Vol. 28, No. 6

JOURNAL OF CLINICAL MICROBIOLOGY, June 1990, p. 1249-1253 0095-1137/90/061249-05$02.00/0 Copyright © 1990, American Society for Microbiology

Enzyme-Linked Immunosorbent Assay for Immunoglobulin M Specific Antibody for the Diagnosis of Melioidosis MONGKOL KUNAKORN,1* PAITOON BOONMA,2 KALAYANEE KHUPULSUP,1 AND BENCHA PETCHCLAI1 Clinical Immunology Laboratory, Department of Pathology, Faculty of Medicine-Ramathibodi Hospital, Mahidol University, Bangkok,' and Department of Internal Medicine, Srinagarind Hospital, Faculty of Medicine, Khonkaen University, Khonkaen,2 Thailand Received 22 November 1989/Accepted 20 March 1990

Indirect hemagglutination (IHA) is commonly used for serodiagnosis of melioidosis. However, in endemic high background titers in normal populations and occasional low titers in patients with septicemic melioidosis prompted a search for a more sensitive and more specific method of serodiagnosis. An indirect fluorescent-antibody test for immunoglobulin M (IgM) specific antibody to Pseudomonas pseudomallei was more sensitive and more specific, but fluorescence microscopes are rarely available in the endemic areas. An enzyme-linked immunosorbent assay (ELISA) for IgM antibody is an attractive alternative. An indirect ELISA for IgM antibody (IgM ELISA) and an IgM antibody capture ELISA for melioidosis were developed. Both tests, together with IHA, were evaluated for 153 serum specimens from blood donors and 16 serum specimens from 16 melioidosis patients. It was found that IHA, the IgM ELISA, and the IgM antibody capture ELISA had sensitivities of 88, 88, and 75%, respectively, with specificities of 97.4, 92.2, and 91.5%, respectively. When IHA was combined with IgM ELISA, a sensitivity of 100% and a specificity of 95.4% were obtained. The IgM ELISA and IHA should be used in combination for serodiagnosis of melioidosis. areas,

by the isolation of P. pseudomallei from hemoculture for 11 patients, sputum cultures for 3 patients, and pus from livers for 2 patients. Normal control serum specimens were collected from 153 blood donors. IHA test for melioidosis. The IHA test for melioidosis was performed as described previously (1, 10, 13). In brief, the supernatant from a 2-week-old protein-free broth culture of a local strain of P. pseudomallei was used to sensitize glutaraldehyde-preserved sheep erythrocytes (SRBCs). The optimal dilution of the antigen was determined by checkerboard titration with positive and negative sera. SRBCs were sensitized by incubating the optimal antigen dilution with SRBCs in 0.15 M phosphate-buffered saline (pH 7.2) at 37°C for 1 h, the excess antigen was removed by washing three times with phosphate-buffered saline, and the sensitized SRBCs were stored as a 0.5% suspension in phosphatebuffered saline diluent. Serum specimens were inactivated at 56°C for 30 min and absorbed with nonsensitized SRBCs to remove heterophile antibody. The treated serum was serially diluted twofold (1:20 to 1:2,560) in phosphate-buffered saline, sensitized SRBCs were added, and the hemagglutination results were read 1 h later. IgM ELISA. Soluble sonicated antigen was prepared as described by Boctor et al. (6) from a strain of P. pseudomallei isolated from a patient with melioidosis admitted to Ramathibodi Hospital. The organism was identified by colonial morphology and biochemical reactions (9). After incubation for 24 h at 37°C on blood agar, the confluent surface growth was harvested and made into a 50% (vol/vol) cell suspension in sterile water. The suspension was autoclaved for 15 min and sonicated at 20 kHz and 125 W for 15 min. The disrupted cells were removed by centrifugation at 20,000 x g for 10 min, and the supernatant was dispensed into 0.5-ml aliquots and stored at -20°C. The antigen remained stable at 4°C for more than 3 months. Microdilution strips with high binding capacities (Microwell U16; Nunc, Roskilde, Denmark) were used. The optimal dilutions of antigen and of anti-human IgM-horseradish

