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Keywords:

  • alveolar echinococcosis;
  • Cystic echinococcosis;
  • Ultrasound;
  • Serology;
  • Ningxia Hui Autonomous Region of China
  • Echinococcose alvéolaire;
  • échinococcose cystique;
  • ultrasons;
  • sérologie;
  • Région A utonome de Ningxia Hui en Chine
  • echinococcosis alveolar;
  • echinococcosis quística;
  • ultrasonido;
  • serología;
  • Ningxia Hui Región Autónoma de China

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We correlated ultrasound (US) imaging classifications for human alveolar echinococcosis (AE) and cystic echinococcosis (CE) with serology (ELISA and immunoblotting (IB) incorporating native and recombinant/purified echinococcal antigens) in community surveys (2001–2003) and follow-up (2002 and 2003) of US-confirmed cases in Ningxia, China. One hundred and seventy-one cases (96 with AE, 75 with CE) were identified; of these, US classification and serological data were obtained for 142 and 112 cases, respectively. Seropositive-rates increased in CE patients with highly viable unilocular cyst lesions (Types CL, CE 1 or CE 2) to degenerating primary lesions (CE 3), but then decreased in subjects with inactive (CE 4) or dead (CE 5) cysts. In contrast, there was a constant increase in seropositivity from the early (P1, P2) to the advanced stages (P3, P4) with AE cases. For US-confirmed cases, follow-up by US combined with serology is invaluable for studying the clinical progression of echinococcosis and for detecting recurrent cysts or reinfection post-treatment.

Nous avons corrélé des classifications d'images des ultrasons pour l’échinococcose humaine alvéolaire (EA) et cystique (EC) avec la sérologie (ELISA et immunoblotting incorporant des antigènes échinococcaux purifiés et de recombinaison) dans des surveillances de communauté (2001 à 2003) et dans le suivi (2002 et 2003) de cas confirmés par ultrasons à Ningxia en Chine. 171 cas (96 avec EA, 75 avec EC) ont été identifiés. Pour ces derniers, la classification des ultrasons et les données sérologiques ont été obtenues pour 142 et 112 cas respectivement. Les taux de séropositifs augmentaient chez les patients à EC avec des lésions à kystes uniloculaires fortement viables (types CL, EC 1 ou EC 2) et des lésions primaires dégénératives (EC 3). Mais ces taux diminuaient chez les sujets avec des kystes inactifs (EC 4) ou morts (EC 5). En revanche, il y avait une augmentation constante de la séropositivité pour les stades initiaux (P1, P2) et stades avancés (P3, P4) des cas d'EA. Pour les cas confirmés par ultrasons, le suivi par les ultrasons combiné avec la sérologie est importante pour étudier la progression clinique de l’échinococcose et pour détecter les kystes récurrents ou la réinfection après traitement.

Hemos correlacionado las clasificaciones por imágenes de ultrasonido (US) para la echinococcosis alveolar humana (EA) y quística (EC) con serología (ELISA e inmunoblotting incorporando antígenos echinococcosicos nativos y recombinantes/purificados) en estudios comunitarios (2001–2003) y durante el seguimiento (2002 y 2003) de casos confirmados por US en Ningxia, China. Se identificaron 171 casos (96 con EA, 75 con EC); de estos, la clasificación por US y los datos serológicos se obtuvieron para 142 y 112 casos respectivamente. Las tasas de seropositividad aumentaron en pacientes con EC con lesiones quísticas uniloculares altamente viables (Tipos CL, CE 1 o CE 2) a lesiones primarias degenerativas (CE 3), pero disminuyeron en sujetos con quistes inactivos (CE 4) o muertos (CE 5). En contraste, se observó un aumento constante en la seropositividad desde las etapas tempranas (P1, P2) a las avanzadas (P3, P4) en casos de EA. Para los casos confirmados por US, el seguimiento de US combinado con serología es invaluable a la hora de estudiar la progresión clínica de la echinococcosis así como para detectar quistes recurrentes o una reinfección post-tratamiento.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Echinococcosis is a zoonosis and one of the most important helminthic diseases of humans worldwide (Craig et al. 2003; Nirmalan & Craig 1997). The two major species responsible for human echinococcosis, Echinococcus granulosus (the cause of cystic echinococcosis, CE) and Echinococcus multilocularis (the cause of alveolar echinococcosis, AE), occur in Ningxia Hui Autonomous Region (NHAR), P.R. China (Yang et al. 2005a,b; 2006a,b), where one-third of the population are Islamic Hui, with the majority of the remainder being Han Chinese. The coexistence of both AE and CE in this area indicates that the environment is amenable to transmission of both forms of the disease (Li et al. 1985; Li 1986; Yang et al. 2005a,c). Previously published data of clinical records and results of mass screening have shown that CE occurs throughout NHAR whereas AE occurs predominantly in a confluence area of three counties (Xiji, Guyuan and Haiyuan) in southern NHAR (Yang et al. 2005b, 2006a,c). Here we report on serodiagnostic correlations with ultrasound (US) images from community surveys and follow-up of echinococcosis cases during the period 2001–2003.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Subjects

