Liver alveolar echinococcosis in China: Clinical aspect with relative basic research

Abstract

This paper deals with all aspects of liver alveolar echin-ococcosis (AE) including epidemiology, pathology, clinical manifestations, imaging examinations, diagnosis and differential diagnosis, surgical treatment and chemotherapy. The review is not only based on personal clinical experiences but also in combination with relative basic research such as proliferation and growth of alveococcus, preclinical studies of a novel compound extracted from TCM for treatment of liver AE, and molecular immunology used for specific AE diagnosis, etc.

Key Words: Echinococcosis, Alveolar echinococcosis, Liver, Clinical aspect, Basic research, China

INTRODUCTION

Echinococcosis is a parasitic zoonosis, consisting of cystic echinococcosis (CE) and alveolar echinococcosis (AE). These two types differ in parasitology, epidemiology, pathology, clinical aspects, treatment and prognosis. They are respectively caused by the larval stage of Echinococcus granulosus (Eg) and E. multilocularis (Em) (Figure 1). Over one hundred years, the disease had been misdiagnosed as liver colloid cancer. The cause of AE was not confirmed until 1856. In China, liver AE was reported respectively in Ningxia, Xinjiang autonomous regions and Gansu Province during 1950s to 1970s[1-3]. Up to now, at least one thousand cases of AE have been reported throughout China, but most of them were mistaken as liver cancer. In order to improve the diagnostic level of AE, a review is comprehensively introduced as follows.

Figure 1 Adult of E. multilocularis.

EPIDEMIOLOGY

Em needs two mammalian hosts for completion of its life cycle. They are respectively called the definite host, arnivore and the intermediate host, mainly rodents. Proglottid containing eggs or free eggs are excreted in the feces of the definite hosts, e.g. dog or fox. Once eggs are ingested by the intermediate hosts including rodents and human beings, AE may occur in liver or other organs. In China, fox, dog and wolf have been found to be as definite hosts and a few species of the rodent such as Citellus dauricus, Myospalax fontanieri, Microtus brandti, were confirmed to be as intermediate hosts. Human body affected by AE is chiefly due to contacting the animals: fox, dog, or occasionally cat. Especially, ladies, herdsmen and hunters are higher risk populations. AE has been found mainly in the west of China including eight provinces or autonomous regions (Figure 2)[4].

Figure 2 Distribution of AE in eight provinces in west China.

Gansu province

During a period from 1985 to 2000, of 89 AE cases originating from Gansu Province, 63 came from Zhang County, which may be considered as a hyper-endemic area[5]. In the three neighbouring counties of central eastern Gansu (Figure 3), human AE epidemiological surveys were conducted four times respectively in three counties (Table 1). The results of comparative analysis showed that the prevalent rate was higher in Zhang County and Ming County than in Lintao County. Factors were also investigated regarding natural geographical condition, local climate, parasitic life cycle, and our experimental study[10].

Table 1 Human AE epidemiological survey in three counties of central eastern Gansu.

Yr	County	Persons investigated	Positive rate of immuno-test(%)		Ultrasonicmorbidity(%)
			Intradermal test	ELISA
1986[6]	Zhang County	380	19.2		2.4
1992[7]	Zhang County	1312		2.8	5.0
1998[8]	Ming County	1200		2.2	5.6
2003[9]	Lintao	1071	1.8		0.7

Figure 3 AE endemic counties in central eastern Gansu. (1) Zhang County, (2) Ming County, (3) Lintao, (4) Jinyuan.

Xinjiang uygur autonomous region

AE was more common in northern Xinjiang with a total of 100 cases.

Ningxia Hui autonomous region

Distribution of AE was limited to a specific endemic area, and chiefly found in Xi Hai Gu’s three Counties which were situated bilaterally along branches of Liupan Mountain. In 1989 during human ultrasonic investigation in Xiji County, AE morbidity was 5.9%(141/2 389). The positive rate by ELISA was 5.8%(138/2 389). In 2003, 263 cases of AE in the past 17 years were followed up in Xiji, of whom the positive rate of intradermal test was 90.2%(174/193) and ELISA in 137 patients were all positive[11].

