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Farthing MJG. Goka AKJ. Immunology of giar- diasis. In: Wright R. Hodgson HJ. eds. Clinical Gastroenterology. Immunological aspects of the gut and liver. In press. Halita M. Isaicu L. Reactia de fizare a comple- mentulai in Lambliaza intestinula. Ardealul Medical 1946; 6: 154. Radulescu S. Iancu L. Simionescu 0, Meyer EA. Serum antibodies in giardiasis (letter). J Clin Path 1976; 29: 863. Ridley MJ, Ridley DS. Serum antibodies and jejunal histology in giardiasis associated with malabsorption. J Clin Path 1976; 29: 30-4. Vinayak UK, Jain P, Naik SR. Demonstration of antibodies in giardiasis using the immunodiffu- sion technique with Giardia cysts as antigen. Ann Trap Med Parasitol 1978: 72: 5X1-2. Moody AH. Ridley DS, Tomkins AM, Wright SG. The specificity of serum antibodies to Giar- &I lumhlia and to enterobacteria in gastrointesti- nal disease. Trans R Sot Trop Med Hyg 1982: 76: 630-632. Visvesvara GS. Smith PD. Healy GR, Brown WR. Serum antibodies to Giardia lamhlia demonstrated by immunofluorescence. Ann Int Med 1980: 93: 802-S. Smith PD, Gillin FD, Brown WR. Nash TE. IgG antibody to Giardia lamblia detected by enzyme- linked immunosorbent assay. Gastroenterology 1981; 80: 147680. Baveja UK, Warhurst DC. Humordl response in giardiasis. J Corn Dis 1983; 15: 25660. Wittner M, Maayan S, Farrer W, Tanowitz HB. Diagnosis of giardiasis by two methods: immu- nofluorescence and enzyme-linked immunosor- bent assay. Arch Path01 Lab Med 1983: 107; 524 7. Islam A. Stoll BJ, Ljungstroem I, Biswas J, Nazrul H, Huldt G. Giardiu lumbliu infections in a cohort of Bangladeshi mothers and infants followed for one year. J Pediatr 1983; 103: 996 1000. Haralabidis ST. Detection of antibodies against Cundida albicans in Giardia lamblia infected indi- viduals. Short communication. Acta Trop (Basel) 1984, 41: 3034. Haralabidis ST. Immunodiagnosis of giardiasis by ELISA and studies on cross-reactivity between the anti-Giurdiu lamb& antibodies and some heterologus parasitic antigens and frac- tions. Ann Trop Med Parasitol 1984; 78: 295- 300. Winiecka J, Kasprazak W, Kociecka W, Plot- kowiak J, Myjak P. Serum antibodies to Giardia intestinalis detected by immunofluorescence using trophozoites as antigen. Trop Med Parasit 1984; 35: 2&-2. Miotti PG, Gilman RH, Pickering LK, Ruiz- Palacios G, Park HS, Yolken RH. Prevalence of serum and milk antibodies to Giardiu lumblia in different populations of lactating women. J In- fect Dis 1985; 152: 1025-31. Gilman RH, Brown KH, Visvesvara GS, Mon-

da1 G. Greenberg B, Bradley Sack R, Brand1 F. Khan MU. Epidemiology and serology of Grar- dia lamblia in a developing country: Bangladesh. Trans R Sot Trop Med Hyg 1985; 79: 469 73

23. Goka AKJ. Rolston DDK, Mathan VI, Farthing MJG. Diagnosis of giardiasis by specific IgM antibody enzyme-linked immunosorbent assay. Lancet 1986; 2: 1846.

24. Isaac-Renton JL. Black WA, Mathias RG. Proc- tor EM, Sherlock CH. Giardiasis in a group of travellers - attempted use of a serological test. Can J Pub1 Health 1986; 77: 868.

25. Oyerinde JPO. Ogunbi 0. Alonge AA. Age and sex distribution of infections with Entumoeho histolytica and Giardia intestinalis in the Lagos population. Int J Epidemiol 1977; 6: 231-4.

26. Farthing MJG. Mata L, Urrutia JJ. Kronmal RA. Natural history of Giardiu infection of in- fants and children in rural Guatemala and its impact on physical growth. Am J Clin Nutr 1986: 43: 393303.

27. Roberts-Thomson IC. Anders RF. Serum anti- bodies in adults with giardiasis. Gastroentero- logy 1981; 80: 1262.

28. El-Badrawy F. El-Hady K, Makled MK, Shawky R. Salem M, Bassiuny W. Determ- nation of immunoglobulin E (IgE) and comple- ment III (C,) in children with giardiasis. J Egypt Sot Parasitol 1983; 13: 261-9.

