Diagnosis and treatment of metastatic alveolar echinococcosis in the brain of a Tibetan patient : A case report

Background: A case of primary alveolar echinococcosis (AE) in the liver with kidney and brain metastases was treated at our hospital. The analysis of this case might provide suggestions for the diagnosis and treatment of other cases of AE in the brain. Methods : To determine the disease status of AE in the liver and brain in this patient, computed tomography (CT) of the abdomen and head magnetic resonance imaging (MRI) were performed before surgery. Additionally, the serum of the patient was analyzed using an enzyme linked immunosorbent assay (ELISA) for antibodies against echinococcus. The left pterional approach was selected for head surgery. Along the edema zone around the lesion, complete excision was performed encompassing an area of about 5 mm surrounding the lesion. After surgery, pathological and immunohistochemical examinations of the lesion were performed, and deoxyribonucleic acid (DNA) was extracted for molecular identification. This patient was followed up regularly post-discharge to evaluate the outcome. Results: Anti-echinococcus antibodies were strongly positive in the patient’s serum, and the eosinophil ratio was 20%. The head MRI showed a single lesion in the left temporal lobe. Multiple nodular and small cystic long T 1 short T 2 signals were seen in the lesions. An enhanced scan indicated multiple ring-enhancements, the boundary was unclear, and the size of lesion was 3.2 mm×1.8 mm. Neurological symptoms improved after the surgery. The result of histopathological examination was consistent with the characteristics of AE. Immunohistochemistry showed that the peripheral glial cells were positive for glial fibrillary acidic protein (GFAP) and the Ki-67 index was <3%. DNA analysis identified the parasite as the Asian strain of Echinococcus multilocularis, and the sequence identity with the reference strain was practically 100.00%. Conclusion: Surgery is still the first choice of treatment for single, superficial AE lesions, and those located in nonfunctional areas of the brain. It is suggested that early radical treatment and standard drug treatment of primary hepatic AE are important steps to avoid secondary encephalic AE. Patients need to take albendazole for a

eosinophil ratio was 20%. The head MRI showed a single lesion in the left temporal lobe. Multiple nodular and small cystic long T 1 short T 2 signals were seen in the lesions. An enhanced scan indicated multiple ring-enhancements, the boundary was unclear, and the size of lesion was 3.2 mm×1.8 mm. Neurological symptoms improved after the surgery. The result of histopathological examination was consistent with the characteristics of AE. Immunohistochemistry showed that the peripheral glial cells were positive for glial fibrillary acidic protein (GFAP) and the Ki-67 index was <3%. DNA analysis identified the parasite as the Asian strain of Echinococcus multilocularis, and the sequence identity with the reference strain was practically 100.00%. Conclusion: Surgery is still the first choice of treatment for single, superficial AE lesions, and those located in nonfunctional areas of the brain. It is suggested that early radical treatment and standard drug treatment of primary hepatic AE are important steps to avoid secondary encephalic AE. Patients need to take albendazole for a long time after the operation, and should be followed-up regularly. Background 3 Alveolar echinococcosis (AE) caused by echinococcus multilocularis is a chronic and potentially lethal parasitic disease. In the 1990s, studies of the distribution of human parasites showed that the Qinghai-Tibet Plateau (including Sichuan, Qinghai and parts of Gansu), Xinjiang and Ningxia Autonomous Region were highly affected endemic areas of echinococcosis [1] . Recent global data showed that there are about 18 000 new AE cases per year, 91% of which were diagnosed in China [2] .
Because AE usually originates in the liver, it often involves the lungs, brain, bone and other organs, while central nervous system involvement accounts for only 1-3% of the cases reported in the literature [3] . For example, Altinors et al. [4] showed that the number of cases involving the nervous system accounted for only 2-3%. AE can lead to high-level disability and even death if it cannot be diagnosed and treated correctly in the early stage [5][6] . Therefore, early radical treatment of liver AE is particularly important. Recently, a case of primary liver AE with kidney and brain metastasis was treated in our hospital. The diagnosis, treatment and outcome of this disease were analyzed as follows.

