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Direct agglutination test (DAT): improvement of biosafety for laboratory diagnosis of visceral leishmaniasis
Transactions of the Royal Society of Tropical Medicine and Hygiene 105 (2011) 414–416
Contents lists available at ScienceDirect
Transactions of the Royal Society of Tropical Medicine and Hygiene journal homepage: http://www.elsevier.com/locate/trstmh
Short Communication
Direct agglutination test (DAT): improvement of biosafety for laboratory diagnosis of visceral leishmaniasis Edward Oliveira, Soraya Wilke Saliba, Camila Filizzola de Andrade, Ana Rabello ∗ Clinical Research Laboratory, Centro de Pesquisas René Rachou–FIOCRUZ, Av. Augusto de Lima 1715, 30190-002 Belo Horizonte, Minas Gerais, Brazil
a r t i c l e
i n f o
Article history: Received 23 November 2010 Received in revised form 13 April 2011 Accepted 13 April 2011 Available online 25 May 2011 Keywords: Visceral leishmaniasis Serological diagnosis DAT Kaolin suspension NAC
a b s t r a c t In this study, the direct agglutination test (DAT), using 2-mercaptoethanol (2-ME), kaolin or N-acetylcysteine (NAC) as sample diluents, was used to assay 89 samples from visceral leishmaniasis (VL) patients and 130 samples from patients with other diseases and healthy individuals. Maintaining a cut-off of 1:100, the DAT assays with 2-ME, kaolin or NAC presented sensitivities of 94.4%, 95.5% and 100% (P = 0.09) and specificities of 99.2%, 100% and 97.7% (P = 0.17), respectively. Based on these results, we suggest that NAC can be used as a replacement for 2-ME in the DAT, increasing biosafety in the diagnosis of VL. © 2011 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.
1. Introduction We recently standardised, optimised and evaluated the direct agglutination test (DAT) using Leishmania chagasi promastigotes for diagnosis of visceral leishmaniasis (VL) in Brazil.1–3 Use of the DAT and rK39 tests has been indicated as the best option for VL diagnosis.4 However, the DAT requires use of 2-mercaptoethanol (2-ME) as a sample diluent to eliminate non-specific agglutination. 2-ME can injure the eyes, respiratory tract and nervous system, thus manipulation of 2-ME requires the use of a cabinet fume hood and personal protection equipment in order to prevent the release of toxic gas into the laboratory environment. To eliminate the use of 2-ME in the DAT assay, we have conducted experiments with alternative reagents such as kaolin and N-acetylcysteine (NAC) for the treatment of serum samples; both are non-toxic, odourless and nonharmful. NAC is used as an antioxidant and for fluidising of
∗ Corresponding author. Tel.: +55 31 3349 7782; fax: +55 31 3295 3115. E-mail address: ana@cpqrr.fiocruz.br (A. Rabello).
mucus in pharmaceutical formulations. On the other hand, kaolin is an aluminium silicate used in making paper, plastics, rubber, paints and many other products. Here we present the results of DAT assays in which 0.15 M 2-ME, 10% kaolin and 5 mM NAC were used as sample diluents. 2. Materials and methods 2.1. Antigen preparation The antigen was prepared from L. chagasi promastigotes recovered (MHOM/BR/2002/LPC-RPV) after 4 days of cultivation in biphasic NNN-LIT medium containing 20% heat-inactivated fetal bovine serum (FBS), following the modifications proposed previously.2 Initially, the suspension was centrifuged for 10 min at 25 ◦ C at 10 × g and was then incubated for 30 min in a biological oxygen demand incubator at 26 ◦ C (FANEM, São Paulo, Brazil) to separate dead from viable parasites. Next, the supernatant was carefully transferred into Falcon tubes and was washed three times with 0.01 M PBS (pH 7.4) supplemented with 2% bovine serum albumin (PBS-BSA). Then the parasites
0035-9203/$ – see front matter © 2011 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.trstmh.2011.04.010
E. Oliveira et al. / Transactions of the Royal Society of Tropical Medicine and Hygiene 105 (2011) 414–416
415
Table 1 Diagnostic validity of the direct agglutination test (DAT) using three sample diluents for patients with visceral leishmaniasis (VL) (n = 89) and for the control group (comprising other diseases, other infectious diseases and healthy individuals) (n = 130) DAT
Sensitivity (n = 89) [% (95% CI)]a
Specificity (n = 130) [% (95% CI)]b
PPV (n = 219) [% (95% CI)]c
NPV (n = 219) [% (95% CI)]d
DAT-Mod DAT-Kaolin DAT-NAC
94.4 (86.8–97.9) 95.5 (83.3–98.6) 100 (94.8–100)
99.2 (95.2–100) 100 (96.4–100) 97.7 (92.9–99.4)
98.8 (92.7–99.9) 100 (94.6–100) 96.7 (90.1–99.2)
96.3 (91.1–98.6) 97.0 (92.1–99.0) 100 (96.3–100)
PPV: positive predictive value; NPV: negative predictive value; DAT-Mod/Kaolin/NAC: direct agglutination test with 2-mercaptoethanol, kaolin and Nacetylcysteine, respectively. a Difference between sensitivity rates, P = 0.09. b Difference between specificity rates, P = 0.17. c Difference between PPVs: DAT-Mod vs. DAT-Kaolin, P = 0.25; DAT-Mod vs. DAT-NAC, P = 0.2; and DAT-Kaolin vs. DAT-NAC, P = 0.02. d Difference between NPVs: DAT-Mod vs. DAT-Kaolin, P = 0.79; DAT-Mod vs. DAT-NAC, P = 0.01; and DAT-Kaolin vs. DAT-NAC, P = 0.02.
