Unique Family Clustering of Human Echinococcosis Cases in a Chinese Community

UNIQUE FAMILY CLUSTERING OF HUMAN ECHINOCOCCOSIS CASES IN A CHINESE COMMUNITY

YU RONG YANG, MAGDA ELLIS, TAO SUN, ZHENGZHI LI, XONGZHOU LIU, DOMINIQUE A. VUITTON, BRIGITTE BARTHOLOMOT, PATRICK GIRAUDOUX, PHILIP S. CRAIG, BELCHIS BOUFANA, YUNHAI WANG, XIAOHUI FENG, HAO WEN, AKIRA ITO, AND DONALD P. MCMANUS* Ningxia Medical College, Yinchuan, Ningxia Hui Autonomous Region, People’s Republic of China: Molecular Parasitology Laboratory, Queensland Institute of Medical Research, Brisbane, Queensland, Australia; Surgery Department of Xiji County Hospital, Ningxia Hui Autonomous Region, People’s Republic of China; World Health Organization Collaborating Centre for Prevention and Treatment of Human Echinococcosis, University of Franche-Comte and University Hospital, Besancon, France; Cestode Zoonoses Research Group, University of Salford, Salford, United Kingdom; Department of Surgery, Xinjiang Medical University Hospital, Xinjiang, People’s Republic of China; Department of Parasitology, Asahikawa Medical College, Asahikawa, Japan

Abstract. We have identified a significant focus and unusual clustering of human cases of cystic echinococcosis (CE) and alveolar echinococcosis (AE) in the village of Nanwan, Xiji County, Ningxia Hui Autonomous Region, in one of the most highly endemic areas for both diseases in China. The village, a Chinese Hui Islamic community, is composed of 167 members of four extended families. A total of 28 people died (12 of echinococcosis) since the village was first settled in the 1950s. Despite similar life patterns, the number of AE and CE cases occurring in each family was different. Overall, the prevalences of AE and CE were 9% (20 cases) and 5.9% (13 cases), with a combined prevalence of 14.9%. In contrast to CE, a comparison of the prevalence of AE indicated significant differences between the four family clusters. Although suggestive that host genotype might play a role in susceptibility to AE, this hypothesis requires further investigation.

INTRODUCTION both AE and CE. The results of the survey showed a very serious focus of human echinococcosis (both CE and AE), a Echinococcosis is a zoonosis caused by adult or larval rare case of AE and CE in a single patient, and an unusual stages of cestodes belonging to the genus Echinococcus (fam- family clustering of AE cases. As a result of the usually low ily Taeniidae). Larval infection is characterized by long term prevalence of AE in disease-endemic areas (mostly western growth of metacestode (hydatid) cysts in the intermediate European countries) studied, family clustering of cases has host. The two major species of public health importance are been considered to be extremely rare.6 Echinococcus granulosus and E. multilocularis, which cause cystic echinococcosis (CE) and alveolar echinococcosis (AE), respectively. They are serious life-threatening diseases, espe- METHODS cially AE, and they have high fatality rates and poor progno- Study area and community. The village of Nanwan (Figure 1–4 sis if careful clinical management is not carried out. 1) is located on the slopes of Yueliang Mountain in north- Echinococcosis is recognized as an increasing health prob- eastern Xiji, part of the Liupan Mountain range. Residents lem in China with 60 million persons at risk of infection and are farmers growing crops (potatoes, carrots, soy beans, 2 1.3 million reported cases, one third of whom are children. wheat). In recent years, with dramatic population growth, low Most of the at-risk population is resident in seven provinces/ economic development and poor communication with the Autonomous regions of central and western China that in- outside, large areas of local forest were cut down for eco- 2,5 clude Ningxia Hui Autonomous Region (NHAR). Unlike nomic purposes and the grassland was transformed into areas other parts of the world, E. granulosus and E. multilocularis for cultivating crops, resulting in serious erosion around Nan- occur sympatrically in most Chinese disease-endemic areas, wan. including southern NHAR; however, few clinical or commu- Domestic dogs and cats were previously very numerous nity studies have been reported from this area. (almost every household had one dog and/or cat) although We describe here a community investigation of AE and CE the numbers had decreased dramatically by 1997 when only undertaken in 2001 and 2002 as part of an ongoing investiga- three dogs remained in the entire village. This was due to the tion of the epidemiology of echinococcosis in NHAR. The introduction in the early 1990s of poisoned baits to control the focus was Nanwan, a small village situated in the northeastern large rodent population present in the village. The poisoned part of Xiji county and inhabited only by Chinese Hui, a baits or rodents were inadvertently eaten by the dogs, result- minority Islamic group (Figure 1). The village and surrounding in a sharp decrease in their numbers. Sheep farming, 2 ing farmland, which occupy an area of approximately 10 km , home slaughter, and throwing viscera to dogs is a common are close to the border of Guyuan, another county. Its unique practice, particularly during religious (Islamic) festival sea- population resulting from a 50-years settlement by only four sons. Other animals such as donkeys, rabbits, cats and horses, families gave us the opportunity to assess potential family although not abundant, were also commonly kept. The whole clustering of echinococcosis in an area highly endemic for village used valley rivulets and two shallow ground springs for its water supply that could be accessed by both animals and humans until 1997. Because of food shortages after frequent * Address correspondence to Donald P. McManus, Molecular Para- sitology Laboratory, Queensland Institute of Medical Research, 300 annual natural disasters, collecting herbs and wild vegetables Herston Road, Brisbane, Queensland 4006, Australia, E-mail: and hunting wild animals, especially large mammals, were [email protected] usual daily occurrences. However, some recent changes have 487 488 YANG AND OTHERS

