although the epidemiological data on the disease is frequently incomplete , it has been recognized as one of the most common zoonoses in the eastern mediterranean region , with more than 45000 cases reported annually . brucellosis is an important health problem in iran , and according to the data derived from active surveillance during 2001 - 2005 , the incidence of the disease is between 120 - 400 per 100,000 people . according to the surveillance program , most of the cases are among farmers , slaughterers and butchers , or those who have an occupational risk factor . furthermore , a large study in 1986 revealed that approximately 7.4% of cows in iran were infected with brucellosis . since 83% of cases with brucellosis in this country are less than 40 years old , the importance of occupational exposure , especially during adolescence and young adulthood , can not be overemphasized . because of its nonspecific and diverse clinical manifestations , the clinical diagnosis of brucellosis must be certainly ascertained with laboratory confirmation . although , culture of the bacteria is the gold standard for a definite diagnosis , attempts at isolation of the bacteria are frequently unsuccessful , and brucellosis is usually diagnosed serologically . furthermore , despite the fact that some laboratories use rapid isolation techniques such as bactec , dupont isolator , polymerase chain reaction methods or immunoblotting techniques , these techniques are not available in most developing countries , and conventional methods of isolation are too slow to be routinely used for diagnosis . consequently , in the absence of bacteriologic confirmation , a presumptive diagnosis can be made on the basis of a single high or rising titer of specific antibodies . among serological methods , serum agglutination test ( sat ) it is the standard and highly sensitive method for the diagnosis of diseases . in a study in which the sensitivity of enzyme linked immunosorbent assay ( elisa ) igg vs positive culture was 81.3% , the higher sensitivity of sat was also demonstrated in other studies , especially in studies from saudi arabia , which demonstrated that the sat sensitivity was 100% . despite the high yields of sat when sat is used to diagnose brucellosis , false - positive reactions occasionally result from cross - reactions with antibodies to salmonella spp . false - positive and false - negative reactions can be avoided by routinely diluting the serum above 1/320 . another problem with using sat is difficult interpretation of the test results . in various regions , different threshold titers , varying from 1:40 to 1:320 , have been taken as an indicator of active brucellosis . in saudi arabia , where brucellosis is endemic , a titer of 1:320 or higher has been found to be indicative of active brucellosis . a positive sat titer of 1:80 was present in 2.4% of the general population , and a 2-mercaptoethanol ( 2me ) test titer of 1:20 was present in less than 1% of the general population . accordingly , in iran a single titer of sat 1:80 or more in the presence of a 2me titer of 1:20 or more can be taken as a positive test result for brucellosis in the general population . the recently - introduced test , elisa , can determine specific class of igg , igm and iga antibodies against brucella . the assay is a sensitive , simple and rapid test with less limitation , and might be an acceptable alternative to sat . nevertheless , there are some contradictory reports regarding the diagnostic ability of elisa in acute brucellosis . therefore , it is reasonable to further evaluate and standardize the test according to the various geographical regions and populations . the objective of the present study was to determine an optimal cut - off point for elisa and compare the test outcome with that of sat . the optimal cut - off was defined as a point at which , the sum of the sensitivity and specificity are the uppermost . the study was approved by the ethics committee of the shahid beheshti university of medical sciences . four hundred and seven patients with fever and other symptoms of brucellosis from an area endemic for brucellosis in northwest of iran were enrolled in the study . after obtaining written informed consent , 5 ml of blood sample was taken from each patient , and a short questionnaire comprising questions regarding the epidemiological information was filled out for everyone . sera of the samples were separated , and were analyzed for brucellosis using sat , 2me and elisa - igg tests . serum agglutination test 
  the procedure was done using serial dilutions from 1/20 to 1/5120 in tubes to overcome possible prozone phenomenon . 2-mercaptoethanol test 