Acute septicemic melioidosis is a serious bacterial infection. Due to its high fatality rate and an improved prognosis when suitable antibiotics are administered, an early diagnosis on admission is critical (7). Early diagnosis depends on clinical awareness and serodiagnosis because the demonstration and identification of Pseudomonas pseudomallei by culture take a minimum of 2 days (7). Presently the indirect hemagglutination test (IHA) (1, 10) is the simplest and most widely used serodiagnostic assay (7, 13, 14). However, in endemic areas, interpretation of IHA is hampered by elevated titers in normal individuals which overlap with titers found in patients with acute melioidosis (3, 7, 13-15). In addition, IHA is sometimes negative in patients with acute septicemic melioidosis (3, 7, 13-15), for whom a sensitive test is urgently needed. The key to successful serodiagnosis is a sensitive method for detecting the antibody that appears early in acute infection and which is distinct qualitatively and quantitatively from antibodies in individuals with subclinical and past infections. The test for specific immunoglobulin M (IgM) antibody by indirect fluorescent-antibody assay (IgM IFA) developed by Ashdown is more specific and more sensitive for acute melioidosis (2, 3, 13) but requires a fluorescence microscope, which is not readily available in the endemic areas. A test for IgM specific antibody to P. pseudomallei by indirect enzyme-linked immunosorbent assay (IgM ELISA) was recently reported (4). We have developed a different IgM ELISA and an IgM antibody capture ELISA (MAC ELISA), aiming for improved specificity and sensitivity. MATERIALS AND METHODS Serum specimens. Serum specimens from patients with melioidosis were collected from 16 patients admitted to Srinagarind Hospital, Khonkaen, and to Ramathibodi Hospital, Bangkok, Thailand. Ail of the patients had clinical pictures compatible with melioidosis, which was confirmed *

Corresponding author. 1249

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peroxidase (HRP) conjugate were determined by a checkerboard titration with a pair of positive and negative control sera.

