Journal of Microbiological Methods 97 (2014) 25–28
Contents lists available at ScienceDirect
Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth
Note
Innovative modifications to Rose Bengal plate test enhance its specificity, sensitivity and predictive value in the diagnosis of brucellosis Shubhada K. Chothe, Hari Mohan Saxena ⁎ Department of Veterinary Microbiology, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Sciences University (GADVASU), Ludhiana, Punjab 141004, India
a r t i c l e
i n f o
Article history: Received 19 October 2013 Received in revised form 8 December 2013 Accepted 8 December 2013 Available online 15 December 2013
a b s t r a c t Current agglutination tests occasionally yield false results. Superagglutination test reduced false results, had higher sensitivity (95.88%) and negative predictive value (95.83%) than Rose Bengal plate test (RBPT), Standard Tube Agglutination test (STAT), ELISA, and Complement Fixation test and specificity (89.32%) and positive predictive value (89.42%) higher than RBPT and STAT. © 2013 Elsevier B.V. All rights reserved.
Keywords: Superagglutination test Brucellosis Sensitivity Specificity False negative False positive
Brucellosis is an important zoonotic disease caused by Brucella organisms. It is of public health significance and causes huge economic losses to the livestock sector due to reproductive losses in animals, abortions, placentitis, epididymitis and orchitis. Brucellosis is endemic in India (Aulakh et al., 2008) where it is estimated to cause a loss of US $58.8 million per year (Kollannur et al., 2007). The Rose Bengal plate test (RBPT) is often used as a rapid screening test in the diagnosis of brucellosis (Ruiz-Mesa et al., 2005). Although the sensitivity of RBPT is reported to be very high, the specificity can be disappointingly low (Barroso et al., 2002). As a result, the positive predictive value of the test is low and a positive test result thus requires confirmation by a more specific test (Smits and Kadri, 2005). The RBPT could sometimes give a false positive result. Suitable modifications of the RBPT are, therefore, required to get accurate results. We have developed a novel Superagglutination test to enhance the sensitivity and minimize false positive and false negative results of RBPT (Saxena and Kaur, 2013). The present study was undertaken to compare the sensitivity and specificity of the novel Superagglutination test with the available serodiagnostic tests RBPT, STAT, CFT, and ELISA to evaluate its efficacy on serum samples that may be either false positive or false negative by RBPT. Guidelines of the Institutional Animal Ethics Committee were followed in the study.
Abbreviations: RBPT, Rose Bengal plate test; STAT, Standard Tube Agglutination test; iELISA, Indirect enzyme linked immunosorbent assay; CFT, Complement fixation test. ⁎ Corresponding author at: Flat No. 9, First Floor, Geetanjali Apartments, E Block, Rishi Nagar, Ludhiana, Punjab 141001, India. Tel.: +91 9417147813 (mobile). E-mail address: [email protected] (H.M. Saxena). 0167-7012/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.mimet.2013.12.005
A total of 200 bovine (181 cattle and 19 buffalo) serum samples were derived from the animals in veterinary clinics, dairy farms and gaushalas (animal shelters) in and around Ludhiana. All the animals were of age two years or more. Brucellosis suspected herds were selected for sampling primarily based on the history of abortions in the herd while normal healthy animals were sampled from the herds of the university dairy farm without the history of abortions and repeatedly RBPT negative status. Common serological tests i.e. the RBPT, STAT, iELISA and CFT along with the Superagglutination test were applied on all the serum samples. RBPT was done as per the standard method (Morgan et al., 1978). For performing Superagglutination test, equal volumes (2.5 μl each) of RBPT colored antigen, test serum stained with 0.1% Coomassie Blue dye, biotinylated anti-bovine IgG (Sigma) and streptavidin (Sigma) were mixed thoroughly on a clean glass slide in the abovementioned sequence. The slide was observed for 4 min for the formation of clumps. Ordinary hand lens was used occasionally for better resolution. The slides were viewed under low power (10×) of an inverted microscope to visualize the composition of clumps in case of doubt. Formation of clear agglutination, within which the blue color (due to the Coomassie Blue dye staining the serum antibodies) and the pink color (due to the Rose Bengal dye stained RBPT antigen) could be differentiated on magnification, were considered as positive, while absence of clear agglutinates was considered as negative. The standard method recommended by the Office International des Epizootes (OIE, 2004) was followed for Standard Tube Agglutination test (STAT). To perform Indirect ELISA (iELISA), a commercial kit was procured from Immunobiological Laboratories IBL-America (Minneapolis, USA).
