300 TRANSACTIONSOPTHE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE (1988) 82, 300-302

Application of the latex test for human paragonimiasis in South America

M. Kobayashi’, S. Kojima’, M. Yokogawa’, M. Tsuji’ and N. Tsubota’ ‘Department of Parasitology, School of Medicine, Chiba Universi~, Chiba, Japan; ‘Department of Parasitology, School of Medicine, Hiroshima University, Hiroshima, Japan; 3ResearchLaboratory, Eiken Chemical Co. Ltd, Katsushika, Tokyo, Downloaded from https://academic.oup.com/trstmh/article/82/2/300/1899078 by guest on 30 September 2021 Japan

Abstract extracted with Verona1buffered saline (VBS), in 0.2 M The latex agglutination test (LAT) was compared aminomethylpropanol-HCI buffer, pH 8.0. with the (CFT), double diffusion test (DDT) and -linked immunosor- Latex agglutination test bent assav (ELISA) for the serodiaenosis of South The latex agglutination test (LAT) was carried out American*paragon&iasis. Sera from i7 Peruvian and according to the procedure of TSUBOTAet al. (1977). Ecuadorian patients with paragonimiasis, from 15 Japanesepatients with other hehninthiases, and from Other immunoserologicaltests 100 healthy students were analyzed. The tests were Complement fixation tests (CFT), double diffusion also compared using serum samples from patients tests (DDT) and enzyme-linked immunosorbent treated with bithionol. There was remarkable agree- assays (ELISA) were carried out as described pre- ment among the methods and it was concluded-that viously (YOKOGAWAet al., 1983). the LAT could be a useful serodiagnostic techniaue for paragonimiasis, especially in fi:ld surveys. A Serum samples Serum samples were collected from patients with Introduction paragonimiasis in Peru and Ecuador in 1981 (YOKO- Various methods have been introduced for the GAWAet al., 1983). Another batch of samples was immunoserological diagnosis of human naraaoni- collected from patients, 12 to 18 months after miasis (YOKO;AWA and TSUJI, 1962: COO eral., treatment with bithionol. Serum samples were 1981: KO~IMA et al.. 19831.However. methods such obtained from Japanesepatients with other hehnin- as the complement iixation test (cm-j, immunoelec- thiases who had been diagnosed by a combination of trophoresis (IEP) and enzyme-linked immunosorbent CFT, ELISA and DDT. Normal serum sampleswere (ELISA) require special equipment and rather collected from 100 students of Chiba University who complicated procedures, although they are excellent were shown to be parasite-free by stool examinations not only for diagnosis but for evaluation of cure. and negative for the intradermal test with P. wester- Therefore, it would be useful if a simple method, mani . easily performed in a field survey, could be de- veloped. Results Recently, the latex agglutination test (LAT) has The LAT was carried out on normal serum been shown to be one of the more useful tools for samples, in order to estimate the extent of false serological diagnosis of in terms of its positive reactions. 99 out of 100 samples had titres sensitivity, simplicity and low cost (TSUBOTAet al., below 1:8. However, one, who was negative by both 1977). CFT and ELISA, had a titre of 1:16 (Table 1). We report here that the LAT is also a useful tool for The results of CFT, ELISA,. DDT and LAT on the diagnosis of South American paragonimiasis, serum samples from proven infections with P. based on results of a comoarison between the LAT mexicanus are shown in Table 2. Titres of the LAT and other immunoserolog~cal techniques. correlated well with those of other examinations. Latex particles coated with P. mexicanuswere also Materials and Methods used with serum samplesobtained from patients with other hehninthiases, ‘shown to contain specific anti- Extracts of Paragonimus mexicanus were prepared bodies to other antigens bv the CFT and ELISA either with veronal buffered saline, carbonate buffer, (Table 3). In addition, serum samples from casesof or 0.1% sodium chloride as described previously both paragonimiasis westermani and miyazakii (YOKOGAWAet al., 1983). Antigens were similarly showed remarkably high titres in LAT and ELISA DreDared from R. westewnuni. P. mivazakii. Anaios- with P. mexicanus antigen, whereas in general low iron&us cantonen+ Trichinel~a spirals, Fa&ola “sp., titres were observed with samples from patients with , Gnathosthomahispidum and Diphyb other helminthiases. However, some samples lobothrium erinacei. obtained from patients with gnathostomiasis or spar- ganosis showed exceptionally high titres in the LAT Antigen coated latex particles (Table 3). Antigen coated latex particles were prepared Treatment with bithionol was given to patients with accord& to the method of TSUBOTA and OZAWA proven paragonimiasis. Typical results in the LAT, (1977). The particles were sensitized with antigen, CFT, ELISA and DDT before and after treatment 301

Table l-Results of the latex agglutination test on are snmmarixed in Table 4. No positive reaction was normal seral obtained in the DDT after treatment, although 2 of 6 patients were still positive in the &I’, and-3 in the Titre Number ELBA. However, titres in the CFT and ELISA <2 68 decreasedsignificantly after treatment. This tendency 19 was also observed in the LAT (Table 4). : Discussion : The results of the LAT on normal seraindicate that 1: 1 agglutination may occur at a serum dilution of 1:16

Total 100 non-specifically. However, 99 out of 100 had titres Downloaded from https://academic.oup.com/trstmh/article/82/2/300/1899078 by guest on 30 September 2021 below 1:8. TSUBOTAet al. (1977) reported that 6 of ‘The donors were negative in the IDT with P. 692 normal sera had a titre of 1: 16 in the LAT for westefvnuni antigen toxoplasmosis, while the remainder were below 1:8.