  the test was performed like serum agglutination test except for the addition of the 2-mercaptoethanol . patients with a sat titer of 1/80 or greater plus a 2me titer of 1/20 or greater were considered to have brucellosis , and the remaining patients were considered to have other febrile illnesses mimicking brucellosis . data including age , sex and antibody titers were analyzed using statistical package for social sciences ( spss version 16 , chicago , illinois , usa , ) . data were analyzed using student t , chi - squared , or mann - whitney u - test . receiver operating characteristic ( roc ) curve was utilized to establish the best cut - off point , which yielded the best sensitivity and specificity for elisa from the 407 sera , 11 had missing data and were not included in data analysis . distribution ( in percentage ) of titers of serum agglutinin test ( sat ) and 2-mercaptoethanol ( 2me ) in brucellosis patients receiver operating characteristic ( roc ) curve distinguishing between patients with brucellosis and those without the disease ( control patients ) diagnosed using elisa igg assay . the power of different elisa titers in diagnosing acute brucellosis lr+ : positive likelihood ratio ; lr- : negative likelihood ratio based on clinical symptoms as well as a sat titer of 1/80 or greater and a 2me titer of 1/20 or greater , 88 patients ( 21.9% ) had brucellosis and 308 patients ( 77.7% ) had other febrile illnesses . in subjects who deemed positive for the disease , the sat titers were between 1/80 to 1/5120 and 2me titers between 1/20 to 1/640 ( table 1 ) . the mean serum level of elisa - igg in the brucellosis - positive group was 103.9611.08 iu / ml , which was significantly ( p<0.001 ) higher than that of the brucellosis - negative group ( 69.103.93 iu / ml ) . the area under the roc curve to differentiate the brucellosis - positive and brucellosis - negative groups was 0.858 , which was significantly ( p<0.001 ) different from 0.5 ( figure 1 ) . compared to other cut - off points elisa - igg , the cut - off point of 53 iu / ml for elisa - igg yielded the highest sensitivity and specificity . therefore , it was considered the best cut - off point of elisa - igg to diagnose acute brucellosis . at this cut - off , the sensitivity , specificity , positive predictive value ( ppv ) , negative predictive value ( npv ) , positive likelihood ratio , and negative likelihood ratio were 84.09% , 85.38% , 62.20 , 94.90 , 5.75 , and 0.18 , respectively ( table 2 ) there was a significant correlation between sat and elisa - igg titers ( r=541 , p<0.001 ) or 2me and elisa - igg titers ( r=0.534 , p<0.001 ) . in another analysis , patients with a sat titer of 1/160 or greater and a 2me titer of 1/40 or greater were considered to have brucellosis , and the remaining patients were considered to have other febrile illnesses mimicking brucellosis , and the maximum sensitivity and specificity was attained at elisa - igg level of 53 iu / ml ( table 3 ) . serum agglutination test is the most widely- used serological test for the diagnosis of brucellosis . it is very sensitive , and considered the most reliable method for the diagnosis of brucellosis . according to ciftci et al . if culture - positivity is accepted as the gold standard in the diagnosis of brucellosis , the sensitivity of the serum agglutination will be 94.3% . although , conflicting studies have been published regarding the diagnostic accuracy of elisa , it has been reported to be rapid , highly sensitive , and specific in determining the brucella igg , igm , and iga antibodies in blood and csf . guneri , compared sat and elisa , and reported that their sensitivities was 80% and 72% , respectively . esalatmanesh , reported that the sensitivity , specificity , ppv , and npv of elisa - igg were 100% , 72.7% , 86.9% and 100% , respectively . etrek and colleagues , reported that the sensitivity and the specificity of elisa were , 81.3% and 95% , respectively . compared the diagnostic ability of sat with brucella elisa - igg and igm tests in patients with brucella bacteremia . sixty eight patients with clinical features suggestive of brucellosis , who had positive blood cultures for brucella species , and a control group including 70 healthy military personnel , who were blood donors and had no symptoms of brucellosis , were enrolled in the study . the sensitivity and specificity of the sat for the bacteremic patients were 95.6% and 100.0% , respectively , while those of the elisa - igg were 45.6% and 97.1% , and those of the elisa - igm were 79.1% and 100.0% , respectively . the sensitivity and specificity of either igg or igm positivity were 94.1% and 97.1% , respectively . assuming that the prevalence of active brucellosis in saudi arabia was 5% , the ppv and npv of sat were 100% and 99.7% , those of elisa - igg were 45.2% and 97.1% , and those of elisa - igm were 100% and 98.9% , respectively . when both the elisa - igg and igm were combined , the ppv and npv were 63% and 99.6% , respectively . in patients with brucella bacteremia , the sensitivity of either elisa - igm or igg were lower than those