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S.K. Chothe, H.M. Saxena / Journal of Microbiological Methods 97 (2014) 25–28
The test was performed according to the instructions provided in the kit manual. Complement Fixation test (CFT) was performed as per the OIE Manual. The positive predictive value (PPV) and negative predictive value (NPV) for each diagnostic test were calculated using the following formulae: PPV ¼
Number of True Positive cases Number of True Positive cases þ Number of False Positive cases
NPV ¼
Number of True Negative cases : Number of True Negative cases þ Number of False Negative cases
Observed proportion of agreement (OPA) and agreement beyond chance (kappa values) were determined using Winepiscope-2 software package with 95% confidence level. Out of the 200 samples, 97 were found to be positive by RBPT (Table 1). The percent prevalence of brucellosis varied with the test
and ranged from 43.50% to 48.50%. The test detected 6% less positive samples than the Superagglutination test and showed a sensitivity of 93.33%, a specificity of 88.18%, a PPV of 86.6% and NPV of 94.17%, respectively (Table 2). In the case of the Superagglutination test, the clumps on the slide had both blue and pink color. When the slide was viewed under the low power of a light microscope, the agglutinate could be very easily differentiated into two parts, the antibodies were blue in color due to the Coomassie blue dye and the antigen was pink in color due to the Rose Bengal dye (Figs. 1 & 2). A total of 104 out of the 200 serum samples were detected positive by Superagglutination test (Table 1). The test detected more positive samples than ELISA (16.5%), CFT (14.5%), RBPT (6%) and STAT (6%) and showed a sensitivity of 95.88% and a specificity of 89.32%. The positive predictive value (PPV) of this test was found to be 89.42% and Negative Predictive Value (NPV) was 95.83% (Table 2). STAT could detect 119 out of the 200 samples as positive. A titer of 1:40 and above was considered as positive. A total of 81 samples had titers below 1:40, 36 samples had a titer of 1:40 and 83 samples had titers more than 1:40 (Table 1).
Table 1 Results of analysis of sera by various serological tests. Sample no. 69(20/4/11), 80(20/4/11), 83(20/4/11), A, B, F, G, BB, BH, BR, DD, DL, K, L, M, N, S, V, AC, AF, AG, AP, AZ, BG, CB, CK, 68(20/4/11), 70(20/4/11), 71(20/4/11), 77(20/4/11) 78(20/4/11), 81(20/4/11), BK AT T 2(1/3/11), 13(1/3/11), 23(1/3/11), 76(T 5/10/11), 10(13/10/11), E, BA, BF, CY, DN, DR, AB,74(20/4/11), 3 (13/10/11) AE AI, BC, BY AN, BW 23(20/4/11), BP, 102(15/9/11), 7(20/4/11) 73(20/4/11), 1(1/3/11), 20(1/3/11), 21(T5/10/11), 5(13/10/11), AD, BL, 66(20/4/11), 3(1/3/11), T7, 6(13/10/11), T2061(25/11/11), J T6 CE, AV CN, AJ CX, AO 7(13/10/11) 29(15/9/11), 40(15/9/11), 15(20/4/11), 17(20/4/11), 20(20/4/11), 34(20/4/11), 42(20/4/11), 51(20/4/11), 53(20/4/11), 54(20/4/11), 72(20/4/11), 76(20/4/11), T1, T3, 4(1/3/11), 92(T 5/10/11), 11(13/10/11) T8, 79(T 5/10/11), T 85 (25/11/11), O DQ, BE, AQ, 2413 T 81 (25/11/11) T 2062 (25/11/11) H, W, X, AS, BT AL, AY, 2379 CI 2218, 2452, 2581, 101(15/9/11), I, AH, CR, DG, DJ, DO 13(20/4/11), 79(20/4/11), 100(20/4/11) 11(1/3/11), 19(1/3/11), 82(T 5/10/11) DU, BI 12(13/10/11), CV, 16(20/4/11), BJ, BM P 2308, 2417, 89(T 5/10/11), U, Y, AK, AW, CT, DB, DC 103(15/9/11), 6(1/3/11), 9(1/3/11) T4 86(T 5/10/11) CM, T5, AA R, BD BX, DP, CZ 2362, Q, AM, AR, AU, BS 82(20/4/11) BO, BQ 2490, T2, AX, BZ, 25(20/4/11) 2574, 2582 2426, 2467, 2554, 2567, 104(15/9/11), 19(20/4/11), 32(20/4/11), 87(T 5/10/11), CD, CH, CS, 2489, 88(T 5/10/11), 80(T 5/10/11), T 29 (25/11/11), T 77 (25/11/11), T 78 (25/11/11), T 84 (25/11/11) T 8 (25/11/11) T 86 (25/11/11)
STAT titer
RBPT
Superagglutination test
iELISA
CFT
00
−
−
−
−
00 00 10
− − −
− + −
− − −
+ − −
10 10 10 20 20
− + + − −
− + + − −
− − + − −
+ − + + −
20 20 20 20 20 40
+ − + + − −
− − + + + −
− + − + − −
− + − + − −
40 40 40 40 40 40 40 80 80 80 80 80 80 160 160 160 160 160 160 160 320 320 320 640 640 N1280
− + − − + + + + − − + + + + − + + + + + + − + + + +
− + + + + + + + + − + + + + + − − + + + + + + + + +
− + − + + − − + − − + − − + − − + − − + + − − + + +
+ − + + + + − + − − − − + + − − + + − − + + + + − +
N1280 N1280
+ +
+ −
− +
+ +
S.K. Chothe, H.M. Saxena / Journal of Microbiological Methods 97 (2014) 25–28
27
Table 2 Sensitivity and specificity of the Superagglutination test and its agreement with other serodiagnostic tests. Serological test
Superagglutination RBPT STAT iELISA CFT
Values (in percent)
Agreement of Superagglutination with other serological tests
Sensitivity
Specificity
PPV
NPV
Prevalence of the disease
OPA (percentage)
Kappa value
Degree of agreement
95.88 93.33 94.25 74.47 82.80
89.32 88.18 68.14 95.24 93.46
89.42 86.60 69.49 93.33 91.67
95.83 94.17 93.90 80.65 86.21
48.50 45.00 43.50 47.24 46.5
92.5 81.5 81.5 81.0
0.850 0.626 0.634 0.623
Almost perfect Substantial Substantial Substantial
STAT detected 6% less positive samples than the Superagglutination test and showed a sensitivity of 94.25% and a specificity of 68.14%. PPV of this test was found to be 69.49% and NPV was 93.90% (Table 2). Out of the 200 samples, 75 were detected as positive by iELISA (Table 1). ELISA detected 16.5% less positive samples than the Superagglutination test. Sensitivity of this test was calculated to be 74.47% and the specificity was found to be 95.24%. PPV of this test was 93.33% and NPV was found to be 80.65% (Table 2). Out of the 200 samples, 86 were detected as positive by CFT (Table 1). In the present study, the CFT detected 14.5% less positive samples than the Superagglutination test and showed a sensitivity of 82.8% and a specificity of 93.46% (Table 2). Statistical agreement between the Superagglutination test and other tests was determined. The kappa values and the observed proportions of agreements are presented in Table 2. The agreement between the Superagglutination test and RBPT was found to be almost perfect, whereas the agreement between the Superagglutination test and other tests was found to be substantial. False positive reactions in RBPT have been attributed to residual antibody activity from vaccination, cross reaction with certain bacteria and laboratory error. False negative reactions may arise during early incubation of disease or immediately after incubation (Radostits et al., 2000). False negative results may be due to a small clump size in sera with low titers of antibodies and false positive results due to the inability to differentiate non-specific aggregates of antigen particles alone from the true agglutinates comprising both antigen and antibody. Cross reacting antibodies have been detected by RBPT and false negative reactions are believed to occur mostly due to prozoning (OIE, 2004).