FIGURE 1. Location of Nanwan in the People’s Republic of China. occurred. After 1997, a reticulated water system was set up to or vegetables, and a medical history of either AE or CE for provide protection of the water source from contamination by subjects and their relatives. All inhabitants ranging from in animals. Current extension of plowing fields has resulted in a age from 7 to 70 years were invited to participate in the survey continuous increase in the local rodent population, and there in 2001 and 2002, usually in family groups. The completion of has been a reduction in the wild fauna because of deforesta- the questionnaire was carried out at an interview mainly with home units in 23 ס tion. Numerous wild animals including foxes, wolves, rodents, the husbands from each family (total and rabbits previously inhabited the area surrounding Nan- 2001), with the help of local county leaders. Additional ques- wan, where most of the slopes were previously covered by tions included the history of the settlement, land use around shrubs, forest and grassland. the village in the past 30 years (forests, shrub or scrub land, Questionnaire and data collection. A single-page question- plowed fields and crops), water source(s) for the community, naire was used to register individuals in the survey and record and presence of animal hosts for both E. granulosus and E. information on each person in the village. The questions were multilocularis in the immediate village environment. All pro- designed to record personal data including name, age, sex, cedures related to the study and the purposes of collecting previous and present address, marital status and family lin- blood were thoroughly explained to the subjects. They were eages, behavioral information, including dog ownership, fox told that this was totally voluntary and the subjects were in- hunting history, and contact with fox skins, eating of wild fruit formed that, if they refused to partake, it would not affect the CLUSTERING OF ECHINOCOCCOSIS CASES 489 standard care given to patients in the study site. All human Bartholomot and others.7 The native Em 18 and recombinant adult participants or the parents or legal guardians of minors Em 18 antigen preparations from E. multilocularis and their were then asked to sign an informed consent. The study was use in an ELISA and immunoblotting assay followed the reviewed and approved by the Ethical Review Board of methods of Ito and others14 and Xaio and others.15 Ningxia Medical College and the Regional Clinical Ethical Data management and analysis. Chi-square values and Committee, Besancon, France, according to National Insti- 95% confidence intervals were calculated using Epi-Info tutes of Health (Bethesda, MD) procedures. (Centers for Disease Control and Prevention, Atlanta, GA) Screening for human AE and CE. After completion of the and SPSS version 11.5 (SPSS, Inc., Chicago, IL) for analyses questionnaire, a liver scan was performed on all volunteers by of prevalence. Multiple variate logistic regression was used to a sonographer using an ultrasound scanner (model 3.5 calculate adjusted odds ratios to assess the relationship of dog MG67N-35F2.4; Aloka, Tokyo, Japan). Sonograms were re- ownership to Echinococcus spp. infections while controlling corded using a thermal printer (Sony, Tokyo, Japan). There is for factors as age, sex, and families. The Statistical Analysis generally no difficulty in differentiating established AE from for Genetic