Volunteer participants came from rural village and urban areas of Xiji, Guyuan and Haiyuan counties in southern NHAR. Community surveys occurred in 2001–2003 and cases were subjected to follow-up in 2002 and 2003. Prior to the surveys, written consent was obtained from all adults and from parents of minors aged five years or older who agreed to participate after approval by the Ethics Committee of Ningxia Medical College.

Methods

All community survey participants underwent a questionnaire survey and upper abdominal US examination. A 5 ml venous blood sample was taken from individuals with a previous surgical history of CE or AE, and from all cases considered abnormal by ultrasonography (US). Venous blood samples were also taken from city-dwelling blood donors unlikely to have come in contact with Echinococcus spp. eggs, as negative controls. Serum samples, collected after separation from blood clots, were tested in two independent laboratories for anti-AE/CE antibodies in serum by ELISA or IB using native antigen B (AgB, also termed EgB) from E. granulosus hydatid cyst fluid (HCF), recombinant/affinity purified antigen B (AgB), a crude extract of E. multilocularis protoscoleces (EmP), recombinant/affinity purified E. multilocularis Em18 antigen, and affinity purified, native E. multilocularis Em18 (Craig et al. 1992, 1995; Ito et al. 1993, 1997, 1999). Details of the assays are as follows.

ELISA method used in laboratory 1

ELISA plates were pre-coated with optimal concentrations of native antigens (AgB, 1:1000 dilution; EmP, 1:4000 dilution; 100 μl per well) in 0.05 m bicarbonate–carbonate buffer pH 9.6 (BCB). Two wells were left uncoated to act as blank controls for the plate. After overnight incubation at 4 °C, the plates were rinsed once with 0.15 m phosphate buffered saline pH 7.2 (PBS) containing 0.1% Tween-20 (PBST 0.1%). Following blocking with 100 μl per well of PBST 0.3% containing 5% (w/v) skimmed powder milk (PBSTM) by incubating for 30 min at room temperature (RT) and rinsing three times with PBST 0.1%, the plate was loaded with 100 μl of diluted serum (1:200 dilution in PBSTM) and incubated for 1.5 h at RT. Each plate included control AE and CE positive and negative serum samples. Serum samples from the plate were discarded into 10% bleach, and the plate rinsed four times with PBST 0.1%. Conjugate solution (anti-human IgG (gamma-chain specific) peroxidase; Sigma A-6029; at 1:2000 dilution for CE assay; 1:8000 for AE assay) was loaded (100 μl per well), kept at RT for 1 h, and then rinsed. The plate was then tapped dry, 100 μl of tetramethyl benzidime (TMB) substrate were added per well and the plate was left to stand for 20 min at RT. All wells, including the two blank wells, were read on a microplate ELISA reader at 490 nm. The positive–negative cut off level was calculated using the mean optical density (OD) value plus three standard deviations of the negative controls (Craig et al. 1992, 1995).

