Journal of Gasrroenrerologv and Hepatologv (1992) 7, 17-21

LIVER A N D BILIARY Relationship between intensity of Opisthorchis viverrini infection and hepatobiliary disease detected by ultrasonography EIMORN MAIRIANG,' DAVID B. ELKINS,Z*4PISALN MAIRIANG,3 JITJAROEN CHAIYAKUM,' NITTAYA CHAMADOL,' VALLOP LOAPAIBOON,' SUMAREE POSRI,3 PAIBOON SITHITHAWORN' AND MELISSA HASWELL-ELKINS'94 Nongrua District Hospital, Nongrua District, Khon Kaen Province, Thailand Abstract Twenty-four locality-, age- and sex-matched groups of village residents with no light, moderate and heavy Opisthorchis vivernni infection were examined by ultransonography. Highly significant differences were observed between the groups in the relative size of the left lobe of the liver and the fasting and post-meal size of the gall-bladder. In addition, indistinct gall-bladder wall, the presence of gall-bladder sludge and strongly enhanced portal vein radicle echoes were most frequently observed in the heavily infected group. Two suspected cases of cholangiocarcinoma were identified from the heavy group. The results highlight the importance of intensity of infection on the frequency and severity of fluke-associated hepatobiliary disease.

Key words: cholangiocarcinoma, community-based study, gall-bladder enlargement, hepatobiliary disease, intensity of infection, Opisthorchis viverrini, ultrasonography.

INTRODUCTION The liver fluke, Opisthorchis viverrini, is an important public health problem in Northeast Thailand, where an estimated 7 million people are infected. I Humans acquire this parasite by eating raw fish containing infective metacercaria. Chronic liver fluke infection of the biliary tract is believed to enhance its susceptibility to carcinogenesi~.~-~ Although cholangiocarcinoma (CHCA) is rare in non-endemic areas, it is a leading cancer in Northeast Thailand6-' and is also more frequent in areas endemic for the related liver fluke, Clonorchis ~inensis."~ Hospital-based and autopsy studies have reported numerous pathological changes in subjects with Opisthorchis infection, including gall-bladder and liver enlargement, cholangitis, cholecystitis and bile duct desquamation and I However, the frequency of these proliferati~n.~-~*'-' changes in apparently healthy individuals in the community and their association with intensity of infection remains unclear. Recently, ultrasonography has been used to examine this question. Pungpak et al. reported a low frequency of biliary tract abnormalities among residents of an area where the anthelmintic drug, praziquantel, had been extensively applied over a 6-year period.12 In contrast, Elkins et al.

'

reported a high prevalence of disease in a small community where a high proportion ( 10%) of residents harboured heavy infections (> 10000 eggs per gram of faeces (EPG); > 100 worms). Diseased individuals had significantly higher intensities of infection and parasite-specific antibody levels than those with normal biliary tracts. ' 3 9 1 5 From the total population of 217 adults, 8 cases (4%) of suspected early CHCA were identified. This striking number suggests that CHCA may be highly clustered,13 because the populationbased cancer registry reports a lower annual incidence (0.12% of ad~lts).'.~ We undertook this case control study in order to confirm and further define the high frequency of ultrasound-diagnosed hepatobiliary abnormalities associated with heavy Opisthorchis infection. Subjects with varying intensities of infection were drawn from 14 villages of a less heavily infected district. In contrast to previous studies, the close agelsex matching within groups eliminates the possibility of age- or sex-related bias in the disease profiles.

METHODS This study is part of a large-scale village-based ultrasound screening programme currently in progress. Fourteen villages in Nongrua district, Khon Kaen Province, Northeast

Present addresses: Departments of Radiology, Parasitology and 'Medicine, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand and Tropical Health Programme, Queensland Institute of Medical Research, Herston, Queensland, Australia Correspondence: Dr David B. Elkins, PO Box 108, Khon Kaen 40000, Thailand. Accepted for publication 12 June 1991.

E . Main'ang et al.