Qinghai province

Until 2000,1 748 cases with echinococcosis were reviewed and 143 of them belonged to AE (8.3%). Another survey was made in 3 702 individuals, of whom 23(0.62%) were ultrasonically diagnosed as liver AE. The distribution of AE appeared with a scattered tendency. During an investigation of human AE in three counties under the jurisdiction of Qingnan plateau, the average morbidity was 0.35%. From 1959 to 1998, 111 cases of AE were surgically treated[12].

West Sichuan province

AE severely affected three counties of Ganzi, Shiqu, and Seda. According to investigations of 3 998 individuals in Ganzi and Shiqu from 1997 to 1998, AE morbidity was 7. 78% and 2.33% respectively.

Tibet autonomous region

In 1993, 12 cases of AE originating from Larsa and three prefectural hospitals of Naqu, Shannan, and Changdu were discovered surgically and pathologically.

Inner Mongolia autonomous region

Although animals of definite host and intermediate host were found in Hulunbeier grassland in 1988, the patient of AE was not clinically reported until 1998[13].

Heilongjiang province

Two scattered patients of liver AE were identified surgically and pathologically. One of them was a native of Nahe County[14] and another case was from Jiamusi city[15].

PROLIFERATION AND GROWTH OF ALVEOCOCCUS AND HISTOGENESIS OF PROTOSCO LEX

Proliferation and growth of alveococcus is very significant as it bears practical relation with clinical aspects especially chemotherapy. As early as in 1950s to 1980s, a few Euroa-merican authors[16-21] observed the proliferation and growth phenomenon of experimental alveococcus respectively in white mice, cotton mice, and jirds (Meriones unguiculatus). Our study was partially carried out microscopically on the pathological sections of human AE[22]. Two modes of proliferation were found, i.e. endogenous budding and exogenous budding (Figure 4). The former (Figure 4A) was characterized by internally protrusive hyperplasia from the mother alveolar wall into the alveolar cavity and then proliferation extending continuously to reach the opposite wall of the cavity. So, septum-like budding was named. Sometimes two or more proliferative sites on the alveolar wall propagated simultaneously into the cavity in opposite directions, and mingled with each other to form a septum dividing the mother to form two or more small alveoli (Figure 4B). Externally protrusive proliferation occurred at one or several sites of the mother alveolar wall, producing single or multiple daughter and granddaughter alveoli under the name of exogenous budding (Figure 4C). These two buddings may coexist not only in one section of liver AE but also in that of metastatic lung or brain AE and metastatic lymph node AE. To date, the mechanism of AE proliferation and growth is not understood completely although it has been studied for decades. In the exogenous proliferative course of liver AE, the daughter alveoli of 1^st grade budding and the granddaughter alveoli of 2^nd grade budding were respectively named. Proliferating grade by grade, a mother alveolus may propagate into numerous new alveoli of multi-grades just like infiltrative dissemination of carcinoma, or it further affects neighbouring organs such as porta hepatis, inferior vena cava or pancreas, rendering radical surgical operation impossible. Euzeby[21] explained that exogenous budding was due to discontinuity of the alveolar cuticular layer, allowing the germinal membrane to escape from the mother alveolus (Figure 4D). As concerns the entire process of protoscolex histogenesis, a stage of the formation of brood capsule must be passed through. Local cellular hyperplasia began in the wall of brood capsule, and elliptical, mushroom-shaped or lingual protrusion protruded into the cavity of brood capsule. After that, the protrusion gradually developed into rostellum and suckers, finally changing from embryonic protoscolex to mature protoscolex of evaginated or invaginated type (Figure 5). The formation of brood capsule expressed two modes. The first was local hyperplasia of germinal membrane of alveococcus wall, forming cellular group. By means of cellular rearrangement, the cells accumulated towards the periphery and a cell-free space appeared in the center, developing further into brood capsule[23] (Figure 5). This is basically similar to other reports at home and abroad[19,25]. The second mode of brood capsule formation was found by us from liver AE of Mus musculus in Zhang County, Gansu Province[24] (Figure 6). Similar report has not been seen yet.