ELISA method used in laboratory 2

ELISA plates were coated with 0.1 μg of recombinant/affinity purified AgB or Em18 proteins. The wells were blocked with 300 μl of casein buffer (20 mm Tris–HCl [pH 7.4], 150 mm NaCl, 1% casein) at 37 °C for 1–2 h. After the wells were rinsed twice with PBST 0.05%, 100 μl serum samples diluted 1:100 in PBST containing 1% bovine serum albumin were added, and the mixtures were incubated at 37 °C for 1 h. The wells were rinsed five times with PBST, incubated with 100 μl of anti-human IgG antibodies conjugated with peroxidise (Cappel, West Chester, PA, USA) at 37 °C for 1 h, and rinsed five times with PBST. After incubation with 100 μl of substrate (0.4 mm 2,2′-azino-di-[3-ethyl-benzthiazoline sulfonate] in 0.2 m citric acid buffer; pH 4.7) for 15 min at RT, the optical density at 405 nm (OD405) of each well was determined. Serum samples giving OD values greater than the three mean OD values for normal human controls were considered seropositive (Sako et al. 2002).

Immunoblotting method used in laboratory 2

Approximately 400 mg of crude antigen (HCF) or 20 mg of affinity purified antigens (recombinant Em18, native Em18 or recombinant AgB) were loaded into one large sample well of a 6-cm width gel (Tefco, Tokyo, Japan). Electrophoresis was carried out at a constant current of 25 mA for approximately 80 min. Transfer, using ImmobilonTM polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA), was carried out at 150 mA for 4 h. The membranes were blocked with 3% (w/v) powdered skim milk, each was cut into 50 strips and probed with diluted human serum (1:50). Bound antibody was detected using peroxidase-conjugated anti-human IgG (Cappel, West Chester, PA, USA) and 4-chloro-l-naphthol (Nacalai Tesque, Kyoto, Japan) at a final concentration of 0.05% (v/v) as the substrate for colour development (Sako et al. 2002).

All cases confirmed by US imaging as having echinococcosis in the community surveys, and select individuals, including case family members or close relatives, seropositive query cases with evidence of calcification or hypo-echo haemangioma-like lesions on US imaging, and subjects consistently seropositive but US-negative, were followed up in the subsequent year by US scanning and IB. Follow-up cases also included some patients identified from hospital records.

Diagnostic criteria

Cystic echinococcosis.

Ultrasonography is the preferred community-based diagnostic tool for hepatic CE. It is readily available, cost-effective and enables classification and confirmation of cyst viability (Macpherson et al. 2003). The typical appearance of a CE cyst by US imaging is a round or ovoid space-occupying lesion or hypoechogenic area (fluid-filled) with a well-defined outer wall and internal septations or is multivesicular (contains daughter cysts). Cysts may have a hyperechoic solid pattern (pseudotumor appearance) or may have a reflective wall suggestive of calcification. A ‘double layer and arc calcification’ can be considered specific for echinococcosis lesions caused by E. granulosus (Wen et al. 2004). Positive serology is used to complement US diagnosis of CE (Pawlowski et al. 2001).

Due to differing cyst morphology and viability of protoscoleces at pre-operative surgery, liver cystic echinococcosis has been classified into six types; an ‘active’ group (Group 1: types CL, CE1 and CE2), a ‘transitional’ group (Group 2: type CE3) and an ‘inactive’ group (Group 3: type CE4 and CE5) (McManus et al. 2003; WHO 2003).

Alveolar echinococcosis.

All cases with hepatic space-occupying masses with irregular or indistinct boundaries, heterogeneous and/or calcified structures were suspected as having liver AE; combined with positive serology, these cases were diagnosed definitively as AE. Lesion(s) with marked hypodense areas (necrotic fluid occupying) in the central mass associated with hyperdensity (calcification) in the outer contours, with positive serology, were also diagnosed as AE. The classification and staging of AE cases used the PNM (P: Hepatic localisation of the parasite; N: Extrahepatic involvement of neighbouring organs; M: Presence of distant metastases) system proposed by The European Network for Concerted Surveillance of AE and the WHO Informal Working Group on Echinococcosis (Pawlowski et al. 2001; Macpherson et al. 2003; Kern et al. 2006). The PNM classification was designed not only to describe the anatomical extent of AE disease but also to provide information on the feasibility and suitability to judge the P stages from 1 to 4 within the liver assessed by US (Kern et al. 2006).

