Acta Tropica, 47(1990)69-77 Elsevier
69
ACTROP 00052
Serological survey of human cysticercosis in Irianese refugee camps in Papua New Guinea M. F r i t z s c h e 1, B. G o t t s t e i n 1, M . C . W i g g l e s w o r t h 2 a n d J. E c k e r t 1 t institute of Parasitology, University of Zurich, Zurich, Switzerland, and aDepartment of Primary Industry, Livestock Division, Boroko, Papua New Guinea (Received 16 January 1989; revised version received 3 July 1989; accepted 4 July 1989) In 1984, over 10 000 refugees left the Indonesian province of Irian Jaya, and thus possibly imported Taenia solium taeniasis/cysticereosis into Papua New Guinea, which was believed to be free of T. solium until 1966. In a serological survey carried out in 1986, 50 refugees originating from areas endemic for 7". soliura and 171 patients from other areas with symptoms suggesting the possibility ofcysticercosis were examined. As a sensitive prescreening technique an ELISA was used with a crude antigen extract obtained from 7". solium metacestodes of pig origin. Of 221 persons investigated, 79 (36%) were positive in ELISA. For excluding frequently occurring cross-reactions in ELISA, Western-blotting (or EITB, enzyme-linked immunoelectrotransferblot) was employed. In this test the demonstration of antibody activity to the 26 or the 8 kilodalton band has been proved to be species-specific for T. solium cysticercosis. One from 79 patients positive in ELISA was simultaneously positive (26 and 8 kDa) in Western blot, corresponding to the first case found in Papua New Guinea with a highly probable T. solium cysticercosis. This patient, originating from an endemic area in Irian Jaya, had.immigrated into Papua New Guinea in 1980. The present work emphasizes the need for using highly specific immunodiagnostictechniques in seroepidemiology of larval cestode infections. 7". solium taeniasis/cysticercosis remains a risk for Papua New Guinea, and refugees originating from endemic areas should be regarded as potential carriers of T. solium. Key words: Taenia so/ium: Human cysticercosis; Immunodiagnosis; Sero-epidemiology; Papua New Guinea
Introduction
Human cysticercosis is due to the infection with the larval form of the tapeworm Taenia solium. The definitive host for T. solium is also man. Until recently, Papua New Guinea was believed to be free of T. solium taeniasis (Kelly and Vines, 1966) (Fig. 1). In Enarotali, however, the capital of the Paniai District in the Wissel Lakes area of Irian Jaya, the Indonesian part of New Guinea, Tumada and Margono (1973) identified human T. solium taeniasis by stool examinations for ova and proglotfids for the first time. In a previous survey on intestinal parasites of the same region around the Wissel Lakes, no taeniasis was reported by Van der Hoeven and Rijpsffa (1957). The source of the new disease could be traced back to a number of pigs that had been imported from Bali and were unfortunately infected with T..solium cysticerci. Subianto et al. (1978) found that extensive infection of th 9 Ekari)tribe ' Correspondence address: J. Eckert, Institute of Parasitology, University of Zfifich, Winterthurerstmsse: 266a, CH-8057 Zfifich, Switzerland. 0001-706X/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
70 living in the Wissel Lakes area led to human cysticercosis with an associated epidemic of epileptic seizures. This manifested clinically at first in severe bums among the highlanders from rolling into house fires around which people slept during cold nights. From its original point of introduction the infection spread rapidly to other places within Irian Jaya (Gajdusek, 1978). As only limited epidemiological surveys have been made, such as those done by Cock er-Vann et al. (1981), the present situation is difficult to assess. The frontier which follows the 141st meridian has never been a barrier against sporadic border crossers between Irian Jaya and Papua New Guinea. In 1984, however, approximately 10 000 Irianese fled to Papua New Guinea and stayed in numerous camps along the border. Among the refugees, who mostly originated from the Muyu tribal area in the Merauke district, which is still free of taeniasis, there are also Ekari people from the Wissel Lakes area and Dani people from the Baliem valley where taeniasis is known to be endemic (Gajdusek, 1978). In order to assess the potential risk of introducing the disease across the border, a serological survey was performed among people from 9 refugee camps. A preliminary analysis of the epidemiologieal situation resulted in the information that the disease could be found (by demonstration of proglottids in human feces and cysticerci in pork meat) up to Anguruk and Kosarek, but not beyond to Nipsan or Nalca. It has to be notified that T. saginata is not prevalent on the whole island. Among all refugees, 221 people were arbitrarily selected for a serological examination, the criteria for selection were based on clinical signs and geographical origin of the persons. The use of non-purified or crude antigens from T. solium cysticerci in ELISA does allow a sensitive but not simultaneously specific immunodiagnosis of human cysticercosis (Gottstein et al., 1986; Gottstein et al., 1987). The serum antibodies of eysticereosis patients show major cross-reactions to antigenic components shared by 7". solium, Echinococcus granulosus and E. multilocularis as shown by Schantz et al. (1980) and Gottstein et al. (1987). Cross-reaetivities between T. solium and Echinococcus or Schistosoma infections have been found in ELISA by Cocker-Vann et al. (1981). Gottstein et al. (1986, 1987) demonstrated two polypeptides (26 and 8 kDa band) by SDS-PAGE and Western-blotting (or enzyme-linked immunoelectrotransfer blot, EITB) to bind serum and cerebro-spinal fluid antibodies only from T. solium cysticercosis patients. This species-specificity offered the opportunity to confirm serologically an infection by T. solium metacestodes. In this way a combination of this new technique together with ELISA as a prescreening test was applied in our survey.
Materials and Methods
Target population From a population of more than 10 000 refugees, two groups were selected for examination in September and October 1986: one group with people originating from areas where T. solium taeniasis is known to be endemic (Fig. 1). The other group, originating from an area not known to be endemic for 7". solium, included persons with a positive medical history for one or more of the following symptoms: epileptic seizures, headache or other neurological deficits. The list of symptoms was completed with syncopy, vertigo, abdominal complaints, cardiopulmonary distress and other symptoms (Table 3). After a short cursory clinical examination, venous blood
71
Area known to be endemic for T. solium
~J
MUyu tribal area
Me fackweler
We ena
K
AngUruk NlPelIn ~.