Fig. 1. The gross view of the superagglutination test on a glass slide by naked eye.
In the present study, Superagglutination test, developed by us recently (Saxena and kaur, 2013) by introducing novel modifications in the RBPT, has been evaluated for its usefulness in the diagnosis of bovine brucellosis by comparing its results with those of RBPT, STAT, iELISA, and CFT. The new test could detect more positive samples than RBPT, STAT, iELISA and CFT. In an earlier report (Akhtar et al., 2010) a low specificity of RBPT had been demonstrated. The high sensitivity of STAT observed in the present study can be attributed to its ability to detect antibody titer as low as 1:40 which was probably missed by other tests. In serum agglutination tests, cross reactions with various bacteria for example Yersinia enterocolitica O:9, E. coli O:157, Francisella tularensis, Salmonella urbana group N, Vibrio cholerae and Stenotrophomonas maltophilia have been reported (Corbel and Brinley-Morgan, 1984). The Complement Fixation test is technically challenging because a large number of reagents must be titrated daily and a large number of controls of all the reagents are required. It is an expensive test and is labor intensive. Other problems include the subjectivity of the interpretation of results, occasional direct activation of complement by serum (anti-complementary activity) and the inability of the test for use with hemolysed serum samples. However, since only IgG1 isotype of antibody fixes complement well, the test specificity is high (Poester et al., 2010). In an earlier study, Stemshorn and Forbes (1985) had demonstrated a sensitivity of 79% for CFT for the diagnosis of bovine brucellosis.
Fig. 2. Microscopic view showing a two colored agglutinate formed in the Superagglutination test with blue colored antibodies bound to the pink colored antigen particles.
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S.K. Chothe, H.M. Saxena / Journal of Microbiological Methods 97 (2014) 25–28
The highest sensitivity of all the tests observed with Superagglutination test in our study can be attributed to the fact that the number of false negative results with Superagglutination test was less when compared to the other tests. The anti-bovine IgG plays a role in increasing the sensitivity since if fewer antibodies against the infectious organism are present in the serum, the anti-globulin cross links these antibodies resulting in an increase in the clump size. Streptavidin binds to the biotinylted anti-globulin increasing the clump size further by up to 4 fold due to the presence of four binding sites for biotin on each molecule of avidin. The specificity of the Superagglutination test was found to be higher than that of RBPT and STAT. In the case of the Superagglutination test, the two colored true agglutinates could be very easily differentiated from non-specific one colored aggregates under the low power of a light microscope. The antibodies were blue in color due to the Coomassie blue dye and the antigen was pink in color due to the Rose Bengal dye. Each agglutinate had both the blue and the pink color, which aided in the differentiation of the true agglutinates from the non-specific aggregates of the antigen of pink color only. The antigen and antibodies which did not participate in agglutination reaction could be viewed under the microscope as aggregates of either blue or pink particles alone lying separately. Ordinary hand lens was generally found to be effective in visualizing the agglutination. The highest agreement with RBPT, combined with the higher specificity and sensitivity of Superagglutination test ensures that it can serve as a more efficient screening test than RBPT. The Superagglutination test had a higher sensitivity and a negative predictive value than the other serodiagnostic tests like RBPT, STAT, ELISA and CFT. Its specificity and PPV were found to be better than RBPT and STAT. The test can be used in the pen side diagnosis of bovine brucellosis with better results than the RBPT which is routinely used as a pen side test for brucellosis. ELISA is not a cost effective test when screening has to be performed on herds with a large number of animals. In such situations, Superagglutination test can offer an advantage of increased sensitivity of screening compared to RBPT and STAT. In our study, the agreement between Superagglutination test and RBPT was found to be higher than the agreement between Superagglutination test and other tests. This agreement combined with the higher specificity and sensitivity of Superagglutination test ensures that it can serve as a more efficient screening test than RBPT. The Superagglutination test showed the highest sensitivity of all the tests (95.88%) which can be attributed to the lesser number of false negative results obtained with Superagglutination test compared to the other tests. The anti-globulin played a role in increasing the sensitivity because if there