Clinical data
The patient is a 52 year-old male Tibetan herdsman, born and living in Gander County, Golog Tibetan Autonomous Prefecture, Qinghai Province of China, which is one of those most highly endemic areas of AE [7][8] . He was admitted to the hospital on January 17, 2019 due to "headache for 1 month, aggravating with convulsions for 1 day ". The patient had no obvious causes of the headache, which had the left temporal side as the main area, and did not receive special treatment. The symptoms were recurrent. One day before admission, the symptoms of headache were more severe than before and accompanied by epilepsy. In July 2009, the patient was diagnosed as having liver AE and received liver hydatid cystectomy and cholecystectomy. The diagnosis based on pathological examination was liver AE, and the patient received no medication such as oral albendazole after the operation. In September 2013, the patient suffered from pain in the right lumbar and costal region. He underwent abdominal CT examination in the hospital in our province. As a result, the patient was diagnosed as 4 having right renal echinococcosis and received a right nephrectomy. After 2 months of intermittent oral administration of albendazole, the patient stopped taking the medicine on his own.

Image analysis
The patient was examined by abdominal CT and brain MRI to confirm the status of intracranial and abdominal echinococcosis according to the diagnosis and expert consensus of echinococcosis in the central nervous system in China [9] . According to the PNM typing of liver alveolar echinococcosis proposed by the WHO [10] and the EMUC-US typing based on ultrasonography [11] , the patient was classified as having alveolar echinococcosis of the liver. The CT was performed using a Revolution CT scanner with 64 detector rows (General Electric Company GE, USA) and the MRI analysis used a MAGNETOM Skyra 3.0T Magnetic Resonance Imaging System (Siemens Healthineers USA). The patient previously had liver and kidney echinococcosis. In order to clarify the systemic organ metastases, a PET/CT examination was suggested, but the patient refused the examination.

Serological Detection
ELISA was used to investigate the presence of parasite-specific antibodies in the serum. Serum samples were collected from 2-5 ml aliquots of venous blood and tested for anti-echinococcus antibodies using an echinococcosis IgG antibody detection kit (Zhuhai Haitai Biopharmaceutical Co., Ltd., batch number 20181102). An XT-1800i hematology analyzer (Sysmex Company, Japan) was used for routine blood tests.

Pathological and Immunological Examination
The lesion samples were fixed with 4% formaldehyde, followed by routine dehydration, paraffin embedding, slicing into 4 mm thick sections, hematoxylin-eosin staining (HE) and GFAP staining for microscopic observation.

Genetic Testing
The tissue DNA extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd) was used to extracted genomic DNA. The specific operation steps were conducted in accordance with the kit's 5 instructions. The extracted genomic DNA was used as template and the specific primers COX1F (5'-TTGAATTTGCCACGTTTGAAT GC-3') and COX1R (5'-GAACCTAACGACATAACATAATGA -3') were used for polymerase chain reaction (PCR) amplification.
After amplification, the PCR product was separated by electrophoresis in a 1.5% agarose gel and photographed. After sequencing and identification, a phylogenetic tree (Kimura 2 parameter model,

repeated bootstrap tests) was constructed using the Neighbor Joining method in Molecular
Evolutionary Genetics Analysis (MEGA) 5.0 software.

Differential diagnosis
Lumbar puncture was performed and 5 ml of cerebrospinal fluid was collected for examination to exclude intracranial infection and tuberculosis. Cerebral angiography (CTA) and chest CT were performed to exclude cerebrovascular diseases and pulmonary metastasis.