~_ "~
C e m ~
:&Kemberetoro serok: AGreen River
~, : -"
let ATolefomln Terekblle
~J~O KlUNGA t..WAIkem" "o:\'Nto0omb.n Kutu
New Guinea -
_
Fig. 1. Geographical aspects and location of investigated refugee camps in Papua New Guinea (shown by open triangles).
samples were drawn from 221 informed and consenting patients or after seeking parental permission. Couriers immediately carried the collected sera to nearby mission stations where kerosene refrigerators were available. The sere were transported by plane from the sites via Port Moresby to Switzerland. The age of subjects, 114 males and 107 females, ranged between 3 weeks and 90 years (average: 34 years). They were settled in 9 refugee camps along the border (Fig. 1): in the North, 50 persons were from the Blackwater camp, near Vanimo; in the South, 7 were from Timkwi, 22 from Tarakbits, 28 from Korokpin, 9 from Dome, 19 from Yogi, 52 from Atkamba, 12 from Niogomban and 22 from Kuiu. The refugees in the South originated from the following villages in the Muyu tribal area close to the border in the district of Merauke (Fig. 1): Amuan, Amupka;'Anggambran, Atmimkim, Imko, Ingembit, Kagenot, Kakuna, Kombut, Kwangtet, Mindiptanah, Mokbiran, Nanait, Ninety, Selih, Sesnukt, Simiram, Timkwa, Tokwebit, Umap, Upkin, Upyetetko, Wambiran, Wametkapo, Wangatkibi, Waropko, Wayrin, Wombon, Womsin. Only two persons left this southern tribal area, which is still free of T. solium, for other places where taeniasis is known to be endemic, but they pretended not to have eaten pork in these places. In the Blackwater camp, 39 of the 50 refugees examined originated from the villages Bokondini, Karubaga, Kelila, Kobakma, Pyramids, Tiom and Warnena in or near the Baliem Valley in the Jayawijaya district. Five originated from Enarotali, 1 from Gakokebo, 2 from Ilaga and 1 from Mulia, in the Paniai district; 1 came from Base a n d 1 from Jayapura, in the Jayapura district. All of them originated from or settled in the highlands where T. solium is known to be endemic (Fig. 1, dotted area). In order to set up a collection of stool samples, efforts were made to overcome superstitious beliefs and fear of witchcraft among the natives. Hence we were successful in collecting only a very limited number of reliable eases.
72
ELISA An ELISA was used to prcselcct patient sera which demonstrated antibody concentrations against crude T. solium metacestode antigen. The procedure was performed as previously described (Gottstein et al., 1987). Briefly,the polystyrene surface of micro-ELISA-plates was sensitized using a solution containing 2.5 ttg (protein) T. solium metacestode extract per ml coating-buffer (overnight incubation at +4°C). The sera were tested at a dilution of 1:200, bound patients' antibodies were detected by a second antibody-conjugate [rabbitanti-human IgG (Fc-fragment) from Dako, Copenhagen, Denmark, No. A 089, covalently bound to alkaline phosphatase (Boehringer Mannheim G m b H , No. 567 752) with the heterobifunctional reagent S P D P (Pharmacia Fine Chemicals, Uppsala, Sweden) according to the manufacturer's recommendations]. The substrate was 4-nitrophenylphosphate at a concentration of I m g per ml solution. The reading was carried out by a Dynatech MR600 photometer at A,toan m. Results were expressed arbitrarily in antibody units (AU), calculated according to the following formula: A4o4 m [testserum]-(~ + 3 S D A4o, .m [neg. control]) x I00 = A U A4o4 .m [pos. control]- (.~+ 3 S D A4o4 .m [neg. control])
Arbitrarily, point decimals regarding the expression of results in A U were rounded down for 0.1 to 0.4 and rounded up for 0.5 to.0.9. The mean value plus 3 S D of sera from healthy blood donors (= ~ + 3 S D A4o4 ,m [neg. control]) living in a European area not endemic for T. solium was determined to be the threshold discriminating between 'positive'and 'negative'.Positivelyreacting sera were further investigated by Western-blotting.
Western-blotting All S D S - P A G E chemicals were obtained from Bio-Rad Laboratories (Glattbrugg/Switzerland). Gradient S D S - P A G E and electrotransfer were performed as described previously (Gottstein et al.,1986, 1987). After elcctrotransfer,the proteinbound SDS was replaced with Tween 20 by washing the nitrocellulosesheets in PBS containing 0.3% Tween 20. The sheets were cut into strips and exposed to the sera diluted 1:250 with PBS containing 0.3% Tween 20. Antigen bands with bound antibodies were visualizedwith enzyme-linked immunosorbent techniques (Peralta et al., 1983), using an anti-human IgG-peroxidase conjugate (Sigma No. A-602A). Bound peroxidase activitieswere demonstrated with a precipitating chromogonic substrate (3,3-diaminobenzidine) and H202. Antigenic bands with their bound antibodies and anti-human IgG-peroxidase conjugate appeared then reddish-brown.
Results
From 221 serum samples tested by ELISA, antibodies were detected in 79 cases (Table 1). The 15 highest absorbance values, arbitrarily selected between 30 antibody units (AU) and 66 AU, were found in sera collected from persons of 8 different refugee camps and were not correlated with T. soliura endemic areas (Table 1 and Figure 1). Thirteen of a total of 15 blood samples with antibody reactivities between
73 TABLE 1 Examination of 221 Irian refugees living in Papua New Guinea (in 1986) for serum antibodies against Taenia solhm~ antigens by ELISA and Western-blotting ELISA: AU (antibody units)
Number of persons
Average number of bands in Western blotting (range)
Percentage of persons originating from T. solium endemic areas
0 ('negative') 1-29 30-66
142 64 15
n.d." 7 (0-26) 7 (0-29)
24 22 13
"n.d., not done.
Mr
~o ~: z~ ~. ~ •
94K
-
66K
-
45 K
-
30K 26K
20 K 14K
m
~-SK
t
•
i
•
Fig, 2, Immunological analysis by Western-blotting of serum from an Irianese refugee in Papua New Guinea (PNG), in comparison to sera from patients with radiologically and/or histologically proven T. solium cysticercosis, originating from South Africa, Mexico and Tibet, respectively. All patients' sera immunoreaeted with the specific 26 and 8 kDa bands (indicated by arrows).
