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Evaluation of a brucelle enzyme immunoassay test (ELISA) in comparison with bacteriological culture and agglutination
Journalof Infection(1998) 36, 197-201
Evaluation of a Brucella Enzyme Immunoassay Test (ELISA) in Comparison with Bacteriological Culture and Agglutination M. O. Gad EI-Rab .1 and A. M. KambaF 1Immunology and 2Bacteriology Units, Department of Pathology, College of Medicine, King Khalid University Hospital, King Saud University, P.O. Box 2925, Riyadh 11461, Saudi Arabia An ELISA test for IgG and IgM antibrucella antibodies was found to be effective in diagnosis of human brucellosis. Assays for IgG and IgM in 30 culture-positive cases gave significant ELISA values. By the standard agglutination test, 10% of these cases gave readings less than 1:160. These are considered insignificant, taking 1:160 as the accepted cut-off value. Moreover, in an extra 135 samples from suspected bruceUa cases, where only serology was requested (77.6% of all cases), 7.4% were found to have IgM brucella antibodies by ELISA. In all of these, the corresponding agglutination titres were less than 1:80 and hence reported as insignificant. We report the detection of IgG and IgM antibodies in samples from patients with both acute and chronic disease. In few patients with acute disease, only IgM was detected. These findings are discussed in comparison with earlier studies. Finally, the ELISAtest, in addition to measuring antibody classes directly, also detects incomplete antibodies. By this, it can efficiently replace the 2 mercaptoethanol test (2ME) and the Coomb's antihuman-globulin test. This saves considerable laboratory cost and time.
Introduction Since the recognition of brucellosis as a major health problem in Saudi Arabia in 1983,1 several studies were undertaken to investigate the clinical and epidemiological aspects of the disease. 2-1° Few studies, however, were directed to serological investigation. So far, in our laboratory, diagnosis relies mainly on bacteriological blood cultures and agglutination tests. Because blood cultures may take weeks and are often unsuccessful in isolating the causative organism, laboratory diagnosis is more frequently made serologically. 11-14 The standard agglutination test (SAT), which is the most commonly used test, is less than ideal. It is labour-intensive, requires large amounts of reagents, takes 2 days and can be falsely negative due to the presence of blocking antibodies, is' i6 Moreover, all immunoglobulin classes (IgG, IgA, IgM) give rise to direct agglutination, 17 therefore the relative proportion of individual immunoglobulins which contribute to a titre may be obscure. Determination of the antibody class seems to be important for diagnosis and management, as we encounter patients in the acute, subacute or chronic stage of the disease, is Up to the present, the estimation of the antibody class is accomplished by testing sera before and after treatment with 2 mercaptoethanol (2ME). 19 Moreover, when blocking or
* Address correspondence to: M. O. Gad E1-Rab. Accepted for publication 21 July 1997. 0163~t453/98/020197+05 $12.00/0
subagglutinating antibodies are suspected, 2° the antih u m a n globulin (Coombs') test is performed. This is not without extra laboratory cost and time. Results of several previous studies suggested that specific antibody levels (IgG, IgA, IgM) could be more simply determined with the enzyme-linked immunosorbent assay (ELISA).21'22 This, being a primary assay, has the advantage over other methods that it can detect any antibody with the ability to combine with the surface antigen of brucella organism. It also avoids all the distractions and complications of incomplete or blocking antibody. The technical difficulties we frequently encounter with agglutination test in serological diagnosis of brucellosis, especially subacute and chronic cases, prompted the present study. We present the evaluation of brucella ELISA test in comparison with conventional tests in a region where brucellosis is prevalent.