18

Thailand, were chosen for study. An evening meeting was held in every village for health promotion and to explain the programme. Labelled stool containers were distributed by volunteer health workers to all residents over 24 years of age and present in the village. These were collected during the following 2 days. Upon collection, the faeces were mixed and 1.Og of stool removed for fixation in 10% formalin. Specimens were processed by the quantitative formalidethyl acetate concentration technique.' The close relationship between EPC and worm burden has been ~a1idated.I~ All parasite eggs in 1 drop of sediment were determined by microscopy. From a total of 2850 individuals examined from Nongrua district, thirty (1%) had egg counts over 10000 EPG (heavy group). For each heavy infection, residents of the same sex and village and of similar age (f5 years) were stratified into intensity groups (negative, 200- 1000 and 2000-7000 EPG). One individual from each group was randomly chosen for inclusion in the study. Thirty groups of four people were invited for ultrasonography ; 24 groups were actually examined (95 total subjects; one lightly infected individual was not examined). A heavily infected case died before reaching ultrasound; the reported cause was leukaemia. Other subjects had left their villages or were unable to attend. Informed signed consent was obtained from each participating subject prior to ultrasonography. Ultrasonography was performed by one radiologist who was unaware of the parasitological status of the subject. Serum samples from each subject were examined for parasite-specific antibody levels in enzyme-linked immunosorbent assay as described .I4-l6 Abdominal ultrasonography was performed using a fullsize, high resolution machine, ALOKA model 650, with a 3.5MHz convex array probe. The maximum height of the left lobe of the liver above the abdominal aorta was determined. Maximum gall-bladder length, width and wall thickness were measured after locating the largest image observed in all planes of view.13917 Abnormalities, such as the presence of sludge and poor definition of the wall, were also recorded.

For contraction studies, the gall-bladder was re-measured 30min after consumption of a fatty mea1.10,'3,18 Data was entered on Paradox2 database and analysed using SPSS PC+ software package. The significance of variation in the frequencies of various abnormalities and organ measurements between the intensity groups was determined by Chi-squared tests and one-way analysis of variance (ANOVA) respectively. Gall-bladder width measurements were log-transformed (LOG) in order to achieve a normal distribution. Relationships between parasite egg counts and specific IgG levels were assessed by Pearson's correlation. Following examination, the subjects were weighed and treated with 40mgkg bodyweight of the anthelmintic, praziquantel (Bayer, They will be re-examined after 9 months to determine whether elimination of infection reverses the observed abnormalities.

RESULTS Gall-bladder and liver size Highly significant variation was observed between the average fasting length (Fig. la) and width (Fig. Ib) of gallbladders of the four groups, with a clear increase with intensity of infection (ANOVA, F = 13.2, 10.8; d.f. = 3, 91; P < 0.0001 for length and width respectively). Postmeal measurements also varied significantly between groups, but not as strongly (Fig. 1; F = 3.4, 4.2; P < 0.05 and 0. 01 for length and width respectively). Parasite-specific IgG levels also showed strong associations with gall-bladder measurements. While fasting measurements showed similar correlations with egg count (Pearson's r = 0.43, 0.44; P < 0.001) for length and width respectively) and IgG level (r = 0.48, 0.50; P < 0.001), post-meal measurements showed a stronger association with

I

7

-E2 -s-

O . 0.4

E

I

t

-

0.3

U P

: -

0.2

m

0 0.1

0

Fasting

Post-fat

Fasting

Post-fat

Figure 1 'The average (a) length and (b) width (LOG) of gall-bladders of age-, sex- and locality-matched individuals stratified according to intensitv of 0. vizwrini infection. The left half shows the mean of measurements taken after fasting for at least 10 h; the right half is based on measurements taken after the consumption of a fatty meal. The intensity groups are: (N) Group 1, negative for faecal eggs; (mGroup 2 , 200-1000 EPG; (El)Group 3; (a)2000-7000 EPG; (53) Group 4, > IOOOOEPG. Sample sizes are as shown in Table 1.

19

0. viverrini and hepatobiliary disease

antibody levels (r = 0.47, 0.42; P < 0.001; Fig. 2a) than with EPG (r = 0.26, 0.24; P < 0.01; Fig. 2b). Although the height of the left hepatic lobe did not vary between groups 1, 2 and 3, a significant increase was recorded in the heavily infected group (F= 5.7, d.f. = 1, 93; P < 0.05). In order to control for the influence of bodyweight on liver size, left lobe height was divided by bodyweight. A highly significant increase (F = 14.7, P < 0.001) in the size of the left lobe relative to bodyweight in the heavy group is shown in Fig. 3. The pancreas and portal vein showed no size increase with severity of infection (ANOVA, P > 0.05).