74 TABLE 2 Western-blotting examination of 79 serum samples of refugees showing reactivity in the ELISA using crude Taenia solium antigen
Number of bands in Western blot (average)
Numberof persons
Numberof persons from endemicareas (T. solium) (%)
Numberof persons positivefor 26 and/or 8 kDa
ELISA:mean antibody units, AU (range)
0 I-I0 (5) I 1-29 (7)
5 58 i 16b
0 3 88
0 0 Ic
16 (2-43) 16 (2-66) 19 (2-64)
*One person with 10 bands in Western-blottingand 66 AU in ELISA had a known lymphaticfilariasis. bone person with 15 bands in Western-blottingand 8 AU in ELISAalso had a knownlymphaticfilariasis. ¢I'his person demonstrated29 bands in Western blot and 33 AU in ELISA. 30 and 66 AU had been taken in the southern camps Kuiu, Niogomban, Satkamba, Yogi, Komopkin, Tarakbits and Timkwi from refugees originating in the Muyu tribal area which is not known to be endemic for T. solium; only 2 of these samples were collected in the northern Blackwater camp from persons originating from the highlands where T. solium is endemic. Among all 79 serum samples with antibody reactivity between 1-66 AU (Table 1), 1 sample was confirmed to be positive for cysticercosis by Western blotting (antibodies detected against 26 and 8 kDa band). Fig. 2 shows the Western-blotting analysis of this person's serum in comparison to serum from patients with proven neurocysticercosis originating from different geographical areas. The person from this study was a 24 year-old Ekari tribesman who left Enarotali in 1980. He had neither a positive history nor overt clinical signs suggesting cysticercosis and might therefore be regarded as a subclinical case of human cysticercosis. From that person, positive in Western-blotting for the specific 26 and 8 kDa bands, stool examination was carded out: Endolimax nana and eggs from hookworms were discovered, but neither ova nor proglottids of T. solium. In 21 stool samples from other persons examined at random we found I I (52%) to be positive for 1 or more intestinal parasites, with eggs of hookworms and Ascaris lumbricoides in 9 (43%) and 3 (14%) samples, respectively; cysts ofgntamoeba cog and Endolimax nana were found in 2 samples each (9%). Table 2 shows the results of Western-blotting in relation to ELISA results and to the origin (endemic or non endemic area) of the persons tested. The persons are grouped according to the number of bands in Western blotting. There is an evident lack of correlation between number of bands (Western blotting) and antibody reactivity in ELISA. Persons with I 1 or more bands in Western blotting mostly originated from endemic areas, while persons with few or no bands (0 to 10) originated mostly from areas not endemic for T. solium. Table 3 summarizes the clinical symptoms, signs and concomitant diseases found among the refugees selected for serological examination.
Discussion
Applying a confirmatory specific serological method, immunodiagnostic evidence for T. solium cysticercosis was found in one Ekari tribesman. This was the first case of serologically diagnosed cysticercosis in Papua New Guinea. For further calculation
75 TABLE 3 Clinical symptons, signs and concomitant diseases (single or in combination) among 221 refugees selected for serologic examination Symptoms, signs and concomitant diseases
Moderate headache' Severe headacheb Vertigo Epileptic fitsc Syncope Mental deteriorationd General paresis with muscular atrophy Myalgias Chronic cough with expectoration Chronic abdominal complaints Angina pectoris Palpable splecn~ Lymphatic filariasis Open pulmonary tb r Symptoms undefined due to communication difficulties
Number (percentage) of persons from endemic areas (7". soliurn) 50 (= 100%)
Number (percentage) of persons from non endemic areas (T. solium) 171 (-- 100%)
Total
18 (36.0) 8 (16.0) 9 (18.0) 4 (8.0) 4 (8.0) 0 -
62 (36.2) 27 (15.8) 22 (12.9) 19 (11.1) 10 (5.8) 7 (4.1)
80 (36.2) 35 (15.8) 31 (14.0) 23 (10.4) 14 (6.3) 7 (3.2)
0 4 (8.0)
1 (0.6) 46 (26.9)
1 (0.5) 50 (22.6)
9 (18.0)
38 (22.2)
47 (21.3)
5 (10.0) 1 (2.0) 41 (82.0) 1 (2.0) 1 (2.0)
28 (16.4) 8 (4.7) 37 (21.6) 4 (2.3) 11 (6.4)
33 (14.9). 9 (4.1) 78 (35.3) 5 (2.3) 12 (5.4)
1 (2.0)
7 (4.0)
8 (3.6)
221 (= 100%)
"Headache was considered moderate if it occurred less than once a week but at least once a fortnight,with a duration not exceeding 24 h. bHeadaehe was considered severe if it occurred at least once a week or once per fortnight with a duration over 24 h. c22 eases of epileptic fits were considered focal Jacksonian seizures, 1 ease was considered temporal epilepsy. In 3 eases epileptic symptoms improved promptly after the institution of tuberculostatic treatment in the Government Hospital at Kiunga. °5 eases developed mental deterioration within 2 years, two of them acquired acute mental deterioration after an episode of heavy headache. In one case the symptomatology started 10 years afterand another ease showed recurrent symptomatology with alternating periods of complete remission. rrhe high rate of palpable spleens with 12-37 cm (mean 18 cm) in longitudinal diameter among peoi)le from 7". solium reflects immunological response to malaria. rOpen pulmonary tuberculosis was diagnosed microscopically by direct sputum smear and Ziehl-Neelsen's staining in the Government hospital at Kiunga. The refugees originating from 2". solium non endemic areas come from the Muyu tribal areas in the south of Irian Jaya where tuberculosis is highly prevalent. This area became the actual port of entry for tuberculosis into New Guinea whan Javanese freedom fighters were held in a Dutch prison camp at Tanah Merah. "