Epidemiology Release 5.0 program (S.A.G.E. CE in the liver by ultrasonography, according to a validated version 5.0, Cleveland, OH)16 was used for family correlation procedure.7,8 The CE cases were recorded using the World estimation and homogeneity tests were used in the correlation Health Organization Informal Working Group classification estimations. of ultrasonographic images.9 A small sample of blood was taken from the ear lobe onto strips of Whatman (Brentford, RESULTS United Kingdom) no. 1 filter paper.10 The strips were air- dried, stored at 4°C, and then transported to the laboratory Community survey. Nanwan village is comprised of four for specific antibody testing. A 5-mL venous blood sample for families with 168 inhabitants resident in late December 2001 serum was taken from any person with an abnormal liver scan when the first survey was undertaken. Of these, one 37-year- image. The serum was kept at 4°C during transport and then old man died of AE before the 2002 follow-up survey. Origi- stored at −20°C before testing. An individual was confirmed nally, three families (H, W, and M) settled in this village in the as having AE or CE because of a pathognomonic image by 1950s. Family H was composed of three brothers who arrived ultrasound (with supporting serology) or after positive sero- with their parents, and produced three family lineages (H1, logic confirmation of a query image. All identified AE cases H2, and H3). A fourth, family L, settled in the village in the were offered a minimum treatment of a three-month course 1960s. A daughter of family M married a son of family L in of albendazole, and CE cases were advised to seek treatment the late 1970s. All individuals who were registered as living at local hospitals. Further follow-up of the patients was orga- village residents for the 2001 survey are listed in Table 1. This nized by the investigators. population included 159 individuals (82 females and 77 males) Serologic tests. A rapid dot immunogold filtration assay with ages ranging from 3 months to 70 years for females and (dot-immunogold filtration assay) combined test was used to 1–60 years for males. There were 68 villagers less than 12 detect antibodies to AE/CE, according to the instructions years of age, 4 in family H1, 28 in family H2, 6 in family H3, provided by the manufacturer (Approval no. [2000] 400004; 16 in family W, 12 in family M, and 2 in family L. Xinjiang Bio. Ltd., Urumqi, Xinjiang, China). The antigens When the 2001 and 2002 community surveys were com- used in this combined test were crude hydatid cystic fluid, bined, 113 villagers (72%) Ն 7 years of age were examined by antigen B from hydatid fluid, and a protoscolex extract de- liver ultrasound scanning. Of these, 100 provided blood rived from E. granulosus metacestodes.11,12 The Em2 antigen samples for serologic testing. The ultrasound results showed was an affinity-purified cyst homogenate antigen from E. mul- that there were 8 individuals with AE, 11 with CE, and 1 with tilocularis.13 An enzyme-linked immunosorbent assay both AE and CE (Table 1). Information obtained from the (ELISA) for serum and filter paper blood samples using E. questionnaire indicated that one additional AE case and one granulosus antigen B or E. multilocularis protoscolex extract additional CE case occurred in two family members that were was conducted using the procedures of Craig and others10 and resident outside the village at the time of the survey (Table 1).