are less number of antibodies against the infectious organism present in the serum, anti-globulin binds to these antibodies resulting in an increase in the clump size. Avidin binds to the biotinylated anti-globulin increasing the clump size further by up to 4 fold due to the four binding sites for biotin present on each molecule of avidin. The specificity of the Superagglutination test was however, found to be somewhat lower than that of iELISA and CFT. This may be due to the cross reactions of anti-globulin with non-specific or low affinity antibodies. Interestingly, the agglutinate in the case of Superagglutination test could be differentiated into two parts, the antibodies were blue in color due to the Coomassie blue dye and the antigen having a pink color due to the Rose Bengal dye. Each clump had both the blue and the pink colors, which aided in the identification of the true
agglutinates. This has not been reported by any other researcher as yet. Antigen and antibodies which did not participate in agglutination reaction could be viewed under the microscope as blue and pink particles lying separately or as single colored aggregates. The results obtained in our study suggest that Superagglutination test has a higher sensitivity than other serodiagnostic tests commonly used for brucellosis. The specificity of Superagglutination test was higher than RBPT and STAT but lesser than that of ELISA and CFT. The new test can be used as a screening test in the diagnosis of bovine brucellosis. As ELISA is not a cost effective test, Superagglutination test can offer the advantages of increased sensitivity, cost effectiveness, no requirement for skilled personnel and ease of performance. Conflict of interest The authors declare that there is no conflict of interest. The innovative methods in the Superagglutination test are patented (Inventor: H M Saxena; Assignees: 1. GADVASU, Ludhiana and 2. The Department of Biotechnology, Ministry of S&T, Govt. of India, New Delhi). The study was funded by GADVASU. SKC conducted the study as a Master's degree student under the supervision of HMS, a Professor of Immunology. HMS conceived the ideas underlying the Superagglutination test. He designed, supervised and coordinated the study, analyzed and interpreted the data and drafted the manuscript. SKC carried out the experimental work and statistical analysis. We are thankful to Dr. Mudit Chandra and Dr. Deepti Narang, Department of Veterinary Microbiology, GADVASU, Ludhiana for their help. References Akhtar, R., Chaudhry, Z.I., Shakoori, A.R., Ahmad, M.D., Aslam, A., 2010. Comparative efficacy of conventional diagnostic methods and evaluation of polymerase chain reaction for the diagnosis of bovine brucellosis. Vet. World 3, 53–56. Aulakh, H.K., Patil, P.K., Sharma, S., Kumar, H., Mahajan, V., Sandhu, K.S., 2008. A study on the epidemiology of bovine brucellosis in Punjab (India) using milk-ELISA. Acta Vet. 77, 393–399. Barroso, G.P., Pelayo, R.C., Extremera, G., 2002. Study of 1595 brucellosis cases in the Almeria province (1972–1998) based on epidemiological data from disease reporting. Rev. Clin. Esp. 202, 577–582. Corbel, M.J., Brinley-Morgan, W.J., 1984. Brucella. In: Krieg, N.R. (Ed.), Bergey's Manual of Systematic Bacteriology, Vol. I. Baltimore: Williams and Wilkins, pp. 377–388. Kollannur, J.D., Rathore, R., Chauhan, R.S., 2007. Epidemiology and Economics of Brucellosis in Animals and its Zoonotic Significance. ISAH Tartu, Estonia 466–468. Morgan, W.J., Mackinnon, D.T., Gill, K.P.W., Gower, S.G.M., Norris, P.I.W., 1978. Brucellosis diagnosis: standard laboratory techniques report. Series no. 1, Weybridge, England. OIE, 2004. Bovine brucellosis, Section 2.3, OIE Manual of Standards for Diagnostic Tests and Vaccines, 5th edition. OIE, Paris. Poester, F.P., Nielsen, K., Samartino, L.E., Yu, W.L., 2010. Diagnosis of brucellosis. Open Vet. Sci. J. 4, 46–60. Radostits, O.M., Gay, C.C., Blood, D.C., Hinchcliff, K.W., 2000. Diseases Caused by Brucella spp. A Textbook of the Disease of Cattle, Sheep, Pigs, Goats and Horses, Ninth edition. Harcourt Publishers Limited, London 881–890. Ruiz-Mesa, J.D., Gonzalez, J.S., Reguera, J.M., Martin, L., Palmero, S.L., Colmenero, J.D., 2005. Rose Bengal test: diagnostic yield and use for the rapid diagnosis of human brucellosis in emergency departments in endemic areas. Clin. Microbiol. Infect. 11, 221–225. Saxena, H.M., Kaur, P., 2013. A new superagglutination test to minimize false negative and false positive results common with plate/slide agglutination tests for the diagnosis of infectious diseases. Int. J. Trop. Dis. Health 3, 199–209. Smits, H.L., Kadri, S.M., 2005. Brucellosis in India: a deceptive infectious disease. Indian J. Med. Res. 122, 375–384. Stemshorn, B.W., Forbes, L.B., Eaglesome, M.D., Nielsen, K.H., Robertson, F.J., Samagh, B.S., 1985. A comparison of standard serological tests for the diagnosis of bovine brucellosis in Canada. Can. J. Comp. Med. 49, 391–394.