Materials and Methods One hundred and seventy-four consecutive serum samples, from patients suspected of having brucellosis were included in this study. In thirty-nine of these patients, blood cultures and serum for serology were requested at the same time. In the remaining 13 5 samples only serology was requested. In addition, one hundred and fifty serum samples from febrile patients not suspected of brucellosis, as well as © 1998 The British InfectionSociety
198
M . O . Gad EI-Rab and A. M. Kambal
healthy individuals, were included as controls. These controls are from the same social class in society, and are thus equally exposed to animal contact and milkdrinking. In all samples microagglutination, standard agglutination test (SAT) and ELISA were performed. Sera from 10 patients were tested more than once, because follow-up samples were available.
incubation period (7 days), they were further incubated at 37 °C for another 5 weeks. Brucella organisms were identified by their coccobacilli Gram-negative appearance, requirement for CQ, production of H20, urease production and reaction with monospecific Brucella abortus or Brucella meltensis sera.
Statistics Agglutination tests Stained brucella suspensions for microagglutination and standard agglutination tests were obtained from Murex Diagnostic Limited (Central Road, Temple Hill, Dartford, England DA1 5LR, U.K.).
The StatPac Gold Statistical Analysis Package was used to calculate correlation coefficient between the standard agglutination test titre and ELISA, IgM and IgG values. Bivariate (scatter) plots were also drawn.
Results The microagglutination method This was performed in rigid u-bottoned microtitre plates exactly as described by Bettelheim et al. (1983). 23
The standard agglutination test (SAT) This was done on doubling dilutions from 1:20 to 1:20 840. The methods used were those described in the instruction sheets supplied.
Enzyme immune assay (ELISA) ELISA was performed using reagents supplied by ALFA BIOTECH (Via Castagnetta, 7, 00040 Promezia, Rome, Italy). The ELISA microplate testing procedure was performed exactly as described in the procedures manual. Automatic washing and absorption reading were done using the Diagnostic Pasteur LP35 multiwash and LP400 reader, respectively.
Bacteriological blood culture For bacteriological diagnosis of brucellosis, paired 10 ml blood samples from adults and 5 ml blood samples from children were collected. The culture was done in a Bactec 9240 automatic blood culture machine (Becton-Dickinson, 7 Loventon Circle Sparks, Maryland, U.S.A.) following the manufacturer's instructions. The blood culture bottles were subcultured on blood agar plates when they signalled positive, or at day 3, day 5 and day 7 of incubation. If the bottles are negative after the recommended
Out of thirty-nine cases where both blood culture and serology were requested, Brucella species were isolated from thirty specimens. The corresponding agglutination titres and IgG, fgM ELISA values appear in Table L Three samples were tested three times and seven samples twice. Samples giving similar readings were grouped together whenever possible. All culture positive cases gave significant agglutination and ELISA values except for samples 5, 7, 23, 24, 26, 27 and 28. Samples 23, 24 and 28 showed insignificant standard agglutination titre of 1:40. These are usually reported negative. However, the corresponding IgG, IgM ELISA values were significantly positive. Samples 5 and 7 gave significant agglutination titres and IgG ELISA, but borderline IgM. Samples 26 and 27 showed a similar pattern with borderline IgM reading. The relationship between standard agglutination titres and IgM ELISA showed a significant positive correlation between the two parameters (r = 0.494, P
Brucella Immunoassay versus Bacteriological Culture
199
Table 1. Agglutination titres and ELISAvalues in 30 patients with culture-positive brucellosis. No. of tests performed
Sample serial no.
Samples tested 3 times (no. = 3) Samples tested 2 times (no.- 7)
Brucella* culture
ELISA~
Agglutination Micro
Tube
IgG
IgM
1,2,3
+
1:20480
1:1280
2.642
2.600
5,7 4,6,8
+ +
1:640 .1:640-1:20480
1:160 1:160-1:10240
2.263 Neg.-1.890
0.635 0.917-2.563
23,24,28 26,27
+ +
11-22,26,9,30
+
1:320 1:1280 1:320 1:20480
1:40 1:320 1:2560 1:160-1:10240
O.559-2.904 1:329-2.447 1.111-2.883
0.769-1.804 0.629 0.644-2.902
Samples tested once (no.= 20)
* + = positive blood culture. "~ELISAcut off-valuesIgG = < O.627, IgM = <0.627.