TABLE 1 All registered inhabitants (1950s–December 2001) and echinococcosis cases identified by ultrasound/questionnaire (2001–2002) in Nanwan village, China*

Questionnaire

Registered villagers Ultrasound Cases living Deceased

Family Living resident Absent Deceased AE CE AE/CE AE CE AE CE H1 10 3 4 2 0 1 0 0 1 0 H2† 61 12 10 6 5 0 1 0 6 0 H3 10 3 3 0 0 0 0 0 1 0 W361040100001 M35750500120 L70‡10000000 Total 159 35‡ 27 8 11 1 1 1 10 1 .mixed/simultaneous infection ס cystic echinococcosis; AE/CE ס alveolar echinococcosis; CE סAE* † One male member of family H2 died of AE after the 2001 survey but before the 2002 follow-up survey. Therefore, family H2 was composed of 72 persons in 2002 and the total number of deceased persons in 2002 was 28. ‡ Nine persons were temporarily absent from the village at the time of the 2001 survey. The remaining 26 persons had left permanently. 490 YANG AND OTHERS

ס P ,3.8 ס In addition, the questionnaire recorded 10 AE cases and 1 CE prevalence of CE prevalence than of AE (␹2 case in deceased Nanwan villagers, with most confirmed by 0.051). local authority records. Therefore, there were 20 AE cases A comparison of accumulative prevalence between and (9%) and 14 CE cases (6%), including one mixed AE/CE within the four families based on the ultrasound and ques- case, in the 221 villagers. tionnaire data is shown in Table 3. The prevalence of AE was ,10.29ס The results of serologic analysis correlated well with those significantly higher in family H than in the others (␹2 P < 0.05), especially when compared with family W ,3 ס obtained by ultrasound for diagnosis of AE but less so for CE df P < 0.05). There were no statistical differences in ,5.61 ס Table 2). Three villagers were seropositive for antibodies (␹2) only to E. multilocularis, 9 were seropositive for antibodies to CE prevalence and combined AE and CE prevalence be- both E. multilocularis and E. granulosus, and 20 were sero- tween the various families, except between the families H and -P < 0.05). The prevalence of AE was signifi ,4.31 ס positive for antibodies only to E. granulosus. W(␹2 > P ,4.95 ס Of the 8 ultrasound-positive AE cases detected by the sur- cantly higher than that of CE within family H (␹2 vey, the one serologically negative individual had fully calci- 0.05), but not in the other families. Multiple comparisons for fied images (1 × 1.28 cm in the right liver lobes and 0.8 × 1cm AE/CE between and within families indicated that the cur- in the left liver lobes). Five of the cases were co-positive by rent (2001–2002) and accumulative prevalences were gener- serologic analysis for antibodies to E. multilocularis and E. ally similar, although some differences were apparent (Table granulosus. Of 11 ultrasound-positive CE cases detected by 3). Within family H, the accumulative prevalence of AE was .(0.026 ס P ,4.9 ס screening, six were seronegative, indicating a false-negative significantly higher than that of CE (␹2 and (0.051 ס P ,3.80 ס rate of 54%. Three were from family H2, with ultrasound Within family M, the current (␹2 CE prevalences were (0.32 ס P ,0.98 ס images from two of the cases showing very early cyst devel- cumulative (␹2 opment, and the third showing a type 5 CE ultrasound image9 higher, although not significantly higher, than those for AE. with multiple, small calcified cysts. The remaining three were Family correlation with echinococcosis in Nanwan villag- males from family M, one elderly person with a type 4 CE ers. The questionnaire data were combined with the male/ ultrasound image (solid mass) and two younger men, one with female resident local government registered documents for type 3 and type 4 CE images, and the other with an irregular compilation of family member information. Ultrasound scan- residual cavity remaining after a previous surgery for CE. ning and serologic analysis showed that the confirmed AE Overall, the false-negative serologic rate for AE and CE was and CE cases were mainly distributed in the second and third 35%. The rate of disagreement between ultrasound and se- generations of each family. Because the L and M families rologic diagnosis was 26%, and this included the seven false were related through marriage, we combined them into one seronegative cases described earlier in this report. In addition, family (L-M); thus, family correlations were determined for there were 19 seropositive individuals who were not con- three families (Table 4). The results indicated that for AE, firmed by ultrasound although cysts in other organs other sibling relationships (brother-brother, sister-brother, and sis- than the liver or abdomen would have been missed. ter-sister) showed significant correlations with infection, as Current situation and family history. The distribution of did the relationship between mother and offspring (son and AE and CE in individuals from the four families representing daughter). In contrast, there was no significant correlation the entire population of Nanwan is shown in Table 3. Up to between infection with CE and any of the various family re- 2002, the overall prevalences for AE and CE were 4.7% and lationships. Using homogeneity tests, we observed that com- 6.2%, respectively, with one simultaneous AE/CE case. The parisons for parents and offspring showed significant differ- combined prevalence of AE and CE was 11.4% for living ences (P < 0.001) in AE infection between mother and son, family members of Nanwan village (Table 3). mother and daughter, father and son, and father and daugh- Analysis of the survey data for AE and CE (Table 3) indi- ter, but not between siblings; no differences were evident cated a higher prevalence of AE infection in family H than in between any of the family relationships for CE. -P < 0.05). Risk factors. The questionnaire data showed that dog own ,3 ס [degrees of freedom [df ,8.36 ס the others (␹2 There was no significant difference for CE prevalence or ership and dog ownership history had changed in the recent combined AE and CE prevalence between the four families. past. Thus, if the questionnaire for teenagers (young children) There was no significant difference in the prevalence for AE showed no dog, while an answer from their parents showed and CE within each family, although family M had a higher that they had a dog several years ago, the risk factor will be

TABLE 2 Results of ultrasound scanning compared with serology from the Nanwan, China village surveys (2001 and 2002)*

