|Year : 2015 | Volume
| Issue : 2 | Page : 136-139
Comparative evaluation of various serological tests in the laboratory diagnosis of Brucellosis
Meena Dias1, Edwin Dias2
1 Department of Microbiology, Fr. Muller Medical College, Mangalore, Karnataka, India
2 Department of Paediatrics, Srinivas Institute of Medical Sciences, Mangalore, Karnataka, India
|Date of Web Publication||16-Mar-2015|
S-3, Casa Leila, S. L. Mathias Road, Falnir, Mangalore - 575 002, Karnataka
Source of Support: None, Conflict of Interest: None
Introduction: Brucellosis is a zoonotic disease of public health importance having an impact on animal husbandry and dairy industry. Isolation of bacteria from blood cultures is very low, and diagnosis mainly depends on serology and molecular methods. The aim of this study is to know the impact of various serological tests on the diagnosis of brucellosis. Materials and Methods: In a prospective study, a total of 180 samples, 90 from pyrexia of unknown origin (PUO) and 90 from high-risk group comprising veterinarians and animal handlers were serologically tested by Rose Bengal plate test, standard tube agglutination (STA) test and enzyme-linked immunosorbent assay (ELISA) and results were analyzed. Results: Out of 90 PUO cases, 28 were positive for brucellosis and in high-risk group, out of 30 veterinarians three were positive and out of 60 animal handlers 14 were positive. Male preponderance was seen. Rose Bengal and STA tests were still efficient methods for brucellosis serodiagnosis. The ELISA was observed to be more efficient in both acute and chronic brucellosis. Conclusion: All the three methods used are efficient methods for detecting Brucella antibody. Rose Bengal card test is the best suited for rapid diagnosis in rural endemic area as STA test is laborious and time-consuming. ELISA is the most sensitive test.
Keywords: Brucellosis, enzyme-linked immunosorbent assay, Rose Bengal card test, serology, standard tube agglutination test
|How to cite this article:|
Dias M, Dias E. Comparative evaluation of various serological tests in the laboratory diagnosis of Brucellosis. CHRISMED J Health Res 2015;2:136-9
|How to cite this URL:|
Dias M, Dias E. Comparative evaluation of various serological tests in the laboratory diagnosis of Brucellosis. CHRISMED J Health Res [serial online] 2015 [cited 2020 May 25];2:136-9. Available from: http://www.cjhr.org/text.asp?2015/2/2/136/153258
| Introduction|| |
Brucellosis, a zoonotic disease continues to be a major public health problem worldwide. Man is an accidental host and contracts the disease directly from infected animals, contaminated secretions and products of animal conception where bacteria invade through small aberrations of skin or by ingestion of raw milk. It is widely recognized as a potential occupational risk among farmers, slaughterhouse workers, butchers, veterinarians, meat processing maintenance workers and families of dairy workers.  Brucella abortus, Brucella melitensis and Brucella suis are the most common organisms responsible for human disease.
A definitive diagnosis of Brucellosis is established by recovering the bacteria in blood or demonstration of elevated levels of humoral antibody. Although automated blood culture systems have shortened the isolation time from weeks to days, unavailability of it in most laboratories in our country makes isolation often unsuccessful and a positive diagnosis usually depends on clinical, serological and epidemiological data. Serum tube agglutination is the most widely used serological method. This method is less than ideal as it is laborious, takes 2 days and can be falsely negative owing to the presence of blocking antibodies.  Hence, this study was taken up to compare the results of immunoglobulin G (IgG) and IgM enzyme-linked immunosorbent assay (ELISA), tube agglutination test and Rose Bengal card test in the serodiagnosis of Brucellosis. The geographical areas already studied in Karnataka are Belgaum, Hubli and Bijapur. ,, As there is no data available in this part of Karnataka, this study was taken up to survey the seroprevalence of Brucellosis in and around Manipal and also to compare the various serological tests.
| Materials and methods|| |
A prospective study was carried out in the Department of Microbiology, at a Tertiary Care Teaching Hospital, for a period of 1½ years after obtaining the Institutional Ethics Committee's approval and informed consents from the patients. The study group included patients with a prolonged history of fever attending Medicine Outpatient Department. The risk group comprising veterinary surgeons and animal handlers were also included. The demographic data like age, sex, duration of fever, any other symptoms, and the risk factors were noted.