There is generally no difficulty in differentiating advanced stage AE from CE in the liver, and US diagnosis of AE includes the presence of echogenic and hyper-reflective lesions, micro-calcifications, and/or pseudocystic necrotic areas with ragged edges (Pawlowski et al. 2001; Macpherson et al. 2003). Small or early lesions and abortive (calcified) lesions of AE require serological confirmation of diagnosis (Pawlowski et al. 2001).

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

AE and CE cases

A full description of all individuals participating in the community surveys has been presented (Yang et al. 2005b, 2006a). Briefly, 4778 people participated in the surveys from 2001–2003. Four thousand seven hundred and seventy-three subjects living in the surveyed areas were from 26 communities within 16 townships in the three counties of Xiji, Guyuan and Longde. Among these, 96 AE (including three simultaneous infections of AE and CE) and 75 CE cases were identified.

US classification

The US classification for 142 of 171 echinococcosis cases is shown in Table 1; 21% CE cases had active (CL, CE1, CE2) lesions, 60% had inactive (CE4, CE5) lesions, 19% had transitional (CE3) lesions. More than half (53%) of the lesions were located in the right liver lobe and 81% cases had single lesions in the liver. Symptoms such as epigastrial pain were reported by 50% of cases. AE classified as early stage (P1 and P2) accounted for 21% of cases while advanced stage (P3 and P4) accounted for 79% of cases (Table 1). Three AE cases had simultaneous infections of AE and CE, and one case had a lesion that had self-absorbed without any treatment as verified by a 2-year interval follow-up. Fifty percent of cases had lesions involving both sides of the liver, 34% involved the right liver and 14% involved the left liver. Only one case in this study had long-distance metastasis to the lung and brain, which was diagnosed from pre-clinical history records. There were 33 (39%) AE cases with lesions characterized by a mass with central necrotic fluid (Table 1). The necrotic lesions accounted for 15% (2/13) in the P2 case group, 40% (22/55) in the P3 case group, and 75% (9/12) in the P4 case group. Sixty-eight per cent of cases had symptoms such as epigastrial pain, intermittent jaundice and fever. Lesion sizes for 144 of the 171 cases are shown in Table 2. Of these, 22% (22 CE and 10 AE) cases had lesions with diameters less than or equal to 5.0 cm, 59% (23 CE and 36 AE) cases had lesions with diameters ranging from 5.0 to 10.0 cm, and 37% (12 CE and 41 AE) cases had lesions larger than 10.0 cm.

Table 1.   Ultrasound classification, lesion number and anatomical location in 142 of 171 human echinococcosis cases with or without symptoms
Type†Number of cases (%) [Necrosis‡‡]Lesion location‡Lesion numberSymptoms
LRBMSingleMultiple§Yes/No
  1. †See text for ultrasound classification for CE and AE.

  2. ‡Lesion location is represented as follows: L, left liver; R, right liver; B, both sides of liver; M, multiple organs involved. The community screening used US abdominal scanning only, so cases involving other organs were identified by their clinical records; this number is likely underestimated for CE.

  3. §The number of lesions was more than two in the US image.

  4. ¶ NR, no record of AE or CE cyst classification.

  5. ††Three of the AE cases were simultaneous infections of AE and CE in the liver.

  6. ‡‡The number of AE cases with necrotic lesions.

  7. §§P4, the lesion was so large that it could not be identified with a lesion boundary, so was regarded as a single lesion.

CE
 CL and CE111 (19)6221927/4
 CE21 (2)0100101/0
 CE311 (19)28101016/5
 CE424 (42)5135117713/11
 CE510 (18)3610912/8
 Sub-total (%)57 (100)16 (28)30 (53)9 (16)2 (3)46 (81)11 (19)29/28
 NR¶1810/8
 Total7539/36
AE††
 P15 (6) [0]2210413/2
 P213 (15) [2]1660859/4
 P355 (65) [22]920260441145/10
 P4§§12 (14) [9]011111118/4
 Sub-total (%)85 (100) [33]12 (14)29 (34)44 (51)1 (1)67 (79)18 (21)65/20
 NR¶110/11
 Total9665/31
Table 2.   CE and AE lesion sizes determined by US imaging
Size of lesion CE† AE†Combined CE and AE†
  1. †Number of cases (%).