Figure 4 Modes of proliferation. A: Endogenous budding of alveococcus (HE stain ×200). Arrow: Septum; B: One mother alveolus (left lower side) dividing into four daughter alveoli (HE stain ×400); C: Exogenous budding of alveococcus (HE stain ×400); D: Model of alveococcus exogenous budding.

Figure 5 The entire process of mouse AE protoscolex histogenesis (HE stain×200-800). A: local cellular hyperplasia of germinal membrane of alveococcus wall; B: re-arrangement of hyperplastic cells and formation of brood capsule; C: local cellular hyperplasia of brood capsule wall; D: elliptical protrusion into cavity; E: mushroom protrusion; F: lingural protrusion; G: showing rostellum and suckers of protoscolex; H: Hooklets on rostellum; I: mature protoscolex (right: evaginated type. Left: invaginated type).

Figure 6 Histogenesis process of brood capsule of liver AE protoscolex in Mus musculus (HE stain ×200 ). A: local hyperplasia of alveococcus wall into the cavity, looking like a reversed pocket; B: gradually approximating and finally closing of two pocket edges; C: formation of brood capsule; D: local cellular hyperplasia of brood capsule wall.

PATHOLOGY

Macroscopical appearance

The lesion was yellowish or gray and felt as firm as cartilage. Superficially, it showed numerous noduli or minute cysts without definite encapsu-lation, but with alveolate structure on cross section (Figure 7A). It was usually classified into three types, of which large circumscribed mass type was more common (67.8%) and the other two were nodular type (16.7%) and mixed type (15.5%) (Figure 7B)[26]. The central area of alveococcus may be complicated by coagulated necrosis due to poor blood supply.

Figure 7 Pathology. A: A cross section of human liver AE showed alveolate appearance; B: Types of liver AE.↑: Nodular type, ↑↑: Mixed type; C: Protoscolices inside brood capsule (HE stain×100); D: Alveococcus nodule (HE stain×200); E: Three arrows indicating metastatic bilateral lung AE; F: ↑: metastatic right brain AE; G: lymph node metastasis near porta hepatis (HE stain×200). ↑: AE. The left upper side: Metastatic lymph nodes.

Microscopic finding The lesion was characterized by many alveoli with different sizes and shapes. Observation of alveolus wall showed that the thick, acellular, laminated outer layer looked bank-like, sometimes folding within the alveolar cavity. The thin, germinal inner membrane lined by a single-layer cell was usually deficient due to detachment. Brood capsule or protoscolices were occasionally seen (Figure 7C). The lesion may be complicated by central necrosis, producing a cavity or pseudocyst after liquidization. In the periphery of alveoli group it showed hyperplasia of fibro-connective tissue and cellular infiltration of eosinocytes, lymphocytes, plasma cells and giant cells, forming a typical alveococcus nodule (Figure 7D).

Metastasis According to a collective analysis of 270 cases with liver AE from five provinces of China (Table 2)[27], general metastatic rate was 3.7%(10/270). The commonest metastatic organ was lung (4.7%)(Figure 7E) and the next one was brain (3.3%) (Figure 7F).

Table 2 Remote metastases of 270 cases with liver AE in China.

Province or autonomousregion (yr)	Number of cases of blood-stream metastasis (%)					Number of cases of lymphatic metastasis (%)
	n	Lung	Brain	Lung+brain	Spleen	Lymph node ofporta hepatis	Lymph node ofperitoneal cavity	Lymph node ofcolonic mesentery
Xinjiang (1985)	43	3	2
Ningxia (1991)	22						1
Sichuan (1994)	24	2		1
Gansu (1995)	70	3	2	1		1		1
Qinghai (2000)	111	2	7		2		1
Total	270	10 (3.7)	11 (4.1)	2 (0.7)	2 (0.7)	1 (0.4)	2 (0.7)	1 (0.4)

Three modes of metastasis were found. The first was direct infiltrative dissemination. The alveococcus lesion infiltrated gradually into the liver parenchyma and formed a large mass. The neighbouring organs outside the liver such as the porta hepatis, diaphragm, pancreas or inferior vena cava were further involved. The second was blood metastasis. A small portion of detached proliferating bud, if involving the branches of portal vein, extensively disseminated within the liver parenchyma resulted in multiple noduli. Involving the branches of hepatic vein, the alveococcus spread along systemic circulation to distal organs such as lung or/and brain. The third was lymphatic metastasis. The liver alveococcus may spread to lymph nodes of the porta hepatis (Figure 7G), peritoneal cavity and colonic mesentery. According to the results of animal experiment in Japan[28], the mechanism of metastasis was due to the detached proliferated bud, even very small or only a few nuclei entering the blood vessels. Nevertheless, it was incapable of causing metastasis if protoscolices were injected into the vessels.