To each well, 0.1 ml of optimally diluted antigen (5 ,ug of protein per ml) in bicarbonate buffer (pH 9.5) was added, and the wells were incubated at 4°C overnight and then washed three times with Tris-buffered saline (TBS) (pH 7.4) containing 0.05% (vol/vol) Tween 20 (Sigma Chemical Co., St. Louis, Mo.). The free binding sites were blocked by adding to each well 125 p1 of 1% (wt/vol) bovine serum albumin (Sigma) in TBS, incubating at 37°C for 0.5 h, and washing. To each test well was added 0.1 ml of serum diluted 1:500 in TBS containing 15% (wt/vol) skim milk (Difco Laboratories, Detroit, Mich.)-20% (vol/vol) normal rabbit serum (Pel-Freez Biologicals, Rogers, Ark.}-0.05% (vol/vol) Tween 20. Positive and negative control sera were similarly added and the mixtures were incubated at 37°C for 1 h and washed. Rabbit F(ab')2 anti-human IgM-HRP conjugate (Dakopatt, Glostrap, Denmark) diluted 1:5,000 in TBS-bovine serum albumin-Tween was added (0.1 ml per well), and the wells were incubated for 1 h more and then washed. Freshly prepared 0.1% (wt/vol) orthophenylenediamine (Sigma)0.01% (vol/vol) H202 in citrate-phosphate buffer (pH 5.0) substrate solution was added (0.1 ml per well). After incubation in the dark at room temperature for 10 min, the reaction was stopped by adding 0.05 ml of 4 N H2SO4, and the optical density (OD) at 492 nm was measured with a Titertek Multiskan microplate reader (Flow Laboratories Inc., McLean, Va.). The ODs of the specimens were divided by the OD of a negative serum pool, and the results were expressed as positive to negative ratios (PIN) (16). The negative serum pool was a pool of serum specimens from 20 donors selected from serum specimens of 153 donors that gave the lowest ODs by the IgM ELISA. MAC ELISA. In principle, in the MAC ELISA (16), anti-human IgM already coated to the microdilution plate will capture IgM which in turn captures antigen of P. pseudomallei conjugated to HRP, and the capture is detectable by a color reaction. P. pseudomallei antigen was prepared as described above in the indirect ELISA. The antigen was conjugated to HRP type V (Sigma) by the periodate technique described by Nakane and Kawaoi (17). A 1-ml volume of 0.4% (wt/vol) HRP was mixed with 0.2 ml of freshly prepared 2.1% (wt/vol) NaIO4 for 20 min and dialyzed in 0.001 M sodium acetate buffer (pH 4.4) at 4°C overnight. A 1-ml volume of antigen (2 mg of protein per ml) was dialyzed in 0.01 M carbonate buffer (pH 9.5) at 4°C overnight, mixed with dialyzed HRP solution and 0.02 ml of 0.2 M carbonate buffer (pH 9.5), and stirred for 2 h, and then 0.1 ml of 0.38% (wt/vol) NaBH4 was added. The mixture was kept at 4°C for 2 h and centrifuged at 3,000 x g for 15 min. The supernatant was made to 2% (wt/vol) bovine serum albumin solution and stored at -20°C. The conjugated antigen remained stable at 4°C for 3 months. Optimal dilutions of anti-human IgM and antigen-HRP conjugate were determined by a checkerboard titration with a pair of positive and negative sera. Rabbit F(ab')2 antihuman IgM (Dakopatt) diluted 1:1,000 in bicarbonate buffer (pH 9.5) was added in 0.1-ml volumes to each well of micro-dilution strips, which were then incubated at 4°C overnight and washed. Test serum diluted 1:100 in TBSbovine serum albumin-Tween containing 10% (vol/vol) normal rabbit serum was added (0.1 ml per well), and the wells were incubated at 37°C for 1 h and washed. Antigen-HRP conjugate diluted 1:1,000 in the same diluent was added (0.1

TABLE 1. ROC analysis of IgM ELISA Cutoff PIN

>1 >2 >3 >4 >5 >6 >7

No. of specimens

% False-

%

True-positive

False-positive

Sensitivitya

positive'

15 14 14 14

90 44 19 12 6 5 2

94 88 88 88 69 63 50

59 29 12 7.8 4.0 3.0 1.3

il 0

8

a Positive cases; 16 melioidosis patients. Negative cases; 153 blood donors. Percent specificity false-positive.

=

100

percent

-

ml per well), and the mixture was incubated at 37°C for 30 min and washed. Orthophenylenediamine substrate solution was added (0.1 ml per well), the mixture was incubated in the dark for 10 min, and the reaction was stopped with 50 pil of 4 N H2SO4. The OD was measured at 490 nm, and then the PIN ratio was calculated. Strongly positive, weakly positive, and negative controls were run together in each set of assays. ROC analysis. The relative operating characteristic (ROC) curve is made by plotting sensitivity rates against falsepositive rates at various cutoff points (5). The ROC analysis was used to determine the appropriate cutoff point of each test and to evaluate and compare the performances of the tests. The areas under ROC curves were calculated by the nonparametric Wilcoxon statistic method (11). Statistical analysis of the area was determined by z score (12).