o f e p i d e m i o l o g i c a l b a s e l i n e d a t a , there is o n e l i m i t i n g f a c t o r o f the p r e s e n t s t u d y w h i c h h a s to be t a k e n i n t o a c c o u n t : we d o n o t k n o w the local d i a g n o s t i c sensitivity o f 26 a n d 8 k D a b a n d d e t e c t i o n i n W e s t e r n b l o t t i n g a m o n g eysticercosis p a t i e n t s o f t h a t g e o g r a p h i c a l area. H o w e v e r , p r e v i o u s w o r k ( G o t t s t e i n et al., 1987), h a s s h o w n t h a t i n all the g e o g r a p h i c a l areas i n v e s t i g a t e d u p to n o w ( M e x i c o , S o u t h A f r i c a , E u r o p e a n d C e n t r a l Asia), t h e relative d i a g n o s t i c sensitivity r a n g e d a r o u n d 92*/. f o r s e r u m
76
samples and 100% for cerebro-spinal fluid. It can be assumed that results in Papua New Guinea would correspond to these percentages. Unfortunately, no sera from a defined Irianese patient group with proven cysticercosis were available to our study, whereby the operating characteristic in question could have been elaborated. All 12 of our 221 sera with 15 or more antibody-binding bands in Western-blotting originated from areas where T. solium is endemic (see Table 2). This stands in contrast to the results of the nonspecific ELISA technique (see Table 1), where the highest absorbance values showed no correlation to endemic areas. In ELISA, the 6 highest values were found in patients originating from areas non-endemic for T. solium. They are most probably related to cross-reactions due to infections with other helminth species. On the other hand we could observe a correlation between the number and the distribution pattern of binding bands in Western-blotting and 7". solium endemicity. Cross-reactions can most likely be ruled out, since the closely related ecstode species (Echinococcus granulosus and E. multilocularis), which may induce some degree of cross-reactivity (Schantz, 1984) are not prevalent on the island. The only species of cestodes described in New Guinea are Hymenolepis nana and H. diminuta. These two parasites are not only limited to the endemic distribution of T. solium in Irian Jaya, but can also be found in other parts of New Guinea as shown by Bearup and Lawrence (1950) and Kelly and Vines (1966). Parasites of other species are unlikely to produce a similar characteristic distribution pattern of antibody-binding bands in Western-blotting. T. soliurn taeniasis and cystiecrcosis still constitute a risk for Papua New Guinea. In one case we found immunodiagnostic evidence for human cysticercosis in the Blackwater refugee camp. Other cases of cysticercosis in the northern camps cannot be excluded at the moment, whereas the occurrence of cysticercosis in the southern camps seems unlikely. Consequently, refugees originating from endemic areas must be regarded as potential carriers of T. solium. Beside prophylactic treatment of refugees with praziquantel for the removal of adult cestodes, the movement of pigs out of refugee camps is prohibited and further surveillance measures are proposed.
Acknowledgements The authors wish to thank Dr. Z.S. Pawlowski, former Senior Medical Officer of the Parasitic Disease Program at the WHO in Geneva, and Dr. W. Schiefenhoevel, ethologist a t the Max Plank Institute for Behavioural Physiology in Secwiesen, for their help and interest in this study. The authors would also like to express gratitude to Dr. B. Subianto, Chief Medical Officer at Kantor Kesehatan Wilaya Jayawijaya in Jayapura, Dr. G.H. Haina, Area Medical Officer at Kiunga, Mrs. E. Morris-Highes, Field Director of the Save the Children Fund, and Dr. B. Patten, Senior Veterinary Microbiologist for the National Veterinary Laboratory in Papua New Guinea. Thanks also to Mrs. E. Hemmeler for her skillful laboratory assistance and Mrs. Helfenstein and Mrs. Oberh~insli for secretarial work. References Beaurup, A.J. and Lawrence, J.J. (1950) A parasitological survey of five New Guinea villages. Meal. J. Austral. l, 724. Cocker-Vann, M.R., Subianto, D.B., Brown, P.; Diwan, A.R., Desowitz, R., Garruto, R.M., Gibbs, C.J.
77 and Gajdusek, D.C. (1981) Elisa antibodies to cysticerciof Taenia solium in human populations in New Guinea, Oceania, and Southeast Asia. Southeast Asian J. Trop. Med. Publ. Hlth. 12, 499-505. Gajdusek, D.C. (1978) Introduction of Taenia solium into West New Guinea with a note on an epidemic of burns from cysticercusepilepsyin the Ekari people of the Wissel Lakes area. Papua New Guinea Med. 3. 21,329-342. Gottstein, B., Tsang, V.C.W. and Schantz, P.M. (1986) Demonstration of species specific and crossreactive components of Taenia solium metacestode antigens. Am. J. Trop. Med. Hyg. 35, 308-313. Gottstein, B., Zini, D. and Schantz, P.M. (1987) Species specific immunodiagnosis of Taenia solium cysticercosis by ELISA and immunoblotting. Trop. Meal. Parasitol. 38, 299-303. Kelly, A. and Vines, A.P. (1966) Highland region survey of intestinal parasites. Meal. J. Austral. 2, 635-640. Peralta, J.M., Simons, A.R. and Tsang, V.C.W. (1983) Enzyme-linked immunoelectrotransfer blot techniques (EITB) for studying the specificitiesof antigens and antibodies separated by gel electrophoresis. Methods Enzymol. 92, 377-391. Subianto, D.B., Tumada, L.R. and Margono, S.S. (1978) Burns and epileptic fits associated with cysticercosis in mountain people of Irian Jaya. Trop. Geogr. Med. 30, 275-278. Tumada, L.R. and Margono, S.S. (1973) Cysticercosis in the area of the Wissel Lakes, West Irian. Southeast Asian J. Trop. Med. Publ. Hlth. 4, 371-376. Van der Hoeven, J.A. and Rijpstra, A.C. (1957) Intestinal parasites in the Central Mountains District of Netherlands New Guinea. Trop. Geogr. Meal. 9, 225-228. Schantz, P.M. (1984) Echinocoocosis(Hydatidosis). In: A.A. Mahmoud and K.S. Warren (Eds.), Tropical and Geographical Medicine. McGraw-Hill, Singapore, pp. 487-497. Schantz, P.M., Shanks, D. and Wilson, M. (1980) Serologic cross-reactions with sera from patients with echinococcosis and cysticercosis. Am, J. Trop. Meal. Hyg. 29, 609-612.