Contents lists available at ScienceDirect
Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth
Note
Innovative modifications to Rose Bengal plate test enhance its specificity, sensitivity and predictive value in the diagnosis of brucellosis Shubhada K. Chothe, Hari Mohan Saxena ⁎ Department of Veterinary Microbiology, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Sciences University (GADVASU), Ludhiana, Punjab 141004, India
a r t i c l e
i n f o
Article history: Received 19 October 2013 Received in revised form 8 December 2013 Accepted 8 December 2013 Available online 15 December 2013
a b s t r a c t Current agglutination tests occasionally yield false results. Superagglutination test reduced false results, had higher sensitivity (95.88%) and negative predictive value (95.83%) than Rose Bengal plate test (RBPT), Standard Tube Agglutination test (STAT), ELISA, and Complement Fixation test and specificity (89.32%) and positive predictive value (89.42%) higher than RBPT and STAT. © 2013 Elsevier B.V. All rights reserved.
Keywords: Superagglutination test Brucellosis Sensitivity Specificity False negative False positive
Brucellosis is an important zoonotic disease caused by Brucella organisms. It is of public health significance and causes huge economic losses to the livestock sector due to reproductive losses in animals, abortions, placentitis, epididymitis and orchitis. Brucellosis is endemic in India (Aulakh et al., 2008) where it is estimated to cause a loss of US $58.8 million per year (Kollannur et al., 2007). The Rose Bengal plate test (RBPT) is often used as a rapid screening test in the diagnosis of brucellosis (Ruiz-Mesa et al., 2005). Although the sensitivity of RBPT is reported to be very high, the specificity can be disappointingly low (Barroso et al., 2002). As a result, the positive predictive value of the test is low and a positive test result thus requires confirmation by a more specific test (Smits and Kadri, 2005). The RBPT could sometimes give a false positive result. Suitable modifications of the RBPT are, therefore, required to get accurate results. We have developed a novel Superagglutination test to enhance the sensitivity and minimize false positive and false negative results of RBPT (Saxena and Kaur, 2013). The present study was undertaken to compare the sensitivity and specificity of the novel Superagglutination test with the available serodiagnostic tests RBPT, STAT, CFT, and ELISA to evaluate its efficacy on serum samples that may be either false positive or false negative by RBPT. Guidelines of the Institutional Animal Ethics Committee were followed in the study.
Abbreviations: RBPT, Rose Bengal plate test; STAT, Standard Tube Agglutination test; iELISA, Indirect enzyme linked immunosorbent assay; CFT, Complement fixation test. ⁎ Corresponding author at: Flat No. 9, First Floor, Geetanjali Apartments, E Block, Rishi Nagar, Ludhiana, Punjab 141001, India. Tel.: +91 9417147813 (mobile). E-mail address: [email protected] (H.M. Saxena). 0167-7012/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.mimet.2013.12.005
A total of 200 bovine (181 cattle and 19 buffalo) serum samples were derived from the animals in veterinary clinics, dairy farms and gaushalas (animal shelters) in and around Ludhiana. All the animals were of age two years or more. Brucellosis suspected herds were selected for sampling primarily based on the history of abortions in the herd while normal healthy animals were sampled from the herds of the university dairy farm without the history of abortions and repeatedly RBPT negative status. Common serological tests i.e. the RBPT, STAT, iELISA and CFT along with the Superagglutination test were applied on all the serum samples. RBPT was done as per the standard method (Morgan et al., 1978). For performing Superagglutination test, equal volumes (2.5 μl each) of RBPT colored antigen, test serum stained with 0.1% Coomassie Blue dye, biotinylated anti-bovine IgG (Sigma) and streptavidin (Sigma) were mixed thoroughly on a clean glass slide in the abovementioned sequence. The slide was observed for 4 min for the formation of clumps. Ordinary hand lens was used occasionally for better resolution. The slides were viewed under low power (10×) of an inverted microscope to visualize the composition of clumps in case of doubt. Formation of clear agglutination, within which the blue color (due to the Coomassie Blue dye staining the serum antibodies) and the pink color (due to the Rose Bengal dye stained RBPT antigen) could be differentiated on magnification, were considered as positive, while absence of clear agglutinates was considered as negative. The standard method recommended by the Office International des Epizootes (OIE, 2004) was followed for Standard Tube Agglutination test (STAT). To perform Indirect ELISA (iELISA), a commercial kit was procured from Immunobiological Laboratories IBL-America (Minneapolis, USA).
