Sensitized-erythrocyte-lysis (SEL) test as an epidemiological tool for human leptospirosis serological surveys

Bull. Org. mond. Santej 1969, 40, 899-902Bull. Wid Hlthi Org.J Sensitized-Erythrocyte-Lysis (SEL) Test as an Epidemiological Tool for Human Leptospirosis Serological Surveys DORA S. K. TAN 1 Epidemiological studies ofhuman leptospirosis have generally been limited to countries with specialized laboratories employing the microscopic-agglutination (MA) test. The sensitized-erythrocyte-lysis (SEL) test is much simpler for routine hospital laboratories to carry out and it has been found valuable in the diagnosis ofhuman leptospirosis. Thispaper reports the results of studies of the SEL test as an epidemiological tool in serological surveys. The results showed that the significant SEL titre was 1:80 and that the sensitivity of the test depended possibly on the antigen preparation and the amount of complement used. Most of the SEL antibodies were found to persist at significant titres for about 1 year after active infection, but less than halfpersisted longer than that. The SEL test is therefore useful for detecting recent infections and for indicating the stability of leptospirosis in an area. The endemicity ofleptospirosis in West Malaysia was confirmed by the SEL test, based on the employment of 1: 80 as the significant titre. The sensitized-erythrocyte-lysis (SEL) procedure, also known as the haemolytic (HL) test (Cox, 1955), has been evaluated extensively by Cox et al. (1957), Chang et al. (1957), Sharp (1958) and in this labora- tory (unpublished) as a means of diagnosing human leptospirosis, and the results have been good. It has advantages over the long-established and widely employed microscopic-agglutination (MA) test, especially in areas where many serotypes of lepto- spires exist. In such places the necessity to test each serum separately against a battery of antigens belonging to 15 or more serotypes, even when pooled, is most tedious and impracticable for the ordinary diagnostic laboratory. Although the SEL test has been used with advan- tage for diagnosis ofhuman cases of leptospirosis, its potential usefulness as an epidemiological tool has not been established. This paper reports the results of studies of the SEL test as an epidemiological tool to determine antibody prevalence ratios in persons from non-endemic areas and also the level of SEL antibodies in healthy persons in West Malaysia. 1Virus Research Officer, Virus Research Laboratory, Institute for Medical Research, Kuala Lumpur, Malaysia. MATERIALS AND METHODS Source ofsera A random collection of 307 normal human sera was made; 100 of these were from the USA and 207 were from the United Kingdom of Great Britain and Northern Ireland. In 1960-61, a total of 4819 sera was collected from healthy persons of different age and sex, and from among the various racial and occupational groups present in West Malaysia. The persons sampled had all been resident for at least 1 year. Cases of leptospirosis In all, 40 patients with proved leptospirosis, from 5 different states of West Malaysia, were selected and follow-up bleeding was done on these patients at various intervals for a maximum of 3 years. Diagno- sis of these cases was based on significant increases in SEL titres in paired sera or on positive blood cultures. All sera were inactivated at 56°C for 30 min prior to testing. 2345 899 D. S. K. TAN Techniques Microscopic-agglutination (MA) test. Of the 307 sera from the USA and from the United Kingdom, 27 were tested in England by Dr L. H. Turner, Director of the FAO/WHO Leptospirosis Reference Laboratory, London, England. He used a formolized leptospiral antigen and screened each serum at a final dilution of 1:10 with 15 pools of antigens (arranged according to the current concept of serogroups). Dr Turner then titrated each serum with the constituent antigen suspensions of the pools that reacted, at final dilutions of 1: 10, 1: 30, 1:100, 1: 300 and at higher dilutions if necessary. The remaining 280 sera were tested in this labora- tory, employing live antigen, each serum being screened at a final dilution of 1: 10 with 24 serotypes representing 14 serogroups. Titration of sera with the reacting serotype was also done at the above- mentioned dilutions. All sera were clarified by filtration through Hem- ming's filter prior to testing to make it easier to read the results. Sensitized-erythrocyte-lysis (SEL) test. The tech- nique employed was based on that described by Chang et al. (1957). The erythrocyte sensitizing substance was prepared from the AM strain of Leptospira biflexa. RESULTS The significant SEL titre To provide an index of non-specific reactions of the SEL test, comparative MA and SEL tests were done on the sera collected from the USA and the United Kingdom. These 2 countries have a low or negligible prevalence (less than 1 %) of leptospiral antibodies in man (Alexander, personal communica- tion, 1966) detected by the MA method employing the