69
ACTROP 00052
Serological survey of human cysticercosis in Irianese refugee camps in Papua New Guinea M. F r i t z s c h e 1, B. G o t t s t e i n 1, M . C . W i g g l e s w o r t h 2 a n d J. E c k e r t 1 t institute of Parasitology, University of Zurich, Zurich, Switzerland, and aDepartment of Primary Industry, Livestock Division, Boroko, Papua New Guinea (Received 16 January 1989; revised version received 3 July 1989; accepted 4 July 1989) In 1984, over 10 000 refugees left the Indonesian province of Irian Jaya, and thus possibly imported Taenia solium taeniasis/cysticereosis into Papua New Guinea, which was believed to be free of T. solium until 1966. In a serological survey carried out in 1986, 50 refugees originating from areas endemic for 7". soliura and 171 patients from other areas with symptoms suggesting the possibility ofcysticercosis were examined. As a sensitive prescreening technique an ELISA was used with a crude antigen extract obtained from 7". solium metacestodes of pig origin. Of 221 persons investigated, 79 (36%) were positive in ELISA. For excluding frequently occurring cross-reactions in ELISA, Western-blotting (or EITB, enzyme-linked immunoelectrotransferblot) was employed. In this test the demonstration of antibody activity to the 26 or the 8 kilodalton band has been proved to be species-specific for T. solium cysticercosis. One from 79 patients positive in ELISA was simultaneously positive (26 and 8 kDa) in Western blot, corresponding to the first case found in Papua New Guinea with a highly probable T. solium cysticercosis. This patient, originating from an endemic area in Irian Jaya, had.immigrated into Papua New Guinea in 1980. The present work emphasizes the need for using highly specific immunodiagnostictechniques in seroepidemiology of larval cestode infections. 7". solium taeniasis/cysticercosis remains a risk for Papua New Guinea, and refugees originating from endemic areas should be regarded as potential carriers of T. solium. Key words: Taenia so/ium: Human cysticercosis; Immunodiagnosis; Sero-epidemiology; Papua New Guinea
Introduction
Human cysticercosis is due to the infection with the larval form of the tapeworm Taenia solium. The definitive host for T. solium is also man. Until recently, Papua New Guinea was believed to be free of T. solium taeniasis (Kelly and Vines, 1966) (Fig. 1). In Enarotali, however, the capital of the Paniai District in the Wissel Lakes area of Irian Jaya, the Indonesian part of New Guinea, Tumada and Margono (1973) identified human T. solium taeniasis by stool examinations for ova and proglotfids for the first time. In a previous survey on intestinal parasites of the same region around the Wissel Lakes, no taeniasis was reported by Van der Hoeven and Rijpsffa (1957). The source of the new disease could be traced back to a number of pigs that had been imported from Bali and were unfortunately infected with T..solium cysticerci. Subianto et al. (1978) found that extensive infection of th 9 Ekari)tribe ' Correspondence address: J. Eckert, Institute of Parasitology, University of Zfifich, Winterthurerstmsse: 266a, CH-8057 Zfifich, Switzerland. 0001-706X/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
70 living in the Wissel Lakes area led to human cysticercosis with an associated epidemic of epileptic seizures. This manifested clinically at first in severe bums among the highlanders from rolling into house fires around which people slept during cold nights. From its original point of introduction the infection spread rapidly to other places within Irian Jaya (Gajdusek, 1978). As only limited epidemiological surveys have been made, such as those done by Cock er-Vann et al. (1981), the present situation is difficult to assess. The frontier which follows the 141st meridian has never been a barrier against sporadic border crossers between Irian Jaya and Papua New Guinea. In 1984, however, approximately 10 000 Irianese fled to Papua New Guinea and stayed in numerous camps along the border. Among the refugees, who mostly originated from the Muyu tribal area in the Merauke district, which is still free of taeniasis, there are also Ekari people from the Wissel Lakes area and Dani people from the Baliem valley where taeniasis is known to be endemic (Gajdusek, 1978). In order to assess the potential risk of introducing the disease across the border, a serological survey was performed among people from 9 refugee camps. A preliminary analysis of the epidemiologieal situation resulted in the information that the disease could be found (by demonstration of proglottids in human feces and cysticerci in pork meat) up to Anguruk and Kosarek, but not beyond to Nipsan or Nalca. It has to be notified that T. saginata is not prevalent on the whole island. Among all refugees, 221 people were arbitrarily selected for a serological examination, the criteria for selection were based on clinical signs and geographical origin of the persons. The use of non-purified or crude antigens from T. solium cysticerci in ELISA does allow a sensitive but not simultaneously specific immunodiagnosis of human cysticercosis (Gottstein et al., 1986; Gottstein et al., 1987). The serum antibodies of eysticereosis patients show major cross-reactions to antigenic components shared by 7". solium, Echinococcus granulosus and E. multilocularis as shown by Schantz et al. (1980) and Gottstein et al. (1987). Cross-reaetivities between T. solium and Echinococcus or Schistosoma infections have been found in ELISA by Cocker-Vann et al. (1981). Gottstein et al. (1986, 1987) demonstrated two polypeptides (26 and 8 kDa band) by SDS-PAGE and Western-blotting (or enzyme-linked immunoelectrotransfer blot, EITB) to bind serum and cerebro-spinal fluid antibodies only from T. solium cysticercosis patients. This species-specificity offered the opportunity to confirm serologically an infection by T. solium metacestodes. In this way a combination of this new technique together with ELISA as a prescreening test was applied in our survey.
Materials and Methods
Target population From a population of more than 10 000 refugees, two groups were selected for examination in September and October 1986: one group with people originating from areas where T. solium taeniasis is known to be endemic (Fig. 1). The other group, originating from an area not known to be endemic for 7". solium, included persons with a positive medical history for one or more of the following symptoms: epileptic seizures, headache or other neurological deficits. The list of symptoms was completed with syncopy, vertigo, abdominal complaints, cardiopulmonary distress and other symptoms (Table 3). After a short cursory clinical examination, venous blood
71
Area known to be endemic for T. solium
~J
MUyu tribal area
Me fackweler
We ena
K
AngUruk NlPelIn ~.
