Serodiagnosis of human employing immunodominant

M A Ribeiro,
Adolf0

leptorrpirosis ant

C S N Assis, E C Romero
Sao Paulo, Brazil

Lutz Institute,

Summary IgM-PK-ELISA, an enzyme-linked immunosorbent assay for immunoglobulin M employing enzymatically treated antigen is described for the serodiagnosis of leptospirosis. It is based on the observation that patients serum samples present IgM antibodies reacting with a diffuse band of 14-22 kDa proteinase-K resistant. The assay was evaluated in serum samples from patients with leptospirosis (n = 89), typhoid fever (n = 81, malaria (n = 19), syphillis (n = 20). hepatitis (n = 16) and clinically healthy individuals (n = 92). The microscopic agglutination test (MAT) served as a reference. IgM-PK-ELISA and MAT presented the sensitivity of 89.89% and 92.13% and the specificity of 97.42% and 94.84% respectively. The overall results of the IgM-PK-ELISA are comparable to those of the MAT. However, the IgM-PK-ELISA detected antibody activity in 38% of acute-phase sera which were negative by the MAT. Our data suggest that IgM-PK-ELISA can be used as a screening test. Key words: Serodiagnosis, antileptospira IgM Serodiagn. Immunother. human leptospirosis, Infect. Disease ELISA, IgM-PK-ELISA, proteinase-K September treated antigen,

1994, Vol. 6, 140-144.

Introduction Leptospirosis, a world-wide infection of zoonotic origin is caused by spirochaetes of Leptospira interrogans complex and occurs in Brazil, chiefly during the rainy season. In Sao Paulo, Brazil, L. interrogans serovars icterohaemorrhagiae and copenhageni are the most frequently isolated serovarsl. These serovars are usually the cause of Weils syndrome. Leptospirosis is an acute, febrile illness, the severity of which varies from mild to rapidly fatal. It may remain undiagnosed because the symptoms are non-specific, especially in the early stages of infection*. Immunity to leptospirosis is mainly humorally mediated3-h. Leptospiral infections in humans may be diagnosed in the laboratory either by isolating the causal organism or by demonstrating a rise in specific serum antibody. Whereas culture is of undoubted epidemiological importance, the time mediated between the culture and the identi-

requests to: Dra Maricy A Ribeiro, lnstituto Adolf0 Lutz, Se@o de Imunologia, Av Dr Arnaldo 355, 11 andar, CEP: 01246-902 S&o Paulo, SP, Brazil 0 1994 Butterworth-Heinemann Ltd 0888-0786/94/030140-05

Received: 5 May 1994 Accepted: 3 June 1994 Correspondence and reprint

fication of the infecting organism permits only a retrospective diagnosis. The microscopic agglutination test (MAT), for the detection of specific antibodies, is still the standard reference test for serology. It remains a specialized test which is not generally performed in routine diagnostic laboratories. Some previous studies showed that IgM was the predominant class of antibody developed during the infection5.8-10. IgM agglutinins may persist for several years. Thus, on the basis of MAT, current or recent infections cannot be readily differentiated from past infections. Although the importance of agglutinating antibodies in immunity has been well established, the nature and identity of the corresponding leptospiral antigens have not so far been greatly studied. Immunoblotting of leptospiral extracts with patients sera revealed antibodies recognizing several components in the molecular weight range of 14.5-105 kDa of both IgM and IgG response12J3. All patients serum samples presented IgM antibodies reacting with a diffuse band of 14.8-22 kDa proteinase-K resistantY,13, whereas sera from healthy individuals presented IgM antibodies reacting with several antigens, but lacked response against those of the diffuse band of 14.8-22 kDa. Chapman et a1.14reported that the nature of this low molecular mass antigen is unknown, but warrants