Surgery and drug therapy
After admission, albendazole tablets were administered, 60 mg once, twice a day. According to the diagnosis and expert consensus on echinococcosis in central nervous system in China [9] , the authors excluded the contraindications to operation. Microsurgery was used and the left pterional approach was selected, with routine craniotomy, suspension of the dura mater, "Y" type incision of the dura mater and positioning of the S88 microscope (Carl Zeiss AG, Germany). The edge of the lesion was distinguished along the edema zone around the lesion, about 5 mm away, and the lesion was completely resected (Figs. 1A and B). The patient was given therapy against inflammation and for the prevention of epilepsy, rehydration and oral albendazole. The liver function of the patient was monitored regularly.

6
The results of brain MRI revealed a single lesion with multiple nodular and small cystic long T1 and short T2 signals in the left temporal lobe. On the contrast-enhanced scan, the lesion showed multiple ring-shaped enhancements with an unclear boundary. The size of the lesion was 32mm × 18 mm

Serology
The serum was strongly positive for antibodies against Echinococcus, and the eosinophil ratio was 20%.

Pathohistology and immunology
The morphological results of HE staining were consistent with the characteristics of alveolar echinococcosis (Figs. 4A and B). The results of immunohistochemical staining showed positive staining for GFAP in peripheral glial cells and the Ki-67 index was less than 3% (Figs. 5A and B).

Molecular genetic analysis
After amplification of the cytochrome C oxidase gene by PCR using primers specific for Echinococcus, a fragment of the expected size of about 880 bp was observed on the agarose gel after electrophoresis (Fig. 6) China. The phylogenetic tree showed that the isolate was closely related to other Chinese isolates, clustered on one branch and far from other isolates (Figure 7).

7
The results of lumbar puncture showed that the CSF was clear and transparent, the number of white cells was 1 × 10 6 /L (1-10 ×10 6 /L), and the CSF fluid pressure was normal. No abnormalities were found upon brain CTA and chest CT.

Surgery and drug therapy
Albendazole tablets were administered twice a day after admission, and the changes of liver function were monitored regularly. Microscopy revealed that the left temporal pole was very full, the sulcus gyri became shallow and flat, the cortex was beige, the boundary of the lesion was unclear, the lesion was firm, and there was no envelope. One piece of toughened grayish red and yellow tissue with a size of 40 mm × 30 mm was excised. The cysts were solid and 0.2-0.4mm in diameter. Gray inclusions were visible in the capsule (Figs. 8A and B).

Outcome
The patient's headache symptoms improved and no other neurological complications occurred. The brain MRI one month later after the discharge showed that the lesion had been fully resected ( Figure   9).

Discussion
The Qinghai-Tibet Plateau, centered around Qinghai Province, is a high incidence area of AE. The patient described here is a herdsman with a history of close contact with cattle, sheep and dogs. The patient was diagnosed with liver AE in 2009 and received surgery. After the operation, the patient was required to take albendazole tablets orally. However, the patient had poor compliance, which is the most likely cause of the disease progression. He was diagnosed with echinococcosis of the right kidney in 2013 and underwent a right nephrectomy. The patient was asked to take albendazole orally again after the operation. After 2 months of intermittent administration, the patient stopped taking albendazole of his own accord. The patient lived in the Qinghai-Tibet Plateau, an economically underdeveloped area with inconvenient transportation and poor hygienic habits. Moreover, understanding of the disease is not high in said geographic area. Therefore, he did not cure the primary liver AE early and did not take albendazole orally as prescribed by the physician. [9,[11][12][13] and WHO [14] recommend long-term oral 8 albendazole for patients with echinococcosis. It is suggested that albendazole should be taken for life in patients with confirmed echinococcosis.