of sat , however , combining igm and igg resulted in a sensitivity and specificity similar to those of sat . the higher sensitivity of sat in comparison with elisa was also documented in other studies by others . however , we found only two published studies that had compared quantitatively these two tests . in the present study , patients with a sat titer of 1/80 or greater and a 2me titer of 1/20 or greater were considered to have brucellosis , and the remaining patients were considered to have other febrile illnesses mimicking brucellosis . such criteria would increase the overall diagnostic specificity at the expense of sensitivity . since we compared patients with brucellosis with patients with other febrile illnesses that should be discriminated from brucellosis , the power of elisa in diagnosing acute brucellosis in different titers of serum agglutinin test gold 1 : sat titer of 1/80 or more plus 2me titer of 1/20 or more ; gold 2 : sat titer of 1/160 or more plus 2me titer of 1/40 or more ; ppv : positive predictive value ; npv : negative predictive value ; lr+ : positive likelihood ratio ; lr- : negative likelihood ratio ; tps : true positive for the disease hasibi et al . studied 37 patients with brucellosis and 78 healthy control individuals , and performed sat and elisa on their sera . furthermore , the optimal cut - off point for elisa at 167.35 iu / ml , which is significantly different from our result . their cut - off point had a sensitivity , specificity , ppv , and npv of 89.2% , 100% , 100% and 795.1% , respectively . compared sat and elisa - igg in 56 brucellosis patients with a control group consisting of healthy individuals and patients with febrile illnesses other than brucellosis , and found that at the igg level of 50 iu / ml , the sensitivity and specificity were 75 and 100% , respectively . at igg level of 10 iu therefore , the first level of elisa - igg was better in terms of sensitivity , and the second level was better in terms of specificity . in the present study , we chose a level of elisa - igg ( 53 iu / ml ) that provided the highest sum of the sensitivity ( 84% ) and specificity ( 85% ) . in soodbakhsh and colleagues ' study , the area under roc curve of elisa - igg for discriminating brucellosis patients from other febrile patients were 0.97 . one reason for the difference between the results of our study and that of soodbakhsh et al . might be the method of selection of patients with brucellosis . in their study , patients who had a sat titer of 1/160 or more and a 2me titer of 1/40 or more in addition to related clinical manifestations were defined to have brucellosis . in the present study , there was a significant correlation between elisa - igg and sat ( r=0.541 , p<0.001 ) , which does not agree with the findings of el - rab and kambal . they found elisa - igm to have a significant positive correlation ( r=0.494 , p<0.001 ) with sat , but found no correlation between elisa - igg and sat . the best cut - off point of elisa - igg is 53 iu / ml , which yields the maximal sensitivity and specificity to diagnose acute brucellosis . at this cut - off , the sensitivity , specificity , ppv , npv , positive likelihood ratio , and negative likelihood ratio are 84.09% , 85.38% , 62.20 , 94.90 , 5.75 , and 0.18 , respectively .
background : brucellosis is a world - wide disease , which has a diverse clinical manifestation , and its diagnosis has to be proven by laboratory data . 
 serum agglutination test ( sat ) is the most - widely used test for diagnosing brucellosis . 
 the enzyme linked immunosorbent assay ( elisa ) can also determine specific antibody classes against brucella . 
 it is a sensitive , simple and rapid test , which could be an acceptable alternative to sat with fewer limitations , however , like any other new test it should be further evaluated and standardized for various populations . 
 this study was planned to determine an optimal cut - off point , for elisa which would offer maximum sensitivity and specificity for the test when compared to sat . 
 methods : four hundred and seven patients with fever and other compatible symptoms of brucellosis were enrolled in the study . 
 serum agglutination test , 2-mercaptoethanol test , and elisa were performed on their sera . 
 results : the cut - off point of 53 iu / ml of elisa - igg yielded the maximal sensitivity and specificity comparing to the other levels of elisa - igg , and was considered the best cut off - point of elisa - igg to diagnose acute brucellosis . at this cut - off , the sensitivity , specificity , positive predictive value , negative predictive value , positive likelihood ratio , and negative likelihood ratio were 84.09% , 85.38% , 62.20 , 94.90 , 5.75 , 0.18 , respectively . 
 conclusion : the best cut - off point of elisa - igg is 53 iu / ml , which yields the maximal sensitivity and specificity to diagnose acute brucellosis .