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further study as it is clearly a major antigen recognized by both infected and vaccinated humans. Furthermore, as this immunodominant antigen appears to be broadly reactive with several serovars and elicits antibodies early in the immune response to leptospiral infection, it may be useful as the basis of a defined crossreactive diagnostic test for human leptospirosist4. Undefined crossreactive preparations have previously been used for this purpose but they are not yet used as a routine test in diagnostic laboratoriesx~15-1x, in part because the antigens detected by antileptospiral antibodies have not been defined and because there is no agreement on the standardized methods of producing these antigens. Bahaman et al.? proposed that further investigation should use a pure and more specific leptospiral fraction, as the test antigen, to lower the background readings of the controls. The main problem is in early diagnosis?, where detection of specific IgM may be important because of its early appearance and long persistence, and because of the failure of some patients to produce specific IgG antibodiesl. The objective of the present study was to an enzyme-linked develop an IgM-PK-ELISA, immunosorbent assay for immunoglobulin M which uses enzymatically treated antigen to detect antileptospiral antibodies and to evaluate its potential usefulness in human serodiagnosis.

Enzymatic treatments The digestion of the soluble antigen by proteinase-K followed the procedure described by Cambiaso et al.2. Briefly, equal volumes of substrate and enzyme (0.2 mg ml- in 0.1 M tris-HCl buffer pH 7.5, containing 20 mM CaCl,) were mixed and incubated overnight at 37 C. The reaction was stopped by the addition of phenylmethylsulfonyl fluoride (1 InM). After proteinase-K treatment, the antigen was treated with 50 ~1 ml-l each of DNase and RNase (Sigma Chemical Co., St Louis, MO, USA) for 3 h at 37 C. This will be referred to as the pK-treated antigen. Desiccated aliquots of this antigen solution were stable, at room temperature. for at least 18 months.

Coating of microtitre plates Thirty ul of antigen diluted to 1 : 500 in absolute ethanol was pipetted into one half of the wells of polystyrene microtitre plates (Corning, New York, USA). The remaining wells were coated only with 30 l~,l of ethanol. The plates were left at 37 C until the fluid had evaporated. The plates were stored at room temperature in a dry place until use.

Materials
Sera

and methods

The following

four collections

of sera were studied:

1. Two serial samples from each of 82 leptospirosis patients who had seroconversion by MAT. 2. Two samples from seven patients with leptospirosis confirmed only on the basis of clinical and epidemiological data. 3. Serial sets of sera collected from seven patients with Weils syndrome who were followed for 4-8 months. 4. Control serum samples obtained from 92 healthy blood donors, 20 patients with syphilis, 19 patients with malaria, 16 patients with hepatitis and eight sera from patients with typhoid fever.
IgM-PK-ELISA

Antigen preparation Strain M20 of serovar copenhageni, was grown for 6-7 days in Korthoff medium. The culture was centrifuged for 20 min at 10 000 X g. The pellet, washed three times with sterile physiological saline to remove adherent proteins present in the medium, was resuspended in 2 ml of distilled water, successively frozen, thawed three times, and sonicated at an output of 1 mA at 4 C for 3 min (MSE Sifam Electrical Instrument Co. Ltd., England). After a centrifugation at 2000 X g, the supernatant had its protein concentration** estimated to be 2 mg ml-, with bovine serum albumin (BSA) as standard.

Test performance Before use the plates were thoroughly washed four times with 0.01 M phosphate buffered saline (PBS) pH 7.2. One hundred l~,l of patients serum was added to the wells in a dilution of 1 : 400 in skimmed-milk 1% in PBS. The incubation period was 1 h at 37 C. The plates were washed four times with PBS. One hundred l~,l of peroxidase-labelled anti-human IgM (goat IgG anti-human IgM p-chain specific, Sigma) was added to the wells in a dilution of 1 : 1000, as determined by checkerboard titration. After an incubation period of 1 h at 37 C the plates were washed as described above. Fifty ~1 of substrate (0.4 mg ml-l o-phenylenediamine in phosphate-citrate buffer, pH 5.0 and 0.012% HZ02) was pipetted into the wells. After 10 min at room temperature in a dark chamber, 50 ~1 of 1 M H2SO4 containing 0.5% sodium sulphite was added to each well to stop the reaction. The intensity of the resulting yellow colour was measured in a spectrophotometer (Titertek Multiskan, MCC Flow Labs. Inc, Finland) at 492 nm. The cutoff points were expressed by means of optical density (OD) of three negative sera included in each plate plus three standard deviations, and these ODs were used to signify the lowest level at which specific IgM was considered to be present, i.e. a positive result.
Microscopic agglutination test