CLINICAL MANIFESTATIONS

Generally speaking, sex difference is not obvious, sometimes males slightly outnumbered females. Most patients were young and in robust years of their life. According to clinical features[29], four types, i.e. simple hepatic enlargement, obstructive jaundice, liver giant node (cancer-like) and remote metastasis were classified. According to clinical course, we divided AE into the early stage with only mild hepatic enlargement, middle stage with progressive hepatomegaly and the advanced stage associated with impaired liver function, portal hypertension, or metastasis[30]. The main symptom was upper abdominal mass or liver mass with stiff and nodular feeling on palpation. The size of the mass varied, with the smallest being not felt or only palpated below right sub-costal margin and the largest reaching the umbilicus level (Figures 8A and B). Another symptom was abdominal pain, but usually not severe. Jaundice was found in some cases, usually due to the impairment of liver function or the invasion and compression of bile duct by liver mass at the advanced stage. Extensive infiltration of the alveococcus lesion or fibrosis caused liver cirrhosis with subsequent portal hypertension such as the superficial varicosity of the thoracic or abdominal wall, ascites and splenomegaly. In case of liver AE associated with lung or brain metastasis, the patient manifested corresponding respiratory or neuropathic symptoms and signs.

Figure 8 The size of upper abdominal mass with the largest reaching the umbilicus level. A: After external drainage of pseudocyst, the patient survived for 23 years. B: The patient survived only for 3 years.

IMAGING EXAMINATIONS

X-ray films

Enlargement of liver and elevation of right diaphragm were shown on abdominal plain film. The shadows of spotted or clustered calcification were visible in the hepatic region. But they must be carefully observed as their appearances were usually not distinct. If multiple shadows of bilateral lung showed on chest film, the possibility of liver AE associated with lung metastasis ought to be considered (Figure 9).

Figure 9 Imaging examinations. A: Ultrasonic scanning of liver AE showed a central necrotic pseudocystic cavity, but the cystic wall was irregular; B: Liver CT showed partial AE calcification (left) and pseudocyst (right) on the left upper side. The patient was confirmed surgically and pathologically.

Ultrasonic scanning

In a series of 141 cases with liver AE[31], the ultrasonic features revealed solid mass in 96 and solid-cystic degeneration in 45. The former comprised 23 cases with local type, 21 with diffuse nodular type and 52 with large circumscribed mass type. If the liver alveococcus was complicated by central necrotic pseudocyst, the ultrasonic scanning showed echo-free area with irregular cystic wall (Figure 9A).

Computerized tomography (CT)

Liver CT showed single or multiple, intrahepatic, hypodense areas with irregular margin, or occasionally partial calcification of alveococcus (Figure 9B).

IMMUNOLOGICAL EXAMINATIONS

As hydatid intradermal test was simple and cheap, it was commonly used, especially in our country. Although the sensitivity was high, false positive reaction may occur. Therefore indirect hemagglutination test and ELISA were reliable methods for immunological diagnosis due to their higher specificity. Along with current advancement of molecular immunology, purification of a specific antigen from Echinococcus multilocularis (Em) has been successful. As early as in 1983, Gottstein et al[32,33] prepared an antigen fraction (Em2) from alveococcus tissue using affinity chromatography. Owing to a high specificity of Em2 for E. multilocularis, a correct serological differential diagnosis was achieved in 95% of 57 confirmed cases of human CE or AE. However, Em2 was isolated from laminated layer of alveococcus, a higher or lower level of antibody titer did not reflect whether the focus was active or not. Em2-ELISA test still showed positive result with a high titer even though the focus became stable or calcified after chemotherapy. As the protoscolex is the most active component of the alveococcus tissues, it was used to isolate protoscolex antigens designated as Em16 and Em18 using Western blotting in a cooperative study between China and Japan[34]. Em18 and Em16, especially the former showed not only a higher sensitivity but also stronger specificity for immunodiagnosis of human AE. We also used the alveococcus protoscolices for isolating Em16 and Em18 by isoelectric focusing analysis[^35]. These protein antigens could not show the best diagnostic value and cross reaction which possibly occurred between AE and CE or between AE and cysticercosis[36]. In short, molecular immunological diagnosis for human AE needs further studies.