Small (≤5 cm)22 (39)10 (12)32 (22)
Medium (5–10 cm)23 (40)36 (41)59 (41)
Large (>10 cm)12 (21)41 (47)53 (37)
Sub-total57 (100)87 (100)144 (100)
No record18927
Total7596171

Correlation of serology with ultrasound classification

A comparison of serology undertaken in the two independent laboratories using the same serum samples collected from 56 US-positive echinococcosis cases in the 2002 survey showed similar results, especially when the antigens EmP and recombinant/affinity purified Em18 were compared. While crude AgB used in one of the laboratories showed highest sensitivity (100%) for detection of CE, it also exhibited the highest level of cross-reactivity (95%) with AE. Although recombinant/affinity purified AgB decreased the cross-reactivity to 48%, the sensitivity also decreased to 67% with the number of false negatives increasing to 33%.

Of the 171 echinococcosis cases, 112 subjects (41 with US-confirmed CE; 71 with US-confirmed AE) volunteered blood samples for serology using recombinant/affinity purified Em18 (r/aEm18) and recombinant/affinity purified antigen B (r/aAgB) by ELISA in 2002 and 2003 (Table 3). All the 112 AE or CE cases confirmed positive by US and ELISA were also positive by IB assay.

Table 3.   Correlation of serology with US classification for CE and AE cases
US diagnosisSerological diagnosis†
CE, n (%)AE, n (%)Negative, n (%)Total, n (%)
  1. †Serological diagnosis used recombinant/affinity purified Em18- and AgB-ELISA (see text for details).

  2. ‡NR, no record of AE or CE cyst classification.

CE types
 CE1, CE25 (63)1 (13)2 (25)8 (100)
 CE36 (86)01 (14)7 (100)
 CE47 (50)2 (14)5 (36)14 (100)
 CE51 (17)3 (50)2 (33)6 (100)
 Sub-total19 (54)6 (18)10 (29)35 (100)
 NR‡3216
 Total (%)22 (54)8 (20)11 (26)41 (100)
AE types
 P1, P21 (11)7 (78)1 (11)9 (100)
 P32 (4)42 (91)2 (4)46 (100)
 P41 (9)10 (91)011 (100)
 Sub-total4 (6)59 (89)3 (5)66 (100)
 NR‡0235
 Total (%)4 (6)61 (86)6 (8)71 (100)

The comparison of US diagnosis with serum ELISA showed that 54% (22/41) of the sera from the CE cases reacted with AgB, 20% (8/41) cross-reacted with Em18, and 26% (11/41) showed no reactivity with either antigen. There was a higher seropositivity rate (86%) among CE cases in the transitional (CE3) group than in the active (CE1, CE2) group (63%). Fewer inactive CE4 and especially CE5 (17%) groups were seropositive. Within the AE cases, 86% (61/71) of sera reacted with Em18; 6% (4/71) exhibited cross-reactivity with AgB; 8% (6/71) showed no reactivity with either antigen (Table 3). The highest seropositivity rate (91%) occurred in AE cases with P3 and P4 classification, with a lower rate (78%) evident in the less developed P1 and P2 AE cases. The highest cross-reactivity (50%) with r/aEm18 antigens in the CE cases occurred with CE5. For AE, higher cross-reactivity with r/aAgB antigens occurred in the earlier (P1 and P2; 11%) and more advanced (P4; 9%) stages. The highest numbers of false negatives for CE cases occurred with CE4 (36%) and for AE (11%) with stages P1 and P2.