26
S.K. Chothe, H.M. Saxena / Journal of Microbiological Methods 97 (2014) 25–28
The test was performed according to the instructions provided in the kit manual. Complement Fixation test (CFT) was performed as per the OIE Manual. The positive predictive value (PPV) and negative predictive value (NPV) for each diagnostic test were calculated using the following formulae: PPV ¼
Number of True Positive cases Number of True Positive cases þ Number of False Positive cases
NPV ¼
Number of True Negative cases : Number of True Negative cases þ Number of False Negative cases
Observed proportion of agreement (OPA) and agreement beyond chance (kappa values) were determined using Winepiscope-2 software package with 95% confidence level. Out of the 200 samples, 97 were found to be positive by RBPT (Table 1). The percent prevalence of brucellosis varied with the test
and ranged from 43.50% to 48.50%. The test detected 6% less positive samples than the Superagglutination test and showed a sensitivity of 93.33%, a specificity of 88.18%, a PPV of 86.6% and NPV of 94.17%, respectively (Table 2). In the case of the Superagglutination test, the clumps on the slide had both blue and pink color. When the slide was viewed under the low power of a light microscope, the agglutinate could be very easily differentiated into two parts, the antibodies were blue in color due to the Coomassie blue dye and the antigen was pink in color due to the Rose Bengal dye (Figs. 1 & 2). A total of 104 out of the 200 serum samples were detected positive by Superagglutination test (Table 1). The test detected more positive samples than ELISA (16.5%), CFT (14.5%), RBPT (6%) and STAT (6%) and showed a sensitivity of 95.88% and a specificity of 89.32%. The positive predictive value (PPV) of this test was found to be 89.42% and Negative Predictive Value (NPV) was 95.83% (Table 2). STAT could detect 119 out of the 200 samples as positive. A titer of 1:40 and above was considered as positive. A total of 81 samples had titers below 1:40, 36 samples had a titer of 1:40 and 83 samples had titers more than 1:40 (Table 1).
Table 1 Results of analysis of sera by various serological tests. Sample no. 69(20/4/11), 80(20/4/11), 83(20/4/11), A, B, F, G, BB, BH, BR, DD, DL, K, L, M, N, S, V, AC, AF, AG, AP, AZ, BG, CB, CK, 68(20/4/11), 70(20/4/11), 71(20/4/11), 77(20/4/11) 78(20/4/11), 81(20/4/11), BK AT T 2(1/3/11), 13(1/3/11), 23(1/3/11), 76(T 5/10/11), 10(13/10/11), E, BA, BF, CY, DN, DR, AB,74(20/4/11), 3 (13/10/11) AE AI, BC, BY AN, BW 23(20/4/11), BP, 102(15/9/11), 7(20/4/11) 73(20/4/11), 1(1/3/11), 20(1/3/11), 21(T5/10/11), 5(13/10/11), AD, BL, 66(20/4/11), 3(1/3/11), T7, 6(13/10/11), T2061(25/11/11), J T6 CE, AV CN, AJ CX, AO 7(13/10/11) 29(15/9/11), 40(15/9/11), 15(20/4/11), 17(20/4/11), 20(20/4/11), 34(20/4/11), 42(20/4/11), 51(20/4/11), 53(20/4/11), 54(20/4/11), 72(20/4/11), 76(20/4/11), T1, T3, 4(1/3/11), 92(T 5/10/11), 11(13/10/11) T8, 79(T 5/10/11), T 85 (25/11/11), O DQ, BE, AQ, 2413 T 81 (25/11/11) T 2062 (25/11/11) H, W, X, AS, BT AL, AY, 2379 CI 2218, 2452, 2581, 101(15/9/11), I, AH, CR, DG, DJ, DO 13(20/4/11), 79(20/4/11), 100(20/4/11) 11(1/3/11), 19(1/3/11), 82(T 5/10/11) DU, BI 12(13/10/11), CV, 16(20/4/11), BJ, BM P 2308, 2417, 89(T 5/10/11), U, Y, AK, AW, CT, DB, DC 103(15/9/11), 6(1/3/11), 9(1/3/11) T4 86(T 5/10/11) CM, T5, AA R, BD BX, DP, CZ 2362, Q, AM, AR, AU, BS 82(20/4/11) BO, BQ 2490, T2, AX, BZ, 25(20/4/11) 2574, 2582 2426, 2467, 2554, 2567, 104(15/9/11), 19(20/4/11), 32(20/4/11), 87(T 5/10/11), CD, CH, CS, 2489, 88(T 5/10/11), 80(T 5/10/11), T 29 (25/11/11), T 77 (25/11/11), T 78 (25/11/11), T 84 (25/11/11) T 8 (25/11/11) T 86 (25/11/11)
STAT titer
RBPT
Superagglutination test
iELISA
CFT
00
−
−
−
−
00 00 10
− − −
− + −
− − −
+ − −
10 10 10 20 20
− + + − −
− + + − −
− − + − −
+ − + + −
20 20 20 20 20 40
+ − + + − −
− − + + + −
− + − + − −
− + − + − −
40 40 40 40 40 40 40 80 80 80 80 80 80 160 160 160 160 160 160 160 320 320 320 640 640 N1280
− + − − + + + + − − + + + + − + + + + + + − + + + +
− + + + + + + + + − + + + + + − − + + + + + + + + +
− + − + + − − + − − + − − + − − + − − + + − − + + +
+ − + + + + − + − − − − + + − − + + − − + + + + − +
N1280 N1280
+ +
+ −
− +
+ +
S.K. Chothe, H.M. Saxena / Journal of Microbiological Methods 97 (2014) 25–28
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Table 2 Sensitivity and specificity of the Superagglutination test and its agreement with other serodiagnostic tests. Serological test
Superagglutination RBPT STAT iELISA CFT
Values (in percent)
Agreement of Superagglutination with other serological tests
Sensitivity
Specificity
PPV
NPV
Prevalence of the disease
OPA (percentage)
Kappa value
Degree of agreement
95.88 93.33 94.25 74.47 82.80
89.32 88.18 68.14 95.24 93.46
89.42 86.60 69.49 93.33 91.67
95.83 94.17 93.90 80.65 86.21
48.50 45.00 43.50 47.24 46.5
92.5 81.5 81.5 81.0
0.850 0.626 0.634 0.623
Almost perfect Substantial Substantial Substantial
STAT detected 6% less positive samples than the Superagglutination test and showed a sensitivity of 94.25% and a specificity of 68.14%. PPV of this test was found to be 69.49% and NPV was 93.90% (Table 2). Out of the 200 samples, 75 were detected as positive by iELISA (Table 1). ELISA detected 16.5% less positive samples than the Superagglutination test. Sensitivity of this test was calculated to be 74.47% and the specificity was found to be 95.24%. PPV of this test was 93.33% and NPV was found to be 80.65% (Table 2). Out of the 200 samples, 86 were detected as positive by CFT (Table 1). In the present study, the CFT detected 14.5% less positive samples than the Superagglutination test and showed a sensitivity of 82.8% and a specificity of 93.46% (Table 2). Statistical agreement between the Superagglutination test and other tests was determined. The kappa values and the observed proportions of agreements are presented in Table 2. The agreement between the Superagglutination test and RBPT was found to be almost perfect, whereas the agreement between the Superagglutination test and other tests was found to be substantial. False positive reactions in RBPT have been attributed to residual antibody activity from vaccination, cross reaction with certain bacteria and laboratory error. False negative reactions may arise during early incubation of disease or immediately after incubation (Radostits et al., 2000). False negative results may be due to a small clump size in sera with low titers of antibodies and false positive results due to the inability to differentiate non-specific aggregates of antigen particles alone from the true agglutinates comprising both antigen and antibody. Cross reacting antibodies have been detected by RBPT and false negative reactions are believed to occur mostly due to prozoning (OIE, 2004).