With proper aseptic precautions, patient's blood was collected in a BD™ (Becton Dickinson) vacutainer by venipuncture and then transported to the laboratory. The patient's serum was tested for standard tube agglutination test (STA), Rose Bengal plate test (RBPT) and Brucella IgG and IgM ELISA. STA test (Tulip diagnostics Pvt, Ltd.) was done according to standard protocol  with double dilution of 1:20-1:160 for initial screening followed by further dilutions to achieve end titer. As there is no data regarding the baseline titer, titers 160 and above were considered significant. Antibodies were detected for B. melitensis and B. abortus. Serum was also tested for Brucella IgG and IgM ELISA (Panbio laboratories Australia) and RBPT.
For RBPT antigen was obtained from the division of biological products, Indian Veterinary Research Institute Izatnagar UP. It is a 8% suspension of pure, smooth, killed cells of B. abortus strain 99 phenolized and stained with Rose Bengal dye and buffered at Ph 3.65 using lactic acid buffer. Test was carried out according to the standard protocol. 
| Results|| |
The sera from a total of 180 persons divided into two groups, pyrexia of unknown origin (PUO) and high-risk group comprising of veterinarians and animal handlers were examined by three serological methods. Out of 90 PUO cases, 28 were positive for brucellosis and in high-risk group, out of 30 veterinarians three were positive and out of 60 animal handlers 14 were positive. Cases were reported between the age groups of 20 and 65 years. Seroprevalence was highest in persons aged 30-40 years in both groups. This may be due to the fact that this is the most active age group, and they come in greater contact with animals. In PUO cases, seropositivity was also noticed in persons more than 60 years. The reason being most of them had cows at home and was consuming milk and its products. But in case of veterinarians they were mostly in the age group of 30-50 years and retire later. So we could not trace them later. One of the patients who had PUO was a vet by profession and was 65-year-old. Males were affected more than females.
[Table 1] shows the positivity of various serological tests in PUO and high-risk groups. The highest titer recorded was 1:5120 in a case of PUO. It is well known that the results given by the serological tests depend upon the stage of the infective process and the degree of contact with a causative agent of the disease. The percentages of positive results were higher for RBPT than for the agglutination test. The ELISA was found to be the most sensitive in both acute and chronic brucellosis. The seroprevalence in PUO cases was 42.20% and in high-risk group was 22.22%. The most common clinical presentation was fever followed by arthritis and backache [Table 2].
|Table 1: Results of various serological tests in PUO and high-risk group|
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There was no case positivity in children hence the seroprevalence was not done except for one neonate who presented with fever and failure to thrive. Investigation of the case revealed presence of Brucella in blood by BACTEC method. The source of infection was traced to milk powder.
Among the positive sera three were positive also for typhoid and two cases were noticed in HIV reactive patients.
| Discussion|| |
Brucellosis is one of the important neglected zoonotic diseases of public health importance. It remains endemic in many developing regions of the world. In India, it was first established early in the previous century and since then there been lot of reports from various states. In India, the serological evidence of Brucellosis in humans has been reported from various parts of the country, where the incidence varies from 0.9% to 18%. ,
The clinical diagnosis of human brucellosis is difficult to establish due to its nonspecific clinical manifestations. In the absence of specific clinical features, serological testing forms the mainstay of diagnosis. This study reveals that brucellosis is not uncommon in this part of small town in coastal Karnataka. This being a rural area, most of them have animals, especially cows at home. This is in conjunction with other studies. ,
It is a matter of experience that Brucellosis presents in a variety of clinical pictures could be missed by clinicians if not considered seriously in the differential diagnosis. This has been confirmed in the present study too. There were only two clinically suspected brucellosis that were serologically positive, others that were positive clinically presented as PUO. The most common clinical feature presented was fever, followed by malaise, myalgia, arthralgia and weight loss. Some presented with only joint pain, low back ache. No complications were noted. The various clinical presentations are shown in [Table 2].
We noticed that the infection was common in the age group of 20-40 years with a male preponderance, as these people take care of animals and come in contact with animals. This in conjuction with Tilak et al. 