RESULTS The cutoff values were based on results for serum specimens from 16 patients with confirmed melioidosis and from 153 blood donors. The cutoff values determined by ROC analysis for the IgM ELISA, the MAC ELISA, and IHA are shown in Tables 1 to 3, respectively. The IgM ELISA showed the best cutoff value at PIN > 4, with a sensitivity of 88% and a specificity of 92.2%. The MAC ELISA showed the best cutoff value at PIN > 2, with a sensitivity of 75% and a specificity of 91.5%. IHA had the best cutoff titer at >1:160, with a sensitivity of 88% and a specificity of 97.4%. The overall performances of the IgM ELISA (area under ROC curve, 0.90; standard error, 0.06) and the MAC ELISA (area under ROC curve, 0.92; standard error, 0.04) were not significantly different (z score, 0.69; P = 0.3). The overall performance of IHA (area under ROC curve, 0.98; standard TABLE 2. ROC analysis of MAC ELISA Cutoff P/N

>0.5 >1.0 >1.5 >2.0 >2.5 >3.0 >3.5

No. of specimens False-positive True-positive

16 16 15 12 10 9 6

153 117 34 13 4 3 2

%

% False-

Sensitivity'

positive'

100 100 94 75 63 56 38

100 76 22 8.5 2.6 1.9 1.3

a Positive cases; 16 melioidosis patients. bNegative cases; 153 blood donors. Percent specificity = 100 false-positive.

-

percent

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MAC-ELISA

TABLE 3. ROC analysis of IHA No. of specimens False-positive

Cutoff titer

True-positive

>20 >40 >80 >160 >320 >640

16 16 16 14 il 7

88 47 18 4 1

O

% Sensitivitya

% False-

100 100 100 88 69 44

56 31 12 2.6 0.6

positive"

O

C

a Positive cases; 16 melioidosis patients. bNegative cases; 153 blood donors. Percent specificity = 100 - percent

false-positive.

error, 0.01) was better than that of the MAC ELISA (z score, 1.69; P < 0.05) but not significantly better than that of the IgM ELISA (z score, 1.44; P = 0.07). Parallel tests of IHA and the IgM ELISA combined were aimed at increasing the sensitivity (8), while the possible loss in specificity of such a combination was reduced by using the IgM ELISA at a higher cutoff PIN ratio. The best results were obtained by the combination of IHA at a cutoff titer of >1:160 and IgM ELISA at a cutoff PIN of >5, achieving a sensitivity of 100% and a specificity of 95.4% (Table 4). The ROC curves of the IgM ELISA, the MAC ELISA, IHA, and the IgM ELISA combined with IHA are shown in Fig. 1. Serum specimens from four patients showed interesting results. Serum specimens from two patients were negative by the IgM ELISA but had elevated IHA titers of 320 and 640 (Fig. 2). One specimen was from a patient with chronic localized melioidosis from whom serum was collected more than 1 month after admission, and the other specimen was from a patient with septicemic melioidosis from whom serum was collected 3 months after the onset of fever. The results were reversed with early serum specimens from two other patients with acute septicemic melioidosis, for which IHA titers were negative at the cutoff titer of 160 but both of which were positive by ELISA (Fig. 2). Both were also positive by the MAC ELISA. DISCUSSION Serodiagnosis of melioidosis can be very demanding, but once these demands are met the results are highly rewarding. This is a rare situation in which serodiagnosis is the key to the life or death of a patient. The sensitivity and specificity of the test used must be close to 100% for sera from acute septicemic patients, who frequently die within 48 h after TABLE 4. ROC analysis of IgM ELISA in combination with IHA at a cutoff titer of >1:160 Cutoff

No. of specimens False-positive

P/N

True-positive

>4.0 >4.5 >5.0 >5.5 >6.0 >6.5 >7.0

16 16 16 16 15 15 15

14 10 8 8 7 7 5

%

% False-

Sensitivitya

positive"

100 100 100 100 94 94 94

9.2 6.5 5.2 5.2 4.6 4.6 3.2

a Positive cases; 16 melioidosis patients. bNegative cases; 153 blood donors. Percent specificity = 100 - percent false-positive.

