%) and 28S rRNA gene (98%) from S. mansoni as a top hit in the BLAST analysis, confirming the diagnosis of sparganosis.

Figure 1.

Magnetic resonance image (MRI) of the patients. The preoperative MRI (A and C) of the brain revealed an enhanced lesion on the frontal lobe of the patient. Postoperative MRI (B) proved a complete resection.

Figure 2.

Histopathological findings: Scolex of carmine-stained intact larva (molecular confirmed as Spirometra mansoni).

PCR amplification was also performed to get sequence of S. mansoni from frogs that were got from the countryside of Chengdu. Moreover, three standard sequences were obtained from the NCBI database (accession numbers: KY114888.1, KY114887.1, and KT376529.1), all of which were sequences of S. mansoni from China. High homology was found between these sequences and that from this case (Supplemental Material), which is consistent with our inference (Figure 3). Considering diet habits of local residents and high homology between PCR-amplified products of the specimen and those of local wild frogs, eating stir-fried frog was considered the most likely source of her infection.

Figure 3.

The results of agar gelatin electrophoresis: Cox1 and 28S rRNA gene fragments of the patient amplified by PCR. Lane 1, the first PCR product using primer pairs for; 28S rRNA gene (179 bp). Lane 2, PCR products using primer pair for COX 1 gene (151 bp). Lane 3, negative control. Lane 4, marker (100–700 bp).

DISCUSSION

China is known to have high endemicity of sparganosis, with more than 1,000 reported cases. A retrospective epidemiological analysis showed that a total of 1,061 valid cases were distributed throughout most of the provinces of mainland China from 1959 to 2012.² Recently, the incidence of sparganosis has gradually risen and sparganosis has even been termed an emerging enzootic disease in several districts.³

In the life cycle of Spirometra, its adult worms can reside in the intestines of dogs and cats, and develop into procercoid larvae when ingested by the first intermediate host cyclops. At the plerocercoid stage, Spirometra is not host restricted, so amphibians including frogs, chickens and fish can act as secondary intermediate hosts, enabling procercoid larvaes to develop into plerocercoid larvaes. The main ways of infection include ingesting uncooked or partially cooked bile, blood of snakes, meat of frogs, snakes, fish, or chickens that contain the plerocercoids; ingestion of larva-containing water; and applying raw plerocercoid-infested flesh from intermediate hosts on conjunctiva, mucosa, or open wounds,¹ which is considered as a traditional treatment of skin ulcers or eye disease. In addition, some patients try to swallow raw frogs for the treatment of cerebral infarction or rheumatism according to the folk prescriptions in China. In fact, it has no help to relief of symptoms, but increases the risk of parasitic infection. Although the way of infection is certain, it is still difficult to know what led to the infection in specific cases if the patients have no absolute potentially risk behaviors. The local diet relies heavily on cooked frogs, of which the most common making method is stir-frying. The meats were heated to high temperature, but the short heating time may be insufficient to fully kill the parasites in it, causing infection of consumers. Consuming wild frog meat, which is a delicacy in Southwestern and Southern China, entails the risk of parasitic infection. Wild frog has higher infection of spargana than dogs, cats, and farmed frog.⁴ A 2009 survey showed that > 25% of the local wild frogs were infected with spargana in central and southern China,⁵ whereas we found the infection rate of S. mansoni pleroceroid in local wild frogs is higher, up to 30%, and they were mainly in the legs of frogs. We used primer pairs to delineate the parasites down to species level, and the homology search of the PCR-amplified product from the case conducted against gene fragments of those from local wild frogs showed high homology, so eating frog was considered the most likely source of infection in this patients.

Cerebral sparganosis is a rare zoonotic infestation that often mimics glioma and metastatic tumors. Although rare, brain sparganosis should be considered in the differential diagnosis of brain mass, especially in southwestern and southern China. Cerebral sparganosis accounts for 12.4% of all S. mansoni infections in China.⁶ Although most of wild frogs have been cooked before eating, the pathogens cannot be fully killed if temperature is not high enough or holding time is not long enough, so stewing is a safer way than stir-frying. The local government, public health officials, and medical practitioners should implement strategies to let people know the risk and advise people not to eat wild frogs.

Note: Supplemental material appears at www.ajtmh.org.