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0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 IgG (ELISA) Figure 1. Correlation between standard agglutination titres and IgM
(ELISA) in brucellosis. IgG a n d IgM; (ii) 17 were positive for IgG only; (iii) 10 were positive for IgM only. The clinical presentations a n d serology values for the latter group with positive IgM appear in Table III. I n the 1 5 0 control samples, 109 were completely negative by both a g g l u t i n a t i o n a n d ELISA. The r e m a i n i n g 41 samples showed low insignificant m i c r o a g g l u t i n a t i o n titres of < 1 : 8 0 a n d reported as negative.
Discussion
A noticeable finding in this study is t h a t s i m u l t a n e o u s blood c u l t u r e a n d serology were requested i n only 22.4%
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0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 IgG (ELISA) Figure 2. Correlation between standard agglutination titres and IgG (ELISA) in brucellosis.
of cases, while in 77.6%, only serology was requested. It appears that blood culture is only ordered w h e n the patient presents with fever. This reflects the need for a satisfactory serological test for diagnosis of brucellosis, particulary in cases with atypical presentations. Out of 30 culture positive cases, three would have been reported as falsely negative due to a low s t a n d a r d a g g l u t i n a t i o n titre. I n these samples (nos. 23, 24 a n d 28), IgG a n d IgM brucella antibodies were positive by ELISA. This a m o u n t s to false-negative a g g l u t i n a t i o n results in 10% of culture-positive cases. It is w o r t h n o t i n g t h a t all culture-positive brucella samples were also positive by ELISA. Moreover, in n i n e suspected brucella cases
200
M. O. Gad EI-Rab and A. M. Kambal Table Ii. Agglutination titres and ELISA values in nine suspected brucella patients with negative blood culture. Sample no.
Agglutination titre
1 4 8 2,3,5,6,7,9
EUSA values*
Micro.
Tube
IgG
IgM
1:320 1:640 1:1280 1:1280-1:20480
1:80 1:80 1:160 1:160-1:1280
1.059 2.676 1.521 0.686-2.883
1.788 0.510 0.611 0.709-2.902
* ELISA cut off-values IgG = <0.627.
Table III. Clinical presentations in brucellosis patients with negative standard agglutination and positive ELISA IgM. Sample serial no.
37 44 65 77 108 127 125 133 140 168
Agglutination titre Micro. Tube (SAT) 1:640 1:320 1:640 1:160 1:320 1:1280 1:1280 1:640 1:160 1:160
1:80 1:40 1:80 1:40 1:40 1:80 1:80 1:80 1:40 1:40
ELISA values
Clinical presentation
IgG
IgM
NEG. NEG. NEG. NEG. NEG. NEG. NEG. NEG. NEG. NEG.
1,110 1.209 1.228 1.937 1.052 0.290 0.784 0.785 1.125 0.640
Joint and bone pain Backache Synovitis left knee Shoulder pain Arthritis Haematemesis, fever, H / 0 drinking raw milk H / 0 drinking raw milk Typhoid + persistent malaria
S A T = s t a n d a r d agglutination test; NEG. =negative.