Serology Ultrasound Ultrasound scanning AE AE/CE† CE Negative positive AE 2 5 1 8 AE/CE‡ 1 1 CE 2 3 6 11 Normal 1 1 17 61 0 Total 3 9 20 68 Total seropositive Total ultrasound positive 32 20 * For definitions of abbreviations, see Table 1. † This indicates positive serologic results for both AE and CE. ‡ Both AE and CE lesions (simultaneous infection) were evident in ultrasound images. CLUSTERING OF ECHINOCOCCOSIS CASES 491

used as a dog owner for all family members. Because of water shortages and limited area for habitation, the entire Nanwan village obtained water from the same valley rivulets and shal- 8 (0) low ground springs; thus, all villagers shared the same water supply until 1997. Although general discussions in the village

nnaire data* indicated a history of hunting by many older villagers, this practice was completed denied in individual questionnaires. This biased answer probably occurred because hunting of large wild animals is forbidden by the Chinese authorities. 0/50 (0) 2/50 (4) Other behavioral traits were comparable because all villagers shared the same religion, poor economy, and education levels. Therefore, logistic regression analysis with dog owner- ) 0/16 (0) 1/16 (6)

13) 0/47 (0) 8/47 (17) ship/ownership history as exposure variables and other po-

7 (0) 1/17 (5.9) 4/17 (24) tential factors such as age, sex, and families/households was

5/83 (6) 0/83 (0) 18/83 (22) used to calculate adjusted odds ratios to assess relationships of the multiple variants to Echinococcus infection (Table 5). There was no significant difference between the three families ) 5/116 (4) 1/116 (0.9) 23/116 (20) for AE or CE infection or echinococcosis when dog ownership/ownership history was compared. The differences that occurred in those less than 30 years of age did not indicate a significant risk factor for CE and echinococcosis. When the

2. family group as a risk factor was compared, the H family, in particular, and the L-M family showed significant associations with AE infection. In contrast, there was no significant association for CE within any of the families. ity (%) Cumulative prevalence (%) DISCUSSION

.7) 11/27 (40.7) 19/221 (8.6) 13/221 (5.9) 1/221 (0.5)This 33/221 (14.9) community survey identified a major echinococcosis focus and an unusual clustering of human cases of CE and AE 3 he four families in Nanwan village, China bassed on ultrasound and questio in Nanwan village in NHAR, People’s Republic of China,

ABLE with a combined prevalence of 11%. Alveolar echinococcosis T was a significant cause of death in this village, which was

combined echinococcosis cases and prevalence. founded in the 1950s. This study confirmed the particular severity of this disease when patients have no access to any ס type of treatment. The village was composed of four family clusters and the inhabitants lived in the same ecologic and social environment and shared a similar life style and religion. However, the prevalence of AE and CE cases in the four families differed significantly. The most striking feature of

02 follow-up survey. Therefore, family H2 was composed of 72 persons in 200 this analysis was the clustering of AE cases in family H and the family composition, which showed a correlation between mother-offspring and sibling-sibling. All AE/CE patients

mixed/simultaneous infection; Echino. were distributed in the second and third generations, which probably reflects higher levels of Echinococcus transmission ס in the past, compared with the current situation, and provides potential insight into the sibling correlation obtained. There were no significant differences in environment between the families/households detected in this regression analysis, indicating that the difference in prevalence between the families may be due to genetic factors, which could also, at least in

cystic echinococcosis. AE/CE part, account for the strong mother-offspring correlation. ס

Living individuals/current prevalence (%) Deceased individuals mortal Other studies have shown only limited correlations of family members being affected by echinococcosis, although village 2002) (based on US), mortality and cumulative prevalence distributed in t – clustering has been reported frequently.5,17,18 The coexistence of both AE and CE in the same commu- AE CE AE/CE Echino.nity AE indicates CE that the environment Echino. AE was conducive CE to the AE/CE Echino.

7/73 (10)9/99 (10) 5/73 (7) 5/99 (5) 0/73 (0) 1/99 (1) 12/73 (17) 15/99 (15) 6/10 (60)transmission 8/17 (47) 0/10 (0) 0/17 (0) 6/10 (60) of 8/17both (47) 13/83 (16) forms 17/116 (15 of the disease. Diagnosis of a combined case of CE and AE in the same patient, albeit not alveolar echinococcosis; CE 19 unique, is an example of the infection pressure by both ס Prevalence (2001 † One male member ofSubtotal family of H2 family dies H. of AE after the 2001 survey but before the 20 Echinococcus species in this community. Total 9/194 (4.7) 12/194 (6.2) 1/194 (0.5) 22/194 (11.4) 10/27 (37) 1/27 (3 ‡ *AE † ‡ Family H1H2 H3H 2/13 (15) 0/13 (0) 0/13 (0) 0/13 (0) 1/13 (8) 0/13 (0) 3/13 (23) 0/13 (0) 1/4 (25) 1/3 0/4 (33) (0) 0/3 (0) 1/4 (25) 1/3 (33) 3/17 (18) 1/16 (6) 0/1 0/16 (0 WML 0/46 (0) 0/42 (0) 0/7 (0) 1/46 (2) 6/42 (14) 0/7 (0) 0/46 (0) 0/42 (0)Among 0/7 (0) 1/46 (2) 6/42 (14) the well 0/7 (0) 0/4 2/5 (0) (40) recognized 0/5 (0) 1/4 (25) 0/1 (0) behavioral 1/4 2/5 (25) (40) 0/1 (0) factors 0/50 2/47 (0) (4) 0/1 (0) for 2/50 6/47 (4) ( both 0/8 (0) CE 0/8 (0) 0/8 (0) 0/ 492 YANG AND OTHERS