Correlation of serology using immunoblotting with ultrasound classification in follow-up group

Nineteen subjects (11 AE cases, 4 CE cases and 4 US-normal individuals) volunteered blood samples for serology in 2003 for follow-up after the 2001 and 2002 surveys (Table 4). Of the AE cases, ten were US stage P3 and one was stage P1; one of the P3 cases (I.D.19; Table 4) was seropositive but US-negative in 2002 according to the survey records, but was inferred a P3 case by US in the 2003 survey. Of the four CE cases, two cases were CE5 and the other two were CE3 and CE4. Nine AE cases were seropositive to r/aEm18 in IB (Table 4; Figure 1); four of the nine AE cases had lesions with central necrosis, two had plaque calcifications and the remaining three cases had AE masses (Table 4). The two serology-negative AE subjects were P3 stage cases with multiple lesions and partial calcification. The CE3 and CE4 cases demonstrated cross-reactivity with r/aEm18 and two CE5 cases were false-negatives. The four US-normal individuals were serology-negative.

Table 4.   Patient data, US imaging and serology of individuals at follow-up in 2003
ID†SexAgeEthnicityUS imaging findings‡Serology§
123451234
  1. †ID, Patient identifier number. ID 19 was an AE case who was US-negative in the 2002 survey but who US-positive in the 2003 follow-up after being seropositive in the 2001 and 2002 surveys.

  2. ‡1, Diagnosis (AE, alveolar echinococcosis; CE, cystic echinococcosis; USN, ultrasound normal); 2, US classification; 3, size of lesion (cm); 4, Lesion location (RL, right liver; LL, left liver; BL, both lobes of liver); (sl), single lesion; (m), multiple lesions; 5, other specific image signs (Mass; Fistulae; Calcific., partial/full/point calcification/s; Necosis; Ascites).

  3. §Serology involved immunoblotting with the following antigens (see text): 1, recombinant/affinity purified Em18; 2, affinity purified native Em18; 3, recombinant/affinity purified AgB; 4, EgCF, Echinococcus granulosus cyst fluid antigen. ‘+’, positive; ‘−’, negative.

 1M41HuiAEP316LL (sl)Necrosis; ascites++++
 2F33HanAEP313RL (sl)Mass++++
 3F52HuiAEP311BL (sl)Necrosis++
 4F38HuiAEP17LL (sl)Calcific.+++
 5F34HanUSN    
 6M33HuiAEP316LL (sl)Necrosis++++
 7F50HuiCECE43LL (sl)Fistulae+
 8F34HuiAEP35RL (sl)Mass++++
 9M39HuiAEP34LL (m)Calcific.
10M16HuiUSN    
11F37HuiCECE52, 1RL (m)Calcific.
12M41HuiAEP33,2,1BL (m)Calcific.
13M26HuiAEP35RL (sl)Calcific.++++
14F50HuiCECE52,1RL (m)Calcific.
15M34HuiUSN    
16F62HuiCECE313BL (sl) ++
17F50HanAEP313LL (sl)Mass++++
18M16HuiUSN    
19M40HanAEP35LL (sl)Necrosis++
image

Figure 1.  Immunoblotting results of sera, full details of which are provided in Table.. 4 and 5, from individuals with suspected echinococcosis in the 2003 community survey. Follow-up cases are numbered 1–19. m, molecular weight marker lane; kDa, kilodalton; AE, control AE serum; CE, control CE serum; rEm18, recombinant/affinity purified Em18; aEm18, affinity purified, native Em18; rAgB, recombinant/affinity purified AgB; EgCF, Echinococcus granulosus cyst fluid antigen (position of AgB is arrowed). The arrows show reactivity of serum antibodies with four different antigen sources.

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Correlation of immunoblotting serology with ultrasound classification in community surveyed individuals.

Eighteen US-screened individuals from the 2003 community survey were tested by serology (Table 5; Figure 1). There were five AE cases, four of whom were at P3 stage (three of the four were seropositive and one was seronegative), and one was a strongly seropositive case although no AE hepatic lesions were detected by US. This patient had received radical surgery for removal of AE lesions from the liver (in 1992) and brain (in 1999) followed by continual recommended albendazole treatment post-surgery. Six CE cases were serologically negative (one was a query CE case, one was stage CE1, one was CE3, and three were CE4). Only one case was seropositive against both E. multilocularis and E. granulosus antigens. This subject had received CE surgery 10 years ago but no hepatic cysts were detectable by US at the time of the survey. There were three individuals with isolated calcified images and three other subjects with abnormal images who were seronegative (Table 5).