10 20 30 40 50 60 70 80 90

100

% FALSE POSITIVE FIG. 1. Comparison of the ROC curves of various melioidosis serodiagnosis tests. The selected cutoff points of the tests are shown (M, A, *, and *).

admission (7). Many are immunocompromised individuals living in an endemic area in which the population has high background antibody titers. The test must be rapid, simple, and readily available in most hospitals, some of which have limited facilities and personnel. The development of such a test has been focused exclusively on the detection of IgM specific antibody by IFA (2, 3), and recently by ELISA (4), with considerable success. The present contribution was concerned with the IgM ELISA and the MAC ELISA. The overall performance of each ELISA was not better than that of IHA. The patients in this study were not confined to acute septicemic melioidosis, for which the IgM ELISA and the MAC ELISA are expected to be more sensitive and more specific than IHA. A recent report (4) of an IgM ELISA using a similar antigen showed excellent sensitivity in active melioidosis without overlapping chronic and subclinical infections. The cutoff value in that study was based on serum dilutions (1:5,120), whereas in the present study it was based on the PIN ratio at a single serum dilution (1:500). A more concentrated serum was used in order to increase the sensitivity while sacrificing some specificity. This study used the identical method for antigen preparation. On the basis of the excellent sensitivity in active disease as reported previously (4), the negative results for some patients in the present study are presumably due to the late collection of sera. This is apparent in the cases of two patients, one recovering from acute septicemic melioidosis and the other from chronic localized melioidosis, for whom the IgM ELISA was negative for serum specimens collected more than 1 month after admission but whose IHA titers were elevated at 1:320 and 1:640 (Fig. 2). In contrast, positive IgM ELISA and MAC ELISA results for two patients with low IHA titers of 1:160 (Fig. 1) confirmed the sensitivity of the ELISA in acute septicemic melioidosis, supporting the previous report on the IgM ELISA (4). It is concluded that the ELISA is insensitive for sera from chronic melioidosis but sensitive for sera from acute septicemic melioidosis. Additional studies of acute septicemic melioidosis will establish the benefits of the

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laboratory and distributed to all hospitals. This ensures consistent, well-controlled quality as well as convenience for

ELISA P/N 251

the users. Both ELISAs can be read with the naked eye when necessary. This is a real advantage over the IgM IFA, which requires a fluorescence microscope, which is rarely available in the endemic areas. Ashdown et al. (4) pointed out that the IFA is subjective, requiring frequent subculture and animal passage of P. pseudomallei to maintain consistent quality. Compared with the IgM IFA, the IgM ELISA is much more applicable in the endemic areas. Previous reports (4) supported by the present one indicated that the IgM ELISA for melioidosis is sensitive and specific for acute septicemic melioidosis. The present study recommends the combined use of IHA and the IgM ELISA for the most sensitive and most specific serodiagnosis of melioidosis. The IgM ELISA identifies the acute stage of disease, and IHA detects chronic as well as some of the acute melioidosis. IHA is also simple, rapid, and readily available. Clinical awareness and the combined use of IHA and the IgM ELISA are the keys to early diagnosis and a more successful treatment of acute septicemic melioidosis.

* MELIOIDOSIS SERA * BLOOD DONOR SERA a

13. a

12il-

a a

10-

9. a

6-

7.1

&

:

ACKNOWLEDGMENTS K. Nuntirut, Department of Internal Medicine, Srinagarind Hospital, kindly provided some sera from patients with melioidosis. W. Panbankret, Department of Microbiology, Faculty of Science, Mahidol University, permitted us to use the sonicator. The staff of Clinical Immunology Laboratory, Ramathibodi Hospital, provided excellent technical assistance when needed. LITERATURE CITED 1. Alexander, A. D., D. L. Huxsoll, A. R. Warner, V. Shepler, and A. Dorsey. 1970. Serological diagnosis of melioidosis with indirect hemagglutination and complement fixation test. Appl.

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Enzyme-linked immunosorbent assay for immunoglobulin M specific antibody for the diagnosis of melioidosis.

Indirect hemagglutination (IHA) is commonly used for serodiagnosis of melioidosis. However, in endemic areas, high background titers in normal populat...
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