full range of leptospiral antigens in the screening -procedure. In Table 1, it is shown that at SEL titre levels of 1: 5 and 1.: 20, antibody reactions were seen in 19% of the sera, whereas, at levels of 1: 80 and above, fewer than I % of the sera were positive. On the basis of these findings, the titre of 1:80 could be selected, with reasonable assurance, as the minimum criterion for a significant positive reaction. At MA titre levels of 1:10 and 1:30 antibody reactions were found in 10% of the sera, but at the titre of 1:100 only 1 % reacted. This indicated that 1:100 may be safely regarded as the significant titre in MA tests, supporting the criterion which had been arbitrarily determined previously. TABLE I PERCENTAGES OF POSITIVE REACTIONS IN THE SEL AND MA TESTS AT DIFFERENT TITRE LEVELS AMONG SERA FROM THE USA AND THE UNITED KINGDOM Positive reactions - Percentage ofSEL test W% total no. of SEL titre a USA sera United Kingdom sera tested (100) b sera (207) b 3 5 9 10 10 20 8 10 9 80 0 2 1 320 0 <1 <1 Positive reactions - MA test Percentage (%) of total no. MA titre a . of sera testedUSA sera United Kingdom (280) b (100) b sera (180) b 10 5 6 6 30 1 5 4 100 1 1 1 a Titres expressed as reciprocals. b Number of sera tested is shown in parentheses. In the MA test some of the United Kingdom sera were tested in Lon- don by Dr L.H. Turner. Persistence ofSEL antibodies The 40 persons with proved leptospirosis were bled at various intervals after infection, some over a period of 3 years, and their sera were tested for SEL antibodies. Significant rises in SEL antibody titres were demonstrated in paired sera from these patients and leptospires were isolated from several. Table 2 summarizes the change in the number of antibody titres of 1: 80 and greater with time, and these data are compared with the findings of Cox et al. (1957), who employed a similar procedure (HL test). The SEL test (as performed in this laboratory) appeared to be more sensitive than the HL test in detecting early antibody rise. From 11 days to 6 months, both tests were equally sensitive, but thereafter (up to 1½/2 years) the HL test appeared to be more sensitive than the SEL test. From 11/2 years to 3 years the SEL antibodies declined gradually. Data on HL antibodies during this period were not available. These differences in sensitivity may well be attributed to the different antigen preparation 900 EPIDEMIOLOGY OF LEPTOSPIROSIS: SENSITIZED-ERYTHROCYTE-LYSIS TEST TABLE 2 PERSISTENCE OF SEL AND HL ANTIBODIES AT 1:80 AND ABOVE Anioya Time after onset of Persistence b' GeometricAntibod a disease (%) mean titre c HL 1-10 days 37 (177) 16 11 days - 6 months 98 (259) >1 677 6 months - 1 year 91 (11) 129 1 year - 1/, years 75 (8) 106 SEL 1-10 days 11 days - 6 months 6 months - 1 year 1 year - 1/ years 1', years - 3 years 53 (43) 100 (49) $:Q 141% 31 >1 111 AO 48 (23) 35 40 (15) 32 a The results for HL antibodies are taken from Cox et al. (1957). b Total number of sera tested is shown in parentheses. c Titres expressed as reciprocals. employed in the two tests as well as to the amount of complement used. SEL antibody prevalence ratio in West Malaysia Of the 4819 survey sera tested, 75% had no detectable antibodies and 13% had low titres of 1: 5 or 1: 20, as shown in Table 3. This is comparable to the rate of " non-specific " titres in non-endemic leptospirosis areas. Assuming 1: 80 to be the TABLE 3 DISTRIBUTION OF SEL TITRES AMONG SERA FROM HEALTHY PERSONS IN WEST MALAYSIA SEL titre a No. of sera 1 Prevalence ( <5 3610 74.91 5 144 3.00 20 497 10.31 80 441 9.15 320 103 2.14 1 280 22 0.46 >5 120 2 0.04 Total 4 819 100.01 a Titres expressed as reciprocals. significant titre, 88% of the population tested were negative and 12% positive. This prevalence rate is about 12 times higher than that found in the sera from the USA and the United Kingdom and supports the fact that leptospirosis is endemic in West Malaysia. DISCUSSION Epidemiological studies of human leptospirosis have generally been limited to countries with spe- cialized laboratories employing the microscopic- agglutination (MA) test. The ordinary routine diagnostic laboratory, with its multiple commit- ments, finds it tedious, if not impossible, to employ a battery of 15 or more different antigens to detect aggltitinins in a single serum specimen. Apart from the possibility of getting false negative results through the omission of an antigen in the test pro- cedure, especially when new serotypes are constantly being identified, the handling of pathogenic cul- tures renders the test too hazardous for general adoption. Since the introduction of a biflexa extract as antigen in a genus-specific test (the SEL or HL test), the routine hospital laboratory has been provided with a much simpler, safer and quicker method of diagnosing leptospirosis in febrile patients. The experiments described in this paper show that it is also possible to employ this test in human serological surveys. It was found, however, that the