T&~tio~Resuks of the latex agglutination test on sera from 17 patients with Paragonimus mexicanus

Patient No. LAT CFT ELBA DDT

: 256512 11832 640160 +++++

I: 1024256 11828 32040 +++ 5 512 + 6 128 :: :8 + 7 8192 115 1280 ++ + i 81;; 1:; 12:: ++ :: 512128 :4 160 +++ if 163841024 10736 1280ISii +++++

:: 4096256 is 1280160 +++++

;: 20: 46 640 +++ >160 >1280 +++ ‘Results expressed as reciprocal titres (except for DDT)

Table 3-Results of the latex agglutination test with P. mexicanus antigen on various hebninthiases’ P. mexicanus antigen Homologous antigen2 Serum LAT ELISA CFT ELISA Paragonimiasis 1 2048 320 640 westermani >640 1:: X40 $ 163841024 320 640 4 128 1:: 320 Paragonimiasis 1 4096 6:: 120 llliyaZakii 320 92 ii $3 20488192 >640 >160 >640 Fascioliasis < 20 160 : < :z 80 1: 320 Dirofilariasis c 20 160 : < :: 20 !8 320 Gnathostomiasis < 20 65 320 : < 10:: 160 >160 640 Sparganosis c 20 65 :3 20:: 80 >160 :zi ‘Results expressed as reciprocal titres. Note that sera from patients with hehninthiases other than paragonimiasis generally showed negative or low titres in the LAT with P. mexicanus antigen, although they contained specific to another antigen. One sample from a caseof gnathostomiasis, and another from one of sparganosis, showed exceptionally high titres in the LAT. 2Antigen prepared from the species listed in the f&t column. 3Pleural effusion. 302 Table 4-Demonstration.of decrease of titres of the latex in South America where both paragonimiasis and agglutination test and other immunological teats after fascioliasis are common. treatment with bithionol of patients infected witb P. After treatment with bithionol, 5 out of 6 patients mukamd becamenegative in the LAT (Table 4). This result is Patient consistent with CFT and DDT, suggesting that the No. LAT CFT ELBA DDT LAT is a useful tool for evaluation of cure of paragonimiasis. 1 Latex particles coated with antigen from Toxoplas- (118) (&) (+T+, 2 ma go& are still efficient after 3 $ars’ preset&ion (TSUBOTA et al.. 1977). We found that latex coated (2326) (32) Go) F+, 3 with antigen from P. hexicarus showed no decline in Downloaded from https://academic.oup.com/trstmh/article/82/2/300/1899078 by guest on 30 September 2021 (lOi4) (l%, (3%) (++I titre after storage for 18 months in a refrigerator 4 (4°C). 5 (2;6) (28) (40) 6 We conclude that the LAT using P. mexicanus antigen is useful for diagnosis and evaluation of cure (5;2) (15) cso) 6 of South American paragonimiasis, although there is a 6 limitation concerning cross reactivity with some, but (128) fp3) &) G) not all, other helminth antigens. ‘Resultsare expressedas reciprocal titres; thosein parenth- Acknowledgements eseswere obtained before treatment. We thank Dr 0. Grados,Nationales de Mud, Peru, Dr H. Miranda, Universidado National de T~jillo, Peru, and Dr J. Rurnbea, Universidado National de Quito, Ecuador Consequently, they regarded titres above 1:32 as for kindly supplying serum samples. positive, and 1:16 as doubtfully positive. Furth- This work was suonorted bv the Grants-in-Aid for ermore, false oositive reactions in the ELISA for Overseas Scientific Red&h from the Ministry of Educa- -wereobserved in a high proportion of tion, Science and Culture, Japan, Nos 404112, 56041013, uninfected residents of tropical countries (VOLLER et 58041013 and 60041048. al., 1977). Since a rate of non-specific reactions of 1% References is considered to be reasonable,we regard the titre of Cho, S. Y., Hong, S. T., Rho, Y. H., Choi, S. & Han, C. (1981). Application of micro-ELBA in serodiagnosts of 1:32 as the lowest indicating a positive reaction in the human paragonimiasis. Korean Journal of Parasiwlogy, LAT for paragonimiasis. 19. 151-156. The LAT titres of 17 caseswith proven paragoni- Kobayashi, M., Niimura, M., Yokogawa,.M. & Yamaguchi, miasis ranged from 1:32 to 1:16 384 (Table 2). Thus, T. (1987). Studies of immunoserologuzal tests for acute if 1:32 is taken as a positive result, all these cases human . JapaneseJmnnal of Parasitology, 36, would be judged to be positive. 248-253. The coefficients of correlation among the titres of Kojima, S., Kobayashi, M. & Yokogawa, M. (1983). the LAT, CFT and ELISA were calculated. There Demonstration of the cross-reactivity among antigens extracted from four species of Paragonimus and its was a correlation (P