Acute allergic phenomena in giardiasis. Lancet 1984: 2: 1428.

29. Farthing MJG. Chong S. Walker-Smith JA.

Immunodiagnosis of Yersinia disease

Infection with Yersinia enterocolitica or Yer- sinia pseudotuberculosis has been recognized as an important cause of gastroenteritis and abdo- minal pain in many countries’. For some years Y. enterocolitica has been associated with dis- ease in continental Europe2, particularly in Scandinavia’. The commonest pathogen is Y. enterocolitica serotype 0:3 and less often sero- type 0:9. This pattern is also seen in Canada. However, in the U.S.A., serotype 0% is the prevalent organism’. A complication of infec- tion is reactive arthritis, some affected patients having features of Reiter’s Syndrome and most having HLA-B274. Yersinia pseudotuberculosis is seen less frequently and causes abdominal pain due to mesenteric lymphadenitis.’

The method of choice for laboratory diagno- sis of yersiniosis is culture, often using cold enrichment techniques6 and prolonged incuba- tion times’. However, serological methods have been proven of value in the investigation of outbreaks of infection with Yersinia SP.“.~, although background data about the preva- lence of Yersinia antibodies in the general popuiation is lacking. Outside epidemiological

236 Leading articles

studies, antibody titres should be interpreted on an individual basislo.

In routine laboratories, screening of human sera is often limited to those organisms asso- ciated usually with human disease, namely Y. enterocolitica 0:3,0X 0:9 and 0:5,27 and the six serogroups. I to VI, of Y. pseudotuberculosis. Most of these organisms elicit a rapid antibody response. The bacterial agglutination test has been used widely and this has been modified to a slide agglutination method which is of value in giving a rapid presumptive diagnosis”. The antigen is a living suspension or a culture killed by 0.25% phenol or formalin. Also an adapted microtitre technique has been described’“. The prozone phenomenon may hinder diagnosis if sera are not screened at a high dilution. For Y. pseudotuberculosis, an antibody titre of I in 160 can be regarded as significant”. The end-point for agglutination of Y. enlerocolitica is more difficult to read but this is facilitated by mech- anical shaking”. Antibody titres of I in I28 or more”’ or 1 in 80 and above” are considered significant, but lower titres may not rule out yersiniosis in an infant or immunocom- promised patientlO. The nature of the antigen in the test is important. With live suspensions data suggest that cells grown at 37°C are not as reactive as at 25°C and that repeated subculture of stock antigen at 25°C may result in loss of a major antigenic determinant”‘.

A problem in interpretation of antibody titres in yersiniosis arises because of well- known cross-reactions with other bacteria. There is an antigenic relationship between the 0 antigens of Y. pseudotuberculosis group II and those of Salmonella B group; also between the 0 antigens of Y. pseudotuberculosis group IV and those of Salmonella groups D and H”. A presumptive positive result must be confirmed by absorption of the patient’s serum with Sal- monella of the corresponding group before being re-tested. Also, Brucella abortus cross- reacts with Y. enterocolitica 0:9” and may present difficulties with the serological diagno- sis of brucellosis and yersiniosis.

Solid phase radioimmunoassay (RIA) for immunoglobulin M (IgM) and IgG antibodies to Yersinia sp. has been compared with tube agglutination in 227 serum samples in Fin- land”. The results agreed well but false posit- ives were proposed in 10 of 202 sera with agglutinins but negative by RIA. This sensitive method has the disadvantages of using radioi- sotopes and requiring expensive laboratory equipment. The more convenient enzyme- linked immunosorbent assay (ELISA) has also

been applied to detect antibodies in yersiniosis. with either whole bacteria”-rh or phenol-water extracted lipopolysaccharide (LPS) as anti- gen”. However, LPS-ELISA has been found to offer no advantage over whole bacterium ELISA, and the latter is preferred with ELISA inhibition to control potential cross-reaction’! The test detects IgA as well as IgM and IgG, and has been found useful in the diagnosis of yersiniosis as a cause of reactive arthritis”. However, false positive reactions with ELISA may occur”‘, as with the tube agglutination method in patients with various thyroid dis- eases”‘. Enzyme immunoassay is considered more sensitive than agglutination in the detec- tion of IgG and IgA antibodies”, although correlations between each immunoglobulin class were found to be good between the tests”.