which were culture-negative, two more would have been reported negative due to low agglutination titres of 1:40 and 1:80. Both of these were positive by ELISA. Furthermore, in 13 5 samples from patients suspected of having brucellosis, but for w h o m only serology was requested, 25 samples negative by the standard agglutination test were positive by F_LISA. Ten of these (7.4%) were brucella IgM antibodies positive and 17 were IgM and [gG positive. This discrepancy could be due to the high sensitivity of the ELISA test being a primary assay. Therefore, careful interpretation of the results will be required, as cross-reactions between brucella and other microbial antigens have been reported. 24 It has also been noted with ELISA tests that strong IgG responses with little or no IgM could be due to secondary rickettsial 2s or typhoid infections. 26 In this study, elevated IgM and IgG brucella antibodies were detected in most of the samples. This is in agreement with reports by Reddin et al, 19 and Sippel et tll. 27 who found elevated IgM and IgG in patients with acute brucellosis. Similar studies by Paratt et ill. 22 and Magee et al. 21 found IgM brucella antibodies alone in acute cases. These inconsistencies could be due to the fact that acute and chronic brucellosis m a y not appear as two distinct immunological entities in our patients as a result of delay in diagnosis or partial antibiotic treatment. In addition,
most of the patients are from rural areas and m a y have long-term illness before diagnosis. Thus, at presentation, they m a y have immunological patterns consistent with both acute and chronic disease. We also report a significant correlation between brucella IgM ELISA values and standard agglutination titres in culture-positive cases. No correlation was observed with IgG. In the control samples, where brucellosis was not suspected, 41 samples out of 150 showed a low microagglutination titre (< 1:80) suggesting that some chronic infections are subclinical. 2s Such presentations would be expected in our region with a high exposure rate of 3 5% and active disease in 2% of the populations. 4' 19 In conclusion, we found ELISA an effective method for diagnosis of brucellosis, particularly in subacute and chronic cases. This includes patients with negative standard agglutination test in presence of a compatible clinical history, as reported previously. 3° Moreover, the ELISA test, by measuring both IgM and IgG, helps in staging the disease.
Acknowledgement We wish to t h a n k Dr Awad Abd E1-Gafar for his critical comments. We also extend our thanks to Mr Mustafa Hussain and Mr A1-Waleed AlH a m a d for technical assistance.
Brucella Immunoassay versus Bacteriological Culture References 1 Kambal AM, Mahgoub ES, ]amioon GA, Chowdhury MNH. Brucellosis in Riyadh, Saudi Arabia: a microbiological and clinical study. Trans Roll Soc Yrop Med tt!tg 1983; 77: 8204. 2 A1 Eissa Y, Kambal A, A1 Nasser Met ca. Childhood Brucellosis: a study of 102 cases. Pediatr Infect Dis J 1990; 2: 749. 3 AI Freihi HM, A1 Mohaya SA, A1 Mohsen MF et ca. Brucellosis in Saudi Arabia: diverse manifestations of an important cause of prexial illness. Ann Saudi Med 1986; 6: 95-100. 4 A1Nasser A, A1Aska A, A1Balla Set ca. Epidemiology of Brucellosis in Saudi Arabia. Ann Saudi Med 1991; 2: 245. 5 AI Zeftawi NM, A11ssa M, Bekairi SI et ca. Epidemiology of Brucellosis among abattoir workers in Saudi Arabia. Ann Sau~ Med 1986; 6 (Suppl. 29): 29-31. 6 Arrighi HM. Brucellosis surveillnce in Saudi Arabia's Eastern Province. Ann Saudi Med 1986; 6 (Suppl.): 5-10. 7 Bilal N, Jamjoom G, Bobo R et al. Brucellosis in the Asir Region of Saudi Arabia. Saudi Med ] 1991; 12: 38-~tl. 8 Kiel F, Khan Y. Analysis of SOB consecutive positive serologic tests for Brucellosis in Saudi Arabia. ] Cfin Micro 1987; 25: 1284-1287. 9 Norton WL. Brucellosis and rheumatic syndromes in Saudi Arabia. Ann Rheum Dis 1984; 43: 810-815. 10 Smith K, Qadri SMH. Serious complications of Brucellosis in a major tertiary care hospital. Med Sci Rev 1991; 19: 311-312. 11 Stokes E] & Ridgway G. Clinical bacteriology (5th edit.) London: Edward Arnold, 19806; pp. 33-42, 205. 12 Kerr WR, Coghlan ]D, Payne MF & Robertson L. The laboratory diagnosis of chronic brucellosis. Lancet 1966b; ii: 1181. 13 Kerr WR, McCaughley W], Coghlan ]D et ca. Techniques and interpretations in the serological diagnosis of brucellosis in man. ] Med Microbiol 1968; 1: 181. 14 Macdonald A, Elmsfie WH. Serological investigations in suspected brucellosis. Lancet 1976; 1: 380-382. 15 Drutz D] & Graybill ]1/. Infectious diseases, brucellosis. In: Funderbeg HH, Sittes DP, Coldwell ]L, Wells IV (eds). Basic and clinical immunology. Los Angeles, California: Lanage Medical Publications, 180; p. 632. 16 Wicher K. In: Milgron F, Abeyounis C] & Kano K (eds). Principals of immunological diagnosis in medicine. Philadelphia: Lea & Febiger 1981: pp. 97-101.