TABLE 4 Family correlation analysis and homogeneity tests among families H, W, andL–M of Nanwan villagers in China

AE CE

Parameters Pairs Correlation SE Correlation SE Family correlation Husband–wife 44 0.23 0.15 −0.14 0.15 Parent–offspring 320 0.08 0.1 −0.1 0.06 Sibling 308 0.53* 0.1 0.11 0.09 Father–son 99 −0.16 0.13 −0.12 0.11 Mother–son 101 0.3* 0.13 −0.10 0.12 Father–daughter 60 −0.18 0.16 −0.07 0.13 Mother–daughter 60 0.59* 0.12 −0.07 0.13 Brother–brother 125 0.46* 0.12 0.20 0.13 Sister–brother 125 0.57* 0.1 0.02 0.11 Sister–sister 58 0.56* 0.17 −0.026 0.14 Homogeneity tests P > 0.05 †0.12 ס P < 0.001 ␹2 †23.8 ס Parent–offspring 320 ␹2 P > 0.05 ‡1.86 ס P > 0.05 ␹2 ‡0.97 ס Sibling 308 ␹2 * indicates a significant association. .3 ס degrees of freedom † .2 ס degrees of freedom ‡ and AE, poor life style of villagers, severe water shortages, population and the requirement for agricultural land have contacts with infected dogs, lack of knowledge regarding pre- resulted in deforestation. Scrub and shrub lands after exten- vention of echinococcosis, and eating of Echinococcus eggs sive deforestation have been shown to favor high densities of could play an important role in the existence of both AE and rodents that may act as intermediate hosts for E. multilocu- CE.3,20–22 Islamic culture could also play a role in increasing laris. Similar factors were noted in surveys of echinococcosis levels of transmission by promoting community sheep slaugh- in southern Gansu Province.5 The influence of permanent tering for religious purposes, as has been suggested in North pasture increasing AE transmission was also reported in Eu- African and Middle East countries.23–25 Dog ownership and rope27 and on the Tibetan plateau.28 With regard to human dog ownership history have been correlated with E. granulo- transmission in the future, further landscape and bioecologic sus transmission.20 In addition, the domestic water supply studies will be required to clarify the transmission factors could have played a major role in CE and AE transmission in involved and the risks for AE and CE infection at the village Nanwan, as shown previously by Dowling and others26 in level in Ningxia. Persons who that are currently less than 12 Jordan for CE. The environmental and behavioral risk factors years of age (40% of the population of Nanwan village) could also explain the unusually high prevalence (5%) for should be screened again because Echinococcus infection human AE in the community we studied, which has one of the may occur early in life and there is a long-lasting latent phase highest prevalence rates for AE. However, these factors could in both diseases. not have influenced the family differences because these risk The unique history of this village, which has a limited num- factors were similar in all four families from the questionnaire ber of families, a common life style and culture, a limited study. Additional environmental factors might have played a mobility outside the county, and an unusually high prevalence specific role in increasing AE prevalence because numerous of both types of echinococcosis ascertained by systematic wild animals, such as foxes, wolves, lagomorphs, and rodents, combined ultrasound and serologic screening on the living and domestic dogs and livestock were present in Nanwan and population and medical records for the deceased, allowed us the neighboring areas in the recent past. Furthermore, the to undertake correlation analysis among family members. To changes in land use due to sharp growth in the local human our knowledge, such a family analysis has never been re-

TABLE 5 Multiple logistic analysis of the relationship of dog ownership and echinococcosis, and other potential risk factors among Nanwan villagers in China*

