Journal of Microbiological Methods 97 (2014) 25–28

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Journal of Microbiological Methods

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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 a b s t r a c t

Article history: Current agglutination tests occasionally yield false results. Superagglutination test reduced false results, had
Received 19 October 2013 higher sensitivity (95.88%) and negative predictive value (95.83%) than Rose Bengal plate test (RBPT), Standard
Received in revised form 8 December 2013 Tube Agglutination test (STAT), ELISA, and Complement Fixation test and specificity (89.32%) and positive pre-
Accepted 8 December 2013
dictive value (89.42%) higher than RBPT and STAT.
Available online 15 December 2013
© 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 or-
ganisms. It is of public health significance and causes huge economic
losses to the livestock sector due to reproductive losses in animals, abor-
tions, 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 dis-
appointingly low (Barroso et al., 2002). As a result, the positive predic-
tive 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 mod-
ifications of the RBPT are, therefore, required to get accurate results. We
have developed a novel Superagglutination test to enhance the sensitiv-
ity 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: hmsaxena@yahoo.com (H.M. Saxena).
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 select-
ed for sampling primarily based on the history of abortions in the herd
while normal healthy animals were sampled from the herds of the uni-
versity 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 se-
quence. 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 mag-
nification, were considered as positive, while absence of clear aggluti-
nates 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).

0167-7012/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.mimet.2013.12.005
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:
Number of True Positive cases
PPV ¼
Number of True Positive cases þ Number of False Positive cases
Number of True Negative cases
NPV ¼
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%, respec-
tively (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 dif-
ferentiated 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 de-
tected 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. STAT titer RBPT Superagglutination test iELISA CFT

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, 00 − − − −

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 00 − − − +
T 00 − + − −
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, 10 − − − −
AB,74(20/4/11), 3 (13/10/11)
AE 10 − − − +
AI, BC, BY 10 + + − −
AN, BW 10 + + + +
23(20/4/11), BP, 102(15/9/11), 7(20/4/11) 20 − − − +
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), 20 − − − −
3(1/3/11), T7, 6(13/10/11), T2061(25/11/11), J
T6 20 + − − −
CE, AV 20 − − + +
CN, AJ 20 + + − −
CX, AO 20 + + + +
7(13/10/11) 20 − + − −
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), 40 − − − −
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 40 − − − +
DQ, BE, AQ, 2413 40 + + + −
T 81 (25/11/11) 40 − + − +
T 2062 (25/11/11) 40 − + + +
H, W, X, AS, BT 40 + + + +
AL, AY, 2379 40 + + − +
CI 40 + + − −
2218, 2452, 2581, 101(15/9/11), I, AH, CR, DG, DJ, DO 80 + + + +
13(20/4/11), 79(20/4/11), 100(20/4/11) 80 − + − −
11(1/3/11), 19(1/3/11), 82(T 5/10/11) 80 − − − −
DU, BI 80 + + + −
12(13/10/11), CV, 16(20/4/11), BJ, BM 80 + + − −
P 80 + + − +
2308, 2417, 89(T 5/10/11), U, Y, AK, AW, CT, DB, DC 160 + + + +
103(15/9/11), 6(1/3/11), 9(1/3/11) 160 − + − −
T4 160 + − − −
86(T 5/10/11) 160 + − + +
CM, T5, AA 160 + + − +
R, BD 160 + + − −
BX, DP, CZ 160 + + + −
2362, Q, AM, AR, AU, BS 320 + + + +
82(20/4/11) 320 − + − +
BO, BQ 320 + + − +
2490, T2, AX, BZ, 25(20/4/11) 640 + + + +
2574, 2582 640 + + + −
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, N1280 + + + +
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) N1280 + + − +
T 86 (25/11/11) 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 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

Superagglutination 95.88 89.32 89.42 95.83 48.50

RBPT 93.33 88.18 86.60 94.17 45.00 92.5 0.850 Almost perfect
STAT 94.25 68.14 69.49 93.90 43.50 81.5 0.626 Substantial
iELISA 74.47 95.24 93.33 80.65 47.24 81.5 0.634 Substantial
CFT 82.80 93.46 91.67 86.21 46.5 81.0 0.623 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 sam-
ples 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 an-
tibody activity from vaccination, cross reaction with certain bacteria and
laboratory error. False negative reactions may arise during early incuba-
tion 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 reac-
tions are believed to occur mostly due to prozoning (OIE, 2004).
In the present study, Superagglutination test, developed by us re-
cently (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 at-
tributed to its ability to detect antibody titer as low as 1:40 which was
probably missed by other tests. In serum agglutination tests, cross reac-
tions 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 interpre-
tation 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 anti-
body fixes complement well, the test specificity is high (Poester et al.,
2010). In an earlier study, Stemshorn and Forbes (1985) had demon-
strated a sensitivity of 79% for CFT for the diagnosis of bovine
brucellosis.

Fig. 1. The gross view of the superagglutination test on a glass slide by naked eye. (For Fig. 2. Microscopic view showing a two colored agglutinate formed in the
interpretation of the references to color in this figure, the reader is referred to the web Superagglutination test with blue colored antibodies bound to the pink colored antigen
version of this article.) particles.
28 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 in-
fectious 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 an-
tibodies were blue in color due to the Coomassie blue dye and the anti-
gen 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 ag-
gregates 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 speci-
ficity 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 in-
creased 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 en-
sures 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 neg-
ative 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 or-
ganism present in the serum, anti-globulin binds to these antibodies
resulting in an increase in the clump size. Avidin binds to the biotinylat-
ed 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 anti-
bodies. 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 parti-
cles 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 innova-
tive 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 su-
pervision of HMS, a Professor of Immunology. HMS conceived the ideas
underlying the Superagglutination test. He designed, supervised and co-
ordinated 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.
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