201
17 Wilkinson PC. Immunoglobulin patterns of antibodies against Brucella in man and animals. ] Immunol 1966; 96: 457. 18 White RG. Immunoglobnlin profiles of chronic antibody response: discussion in relation to brucellosis infection. Postgrad Med J 1978; 54: 595-602. 19 Reddin JL, Anderson RK, Jenness R & Spink WW. The significance of 7S and macrogubulin Brucella agglutinins in human brucellosis. New Engl J Med 1965; 272-1263. 20 Spink WW. The nature of Brucellosis. Minneapolis, USA: University of Minnesot Press, 1986. 21 Magee JT. An enzyme-labelled immunosorbent assay for Brucella abortus antibodies. J Med Microbiol 1980; 13: 167-172. 22 Parrat D, Nielsen KH, White RG, Payne DJH. Radioimmunoassay of IgM, IgG and IgA brucella antibodies. Lancet 1977; 1:1075 1078. 23 Bettelheim KA, Maskill WJ, Pearce J. Comparison of standard tube and microagglutination techniques for determining Brucella antibodies. J Hygiene 1983: 90: 33-39. 24 Norman B, McCullough. Immune response to brucella. In: Rose MR, Frichman H (eds). Manual of clinical immunology (2nd edition). Washington: American Society for Microbiology, pp. 489-i95. 25 Murray ES, O'Connor JM, Gaon JA. Serologic studies of primary epidemic typhus and recrudescent typhus (Brill-Zinsser disease) II. Difference in immunoelectrophoretic patterns, response to 2mercaptoethanol and relationships 1 to 19S and 7S antibodies. ] Immunol 1965; 94: 734-740. 26 Chernoldavosotova E, Luxemburg KI, Starshinova V, Andreera N, German G. Study on the production of IgG, fgA-antibodies to somatic antigens of Scamonella typhi in human. CIin Exp Immunol 1969; 4: 407-421. 27 John E, Sippel N, Ayad E-M & Zohair F. Diagnosis of human brucellosis wRh ELISA. Lancet 1982; 19-21. 28 Henderson RJ, Hill DM. Subclinical Brucella infection in man. Bri IVied ] 1972; iii: I54-156. 29 A1 Balla SR, A1 Balla SR, A1 Aska Abdulkarim, Kambal AM, AI Hedaithy MA. Seasonal variations of culture positive brucellosis at a major teaching hospital. Ann Saudi Med 1994; 14: 12-15. 30 Carpenter JL, Transmont EC, Branche W. Failure of routine methods in the diagnosis of chronic brucellosis. South Med f 1979; 72: 90-92.
Evaluation of a Brucella Enzyme Immunoassay Test (ELISA) in Comparison with Bacteriological Culture and Agglutination M. O. Gad EI-Rab .1 and A. M. KambaF 1Immunology and 2Bacteriology Units, Department of Pathology, College of Medicine, King Khalid University Hospital, King Saud University, P.O. Box 2925, Riyadh 11461, Saudi Arabia An ELISA test for IgG and IgM antibrucella antibodies was found to be effective in diagnosis of human brucellosis. Assays for IgG and IgM in 30 culture-positive cases gave significant ELISA values. By the standard agglutination test, 10% of these cases gave readings less than 1:160. These are considered insignificant, taking 1:160 as the accepted cut-off value. Moreover, in an extra 135 samples from suspected bruceUa cases, where only serology was requested (77.6% of all cases), 7.4% were found to have IgM brucella antibodies by ELISA. In all of these, the corresponding agglutination titres were less than 1:80 and hence reported as insignificant. We report the detection of IgG and IgM antibodies in samples from patients with both acute and chronic disease. In few patients with acute disease, only IgM was detected. These findings are discussed in comparison with earlier studies. Finally, the ELISAtest, in addition to measuring antibody classes directly, also detects incomplete antibodies. By this, it can efficiently replace the 2 mercaptoethanol test (2ME) and the Coomb's antihuman-globulin test. This saves considerable laboratory cost and time.