Fig. 1. The gross view of the superagglutination test on a glass slide by naked eye.
In the present study, Superagglutination test, developed by us recently (Saxena and kaur, 2013) by introducing novel modifications in the RBPT, has been evaluated for its usefulness in the diagnosis of bovine brucellosis by comparing its results with those of RBPT, STAT, iELISA, and CFT. The new test could detect more positive samples than RBPT, STAT, iELISA and CFT. In an earlier report (Akhtar et al., 2010) a low specificity of RBPT had been demonstrated. The high sensitivity of STAT observed in the present study can be attributed to its ability to detect antibody titer as low as 1:40 which was probably missed by other tests. In serum agglutination tests, cross reactions with various bacteria for example Yersinia enterocolitica O:9, E. coli O:157, Francisella tularensis, Salmonella urbana group N, Vibrio cholerae and Stenotrophomonas maltophilia have been reported (Corbel and Brinley-Morgan, 1984). The Complement Fixation test is technically challenging because a large number of reagents must be titrated daily and a large number of controls of all the reagents are required. It is an expensive test and is labor intensive. Other problems include the subjectivity of the interpretation of results, occasional direct activation of complement by serum (anti-complementary activity) and the inability of the test for use with hemolysed serum samples. However, since only IgG1 isotype of antibody fixes complement well, the test specificity is high (Poester et al., 2010). In an earlier study, Stemshorn and Forbes (1985) had demonstrated a sensitivity of 79% for CFT for the diagnosis of bovine brucellosis.
Fig. 2. Microscopic view showing a two colored agglutinate formed in the Superagglutination test with blue colored antibodies bound to the pink colored antigen particles.
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S.K. Chothe, H.M. Saxena / Journal of Microbiological Methods 97 (2014) 25–28
The highest sensitivity of all the tests observed with Superagglutination test in our study can be attributed to the fact that the number of false negative results with Superagglutination test was less when compared to the other tests. The anti-bovine IgG plays a role in increasing the sensitivity since if fewer antibodies against the infectious organism are present in the serum, the anti-globulin cross links these antibodies resulting in an increase in the clump size. Streptavidin binds to the biotinylted anti-globulin increasing the clump size further by up to 4 fold due to the presence of four binding sites for biotin on each molecule of avidin. The specificity of the Superagglutination test was found to be higher than that of RBPT and STAT. In the case of the Superagglutination test, the two colored true agglutinates could be very easily differentiated from non-specific one colored aggregates under the low power of a light microscope. The antibodies were blue in color due to the Coomassie blue dye and the antigen was pink in color due to the Rose Bengal dye. Each agglutinate had both the blue and the pink color, which aided in the differentiation of the true agglutinates from the non-specific aggregates of the antigen of pink color only. The antigen and antibodies which did not participate in agglutination reaction could be viewed under the microscope as aggregates of either blue or pink particles alone lying separately. Ordinary hand lens was generally found to be effective in visualizing the agglutination. The highest agreement with RBPT, combined with the higher specificity and sensitivity of Superagglutination test ensures that it can serve as a more efficient screening test than RBPT. The Superagglutination test had a higher sensitivity and a negative predictive value than the other serodiagnostic tests like RBPT, STAT, ELISA and CFT. Its specificity and PPV were found to be better than RBPT and STAT. The test can be used in the pen side diagnosis of bovine brucellosis with better results than the RBPT which is routinely used as a pen side test for brucellosis. ELISA is not a cost effective test when screening has to be performed on herds with a large number of animals. In such situations, Superagglutination test can offer an advantage of increased sensitivity of screening compared to RBPT and STAT. In our study, the agreement between Superagglutination test and RBPT was found to be higher than the agreement between Superagglutination test and other tests. This agreement combined with the higher specificity and sensitivity of Superagglutination test ensures that it can serve as a more efficient screening test than RBPT. The Superagglutination test showed the highest sensitivity of all the tests (95.88%) which can be attributed to the lesser number of false negative results obtained with Superagglutination test compared to the other tests. The anti-globulin played a role in increasing the sensitivity because if there