CE AE AE/CE Parameters OR (95% CI) OR (95% CI) OR (95% CI) Individual information Females 0.38 (0.09–1.71) 0.21 (0.03–1.51) 0.39 (0.12–1.27) Յ30 years old 0.18 (0.04–0.81)†–0.05 (0.01–0.18)† Family code H 0.42 (0.08–2.11) 11.81 (1.23–114.43)‡ 1.46 (0.40–5.29) L–M 2.37 (0.47–11.86) 0.08 (0.009–0.81)† 0.68 (0.19–2.48) W 0.29 (0.03–2.86) – 0.23 (0.02–2.37) Dog owner information No dog 0.84 (0.17–4.21) 3.51 (0.47–26.15) 1.33 (0.40–4.44) no values shown because no AE cases reported in the W family and there were too few AE cases in villagers less than 30 years of age. For ס – .confidence interval ס odds ratio, CI סOR* definitions of other abbreviations, see Table 1. † indicates a significant association with decreasing risk. ‡ indicates a significant association with increasing risk. CLUSTERING OF ECHINOCOCCOSIS CASES 493 ported for echinococcosis. Genetic variations might account Franche-Comte and University Hospital, Besancon, France. Philip S. for the prevalence differences evident in this rural group. Craig, Belchis Boufana, and Xiaohui Feng, Cestode Zoonoses Re- search Group, University of Salford, Salford M5 4WT, United King- Genetic correlations with resistance or susceptibility to other, dom. Yunhai Wang and Hao Wen, Department of Surgery, Xinjiang including parasitic, diseases have been recognized and exten- Medical University Hospital, Xinjiang, People’s Republic of China. sively investigated,29–37 but they have been largely ignored Akira Ito, Department of Parasitology, Asahikawa Medical College, for echinococcosis. No genetic studies have been reported for Asahikawa, Japan. CE, although two family-aggregated CE cases have been re- Reprint requests: Donald P. McManus, Molecular Parasitology Labo- ported17,18 and only population studies are available for ratory, Queensland Institute of Medical Research, 300 Herston Road, AE.38–40 Cases of AE observed in central China were shown Brisbane, Queensland 4006, Australia, Telephone: 61-7-3362-0401. Fax: 61-7-3362-0104; E-mail: [email protected]. to be associated with HLA-DR4, but no immunologic correlations were reported.41 Although immunogenetic correlations with AE have been REFERENCES made, until now the absence of a well-studied community 1. WHO, 1996. Guidelines for treatment of cystic and alveolar echi- with a high prevalence of the disease that shared common risk nococcosis in humans. WHO Informal Working Group on factors for a prolonged period of time has been an obstacle to Echinococcosis. Bull World Health Organ 74: 231–242. any further analysis. In this community analysis, there was a 2. Ito A, Urbani C, Jiamin Q, Vuitton DA, Dongchuan Q, Health highly heterogenous distribution of prevalence in four fami- DD, Craig PS, Zheng F, Schantz PM, 2003. Control of echi- lies and evidence of segregation of echinococcosis infection in nococcosis and cysticercosis: a public health challenge to in- ternational cooperation in China. Acta Trop 86: 3–17. two of the family clusters. This suggests that this familial pat- 3. Vuitton DA, Zhou H, Bresson-Hadni S, Wang Q, Piarroux M, terning is most likely due to genetic factors. Future linkage Raoul F, Giraudoux P, 2003. Epidemiology of alveolar echi- analysis designed to localize the specific loci involved would nococcosis with particular reference to China and Europe. be informative. Furthermore, whole genome scanning using Parasitology 127 (Suppl): S87–107. 4. McManus DP, Zhang W, Li J, Bartley PB, 2003. Echinococcosis. highly polymorphic genetic markers to define specific loci Lancet 362: 1295–1304. involved in susceptibility/resistance to AE and CE, may per- 5. Craig PS, Giraudoux P, Shi D, Bartholomot B, Barnish G, Delat- mit identification of the individual genes influencing the pro- tre P, Quere JP, Harraga S, Bao G, Wang Y, Lu F, Ito A, gression of these serious diseases. The combination of ultra- Vuitton DA, 2000. An epidemiological and ecological study of sound examination, which may assess the actual presence of human alveolar echinococcosis transmission in south Gansu, China. Acta Trop 77: 167–177. the disease, and highly specific serologic analysis, which may 6. Pelletier F, Beurton I, Bardonnet K, Bardonnet K, 2000. Three reflect the contamination pressure and individual resistance/ family observations of alveolar echinococcosis. Rev Fr Med