DIAGNOSIS

Until now, liver AE is always mis-diagnosed. To make a correct diagnosis of liver AE, we should not ignore the past histories of the patients. First, where did the patient come from, AE endemic area? Next, did the patient contact dog, cat, or fox-fur? Thirdly, what is the occupation of the patient, fox-hunter or dog-raiser? These data would be helpful for the diagnosis of liver AE.

DIFFERENTIAL DIAGNOSIS

Liver cancer

In our collective review of 274 cases with liver AE, the determination of serum AFP was negative in all the patients[27]. So it is significant for differential diagnosis of AE. As a rule, liver AE pursues a slow, but progressive course. It obviously differs from liver carcinoma which develops faster or very rapidly, leading to patient death in a short time. AFP test is a reliable differential diagnostic method. Sometimes, gross findings of liver AE may be confused with carcinoma on the operation table and frozen section is necessary. When the diagnosis of liver AE is still difficult before operation, liver needle biopsy may be considered (Figure 10). Occasionally liver AE also needs to be differentiated from cystic echinococcosis (CE) or some nonparasitic diseases such as tuberculosis. If chest X-ray film showed multiple shadows of bilateral lung or brain CT showed cerebral lesion, it might be possible that liver AE was associated with lung or/and brain metastasis[37]. Clinically unknown nature of malignant tumor was always determined.

Figure 10 Liver needle biopsy. Arrow: alveococcus (HE stain×200).

TREATMENT

Surgical operation

The rational treatment for liver AE is radical hepatectomy and the prognosis of the patient may be good. An individual case had been postoperatively followed up for 21 years without recurrence[2]. However, the rate of hepatectomy is very low due to the difficulty of early diagnosis. According to our collective analysis of 258 cases of liver AE in China[27], only 27(10.5%) and 16 (6.2%) had performed radical liver lobectomy and partial hepatectomy respectively. In order to increase the percentage of hepatectomy, an epidemiological survey in AE endemic area is very significant to screen out early cases. For liver unresectable case, a palliative operation may be indicated such as surgical drainage for a large pseudocyst in order to decompress pericystic liver tissues. As in Figure 8A, the patient was alive for 23 years after external drainage of liver pseudocyst.

Chemotherapy

Albendazole (ABZ) is considered by WHO as the best anti-AE drug. Experimental studies showed that ABZ was able to inhibit alveococcus proliferation and growth. Considering the clinical effects, it was shown to alleviate the symptoms, to prolong the survival, and diminish lung or/and brain metastasis after therapy. In our 23 cases of liver AE treated with ABZ, symptoms were improved, appetite increased, jaundice relieved, and liver enlargement decreased in 15 (65.2%)[38] cases. Liu et al[39] reported that in 11 cases of liver AE followed-up for 2-7 years after long-term continuous ABZ therapy, 7 showed calcification on liver CT film. Thus ABZ may be lethal to the parasite. In addition, we found a novel compound extracted from traditional Chinese medicine, Xiao-Bao capsule for treatment of AE. Experimental results showed that the inhibitory rate of mouse AE was 65.7% and 80.6% respectively in two groups of mice, i.e., 1-week and 10-weeks after innoculative infection[40]. In our 21 cases of liver AE, symptoms improved in 16 (76.1%)[27] cases. Both acute toxicity test in mice and chronic toxicity test in rats showed no toxic reactions[41]. We postulate that combination of Xiao-Bao capsule and ABZ would be more effective than single medication.