The microscopic agglutination test (MAT) was performed as described by Sulzer and Jones24. Live cultures of strains of the following 20 serovars:
australis, autumnalis, bataviae, butembo, canicola, castellonis, celledoni, copenhageni, cynopteri, djasiman,

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1994; 6: No 3 Table 1. Comparison ELISA for serodiagnosis MAT Pos. Patients* Controls 82 8 Neg. 7 147 between MAT and IgM-PKof human leptospirosis IgM-PK-ELBA Pos. 80 4
1511155

grippotyphosa, hebdomadis, javanica, Panama, pomona, tarassovi and wolffi were

icterohaemorrhagiae, pyrogenes, shermani,

used as antigen; these serovars represent all the serogroups known to be prevalent in Stio Paula, Brazil. Agglutination at a serum dilution of 1 : 200 was considered positive.

Neg. 9 151
= 97.4%

Toral 89 155

Gel electrophoresis and immunoblotting Antigenic extracts were subjected to sodium dodecylsulphate polyacrylamide gel electrophoresis (SDSPAGE) and immunostained with serum samples as described in Ribeiro et al . I3.
Statistical analysis

Sensitivity
Specificity *Collections

82189 = 92.1% 147/l% = 94.8%

1 and 2

80189 = 89.9%

Table 2. Results of the MAT and the IgM-PK-ELBA in the first serum samples collected from patients with leptospirosis* MAT Positive Positive Negative Total
*Collections McNemar

The data obtained from the MAT and IgM-PK-ELISA were analysed using a McNemars test as described by Fleiss25.
Results Immunoblotting of the PK-treated antigen

IgM-PK-ELISA Negative 3 39 42 Total 26 63 89

23 24 47
1 and 2 x2 = 14.81; kO.01.

Our data from the immunoblot analysis indicated that DNase and RNase treatment after the PK digestion was important to reduce the background, and the marked reaction of serum samples of the control group with the 4@-42 kDa band (Figure 1).
Reactivity of sera in MAT and IgM-PK-ELISA

Table 1 presents the results obtained with serum samples from 89 leptospirosis patients and 155 control sera, analysed by MAT and IgM-PK-ELISA and expressed as a positive or negative diagnosis.

IgM-PK-ELISA results did not differ from MAT results when paired sera were assayed. However, IgMPK-ELISA differed statistically (x2 > 3.77) from MAT on the positivity of the first sample (Table 2) through which 38% (24 out of 63) of the patients with leptospirosis had the infection detected earlier than by MAT. From the nine leptospirosis patients who had IgMPK-ELISA-negative results (see Table l), four were positive with the MAT for the serovar austrafis and the other five had the infection confirmed only on the basis of clinical and epidemiological data. Three cases had their first sample positive for leptospira antibodies only by MAT (see Table 2). However, their subsequent sera became positive in the IgM-PK-ELISA. Three control sera were positive by both procedures.
Discussion

Figure 1. lmmunoblot of the PK-digested preparations of Leptospira serovar copenhageni strain M20 before (I) and after (21 DNase and RNase treatment for the detection of the IgM antibodies with the following human sera: leptospirosis patients, lanes a and g; healthy blood donors, lanes b, c and d; patients with syphilis, lanes e and f. Numbers on the left indicate molecular mass markers (kDa).