The veterinartians showed Brucella agglutinins in 3 (10%) whereas in animal handlers 14 (23.33%). In a study conducted by Hemashettar et al.  24 (8.2%) veterinary workers showed Brucella specific antibodies in significant titers. None of the vets were symptomatic, but they are exposed to infected animals owing to their occupation. Similar findings were noticed elsewhere. ,
Some researchers have screened PUO cases for evidence of brucellosis. Handa et al. identified 4 (3.3%) cases with acute brucellosis in a group of 121 patients with PUO. Sen et al. identified 28 (6.8%) seropositive cases in a group of 414 patients with PUO and Kadri et al. identified 28 (0.8%) seropositive cases in a group of 3,532 patients with PUO. Varied percentages were also noticed in other studies. , In our study, out of 90 PUO cases, 28 (31.11%) were positive for brucellosis.
Brucellae have slow growth rate, and the culture result are not available for several days or weeks. The number of bacteria in clinical samples may vary widely, with the isolation of Brucella being highly dependent on the stage of disease (acute vs. chronic), antibiotic pretreatment, the existence of an appropriate clinical specimen and the culturing methods used.  Recently, lysis centrifugation technique  and automated blood culture systems enhanced the speed of detection but are still too slow to make a rapid diagnosis,  polymerase chain reaction (PCR) is fast and can be performed on any clinical specimen.  Although PCR is very promising, standardization of extraction methods, infrastructure, equipment and expertise are lacking, and a better understanding of the clinical significance of the results is still needed.  PCR-based laboratory tests have been proposed, they cannot be considered a routine diagnostic method yet. These limitations make serology for antibody detection the most useful tool for the laboratory diagnosis of brucellosis.
Serological tests are used for the initial diagnosis of human brucellosis as well as during treatment follow-up. In our study, RBPT had a sensitivity of 55.55% and specificity of 98.6% compared to serum agglutination test (SAT) where sensitivity and specificity were 21.65% and 92.95% respectively. The RBPT can be used as a screening test in endemic area especially in rural population. But one should remember its performance is poor in patients formerly and/or repeatedly exposed to the agent.  In high-risk populations, testing of diluted sera using the RBT might be a reasonable alternative, as this would reduce the need for a considerable number of confirmatory tests. 
Serum agglutination test is the best diagnostic tool for the diagnosis of brucellosis because it is easy to perform, does not need expensive equipment and training especially in resource-limited settings. Affordable laboratories can use ELISA as it is the most sensitive test. Though SAT is cheaper and easier test turnaround time is longer. In chronic and convalescent cases, ELISA is more results in acute cases. The detection of the IgG antibody class by ELISA is more sensitive than IgM detection. 
| Conclusion|| |
Brucellosis is seldom suspected and rarely confirmed. The indiscriminate use of antitubercular treatment for prolonged fevers in India further complicates the diagnosis because streptomycin and rifampicin are effective against Brucella also. 22% of occupationally exposed individuals listed positive in this study highlights the continuing existence of this infection in the animal reservoir. Hence, persistence of the animal reservoir of infection, low physician awareness, poor availability of the diagnostic facilities may contribute toward the seroprevalence of this infection in rural India.
Limitations of this study are we could not compare the results with culture or any molecular methods due to the unavailability of these tests and equipment. Culturing is a hazardous procedure, which requires highly secured laboratory with level 3 biosafety containment. We would like to conclude in the absence of culture and PCR, serological tests like ELISA and STA test can be used successfully in resource poor laboratories with financial constraints and RBPT can be used as a screening test.
| References|| |
Mantur BG, Amarnath SK, Shinde RS. Review of clinical and laboratory features of human brucellosis. Indian J Med Microbiol 2007;25:188-202.
Almuneef M, Memish ZA. Prevalence of Brucella
antibodies after acute brucellosis. J Chemother 2003;15:148-51.
Bhat K, Hemashettar BM, Jain R, Andrade AT, Patil CS. Latex agglutination test for antigen detection in human brucellosis. Indian J Med Microbiol 1997;15:210-4.
Mantur BG, Biradar MS, Bidri RC, Mulimani MS, Veerappa K, Kariholu P, et al
. Protean clinical manifestations and diagnostic challenges of human brucellosis in adults: 16 years′ experience in an endemic area. J Med Microbiol 2006;55:897-903.