~_ "~
C e m ~
:&Kemberetoro serok: AGreen River
~, : -"
let ATolefomln Terekblle
~J~O KlUNGA t..WAIkem" "o:\'Nto0omb.n Kutu
New Guinea -
_
Fig. 1. Geographical aspects and location of investigated refugee camps in Papua New Guinea (shown by open triangles).
samples were drawn from 221 informed and consenting patients or after seeking parental permission. Couriers immediately carried the collected sera to nearby mission stations where kerosene refrigerators were available. The sere were transported by plane from the sites via Port Moresby to Switzerland. The age of subjects, 114 males and 107 females, ranged between 3 weeks and 90 years (average: 34 years). They were settled in 9 refugee camps along the border (Fig. 1): in the North, 50 persons were from the Blackwater camp, near Vanimo; in the South, 7 were from Timkwi, 22 from Tarakbits, 28 from Korokpin, 9 from Dome, 19 from Yogi, 52 from Atkamba, 12 from Niogomban and 22 from Kuiu. The refugees in the South originated from the following villages in the Muyu tribal area close to the border in the district of Merauke (Fig. 1): Amuan, Amupka;'Anggambran, Atmimkim, Imko, Ingembit, Kagenot, Kakuna, Kombut, Kwangtet, Mindiptanah, Mokbiran, Nanait, Ninety, Selih, Sesnukt, Simiram, Timkwa, Tokwebit, Umap, Upkin, Upyetetko, Wambiran, Wametkapo, Wangatkibi, Waropko, Wayrin, Wombon, Womsin. Only two persons left this southern tribal area, which is still free of T. solium, for other places where taeniasis is known to be endemic, but they pretended not to have eaten pork in these places. In the Blackwater camp, 39 of the 50 refugees examined originated from the villages Bokondini, Karubaga, Kelila, Kobakma, Pyramids, Tiom and Warnena in or near the Baliem Valley in the Jayawijaya district. Five originated from Enarotali, 1 from Gakokebo, 2 from Ilaga and 1 from Mulia, in the Paniai district; 1 came from Base a n d 1 from Jayapura, in the Jayapura district. All of them originated from or settled in the highlands where T. solium is known to be endemic (Fig. 1, dotted area). In order to set up a collection of stool samples, efforts were made to overcome superstitious beliefs and fear of witchcraft among the natives. Hence we were successful in collecting only a very limited number of reliable eases.
72
ELISA An ELISA was used to prcselcct patient sera which demonstrated antibody concentrations against crude T. solium metacestode antigen. The procedure was performed as previously described (Gottstein et al., 1987). Briefly,the polystyrene surface of micro-ELISA-plates was sensitized using a solution containing 2.5 ttg (protein) T. solium metacestode extract per ml coating-buffer (overnight incubation at +4°C). The sera were tested at a dilution of 1:200, bound patients' antibodies were detected by a second antibody-conjugate [rabbitanti-human IgG (Fc-fragment) from Dako, Copenhagen, Denmark, No. A 089, covalently bound to alkaline phosphatase (Boehringer Mannheim G m b H , No. 567 752) with the heterobifunctional reagent S P D P (Pharmacia Fine Chemicals, Uppsala, Sweden) according to the manufacturer's recommendations]. The substrate was 4-nitrophenylphosphate at a concentration of I m g per ml solution. The reading was carried out by a Dynatech MR600 photometer at A,toan m. Results were expressed arbitrarily in antibody units (AU), calculated according to the following formula: A4o4 m [testserum]-(~ + 3 S D A4o, .m [neg. control]) x I00 = A U A4o4 .m [pos. control]- (.~+ 3 S D A4o4 .m [neg. control])
Arbitrarily, point decimals regarding the expression of results in A U were rounded down for 0.1 to 0.4 and rounded up for 0.5 to.0.9. The mean value plus 3 S D of sera from healthy blood donors (= ~ + 3 S D A4o4 ,m [neg. control]) living in a European area not endemic for T. solium was determined to be the threshold discriminating between 'positive'and 'negative'.Positivelyreacting sera were further investigated by Western-blotting.
Western-blotting All S D S - P A G E chemicals were obtained from Bio-Rad Laboratories (Glattbrugg/Switzerland). Gradient S D S - P A G E and electrotransfer were performed as described previously (Gottstein et al.,1986, 1987). After elcctrotransfer,the proteinbound SDS was replaced with Tween 20 by washing the nitrocellulosesheets in PBS containing 0.3% Tween 20. The sheets were cut into strips and exposed to the sera diluted 1:250 with PBS containing 0.3% Tween 20. Antigen bands with bound antibodies were visualizedwith enzyme-linked immunosorbent techniques (Peralta et al., 1983), using an anti-human IgG-peroxidase conjugate (Sigma No. A-602A). Bound peroxidase activitieswere demonstrated with a precipitating chromogonic substrate (3,3-diaminobenzidine) and H202. Antigenic bands with their bound antibodies and anti-human IgG-peroxidase conjugate appeared then reddish-brown.
Results
From 221 serum samples tested by ELISA, antibodies were detected in 79 cases (Table 1). The 15 highest absorbance values, arbitrarily selected between 30 antibody units (AU) and 66 AU, were found in sera collected from persons of 8 different refugee camps and were not correlated with T. soliura endemic areas (Table 1 and Figure 1). Thirteen of a total of 15 blood samples with antibody reactivities between
73 TABLE 1 Examination of 221 Irian refugees living in Papua New Guinea (in 1986) for serum antibodies against Taenia solhm~ antigens by ELISA and Western-blotting ELISA: AU (antibody units)
Number of persons
Average number of bands in Western blotting (range)
Percentage of persons originating from T. solium endemic areas
0 ('negative') 1-29 30-66
142 64 15
n.d." 7 (0-26) 7 (0-29)
24 22 13
"n.d., not done.
Mr
~o ~: z~ ~. ~ •
94K
-
66K
-
45 K
-
30K 26K
20 K 14K
m
~-SK
t
•
i
•
Fig, 2, Immunological analysis by Western-blotting of serum from an Irianese refugee in Papua New Guinea (PNG), in comparison to sera from patients with radiologically and/or histologically proven T. solium cysticercosis, originating from South Africa, Mexico and Tibet, respectively. All patients' sera immunoreaeted with the specific 26 and 8 kDa bands (indicated by arrows).