are less number of antibodies against the infectious organism present in the serum, anti-globulin binds to these antibodies resulting in an increase in the clump size. Avidin binds to the biotinylated anti-globulin increasing the clump size further by up to 4 fold due to the four binding sites for biotin present on each molecule of avidin. The specificity of the Superagglutination test was however, found to be somewhat lower than that of iELISA and CFT. This may be due to the cross reactions of anti-globulin with non-specific or low affinity antibodies. Interestingly, the agglutinate in the case of Superagglutination test could be differentiated into two parts, the antibodies were blue in color due to the Coomassie blue dye and the antigen having a pink color due to the Rose Bengal dye. Each clump had both the blue and the pink colors, which aided in the identification of the true
agglutinates. This has not been reported by any other researcher as yet. Antigen and antibodies which did not participate in agglutination reaction could be viewed under the microscope as blue and pink particles lying separately or as single colored aggregates. The results obtained in our study suggest that Superagglutination test has a higher sensitivity than other serodiagnostic tests commonly used for brucellosis. The specificity of Superagglutination test was higher than RBPT and STAT but lesser than that of ELISA and CFT. The new test can be used as a screening test in the diagnosis of bovine brucellosis. As ELISA is not a cost effective test, Superagglutination test can offer the advantages of increased sensitivity, cost effectiveness, no requirement for skilled personnel and ease of performance. Conflict of interest The authors declare that there is no conflict of interest. The innovative methods in the Superagglutination test are patented (Inventor: H M Saxena; Assignees: 1. GADVASU, Ludhiana and 2. The Department of Biotechnology, Ministry of S&T, Govt. of India, New Delhi). The study was funded by GADVASU. SKC conducted the study as a Master's degree student under the supervision of HMS, a Professor of Immunology. HMS conceived the ideas underlying the Superagglutination test. He designed, supervised and coordinated the study, analyzed and interpreted the data and drafted the manuscript. SKC carried out the experimental work and statistical analysis. We are thankful to Dr. Mudit Chandra and Dr. Deepti Narang, Department of Veterinary Microbiology, GADVASU, Ludhiana for their help. References Akhtar, R., Chaudhry, Z.I., Shakoori, A.R., Ahmad, M.D., Aslam, A., 2010. Comparative efficacy of conventional diagnostic methods and evaluation of polymerase chain reaction for the diagnosis of bovine brucellosis. Vet. World 3, 53–56. Aulakh, H.K., Patil, P.K., Sharma, S., Kumar, H., Mahajan, V., Sandhu, K.S., 2008. A study on the epidemiology of bovine brucellosis in Punjab (India) using milk-ELISA. Acta Vet. 77, 393–399. Barroso, G.P., Pelayo, R.C., Extremera, G., 2002. Study of 1595 brucellosis cases in the Almeria province (1972–1998) based on epidemiological data from disease reporting. Rev. Clin. Esp. 202, 577–582. Corbel, M.J., Brinley-Morgan, W.J., 1984. Brucella. In: Krieg, N.R. (Ed.), Bergey's Manual of Systematic Bacteriology, Vol. I. Baltimore: Williams and Wilkins, pp. 377–388. Kollannur, J.D., Rathore, R., Chauhan, R.S., 2007. Epidemiology and Economics of Brucellosis in Animals and its Zoonotic Significance. ISAH Tartu, Estonia 466–468. Morgan, W.J., Mackinnon, D.T., Gill, K.P.W., Gower, S.G.M., Norris, P.I.W., 1978. Brucellosis diagnosis: standard laboratory techniques report. Series no. 1, Weybridge, England. OIE, 2004. Bovine brucellosis, Section 2.3, OIE Manual of Standards for Diagnostic Tests and Vaccines, 5th edition. OIE, Paris. Poester, F.P., Nielsen, K., Samartino, L.E., Yu, W.L., 2010. Diagnosis of brucellosis. Open Vet. Sci. J. 4, 46–60. Radostits, O.M., Gay, C.C., Blood, D.C., Hinchcliff, K.W., 2000. Diseases Caused by Brucella spp. A Textbook of the Disease of Cattle, Sheep, Pigs, Goats and Horses, Ninth edition. Harcourt Publishers Limited, London 881–890. Ruiz-Mesa, J.D., Gonzalez, J.S., Reguera, J.M., Martin, L., Palmero, S.L., Colmenero, J.D., 2005. Rose Bengal test: diagnostic yield and use for the rapid diagnosis of human brucellosis in emergency departments in endemic areas. Clin. Microbiol. Infect. 11, 221–225. Saxena, H.M., Kaur, P., 2013. A new superagglutination test to minimize false negative and false positive results common with plate/slide agglutination tests for the diagnosis of infectious diseases. Int. J. Trop. Dis. Health 3, 199–209. Smits, H.L., Kadri, S.M., 2005. Brucellosis in India: a deceptive infectious disease. Indian J. Med. Res. 122, 375–384. Stemshorn, B.W., Forbes, L.B., Eaglesome, M.D., Nielsen, K.H., Robertson, F.J., Samagh, B.S., 1985. A comparison of standard serological tests for the diagnosis of bovine brucellosis in Canada. Can. J. Comp. Med. 49, 391–394.