Renukaradhya GJ, Isloor S, Rajasekhar M. Epidemiology, zoonotic aspects, vaccination and control/eradication of brucellosis in India. Vet Microbiol 2002;90:183-95.
Wright AE, Smith F. On the application of the serum test to the differential diagnosis of typhoid fever and Malta fever. Lancet 1897;1:656-9.
Brinley Morgan WJ, Mackinnon DJ, Gill KP, Gower SGM and Norris PIW (1978). Brucellosis diagnosis - standard laboratory techniques. Ministry of Agriculture, Fisheries and Food, London. MAFF Publication RVC 21, reprinted 1981.
Sehgal S, Bhatia R. Zoonoses in India. J Commun Dis 1990;22:227-35.
Patel PR, Anjaria JM, Dave MR, Desai H. Serological evidence of brucellosis in human beings in Kaira District of Gujarat. Indian J Public Health 1986;30:197-200.
Chauhan RS. Brucellosis in India and its impact on export of buffalo meat. Indian J Anim Prod 1999;31:316-7.
Tilak R, Tilak V, Bhat KG, Hemashettar BM. Evaluation of different serological techniques in laboratory diagnosis of brucellosis. Indian J Prev Soc Med 2007;38:135-41.
Hemashettar BM, Patil CS. Brucellosis among practicing veterinarians. Indian J Med Microbiol 1991;9:45-7.
Pathak AD, Dubal ZB, Doijad S, Raorane A, Rodrigues S, Naik R, et al
. Human brucellosis among pyrexia of unknown origin cases and occupationally exposed individuals in Goa Region, India. Emerg Health Threats J 2014;7:23846.
Handa R, Singh S, Singh N, Wali JP. Brucellosis in north India: Results of a prospective study. J Commun Dis 1998;30:85-7.
Sen MR, Shukla BN, Goyal RK. Seroprevalence of brucellosis in and around Varanasi. J Commun Dis 2002;34:226-7.
Kadri SM, Rukhsana A, Laharwal MA, Tanvir M. Seroprevalence of brucellosis in Kashmir (India) among patients with pyrexia of unknown origin. J Indian Med Assoc 2000;98:170-1.
Agasthya AS, Isloor S, Prabhudas K. Brucellosis in high risk group individuals. Indian J Med Microbiol 2007;25:28-31.
Mangalgi SS, Sajjan AG, Mohite ST. Brucellosis: A cause of pyrexia of unknown origin. Int J Biol Med Res. 2012;3:2054-8.
Al Dahouk S, Sprague LD, Neubauer H. New developments in the diagnostic procedures for zoonotic brucellosis in humans. Rev Sci Tech 2013;32:177-88.
Mantur BG, Mangalgi SS. Evaluation of conventional castaneda and lysis centrifugation blood culture techniques for diagnosis of human brucellosis. J Clin Microbiol 2004;42:4327-8.
Bannatyne RM, Jackson MC, Memish Z. Rapid diagnosis of Brucella
bacteremia by using the BACTEC 9240 system. J Clin Microbiol 1997;35:2673-4.
Queipo-Ortuño MI, Colmenero JD, Muñoz N, Baeza G, Clavijo E, Morata P. Rapid diagnosis of Brucella
epididymo-orchitis by real-time polymerase chain reaction assay in urine samples. J Urol 2006;176:2290-3.
Navarro E, Casao MA, Solera J. Diagnosis of human brucellosis using PCR. Expert Rev Mol Diagn 2004;4:115-23.
Ruiz-Mesa JD, Sánchez-Gonzalez J, Reguera JM, Martín L, Lopez-Palmero S, Colmenero JD. Rose Bengal test: Diagnostic yield and use for the rapid diagnosis of human brucellosis in emergency departments in endemic areas. Clin Microbiol Infect 2005;11:221-5.
Díaz R, Casanova A, Ariza J, Moriyón I. The Rose Bengal Test in human brucellosis: A neglected test for the diagnosis of a neglected disease. PLoS Negl Trop Dis 2011;5:e950.
Fadeel MA, Hoffmaster AR, Shi J, Pimentel G, Stoddard RA. Comparison of four commercial IgM and IgG ELISA kits for diagnosing brucellosis. J Med Microbiol 2011;60:1767-73.
[Table 1], [Table 2]