Antibodies of the IgM class to Y. enterocoli- tica have been found to persist for l-3 months following acute infection14, although, in some patients, antibodies were detected for up to 6 months15. This indicates a probable time of infection. In sera of patients with obscure arth- ritis, IgA has been demonstrated between 2 and 3 years after initial symptoms”. However, other workers by diffusion-in-gel ELISA (DIG- ELISA) have not been able to verify these findings and suggest that studies on serum antibody levels to Y. enterocolitica should in- clude only culture verified cases of infection”. This seems to be particularly important because of reported high prevalence back- ground levels of antibody to Yersinia sp.“‘.

Anti-yersinia antibodies have been assessed in sera from patients presenting with acute abdominal pain by tube agglutination and ELISA techniques. In 194 patients, agglutinins were detected in 23% of symptomatic indivi- duals compared with 2% of control subjects. High antibody titres occurred in 31% ol patients with acute appendicitis.” ELISA has been used to examine the sera from 630 adults with acute abdominal disease. Elevated anti- yersinia antibody levels were demonstrated in 21 patients, of whom 8 had an appendicectomy performed. In all patients with antibodies against Y. enterocolitica 0:9 or Y. pseudotuber- culosis IA. a true appendicitis was found at operation. A variety of acute abdominal inflammations including terminal ileitis, chole- cystitis and pancreatitis were noted in other patients with serum antibodies’“. However, fae- cal culture for yersinia was not attempted in either of these studies. In 286 children with acute gastroenteritis, paired sera were exa- mined by ELISA for yersinia antibodies and

Leading articles 23-l

stool culture was performed. In two patients shown to have Y. enterocolitica 0:3 in faeces, a significant rise was seen in each of IgM, IgG and IgA antibodies to the organism. Culture did not confirm the diagnosis in six patients with elevated IgM alone, indicating possible false positive reactions”.

Other techniques to investigate the antibody response to Y. enterocolitica are radioimmuno- precipitation, (RIP), sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS- PAGE) and Western blottingz4. With rabbit antisera to Y. enterocolitica 0:3, these methods have demonstrated a 92 kDa antigen. protein in nature, present on the bacterial outer mem- brane. This antigen cross-reacts with two strains of Shigefla jexneri of known arthritis causing potential”.

These newer tests have been used to investi- gate the antibody response to Y. enterocolitica in humans with joint disease. In U.S. patients with Reiter’s Syndrome, RIP and SDS-PAGE demonstrated that most serum samples precipi- tated three to five radioactively labelled Yersi- niu molecules and an 88 kDa antigen is of possible discriminatory valuez6. British patients with ankylosing spondylitis do not have anti- bodies against Y. enterocolitica 0:3 as measured by SDS-PAGE and immunoblotting. The pat- tern was completely different from that obtained with Finnish sera containing anti- bodies against several different bacterial components. with molecular weights of 15.~ 89 kDa”.

Classical serological tests such as comple- ment fixation and immunofluorescence are of limited value, offering no advantage over the tube agglutination method”. In cattle, indirect haemagglutination has been employed to dif- ferentiate between brucellosis and infections with cross-reacting Y. enterocolitka. but with inconclusive results’“.

Currently the bacterial agglutination test is performed most often in diagnostic labora- tories. with more sensitive whole bacteria ELISA in use in Finland. However. serology remains a secondary technique with definitive cultural procedures the method of choice in diagnosis.