74 TABLE 2 Western-blotting examination of 79 serum samples of refugees showing reactivity in the ELISA using crude Taenia solium antigen
Number of bands in Western blot (average)
Numberof persons
Numberof persons from endemicareas (T. solium) (%)
Numberof persons positivefor 26 and/or 8 kDa
ELISA:mean antibody units, AU (range)
0 I-I0 (5) I 1-29 (7)
5 58 i 16b
0 3 88
0 0 Ic
16 (2-43) 16 (2-66) 19 (2-64)
*One person with 10 bands in Western-blottingand 66 AU in ELISA had a known lymphaticfilariasis. bone person with 15 bands in Western-blottingand 8 AU in ELISAalso had a knownlymphaticfilariasis. ¢I'his person demonstrated29 bands in Western blot and 33 AU in ELISA. 30 and 66 AU had been taken in the southern camps Kuiu, Niogomban, Satkamba, Yogi, Komopkin, Tarakbits and Timkwi from refugees originating in the Muyu tribal area which is not known to be endemic for T. solium; only 2 of these samples were collected in the northern Blackwater camp from persons originating from the highlands where T. solium is endemic. Among all 79 serum samples with antibody reactivity between 1-66 AU (Table 1), 1 sample was confirmed to be positive for cysticercosis by Western blotting (antibodies detected against 26 and 8 kDa band). Fig. 2 shows the Western-blotting analysis of this person's serum in comparison to serum from patients with proven neurocysticercosis originating from different geographical areas. The person from this study was a 24 year-old Ekari tribesman who left Enarotali in 1980. He had neither a positive history nor overt clinical signs suggesting cysticercosis and might therefore be regarded as a subclinical case of human cysticercosis. From that person, positive in Western-blotting for the specific 26 and 8 kDa bands, stool examination was carded out: Endolimax nana and eggs from hookworms were discovered, but neither ova nor proglottids of T. solium. In 21 stool samples from other persons examined at random we found I I (52%) to be positive for 1 or more intestinal parasites, with eggs of hookworms and Ascaris lumbricoides in 9 (43%) and 3 (14%) samples, respectively; cysts ofgntamoeba cog and Endolimax nana were found in 2 samples each (9%). Table 2 shows the results of Western-blotting in relation to ELISA results and to the origin (endemic or non endemic area) of the persons tested. The persons are grouped according to the number of bands in Western blotting. There is an evident lack of correlation between number of bands (Western blotting) and antibody reactivity in ELISA. Persons with I 1 or more bands in Western blotting mostly originated from endemic areas, while persons with few or no bands (0 to 10) originated mostly from areas not endemic for T. solium. Table 3 summarizes the clinical symptoms, signs and concomitant diseases found among the refugees selected for serological examination.
Discussion
Applying a confirmatory specific serological method, immunodiagnostic evidence for T. solium cysticercosis was found in one Ekari tribesman. This was the first case of serologically diagnosed cysticercosis in Papua New Guinea. For further calculation
75 TABLE 3 Clinical symptons, signs and concomitant diseases (single or in combination) among 221 refugees selected for serologic examination Symptoms, signs and concomitant diseases
Moderate headache' Severe headacheb Vertigo Epileptic fitsc Syncope Mental deteriorationd General paresis with muscular atrophy Myalgias Chronic cough with expectoration Chronic abdominal complaints Angina pectoris Palpable splecn~ Lymphatic filariasis Open pulmonary tb r Symptoms undefined due to communication difficulties
Number (percentage) of persons from endemic areas (7". soliurn) 50 (= 100%)
Number (percentage) of persons from non endemic areas (T. solium) 171 (-- 100%)
Total
18 (36.0) 8 (16.0) 9 (18.0) 4 (8.0) 4 (8.0) 0 -
62 (36.2) 27 (15.8) 22 (12.9) 19 (11.1) 10 (5.8) 7 (4.1)
80 (36.2) 35 (15.8) 31 (14.0) 23 (10.4) 14 (6.3) 7 (3.2)
0 4 (8.0)
1 (0.6) 46 (26.9)
1 (0.5) 50 (22.6)
9 (18.0)
38 (22.2)
47 (21.3)
5 (10.0) 1 (2.0) 41 (82.0) 1 (2.0) 1 (2.0)
28 (16.4) 8 (4.7) 37 (21.6) 4 (2.3) 11 (6.4)
33 (14.9). 9 (4.1) 78 (35.3) 5 (2.3) 12 (5.4)
1 (2.0)
7 (4.0)
8 (3.6)
221 (= 100%)
"Headache was considered moderate if it occurred less than once a week but at least once a fortnight,with a duration not exceeding 24 h. bHeadaehe was considered severe if it occurred at least once a week or once per fortnight with a duration over 24 h. c22 eases of epileptic fits were considered focal Jacksonian seizures, 1 ease was considered temporal epilepsy. In 3 eases epileptic symptoms improved promptly after the institution of tuberculostatic treatment in the Government Hospital at Kiunga. °5 eases developed mental deterioration within 2 years, two of them acquired acute mental deterioration after an episode of heavy headache. In one case the symptomatology started 10 years afterand another ease showed recurrent symptomatology with alternating periods of complete remission. rrhe high rate of palpable spleens with 12-37 cm (mean 18 cm) in longitudinal diameter among peoi)le from 7". solium reflects immunological response to malaria. rOpen pulmonary tuberculosis was diagnosed microscopically by direct sputum smear and Ziehl-Neelsen's staining in the Government hospital at Kiunga. The refugees originating from 2". solium non endemic areas come from the Muyu tribal areas in the south of Irian Jaya where tuberculosis is highly prevalent. This area became the actual port of entry for tuberculosis into New Guinea whan Javanese freedom fighters were held in a Dutch prison camp at Tanah Merah. "