MAT is the current WHO standard reference test for serology. The cumbersome nature of the MAT precludes its use in most clinical settings due to the need to use live cultures of virulent leptospires, the use of microscope endpoint determinations and of its hazardous potential to laboratory staff. The biggest drawback is the insensitivity of the test. High antibody titres are usually not detected before the second week after the onset of the clinical symptoms, the maximum being reached during the third week7. To confirm the clinical diagnosis it is necessary to analyse a second serum sample taken a few days later, to demonstrate a significant rise of the antibody titre. Administration of

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antibiotics, as usually practised, results in the development of low antibody titres. Nevertheless, these very often observed low antibody titres can also be the result of a past infection. These difficulties have led to the development of alternative tests for the diagnosis of leptospirosis in recent years. In this study, the diagnostic potential of the IgM-PK-ELISA was compared with the MAT. We have used PK antigen prepared from strain M20 serovar copenhageni and we have employed it to detect specific antibodies in the sera of leptospirosis patients in S5o Paulo, Brazil, infected by various serovars/serogroups. This leptospiral treatment yielded a purified fraction recognized by IgM antibodies of leptospirosis patients. Immunoblot analysis (see Figure 1) confirmed the purity of the PK antigen. The overall results of the MAT are comparable to those of the IgM-PK-ELISA. However, the IgM-PKELISA was capable of detecting antibody activity in 38% of acute-phase sera which were negative by the MAT. It was observed that the sensitivity for serogroup australis was low. This fact was already reported by Cinco et al.y, who suggested that strains of uustralis should be used in immunoenzymatic assay in order to ensure the detection of antileptospira antibodies more effectively. Blackmore et al. detected agglutinating antibodies up to 20 vr after infection, with a marked variation occurring -in the maximum titres between individuals. These authors indicated that it is therefore not usually possible from a single MAT result to estimate retrospectively the time at which infection occurred. With the IgM-PK-ELISA, we also found that antibody activity persists in serum long after seroconversion. Serial sets of sera obtained from patients with Weils syndrome and followed up for 4-8 months had significant antibody activity detected by the IgM-PK-ELISA from the fifth day of illness, which remained up to the eighth month when its reactivity is usually decreased fourfold (data not shown). Nevertheless, the observation that 52.4% of patients who seroconverted in MAT had an elevated activity in the IgM-PK-ELISA (OD 2 0.800) indicates a strong initial antibody response to infection and suggests that the magnitude of this response in a single serum sample may be indicative of recent infection (data not shown). This is important in cases of fatal outcomeZh when it is not possible to obtain subsequent serum samples. We tested a sample from an individual whose death was attributed to leptospirosis only on the basis of clinical and epidemiological data. The MAT titre was negative but the IgM-PK-ELISA detected an OD of 1.091, indicative of recent leptospiral infection (data not shown). Sera from the control group showed a positive reaction rate of 5.2% and 2.6% using respectively the MAT and the IgM-PK-ELISA. However, as leptospirosis is widespread in Brazil it is possible that some of these reactions were due to subclinical recent or past infection with leptospires.

In conclusion, the IgM-PK-ELISA for the detection of antileptospira IgM is an easy procedure and achieves adequate sensitivity, specificity and reproducibility. The use of a single test serum dilution can discriminate between the presence or absence of an antibody response which enables the testing of 36 test samples together with the positive and negative standards on each microplate. Results can be read visually or automatically. The IgM-PK-ELISA is especially suitable for laboratories that do not have facilities for continuous culture of leptospira since its desiccated antigen is stable for at least 18 months at room temperature. The broad spectrum of serovars detected by the IgM-PK-ELISA could enable general clinical laboratories to perform reliable screening tests for leptospirosis, including atypical and milder forms such as influenza-like leptospirosis. which frequently escape diagnosis and remain unnoticed as fevers of unknown origin. Acknowledgements The authors are grateful to Prof Thales De Brito and Dra Tiyo Sakurai for their suggestions in the preparation of this manuscript. References 1 Sakata EE, Yasuda PH, Romero EC. Silva MV, Lomar AV. Serovarsof Leptospiru interrogans isolated from human leptospirosis in SBo Paulo. Brazil. Rev lnst Med Trop SHo Paul0 1992; 34: 217-21 2 Faine S (ed.). Guidelines for the Control of Leptospirosis. World Health Organization. Offset Publication No. 67, Geneva. 1982 3 Adler B, Faine S. The susceptibility of mice treated with cyclophosphamide to lethal infection with Leptospira interrogans serovar pomona. In,fect Immun
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