Frequency of gall-bladderabnormalities Table 1 shows the biliary tract abnormalities that showed increasing frequencies andor severity with intensity of 0. vivem’ni infection. While actual measurements of the

lp

4 6 p II

I

2 I

I

V I

2

I

I

I

I

I

I

2.5

3

3.5

4

4.5

5

thickness of the gall-bladder wall did not vary between groups, poor definition of the wall was noted more frequently in moderate and heavily infected groups (x2 = 11.2, 3 d.f., P = 0.01), as were enhanced echogenicity of the portal vein radicles (PVR; x2 = 21.9,6 d.f., P < 0.001) and the presence of gall-bladder sludge (x2= 14.1, 3 d.f., P < 0.01). Table 1 also records the increasing frequency and severity of gall-bladder enlargement in the higher intensity groups (x2= 31.1,6 d.f., P < 0.0001). Other measurements were not found to be associated with Opisthorchis EPG or specific antibody levels. Increased echoes throughout the liver (parenchymal disease) were observed in 18 subjects, with similar frequencies among the groups (x2= 5.3, 6 d.f., P > 0.05). No significant association was observed between increased echoes and liver size or hepatitis B carrier status (only two cases were positive for surface antigen). Five cases of cholelithiasis and two of gall-bladder polyps were identified in three of the four groups, but with no clear association with intensity of infection (Table 2). Dilated intrahepatic andor common bile duct was observed in two subjects; in a moderately infected patient the ducts were normal-sized after fatty meal consumption. Two suspected cholangiocarcinoma cases were identified. One was a 42 year old man with heavy infection and dilation of both common bile duct and intrahepatic ducts. A mass at the porta hepatis was observed in another heavily infected woman aged 50 years. These observations have been confirmed by endoscopic retrograde cholangiopancreatography (ERCP), but both patients refused surgery, so histopathological confirmation was not possible. The frequency distribution of hepatobiliary diagnoses based on these ultrasound findings among intensity groups is shown in Table 2. A significant association between intensity of infection and severity - of disease was observed = 28.7, 12 d.f., P < 0.01).

Opisthorchis EPG (log)

‘“I 8

- I I

I

I

=

I-II I

0 036 0.034 0.032 0.03 0.028 0.026

0.024 0.022 0

I

1

I

I

I

0.02 1

2

3

4

Intensity group

Figure 2 The relationship between the post-meal gall-bladder width and (a) Opisrhorchis egg counts, and (b)parasite-specific IgG levels within individuals of the sample group (n = 95).

Figure 3 The average height of the left hepatic lobe divided by total hodvweight of individuals according to Opisrhorchis intensity group (see legend of Fig. 1 for grouping criteria).

E . Mairiang et al.

20

Table 1 Frequencies of gall-bladder abnormalitiesobserved in the four infection groups by uhasonogrdphy

Description Gall-bladderwall Sharply defined Indistinct Gall-bladdersludge N o (normai) Yes Portal vein radicles Normal echoes Mild increase Severe increase Echoes in liver parenchyma Normal Increased Gall-bladder length < 6.7cm 6.7-8.0cmt > 8.0cmS

I

Intensitv group 2 3 4

II 13

8 16

18 6

16

19

16 7

15 7 9 1 7

I

17 3 3

13 10 5 1 6 1 3

21 3

20 3

20

23

18 4 1

15 8 7 6 2 1 0

5 21 2

1 0

7

Less than the mean of the uninfected group (5.4cm) t2-4 s.d. above the mean. $More than 4 s.d. above the mean.

4

18 6

2s.d.

Table 2 Hepatobiliary diagnoses of 95 subjects stratified by intensity of 0. vivem'ni infection

Diagnosis

1

Normal Mild gall-bladder disease GB stones or polyps Chronic cholecystitist Suspected CHCA

16 4

Total

Intensity group 2 3 4

4 0

12 5 2 4 0

5 5 3 II 0

3 6 1 12 2

24

23

24

24

0

.'Diagnosed if gall-bladder had indistinct wall, contained sludge and/or was enlarged ( 2 6.7cm; 2s.d. above mean of group I), but collapsed after fatty meal. tAs for ' , except that contraction was poor following fatty meal consumption.

DISCUSSION This study confirms our previous report that Opisthorchis vivem'ni infection is associated with gall-bladder abnormalities, particularly enlargement, indistinct wall and sludge in apparently healthy a d ~ 1 t s . Although I~ these abnormalities occur in diseases other than opisthorchia~is,'~ their significantly higher frequency among moderately and heavily infected groups than among closely matched non- and lightly infected groups is strong evidence that the liver fluke is the causative agent. In addition, a direct relationship was observed between the frequency and severity of the changes and intensity of infection.

