World Journal of Microbiology and Biotechnoiogy 7, 29-36

Diagnosis of melioidosis by means of an ELISA detecting antibodies to Pseudornonas pseudomallei exotoxin. Preliminary assay evaluation

C.J. S m i t h , M . J o n e s , D . A . B . D a n c e , W . C h a o w a g u l

and N . J . W h i t e

An ELISA system for the detection of human antibodies to Pseudomonas pseudomallei exotoxin is described. Analysis of control sera and sera from patients with culturepositive melioidosis showed that both IgG and IgM antibodies to exotoxin were detectable in patients with melioidosis. This implies that the exotoxin is produced in vivo by P. pseudomallei, and may be involved in the pathogenesis of melioidosis. Furthermore, these rapid and simple assays may prove to be useful for the serodiagnosis of the disease.

C.J. Smith is with the Faculty of Science and Innovation, The North East Wales Institute, Deeside, Clwyd CH5 4BR, UK; D.A.B, Dance and N,J. White are with the Wellcome-Mahidol University-Oxford Tropical Medicine Research Programme, Faculty of Tropical Medicine, Mahidol University, 420/6 Rajvithi Road, Bangkok 10400, Thailand, and Nuffield Department of Clinical Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DU, UK; W. Chaowagul is with the Department of Medicine, Sappasitprasong Hospital, Ubon Ratchatani, Thailand. D.A.B. Dance's present address is: Department of Clinical Sciences, London School of Hygiene and Tropical Medicine, Keppel Street, London, WCIE 7HT, UK. C.J. Smith is the corresponding author.

© t991 Rapid Communications of Oxford Ltd.

Metioidosis, a disease of humans and animals caused by Pseudomonas pseudomallei, is endemic in South-East Asia and tropical Australia. The causative organism produces a lethal exotoxin during in vitro culture (Heckly 1964), but it is not known whether this occurs during natural infection or what role is played by the toxin in the pathogenesis of the disease. We recently isolated and partially purified the exotoxin from P. pseudomallei to yield a single band on electrophoresis (Ismail et aL 1987a). This material was used to prepare a monoclonal antibody, and also to develop sensitive ELISA systems capable of detecting the toxin and its corresponding antibody in mice (Ismail et al. 1987a; b). We now report the development of ELISA techniques for the detection of human antibodies to P. pseudomallei exotoxin, and the results of their application to sera from patients with melioidosis and normal controls.

Materials and M e t h o d s Serum Specimens The human sera used in this study came from two groups of individuals. 1. Melioidosis patients (23 samples from 8 patients). These samples were collected from patients with acute culture-positive melioidosis seen in Sappasitprasong Hospital, Ubon Ratchatani, Thailand, during a prospective clinical study of melioidosis (White & Dance 1988). Serum was separated, stored at --20°C, and transported frozen to the UK. Each sample was tested in Thailand for antibodies to P. pseudomallei by indirect haemagglutination (IliA) and immunofluorescent (IFAT) techniques. The methods used were similar to those previously described (Alexander et aL 1970; Ashdown 1981), except that three locally isolated strains of P. pseudomallei were used to produce antigen, and the sera were pre-treated with RF Absorbent (Behring Diagnostics, UK) before testing for IgM antibody. Brief clinical summaries for these patients are given in Table 1.

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C.J. Smith et al. Table 1. Clinical details of the melioidosis culture positive patients from Thailand. Patient

Age

Sex

Underlying disease

Mel 3

55

M

Chronic renal failure with calculi

Localized (pneumonia)

Mel 4

53

F

Chronic renal failure

Mel 13

37

M

Renal calculi

Septicaemic (with secondary pneumonia and liver abscesses) Septicaemic (with no localization)

Mel 18

21

F

Mel 19

61

F

Mel 21

21

M

Organophosphate poisoning Ventilated HbE trait Diabetic Cardiac arrest Ventilated None known

Met 27

46

M

Diabetic

Ubon 18

55

M

None known

Type of disease

Septicaemic (with pneumonia)