Table 5.   Patient data, US imaging and serology of individuals from the 2003 community survey
ID†SexAgeEthnicityUS imaging findings‡Serology§
123451234
  1. †ID, Patient identifier number.

  2. ‡1, Diagnosis (AE, alveolar echinococcosis; CE, cystic echinococcosis; AP, abnormal presentation (ID 28, simple cyst in the right kidney; ID 30, multiple simple cysts in both kidneys; ID 33, a 9.5 cm hemangioma in the right liver); IC, isolated calcification; ‘?’, query case; 2, US classification; 3, size of lesion (cm); 4, Lesion location and type (RL, right liver; LL, left liver; BL, both lobes of liver); sl, single lesion; m, multiple lesions; 5, Other specific image signs (calcific., partial/full/point calcification/s).

  3. §Serology used immunoblotting with the following antigens (see text): 1, recombinant/affinity purified Em18; 2, affinity purified native Em18; 3, recombinant/affinity purified AgB; 4, EgCF, Echinococcus granulosus cyst fluid antigen. ‘+’, positive; ‘−’, negative.

20F38HuiCECE16RL (sl)Residual cavity; no pathology
21M51HuiCECE33RL (sl)Daughter cysts
22M60HuiAEP39,3BL (m) +++
23M40HuiCECE46LL (sl) 
24M50HuiCE   Post-operation; no liver lesion++++
25F60HuiAEP310LL (sl)2 cm-dimension calcific. in RL+++
26F33HuiAEP312BL (sl) ++
27M32HuiIC 2LL (sl)Calcific.
28M66HuiAP 4RK (sl) 
29F65HuiCECE49RL (sl) 
30M35HanAP  BL (m) 
31F38HanIC 1RL (sl) 
32M72HanCECE48, 4RL (m)Hemangiomas in LL
33F44HanAP   Hemangiomas in RL
34F60HuiAEP37RL (sl) 
35F44HanIC 1RL (sl) 
36F35HuiAE1992/1999 liver/brain AE surgically removedMultiple simple cysts in RL++++  
37M71HanCE? 3L (sl) 

Specificity of the immunoblotting assays

Serum samples from healthy (i.e. ultrasound normal, N = 4) subjects (Table 4) or patients with pathology not due to AE or CE, including three with isolated calcification in the liver, one with a simple cyst in the right kidney, one with multiple simple cysts in both liver lobes and another with hemangiomas (N = 6) (Table 5), were negative by both IB and ELISA (data not shown), confirming previous reports of the specificity of the ELISA and IB test assays (Sako et al. 2002; Xiao et al. 2003).

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Ultrasonography is recognized as a reliable, portable, cheap and non-invasive method for diagnosis of symptomatic and asymptomatic echinococcosis cases in clinical settings or in mass screening surveys (Macpherson et al. 1989, 2003; Craig et al. 1991,2000; Shambesh et al. 1992; Pawlowski et al. 2001; Bartholomot et al. 2002; Tiaoying et al. 2005; Rogan et al. 2006). US can alone correctly differentiate hepatic AE from CE (Macpherson & Milner 2003). Serology can also be used for complementary diagnosis both clinically and in community surveys, and it is important to correlate US and serological prevalence for both human AE and CE. A standardized clinical classification has been elaborated for CE by the WHO Informal Working Group on Echinococcosis (Pawlowski et al. 2001; WHO 2003). The PNM system, which is based on the Tumor-Node-Metastasis (TNM) system of liver cancer, has been proposed for AE disease standardization (Pawlowski et al. 2001; Macpherson et al. 2003; Kern et al. 2006). These classification systems are important to provide some indication of developmental or regressive changes in the lesion either naturally or after therapy (Macpherson et al. 2003; WHO 2003). For both CE and AE cases, it has been reported that echinococcosis lesions can spontaneously regress, disappear naturally over time (Romig et al. 1986; Rausch et al. 1987; Gottstein et al. 1991), as our study has also shown.