Introduction Since the recognition of brucellosis as a major health problem in Saudi Arabia in 1983,1 several studies were undertaken to investigate the clinical and epidemiological aspects of the disease. 2-1° Few studies, however, were directed to serological investigation. So far, in our laboratory, diagnosis relies mainly on bacteriological blood cultures and agglutination tests. Because blood cultures may take weeks and are often unsuccessful in isolating the causative organism, laboratory diagnosis is more frequently made serologically. 11-14 The standard agglutination test (SAT), which is the most commonly used test, is less than ideal. It is labour-intensive, requires large amounts of reagents, takes 2 days and can be falsely negative due to the presence of blocking antibodies, is' i6 Moreover, all immunoglobulin classes (IgG, IgA, IgM) give rise to direct agglutination, 17 therefore the relative proportion of individual immunoglobulins which contribute to a titre may be obscure. Determination of the antibody class seems to be important for diagnosis and management, as we encounter patients in the acute, subacute or chronic stage of the disease, is Up to the present, the estimation of the antibody class is accomplished by testing sera before and after treatment with 2 mercaptoethanol (2ME). 19 Moreover, when blocking or
* Address correspondence to: M. O. Gad E1-Rab. Accepted for publication 21 July 1997. 0163~t453/98/020197+05 $12.00/0
subagglutinating antibodies are suspected, 2° the antih u m a n globulin (Coombs') test is performed. This is not without extra laboratory cost and time. Results of several previous studies suggested that specific antibody levels (IgG, IgA, IgM) could be more simply determined with the enzyme-linked immunosorbent assay (ELISA).21'22 This, being a primary assay, has the advantage over other methods that it can detect any antibody with the ability to combine with the surface antigen of brucella organism. It also avoids all the distractions and complications of incomplete or blocking antibody. The technical difficulties we frequently encounter with agglutination test in serological diagnosis of brucellosis, especially subacute and chronic cases, prompted the present study. We present the evaluation of brucella ELISA test in comparison with conventional tests in a region where brucellosis is prevalent.
Materials and Methods One hundred and seventy-four consecutive serum samples, from patients suspected of having brucellosis were included in this study. In thirty-nine of these patients, blood cultures and serum for serology were requested at the same time. In the remaining 13 5 samples only serology was requested. In addition, one hundred and fifty serum samples from febrile patients not suspected of brucellosis, as well as © 1998 The British InfectionSociety
198
M . O . Gad EI-Rab and A. M. Kambal
healthy individuals, were included as controls. These controls are from the same social class in society, and are thus equally exposed to animal contact and milkdrinking. In all samples microagglutination, standard agglutination test (SAT) and ELISA were performed. Sera from 10 patients were tested more than once, because follow-up samples were available.
incubation period (7 days), they were further incubated at 37 °C for another 5 weeks. Brucella organisms were identified by their coccobacilli Gram-negative appearance, requirement for CQ, production of H20, urease production and reaction with monospecific Brucella abortus or Brucella meltensis sera.
Statistics Agglutination tests Stained brucella suspensions for microagglutination and standard agglutination tests were obtained from Murex Diagnostic Limited (Central Road, Temple Hill, Dartford, England DA1 5LR, U.K.).
The StatPac Gold Statistical Analysis Package was used to calculate correlation coefficient between the standard agglutination test titre and ELISA, IgM and IgG values. Bivariate (scatter) plots were also drawn.
Results The microagglutination method This was performed in rigid u-bottoned microtitre plates exactly as described by Bettelheim et al. (1983). 23
The standard agglutination test (SAT) This was done on doubling dilutions from 1:20 to 1:20 840. The methods used were those described in the instruction sheets supplied.