While some changes, such as indistinct gall-bladder wall and enhanced portal vein echoes, show a gradual increase in frequency with intensity group, others such as severe gallbladder and liver enlargement appear to be largely confined to the heavily infected group. This may suggest that mild inflammation in the wall (leading to poor definition by ultrasound) and mild enlargement may be initial steps in the pathogenesis of fluke-associated gall-bladder disease. More severe gall-bladder damage leading to sludge accumulation, distension and liver enlargement may follow the initial damage when infection is heavy. Duration of infection may also be important; however, no association between age and disease parameters was observed in this or the previous study. A strong enhancement of portal vein radicle echoes is evident in a large proportion of the heavily infected group, while mildly increased echoes were fairly evenly distributed among intensity groups. The histopathological equivalent of enhanced PVR is not yet clear, it may reflect fibrotic change or bile lake di1atation.l" We previously reported an association between enhanced PVR and gall-bladder wall irregularity;15 a similar relationship was observed in this series. In addition, enhanced PVR is a frequent finding among suspected early CHCA cases; there may be an aetiological association. The pathogenic mechanisms of opisthorchiasis in humans are not well defined. The finding that post-meal gall-bladder length and width are strongly associated with parasitespecific IgG levels, rather than with intensity of infection, is consistent with the hypothesis that the pathogenesis, particularly the events leading to functional impairment, may be immunological in Suggested mechanisms include complement-mediated damage or immune complex deposition. O n the other hand, mast cells bearing parasitespecific surface IgE may release vasoactive compounds which cause tissue damage and increase antigen leakage into the tissue which, as a consequence, further enhance IgG levels. We are presently examining the role of parasitespecific T cells and IgG subclass distributions in infected individuals with normal and abnormal hepatobiliary tracts to clarify this relationship. In conclusion, intensity of Opisthorchis infection is an important determinant of several hepatobiliary abnormalities that may enhance susceptibility to cholangiocarcinoma. Two suspected cases of early CHCA were identified from 24 heavily infected people; this further supports the high pathogenicity of heavy Opisthorchis infections. Estimating the degree of morbidity and mortality associated with this parasiste requires knowledge of its distribution and abundance within the c o m m ~ n i t y . ' ~ - ' ~ , ~ '

'

ACKNOWLEDGEMENTS This study was funded by the Tropical Health Program and the National Health and Medical Research Council of Australia and by the Foster Parents Plan International, who generously donated the ultrasound machine. The procedures

0. viverrini and hepatobiliary disease

were approved by the ethical committees of the faculty of Medicine, Khon Kaen University and of the Queensland Institute of Medical Research. We express our sincere thanks to the Director, Dr C. Waratharakul, and staff of Nongrua District Hospital, the Plan field staff and the volunteer health workers of each village for their excellent co-operation. Finally, we thank the fourteen members of the project team for careful diligence in the field and laboratory.

21

infection: a comparison of adults and children. Souiheasr Asian

9. Trop. Med. Public Health 1989;20: 95-100. 12. PUN(;I'AK s., SORN.MANI s., SL'NTHAKASAMAI P. & VIVATANASI~SI'H 1'. Ultrasonographic study of the biliary system in opisthorchiasis patients after treatment with praziquantel. Sourheasr AsianJ. Trop. M e d . Public Health 1989;20: 157-62. 13. ELKINSD. B., HASWELL-ELKINS M. R., MAIRIAN(; E. er al. A high frequencv of hepatobilkarv disease and cholangiocarcinoma associated with heavv Opisihurchis vivemni infection in a small communitv in Northeast Thailand. Trans. H. Soc. lrop. M r d . Hvg. 1990;84: 715-19. I4 EI.KISSD. B.,St.rtitmAwoKx P.,H.ASW,I~LI.-EI.KINS M. R., K.\I:WKI:\S.. A\Y,u:II,\K,\L,As 1'. & W ~ S I ~I K ' . AA S . A ( : I ~ I ~ I ~ \ VS.I N ()pisrhordi. P. F. Roentgenographically controlled healing of burden-related biliary pathology. Kun-TunSui 1991; 22: I 1 1-20. gall bladder lesions in opisthorchiasis after praziquantel treat21. HASWI