Many guidelines of the Expert Consensus in China
In this case, echinococcosis lesions invaded the left branch of the portal vein and the second hepatic portal, indicating that the disease should be classified as having reached P 3 N 1 M 1 and P I−V,VIII I 1 V 1 M 2 P I stage. The diagnosis of intracranial metastasis was clear and indicated an advanced stage of disease [15] . According to the expert consensus on the diagnosis and treatment of hepatic hydatidosis type 2 (2015 edition) [12] , patients with liver AE accompanied by intracranial metastasis are considered to have lost the opportunity for operation, and should be treated mainly with medication (albendazole). However, hepatectomy may also be considered for patients with complications of liver AE and stable intracranial lesions can be treated through medication. Therefore, the patient was treated with albendazole tablets on admission and the brain lesions were treated first.
Primary liver alveolar echinococcosis usually results in multiple intracranial metastases [16] . In recent years, many cases of multiple brain AE in different anatomical regions have been reported [17,18] .
This case presented with a single brain metastasis, which is rare. Algros et al. [19] showed that patients with AE should receive surgery as much as possible. The consensus of experts in China [9] also indicates that the principal surgical treatment for brain AE should be selective excision of intracranial lesions, control of intracranial hypertension and relief of symptoms related to structural compression of nerves while anti-echinococcal drugs should be used at the same time. Microsurgery induces less damage to brain tissue, and makes it easy to identify and completely resect lesions. It also reduces the possibility of anaphylactic shock due to leakage of cystic fluid and might cause less complications after operation, which could improve the therapeutic effect of surgery. The lesion was relatively superficial and located in a non-critical functional area. Therefore, we selected the microsurgical approach with resection along the edema zone around the lesion, and removed the lesion completely. The headache symptoms improved after the operation and no other neurological complications occurred. Therefore, this study suggested that if there is a single brain lesion in AE, especially if it is located superficially and in a non-critical functional region, it should be removed as 9 soon as possible. In the case of AE with multiple brain lesions, the safety and necessity of surgical treatment should be considered comprehensively according to the size, number, location of the lesions, as well as the amount of edema. The recurrence rates of brain echinococcosis after single surgery or chemotherapy were reported as 13 and 4%, respectively [20] . Therefore, patients should be followed up regularly after discharge. When the intracranial condition is stable, the patients' liver condition should be further evaluated to determine whether the liver can receive surgical treatment.
Bardonnet et al. [21] reported the case of a 30-year-old AE patient with multiple metastases who received surgery and continued high-dose albendazole treatment, and achieved a good outcome. Our diagnostic and therapeutic program supports this study.
In addition, molecular genetic analysis of the lesion was carried out in this study and unequivocally identified the etiological agent as the Asian strain of Echinococcus multilocularis. In the era of increasing emphasis on precise medical care, this may provide a new approach for early disease screening of susceptible populations.
Although great progress has been made in basic research, clinical diagnosis and treatment of echinococcosis, AE is still a challenging disease with high morbidity, disability and mortality.
Therefore, in areas with high incidence of the disease, it is very important to carry out extensive screening, and thereby enable early diagnosis and treatment of echinococcosis for the local residents.

Conclusion
Surgery is still the preferred treatment for AE patients with single or superficial lesions, especially in areas that are not functionally critical. Long-term albendazole treatment and regular follow-up are needed after operation. Early radical treatment and standardized drug treatment of primary liver AE are the key to avoid secondary brain echinococcosis. These aspects should be emphasized in the clinical practice to strengthen the comprehensive treatment of echinococcosis Availability of data and materials: Not applicable.

Competing interests:
The authors declare that they have no competing interests.        Representative micrograph of histological straining A: HE staining of lesion tissue at primary magnification, 2 × 10 times, B: Zoom of the selected cutout in image A (the original magnification was 10 × 10 times). In the middle of the picture, there is a lamellar structure, which has no cellular components and is presumably a polysaccharide-protein complex.

Figure 4
Representative micrograph of histological straining A: HE staining of lesion tissue at primary magnification, 2 × 10 times, B: Zoom of the selected cutout in image A (the original magnification was 10 × 10 times). In the middle of the picture, there is a lamellar structure, which has no cellular components and is presumably a polysaccharide-protein complex.

Figure 5
The results of immunohistochemical staining A, B: Immunohistochemical staining of glial cells around the lesion was positive for GFAP (blue arrow), confirming that the lesion was located in the intracranial region.  Brain MRI one month later after the discharge 25 Figure 9 Brain MRI one month later after the discharge

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