o f e p i d e m i o l o g i c a l b a s e l i n e d a t a , there is o n e l i m i t i n g f a c t o r o f the p r e s e n t s t u d y w h i c h h a s to be t a k e n i n t o a c c o u n t : we d o n o t k n o w the local d i a g n o s t i c sensitivity o f 26 a n d 8 k D a b a n d d e t e c t i o n i n W e s t e r n b l o t t i n g a m o n g eysticercosis p a t i e n t s o f t h a t g e o g r a p h i c a l area. H o w e v e r , p r e v i o u s w o r k ( G o t t s t e i n et al., 1987), h a s s h o w n t h a t i n all the g e o g r a p h i c a l areas i n v e s t i g a t e d u p to n o w ( M e x i c o , S o u t h A f r i c a , E u r o p e a n d C e n t r a l Asia), t h e relative d i a g n o s t i c sensitivity r a n g e d a r o u n d 92*/. f o r s e r u m
76
samples and 100% for cerebro-spinal fluid. It can be assumed that results in Papua New Guinea would correspond to these percentages. Unfortunately, no sera from a defined Irianese patient group with proven cysticercosis were available to our study, whereby the operating characteristic in question could have been elaborated. All 12 of our 221 sera with 15 or more antibody-binding bands in Western-blotting originated from areas where T. solium is endemic (see Table 2). This stands in contrast to the results of the nonspecific ELISA technique (see Table 1), where the highest absorbance values showed no correlation to endemic areas. In ELISA, the 6 highest values were found in patients originating from areas non-endemic for T. solium. They are most probably related to cross-reactions due to infections with other helminth species. On the other hand we could observe a correlation between the number and the distribution pattern of binding bands in Western-blotting and 7". solium endemicity. Cross-reactions can most likely be ruled out, since the closely related ecstode species (Echinococcus granulosus and E. multilocularis), which may induce some degree of cross-reactivity (Schantz, 1984) are not prevalent on the island. The only species of cestodes described in New Guinea are Hymenolepis nana and H. diminuta. These two parasites are not only limited to the endemic distribution of T. solium in Irian Jaya, but can also be found in other parts of New Guinea as shown by Bearup and Lawrence (1950) and Kelly and Vines (1966). Parasites of other species are unlikely to produce a similar characteristic distribution pattern of antibody-binding bands in Western-blotting. T. soliurn taeniasis and cystiecrcosis still constitute a risk for Papua New Guinea. In one case we found immunodiagnostic evidence for human cysticercosis in the Blackwater refugee camp. Other cases of cysticercosis in the northern camps cannot be excluded at the moment, whereas the occurrence of cysticercosis in the southern camps seems unlikely. Consequently, refugees originating from endemic areas must be regarded as potential carriers of T. solium. Beside prophylactic treatment of refugees with praziquantel for the removal of adult cestodes, the movement of pigs out of refugee camps is prohibited and further surveillance measures are proposed.
Acknowledgements The authors wish to thank Dr. Z.S. Pawlowski, former Senior Medical Officer of the Parasitic Disease Program at the WHO in Geneva, and Dr. W. Schiefenhoevel, ethologist a t the Max Plank Institute for Behavioural Physiology in Secwiesen, for their help and interest in this study. The authors would also like to express gratitude to Dr. B. Subianto, Chief Medical Officer at Kantor Kesehatan Wilaya Jayawijaya in Jayapura, Dr. G.H. Haina, Area Medical Officer at Kiunga, Mrs. E. Morris-Highes, Field Director of the Save the Children Fund, and Dr. B. Patten, Senior Veterinary Microbiologist for the National Veterinary Laboratory in Papua New Guinea. Thanks also to Mrs. E. Hemmeler for her skillful laboratory assistance and Mrs. Helfenstein and Mrs. Oberh~insli for secretarial work. References Beaurup, A.J. and Lawrence, J.J. (1950) A parasitological survey of five New Guinea villages. Meal. J. Austral. l, 724. Cocker-Vann, M.R., Subianto, D.B., Brown, P.; Diwan, A.R., Desowitz, R., Garruto, R.M., Gibbs, C.J.
77 and Gajdusek, D.C. (1981) Elisa antibodies to cysticerciof Taenia solium in human populations in New Guinea, Oceania, and Southeast Asia. Southeast Asian J. Trop. Med. Publ. Hlth. 12, 499-505. Gajdusek, D.C. (1978) Introduction of Taenia solium into West New Guinea with a note on an epidemic of burns from cysticercusepilepsyin the Ekari people of the Wissel Lakes area. Papua New Guinea Med. 3. 21,329-342. Gottstein, B., Tsang, V.C.W. and Schantz, P.M. (1986) Demonstration of species specific and crossreactive components of Taenia solium metacestode antigens. Am. J. Trop. Med. Hyg. 35, 308-313. Gottstein, B., Zini, D. and Schantz, P.M. (1987) Species specific immunodiagnosis of Taenia solium cysticercosis by ELISA and immunoblotting. Trop. Meal. Parasitol. 38, 299-303. Kelly, A. and Vines, A.P. (1966) Highland region survey of intestinal parasites. Meal. J. Austral. 2, 635-640. Peralta, J.M., Simons, A.R. and Tsang, V.C.W. (1983) Enzyme-linked immunoelectrotransfer blot techniques (EITB) for studying the specificitiesof antigens and antibodies separated by gel electrophoresis. Methods Enzymol. 92, 377-391. Subianto, D.B., Tumada, L.R. and Margono, S.S. (1978) Burns and epileptic fits associated with cysticercosis in mountain people of Irian Jaya. Trop. Geogr. Med. 30, 275-278. Tumada, L.R. and Margono, S.S. (1973) Cysticercosis in the area of the Wissel Lakes, West Irian. Southeast Asian J. Trop. Med. Publ. Hlth. 4, 371-376. Van der Hoeven, J.A. and Rijpstra, A.C. (1957) Intestinal parasites in the Central Mountains District of Netherlands New Guinea. Trop. Geogr. Meal. 9, 225-228. Schantz, P.M. (1984) Echinocoocosis(Hydatidosis). In: A.A. Mahmoud and K.S. Warren (Eds.), Tropical and Geographical Medicine. McGraw-Hill, Singapore, pp. 487-497. Schantz, P.M., Shanks, D. and Wilson, M. (1980) Serologic cross-reactions with sera from patients with echinococcosis and cysticercosis. Am, J. Trop. Meal. Hyg. 29, 609-612.