SEL test could determine only recent incidence of leptospirosis in a particular locality. The conven- tional MA test is still the only method for detecting antibody that has been present for a longer period. Moreover, caution must be exercised in the inter- pretation of results when the SEL test is used for screening sera before eventual grouping and sero- typing by the MA test. Many sera with longer- lasting agglutinins may well be missed when the SEL test is employed for this purpose. It is to be expected, therefore, that prevalence ratios obtained through the use of the MA test would be greater than cor- responding results from the SEL test. Nevertheless, there is one notable advantage to be derived from this limitation. The test is especially valuable in the investigation of recent outbreaks of fever both in non-endemic and endemic places. In endemic areas, where the incidence of leptospirosis is not expected to vary much from year to year, a reasonably correct picture of the incidence of the disease in that area can be obtained, if a knowledge 901 902 D. S. K. TAN of the particular infecting serotypes is not important. Serial sampling of the same locality by this method will indicate the stability of the disease and pick out fluctuations more effectively than the MA test, because changes in incidence of leptospirosis will show greater relative fluctuations in the prevalence ratios obtained by the SEL test than by the MA test. It may therefore be concluded that, despite its disadvantages, the SEL test can help to improve our knowledge of leptospirosis, especially in some Asian countries where facilities for MA testing are non- existent, as long as the worker is aware of its limita- tions and interprets his findings accordingly. Since the completion of this study, the SEL test has been simplified by the use of microtitre techni- ques (Meers & Ringrose, 1968). Although the modi- fication is suitable for the diagnosis of recent leptospiral infections, it has yet to be evaluated as an epidemiological tool for human serological surveys. It is probably safe to assume that the life-span of the SEL antibodies in both the original and simplified tests is comparable, but the significant titre in the latter test, which varies with the type of antigen used, will have to be determined independently, employing sera from non-endemic areas as a baseline control. ACKNOWLEDGEMENTS The author wishes to thank Dr A. D. Alexander, Chief, WHO Leptospirosis Reference Laboratory, Washington, USA, and Dr L. H. Turner, Director, FAO/WHO Leptospirosis Reference Laboratory, Lon- don, England, for the supply of sera and for their invalu- able criticism and advice. She is also grateful to Dr. V. Thuraisingham and his staff at the General Hospital in Penang, the staff of the Institute for Medical Research Branch Laboratory, Penang, and the staff of various hospitals and health offices throughout West Malaysia who assisted her in obtaining blood specimens. The technical assistance rendered by Mr. Mohamed Omar and Mr Johan Hadji Adam is much appreciated. RE'SUMI INTtRtIT EPIDEMIOLOGIQUE DE L'EPREUVE DE LYSE DES ERYTHROCYTES SENSIBILIStS (LES) DANS LES ENQUETES SEROLOGIQUES SUR LA LEPTOSPIROSE HUMAINE Deux tests utilis6s pour le diagnostic de la leptospirose, 1'epreuve d'agglutination microscopique et l'epreuve de lyse des erythrocytes sensibilit6s (LES), ont dans un premier temps fait l'objet d'une etude comparative. L'examen par ces deux techniques d'environ 300 serums pr6lev&s chez des personnes bien portantes a permis de retenir pour l'epreuve LES le titre de 1: 80 comme premiere dilution significative. On a ensuite suivi l'evolution des anticorps s6riques chez 40 habitants de Malaisie occidentale atteints de leptospirose confirm6e. II est apparu que, dans une pro- portion notable des cas, les anticorps LES persistaient a des titres significatifs pendant un an environ apres l'infection, mais disparaissaient ensuite chez plus de la moitie des malades. Enfin, en 1960-1961, on a preleve 4819 serums chez des personnes bien portantes de Malaisie occidentale: 75% des echantillons ne contenaient pas d'anticorps speci- fiques, 13% 6taient faiblement positifs (titres de 1: 5 ou 1: 20) et 12% etaient nettement positifs si l'on choisit comme seuil de specificit6 un titre de 1: 80. On peut en d6duire que la leptospirose est end6mique en Malaisie occidentale. L'auteur met 1'accent sur l'interet de 1'epreuve LES qui apparait comme une methode simple, fiable et rapide de diagnostic, particulierement utile pour l'etude epidemiologique des foyers r6cents de leptospirose. REFERENCES Chang, R. S., Smith, D. F. W., McComb, D. E., Sharp, C. F. & Tonge, J. I. (1957) Amer. J. trop. Med. Hyg., 6, 101-107 Cox, C. D. (1955) Proc. Soc. exp. Biol. (N.Y.), 90, 610-615 Cox, C. D., Alexander, A. D. & Murphy, L. C. (1957) J. infect. Dis., 101, 210-218 Meers, P. D. & Ringrose, M. A. (1968) Trans. roy. Soc. trop. Med. Hyg., 62, 105-108 Sharp, C. F. (1958) J. Path. Bact., 76, 349-356