Enzyme immune assay (ELISA) ELISA was performed using reagents supplied by ALFA BIOTECH (Via Castagnetta, 7, 00040 Promezia, Rome, Italy). The ELISA microplate testing procedure was performed exactly as described in the procedures manual. Automatic washing and absorption reading were done using the Diagnostic Pasteur LP35 multiwash and LP400 reader, respectively.
Bacteriological blood culture For bacteriological diagnosis of brucellosis, paired 10 ml blood samples from adults and 5 ml blood samples from children were collected. The culture was done in a Bactec 9240 automatic blood culture machine (Becton-Dickinson, 7 Loventon Circle Sparks, Maryland, U.S.A.) following the manufacturer's instructions. The blood culture bottles were subcultured on blood agar plates when they signalled positive, or at day 3, day 5 and day 7 of incubation. If the bottles are negative after the recommended
Out of thirty-nine cases where both blood culture and serology were requested, Brucella species were isolated from thirty specimens. The corresponding agglutination titres and IgG, fgM ELISA values appear in Table L Three samples were tested three times and seven samples twice. Samples giving similar readings were grouped together whenever possible. All culture positive cases gave significant agglutination and ELISA values except for samples 5, 7, 23, 24, 26, 27 and 28. Samples 23, 24 and 28 showed insignificant standard agglutination titre of 1:40. These are usually reported negative. However, the corresponding IgG, IgM ELISA values were significantly positive. Samples 5 and 7 gave significant agglutination titres and IgG ELISA, but borderline IgM. Samples 26 and 27 showed a similar pattern with borderline IgM reading. The relationship between standard agglutination titres and IgM ELISA showed a significant positive correlation between the two parameters (r = 0.494, P
Brucella Immunoassay versus Bacteriological Culture
199
Table 1. Agglutination titres and ELISAvalues in 30 patients with culture-positive brucellosis. No. of tests performed
Sample serial no.
Samples tested 3 times (no. = 3) Samples tested 2 times (no.- 7)
Brucella* culture
ELISA~
Agglutination Micro
Tube
IgG
IgM
1,2,3
+
1:20480
1:1280
2.642
2.600
5,7 4,6,8
+ +
1:640 .1:640-1:20480
1:160 1:160-1:10240
2.263 Neg.-1.890
0.635 0.917-2.563
23,24,28 26,27
+ +
11-22,26,9,30
+
1:320 1:1280 1:320 1:20480
1:40 1:320 1:2560 1:160-1:10240
O.559-2.904 1:329-2.447 1.111-2.883
0.769-1.804 0.629 0.644-2.902
Samples tested once (no.= 20)
* + = positive blood culture. "~ELISAcut off-valuesIgG = < O.627, IgM = <0.627.
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0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 IgG (ELISA) Figure 1. Correlation between standard agglutination titres and IgM
(ELISA) in brucellosis. IgG a n d IgM; (ii) 17 were positive for IgG only; (iii) 10 were positive for IgM only. The clinical presentations a n d serology values for the latter group with positive IgM appear in Table III. I n the 1 5 0 control samples, 109 were completely negative by both a g g l u t i n a t i o n a n d ELISA. The r e m a i n i n g 41 samples showed low insignificant m i c r o a g g l u t i n a t i o n titres of < 1 : 8 0 a n d reported as negative.
Discussion
A noticeable finding in this study is t h a t s i m u l t a n e o u s blood c u l t u r e a n d serology were requested i n only 22.4%
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0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 IgG (ELISA) Figure 2. Correlation between standard agglutination titres and IgG (ELISA) in brucellosis.
of cases, while in 77.6%, only serology was requested. It appears that blood culture is only ordered w h e n the patient presents with fever. This reflects the need for a satisfactory serological test for diagnosis of brucellosis, particulary in cases with atypical presentations. Out of 30 culture positive cases, three would have been reported as falsely negative due to a low s t a n d a r d a g g l u t i n a t i o n titre. I n these samples (nos. 23, 24 a n d 28), IgG a n d IgM brucella antibodies were positive by ELISA. This a m o u n t s to false-negative a g g l u t i n a t i o n results in 10% of culture-positive cases. It is w o r t h n o t i n g t h a t all culture-positive brucella samples were also positive by ELISA. Moreover, in n i n e suspected brucella cases
200
M. O. Gad EI-Rab and A. M. Kambal Table Ii. Agglutination titres and ELISA values in nine suspected brucella patients with negative blood culture. Sample no.