ANN LEWIS Department qf‘Medica1 Microbiology,

St Bartholomew’s Hospital, London EClA 7BE

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References

Winblad S. Nilehn B, Sternby NH. Yrrs~rliu enterocolitica (Pasteurella X) in human enteric infections. Br Med J 1966; ii: 1363-6. Vantrappen G. Ponette E, Geboes K. e/ II!. Yu.vinia enteritis and enterocolitis: gastroentero- logical aspects. Gastroenterology 1977: 72 220 7. Bottone EJ. Yer.~inirr enterocolilica: a panoramic view of a charismatic microorganism. Crit Rev Microbial 1977: 5: ?ll-41. Keat A. Reiter’s Syndrome and reactive arthritis in perspective. N Engl J Med 1983: 309: 1606 15. Mair NS. Mair HJ, Stirk EM. Corson JG. Three cases of acute mesenteric lymphadenitis due to Pavrcwr~llu p.crudrlruhPrculosis. J Clin Pathol 1960: I!: 432 9 Werssfeld AS, Sonnenwirth AC. Yer.5 irh enrcrocoli/icn in adults with gastrointestinal dts- turhances: need for cold enrichment. J Clin Mic- robiol 1980; II: I96 7. Lewrs AM, Chattopadhay B. Faecal carriage rate of Yminiu speaes. J Hyg, Camb 1986; 97: 78 I 7. Tertti R. Granfors K. Lehtonen O-P. er ui. .An out break of Ymsbru psrudotzrhrr~,ult~.vi\ infcc- non J Infect DIS 1984; 149: 245550. Black RE. Jackson RJ. Tsai T, Y/ rrl. Epidemic ~Pr.tmiu mmwc olirica infection due to contami- nated chocolate mrlk. N Engl Med 1978. 29X: 76 9). Bottom EJ. Sheehan DJ. ~crsinrrr enterowli~rw Guidelines for serologic diagnosis of human Infection. Rev Inf Dis 1983; 5: 8988906. Mail-. NS. Fox E. Yersiniosis. London: Public Health Laboratory Services Board. 1986: I4 X. C‘orhel M. The serological relationship between Rrwclltr sp.. Ycrsinia entrrocolifiu serotype IX and Salmonella serotypes of Kauffmann-White group N. J Hyg Camh 1975; 75: 151 71. Granfors K. Viljanen M. Ahvonen P. Toibanen P. hleasurement of IgM and IgG antibodres to >‘cr.vin;cr by soltd-phase radioimmunoass;tv J In- fect Dis 197X. 1.1’. 731 6. . - Granfors K Mcaurement of immunoglobulin M (Ighl). IgG and 1g.A antibodies against ~‘<~r.\/nro ~,~~~(‘ro(,~)li/i(,u by enzyme-linked immunosorhent assay: persistence of serum antibodies during disease. J Clin Microbrol 1979: 9: 336 41. Granfors K. Viljanen M, Tiilikainen A, Torva- nen A. Persistence of IgM. IgG and IgA antrho- dies to I.cr.viniu m versinia arthritis. J Infect Dis 19x0: 141: 414 9. Lcino R. Granfors K. Havia T. c’t uI. Yersrm\rsis as a gastrointestinal disease. Stand J Infect Drs 19x7; I’): 63 8. Gripenherg M. Nissinen A. Vaisanen E. Lmder E. Demonstration of antibodies against Yer.tiniu c~rrtc~roc~uliri~ lipopolysaccharide in human sera by enzyme-linked immunosorhent assay. J (.‘lin Mrcrobiol 1979; IO: 279-84. Grdnfors K. Viljanen M. Toivanen A. Measure- ment of rmmunoglubulin M. immunoglohulin G ,md immunoglohulm A antibodies against I’,,rri-

238 Leading articles

nia enterocolitica by enzyme-linked immunosor- bent assay: comparison of lipopolysaccharidc and whole bacterium as antigen. J Clin Microbiol 1981: 14: 6 14.

19. Vesikari T. Granfors K, Maki M, Gronroos P. Evaluation of ELISA in the diagnosis of Yersinia enterocolirica diarrhoea in children. Acta Path Microbial Stand 1980 (B); 88: 139~42.

20. Shenkman L. Bottone EJ. Antibodies to Yersiniu enterocolitica in thyroid disease. Ann Intern Med 1976; 85: 735-9.

21. Bottone EJ, Sheehan DJ. Reply. Rev Infect Dis 1984; 6: 42334.

22. Lange S. Larsson P. What do serum antibodies to Yersinia enterocolitica indicate? Rev Infect Dis 1984; 6: 88&l.

23. Attwood SEA, Mealy K, Cafferkey MT. et al. Yersinia infection and acute abdominal pain. Lancet 1987; i: 529933.

24. Ogasawara M, Kobayashi S, Hill JL, Kono DH. Yu DTY. Rabbit antisera against three different

bacteria which can induce reactive arthritib. analysis by ELISA. immunoprecipitation and Western blot. Immunology 19X5; 54: 665 76

25. Simon DF, Kaslow RA. Rosenbaum J. Kayc RI.. Calin A. Reiter’s Syndrome following epide- mic shigcllosis. J Rheumatol 19x1; 8: 969 1004.

26. Kobayashi S, Ogasawara M, Maeda K. Yu DTY. Antibodies against Yer.smiu entcrocoliti~~cr in patients with Reiter‘s Syndrome. J Lab C‘lin Med 1985; 105: 380 9.

27. Toivanen A. Stahlberg T, Granfors K, Ebringcr A. Absence of antibodies to Yersiniu mtrwmd- ticu in patients with ankylosing spondylitis in London. Lancet 1986; 2: 1400.

28. Mittal K. Ricciardi ID. Tizard IR. Indirect hae- magglutination employing enterobdcterial com- mon antigen and Ycr.yinia somatic antigen: a technique to differentiate brucellosis from infec- tions involving cross-reacting Yersinio mterodr- tics. J Clin Microbial 1980: I I : I49 52.