susceptibility to disease development after Echinococcus in- Interne 21 (Suppl 22): S263. fection, is particularly appropriate for investigating the vari- 7. Bartholomot G, Vuitton DA, Harraga S, Shi D, Giraudoux P, Barnish G, Wang Y, Macpherson CN, Craig PS, 2002. Com- ety of factors influencing the etiology of echinococcosis in a bined ultrasound and serologic screening for hepatic alveolar community such as Nanwan. The high frequency of positive echinococcosis in central China. Am J Trop Med Hyg 66: 23– serologic results without lesions or associations with calcified 29. lesions in healthy subjects living in areas endemic for AE has 8. Macpherson CN, Bartholomot B, Frider B, 2003. Application of been stressed in several studies7,42,43 and was found in ours. ultrasound in diagnosis, treatment, epidemiology, public health and control of Echinococcus granulosus and E. multilocularis. These subjects and their particular response to various anti- Parasitology 127 (Suppl): S21–S35. genic components of the parasite should also be carefully 9. World Health Organization Informal Working Group, 2003. In- taken into account in further family immunogenetic studies ternational classification of ultrasound images in cystic echino- because they may represent examples of genetically resistant coccosis for application in clinical and field epidemiological settings. Acta Trop 85: 253–261. hosts.44,45 10. Craig PS, Deshan L, MacPherson CN, Shi D, Reynolds D, Bar- nish G, Gottstein B, Wang Z, 1992. A large focus of alveolar Received March 8, 2005. Accepted for publication July 9, 2005. echinococcosis in central China. Lancet 340: 826–831. Acknowledgments: Some of the results of this paper were analyzed 11. Ito A, Nakao M, Kutsumi H, Lightowlers MW, Itoh M, Sato S, using the program package S.A.G.E., which is supported by a U.S. 1993. Serodiagnosis of alveolar hydatid disease by western Trans R Soc Trop Med Hyg 87: Public Health Service Resource Grant (RR 03655) from the National blotting. 170–172. Center for Research Resources. We thank all staff at Ningxia Medical 12. Ito A, Ma L, Schantz PM, Gottstein B, Liu Y, Chai JJ, Abdel- College who participated in the community surveys. We are grateful Hafez SK, Altintas N, Joshi DD, Lightowlers MW, Pawlowski to Heather Matthews and Megan Campbell (Queensland Institute of ZS, 1999. Differential serodiagnosis for cystic and alveolar Medical Research) for excellent technical assistance. echinococcosis using fractions of Echinococcus granulosus cyst fluid (antigen B) and E. multilocularis protoscolex (EM18). Financial support: This study was supported in part by Ningxia Medi- Am J Trop Med Hyg 60: 188–192. cal College, the University of Queensland, and an National Institutes 13. Gottstein B, 1985. Purification and characterization of a specific of Health/National Science Foundation Ecology of Infectious Dis- antigen from Echinococcus multilocularis. Parasite Immunol 7: eases Transech project on echinococcosis. 201–212. 14. Ito A, Xiao N, Liance M, Liance M, Sato MO, Sako Y, Mamuti Disclaimer: None of the authors has any conflicts of interest. W, Ishikawa Y, Nakao M, Yamasaki H, Nakaya K, Bardonnet Authors’ addresses: Yu Rong Yang, Magda Ellis, and Donald P. K, Bresson-Hadni S, Vuitton DA, 2002. Evaluation of an en- McManus, Molecular Parasitology Laboratory, Queensland Institute zyme-linked immunosorbent assay (ELISA) with affinity- of Medical Research, 300 Herston Road, Brisbane, Queensland purified Em18 and an ELISA with recombinant Em18 for dif- 4006, Australia. Tao Sun, Zhengzhi Li, and Xongzhou Liu, Ningxia ferential diagnosis of alveolar echinococcosis: results of a blind Medical College, Yinchuan, Ningxia Hui Autonomous Region, test. J Clin Microbiol 40: 4161–4165. 750004, People’s Republic of China, Telephone: 86-951-4091488. 15. Xiao N, Mamuti W, Yamasaki H, Sako Y, Nakao M, Nakaya K, Dominique A. Vuitton, Brigitte Bartholomot, and Patrick Girau- Gottstein B, Schantz PM, Lightowlers MW, Craig PS, Ito A, doux, World Health Organization Collaborating Centre for Preven- 2003. Evaluation of use of recombinant Em18 and affinity- tion and Treatment of Human Echinococcosis, University of purified Em18 for serological differentiation of alveolar echi- 494 YANG AND OTHERS

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