Agglutination titre
1 4 8 2,3,5,6,7,9
EUSA values*
Micro.
Tube
IgG
IgM
1:320 1:640 1:1280 1:1280-1:20480
1:80 1:80 1:160 1:160-1:1280
1.059 2.676 1.521 0.686-2.883
1.788 0.510 0.611 0.709-2.902
* ELISA cut off-values IgG = <0.627.
Table III. Clinical presentations in brucellosis patients with negative standard agglutination and positive ELISA IgM. Sample serial no.
37 44 65 77 108 127 125 133 140 168
Agglutination titre Micro. Tube (SAT) 1:640 1:320 1:640 1:160 1:320 1:1280 1:1280 1:640 1:160 1:160
1:80 1:40 1:80 1:40 1:40 1:80 1:80 1:80 1:40 1:40
ELISA values
Clinical presentation
IgG
IgM
NEG. NEG. NEG. NEG. NEG. NEG. NEG. NEG. NEG. NEG.
1,110 1.209 1.228 1.937 1.052 0.290 0.784 0.785 1.125 0.640
Joint and bone pain Backache Synovitis left knee Shoulder pain Arthritis Haematemesis, fever, H / 0 drinking raw milk H / 0 drinking raw milk Typhoid + persistent malaria
S A T = s t a n d a r d agglutination test; NEG. =negative.
which were culture-negative, two more would have been reported negative due to low agglutination titres of 1:40 and 1:80. Both of these were positive by ELISA. Furthermore, in 13 5 samples from patients suspected of having brucellosis, but for w h o m only serology was requested, 25 samples negative by the standard agglutination test were positive by F_LISA. Ten of these (7.4%) were brucella IgM antibodies positive and 17 were IgM and [gG positive. This discrepancy could be due to the high sensitivity of the ELISA test being a primary assay. Therefore, careful interpretation of the results will be required, as cross-reactions between brucella and other microbial antigens have been reported. 24 It has also been noted with ELISA tests that strong IgG responses with little or no IgM could be due to secondary rickettsial 2s or typhoid infections. 26 In this study, elevated IgM and IgG brucella antibodies were detected in most of the samples. This is in agreement with reports by Reddin et al, 19 and Sippel et tll. 27 who found elevated IgM and IgG in patients with acute brucellosis. Similar studies by Paratt et ill. 22 and Magee et al. 21 found IgM brucella antibodies alone in acute cases. These inconsistencies could be due to the fact that acute and chronic brucellosis m a y not appear as two distinct immunological entities in our patients as a result of delay in diagnosis or partial antibiotic treatment. In addition,
most of the patients are from rural areas and m a y have long-term illness before diagnosis. Thus, at presentation, they m a y have immunological patterns consistent with both acute and chronic disease. We also report a significant correlation between brucella IgM ELISA values and standard agglutination titres in culture-positive cases. No correlation was observed with IgG. In the control samples, where brucellosis was not suspected, 41 samples out of 150 showed a low microagglutination titre (< 1:80) suggesting that some chronic infections are subclinical. 2s Such presentations would be expected in our region with a high exposure rate of 3 5% and active disease in 2% of the populations. 4' 19 In conclusion, we found ELISA an effective method for diagnosis of brucellosis, particularly in subacute and chronic cases. This includes patients with negative standard agglutination test in presence of a compatible clinical history, as reported previously. 3° Moreover, the ELISA test, by measuring both IgM and IgG, helps in staging the disease.
Acknowledgement We wish to t h a n k Dr Awad Abd E1-Gafar for his critical comments. We also extend our thanks to Mr Mustafa Hussain and Mr A1-Waleed AlH a m a d for technical assistance.
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