Serological studies measuring levels of circulating antigens, antibodies and cytokines have been widely used for diagnosis of echinococcosis and characterization of the host–parasite relationship in E. granulosus and E. multilocularis infections (Rogan & Craig 1997; Rigano et al. 2001; Zhang & McManus 2006). Serological monitoring by subclass-antibody detection for clinical management of patients with echinococcosis has also been described (Ortona et al. 2002, 2003; Rigano et al. 2002; Lawn et al. 2004). Serological correlations with US imaging classification have been described for CE (Craig et al. 1995; Rogan & Craig 1997; Cohen et al. 1998) but not for AE. Such studies can provide useful insights on the immunological events that are associated with different clinical outcomes of the disease. This antibody-based study has demonstrated that different morphological cyst types are associated with serological profile changes, which contrasts with the results of Carmona et al. (1998) who found no such correlation. For CE, seropositive rates increased in the active (early) stage to the transitional (middle) stage, and then decreased in the inactive (later) stages. It is possible that these morphological types, which represent a progression in the development of cysts from a highly viable unilocular cyst (Types CE 1 and CE 2) to a degenerating primary cyst (Type CE 3) and finally to an inactive (Type CE 4) or dead (Type CE 5) cyst, may reflect the differing profiles of antigens produced and released to the host immune system by different developing stages of human CE. If this progression in cyst degeneration does occur, then immune responses may play an important role in spontaneous regression and eventual immunological killing of the infection, resulting in self-cure (Rogan & Craig 1997). These cystic lesions will likely present as a partially or completely calcified image on US. Such cases were identified in our study with their serology being antibody-negative. The correlation evident in the current study between serology and US classification of CE is similar to that observed by Craig et al. (2003) and Cohen et al. (1998). The overall low sensitivity (29%) of CE serology probably reflects, in part, the fact that more than half the number of the patients (57%) were in the late stage of development (CE 4 and CE 5).

Unlike CE, the constant increase in seropositivity rates from the early (P1, P2) to the advanced (P3, P4) stages for AE may be due to the multivesiculated infiltrative exogenous budding of the parasite lesion and the lack of any well-defined host capsule formation. This would result in continual and increased release of parasite antigens to the immune system and the generation of a pronounced antibody response (Craig et al. 1992).

The reason why higher serological cross-reactivity occurred with the later developmental stages of either AE or CE is unclear, but it may be because neither antigen AgB (Ito et al. 1999) nor antigen Em18 (Ito et al. 2004) are species-specific for E. granulosus or E. multilocularis but are genus-specific for Echinococcus, as has been suggested previously (Nirmalan & Craig 1997).

Individuals who were seropositive for echinococcosis but negative by US detection in the community surveys were selected for follow-up because they might have been infected with Echinococcus eggs, but neither echinococcal lesion had begun to develop at the time of serum collection nor they had grown to a size detectable by US (Romig et al. 1999; Jensen et al. 2001). For US-confirmed cases, follow-up by US combined with serology is useful for studying the natural history and progression of echinococcosis and/or the response to drug treatment. The reason is that both natural history and treatment efficiency are often unpredictable by imaging findings alone because cysts undergo relatively small changes that cannot be visualized by US. In addition, follow-up of cases by combining US and serology is important to monitor possible recurrent cysts or reinfection post-treatment.

Although antibody detection has limited scope for distinguishing between a previous and current infection, it does indicate natural exposure to echinococcal antigens. Serological detection of infection is useful in the context of control (Doenhoff et al. 2004), where antibody responses to parasite antigen can be mapped, because exposed individuals can be readily identified (Booth & Dunne 2004; Yang et al. 2006a) by serological markers, particularly in individuals below 15 years of age (Yang et al. 2006a). The current study reinforces the usefulness of serology not only for assessing immunological responses during the clinical progression of human echinococcosis, if combined with US classification, but also in helping to monitor environmental exposure of humans to Echinococcus eggs.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This work received financial support from Ningxia Medical College, The University of Queensland and the NIH Echinococcosis China Working Group. We thank the participation of people of rural communities for their invaluable cooperation. We are also particularly grateful to rural medical and nursing staff for assistance and support in the community surveys. We thank staff of Ningxia Medical College who provided expert technical assistance.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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