Outcome Recovered Lost to follow-up Recovered Died later

Recovered Possible relapse and death 6 months later Recovered Died later at home

Septicaem ic (with pneumonia)

Died

Septicaemic (pneumonia with pyopneumothorax) Septicaemic (with soft tissue infection of elbow and secondary pneumonia) Septicaemic (with liver abscess)

Recovered Well at follow-up Recovered Well at follow-up

Recovered Well at follow-up

2. Normal controls (16 samples from 8 individuals). These samples were collected from volunteers in the UK who had not lived in an area endemic for melioidosis. Anti-exotoxin E L I S A Plate preparation. Exotoxin was prepared from a clinical isolate of P. pseudomallei obtained from the Microbiology Department, Faculty of Medicine, University of Malaysia, Kuala Lumpur, as described previously (Ismail et al. 1987a). Polystyrene, flat-bottomed, 96-well microtitre plates (Dynatech) were coated with exotoxin (5/~g/ml in 0.1 M carbonate/bicarbonate binding buffer, p H 9.6, 375 #t/well). After overnight incubation at 4°C the plates were washed three times with binding buffer and post-coated with bovine serum albumin (BSA: 1% w/v in binding buffer, 200 #t/well). The plates were then incubated at room temperature for 30 min, emptied and dried at 37°C for 2 h. Finally, the plates were sealed with tape and stored at 4°C. IgG assay system. The ELISA was performed according to a modification of the method of Voller et al. (1976). In preliminary studies using undiluted serum samples, there were considerable non-specific reactions, but at dilutions of serum of 1 : 100 or greater these were eliminated. A similar observation has been reported for an ELISA system to detect antibodies to Brucella species (Saz et al. 1987). Consequently, all assays were performed on diluted sera. Briefly, serial dilutions of serum samples (1:100 to 1:12800) were prepared (dilution buffer 0.01 M phosphate buffered saline [PBS], 0.5% Tween 80, 1% BSA, p H 7.8). Diluted samples (100 #1) were added to previously prepared plates and incubated with

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Metioidosis detection by E L I S A constant shaking for 1 h at room temperature. The wells were then washed five times with wash buffer (0.01 M PBS, 0.5% Tween 80, pH 7.8) and blot-dried. Peroxidase-labelled rabbit anti-human I g G (ICN Immuno-Biologicals, UK) diluted 1 : 1000 in dilution buffer (100 pl/well) was added. The plates were incubated for 1 h with constant shaking, then washed five times with wash buffer and blot dried. The enzyme substrate (100#t/well) was then added, the plates were incubated with constant shaking for 10 min after which the A405 was measured using a Titertek Multiscan (Flow Laboratories, UK). The enzyme substrate used was 2,2'-azino-bis-(3-ethyl benzthiazoline sulphonic acid) [ABTS], 0.6 mg/ml, in citrate buffer (pH 4.0) containing 0.015% hydrogen peroxide. Blank wells were treated in the same way as the test wells except that the dilution buffer replaced the serum samples. The corrected optical density for each serum dilution was calculated by subtracting the mean blank optical density from the measured optical density of the test samples.

IgM assay system. This was essentially the same as the I g G assay system except that peroxidase-labelled goat anti-human IgM (Sigma) was used as the second antibody, and the optical density was measured after 30 min incubation. Determination of Serum Titres Serum titre was determined using the system of cut-off optical densities (Araj et al. 1986). The titre of the melioidosis patients' serum was taken as the highest dilution giving a corrected absorbance more than two standard deviations greater than the mean of corrected optical densities of control sera at that dilution. Statistical A natysis Titres of control and melioidosis samples were compared using the Mann-Whitney U test (Mann & Whitney 1947).

Results Anti-exotoxin IgG E L I S A Titration curves of optical density versus reciprocal titre were prepared for each sample. Figure l(a) shows examples of two typical titration curves obtained. A

a)

b)

2.0

2.0

E tO o r-

E ttO 0

1.5-

o o ¢-

1.0-

1.5

1.0 ~"

.O 0

0




80to •~

60-

I ID

n

O

n

40-

40-

0

20-

20-

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