were treated with 1.2% 2-ME (Sigma Chemical Co., St Louis, MO, USA) and fixed with 2% formaldehyde diluted in PBS (w/v) at 37 ◦ C under constant agitation. Next, the promastigotes were washed with PBS and stained with 0.1% Coomassie Brilliant Blue R-250 diluted in PBS. The concentration of promastigotes was adjusted to an optical density at 590 nm (OD590 ) of 0.500, which corresponds to a concentration of 2 × 108 parasites/ml. The antigen was stored in a refrigerator (4–8 ◦ C) and was used in DAT assays referred to as DAT-Mod, DAT-Kaolin and DAT-NAC as detailed below. 2.2. Performance of test 2.2.1. Direct agglutination test with 2-mercaptoethanol (DAT-Mod) Sera were diluted in PBS containing 1% heat-inactivated FBS and 0.15 M 2-ME solution and a two-fold dilution series was made from 1:100 to 1:102 400. Then, 50 l of DAT antigen suspension was added to each well of a Vshaped microtitre plate (Sarstedt Inc., Newton, NC, USA) containing 50 l of diluted serum. After 18 h of incubation at room temperature, the end titre was read as the dilution corresponding to the well immediately before the first well with a clear sharp-edged blue spot identical in size to the reaction-negative control. Appropriate negative and positive control samples with known DAT titres were included. The cut-off for interpretation of positive and negative results was 1:100, as determined previously.2 2.2.2. Direct agglutination test with kaolin (DAT-Kaolin) Sera were diluted 1:100 in PBS containing 10% kaolin, homogenised, incubated for 15 min at room temperature and centrifuged for 15 min at 1500 × g. Then, a two-fold dilution series in PBS containing 1% FBS was made from 1:100 to 1:102 400, considering the supernatant to be 1:100. Next, 50 l of DAT antigen suspension was added to each well of a V-shaped microtitre plate containing 50 l of diluted serum. The reading was taken after 18 h of incubation following the same criteria used for the DAT-Mod assay. 2.2.3. Direct agglutination test with N-acetylcysteine (DAT-NAC) Sera were diluted in PBS containing 1% FBS and 5 mM NAC and a two-fold dilution series was made from 1:100
to 1:102 400 following the same procedure used for the DAT-Mod assay. 2.3. Diagnostic validity The internal diagnostic validity of the DAT, using the three reagents as sample diluents, was evaluated using serum samples from the following individuals: 89 VL patients with a clinically and parasitologically confirmed diagnosis of VL; and 130 control serum samples, comprising 24 patients with a firm diagnosis of another disease such as neoplasia (41.7%), fungal, viral or bacterial infection (20.8%), autoimmune disease (16.7%), blood disease (12.5%) or hepatic disease (8.3%), 12 patients with schistosomiasis, 21 patients with Chagas disease, 30 patients with malaria, 10 patients with tegumentary leishmaniasis and 33 healthy individuals. Epi Info 6.04 software (CDC, Atlanta, GA, USA) was used to calculate the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV). The 2 test was used for comparison of the sensitivities, specificities, PPVs and NPVs and P-values of <0.05 were considered significant. 3. Results and discussion The DAT using 2-ME, kaolin or NAC as sample diluents did not show any statistically significant differences in sensitivity and specificity (P = 0.09 and P = 0.17, respectively). The DAT-Kaolin assay presented a higher PPV than DATNAC (100% vs. 96.7%; P = 0.02). DAT-NAC demonstrated a higher NPV than DAT-Mod (100% vs. 96.3%; P = 0.01) and DAT-Kaolin (100% vs. 97.0%; P = 0.02) (Table 1). In addition, dithiothreitol (DTT) was tested at a concentration of 0.15 M as a sample diluent but the results were not as good and the smell of the reagent was strong and unpleasant. Treatment of the samples with 10% kaolin suspension requires incubation and centrifugation steps before dilution of the supernatant, which makes its use less practical than the use of 2-ME or NAC. In relation to the cost, it was observed that 2-ME, kaolin and NAC are cheap and there are no differences in the costs associated with the use of any of these reagents. Thus, based on practicality and the results obtained, we suggest substitution of 2-ME by NAC as a sample diluent. This substitution can increase the biosafety of the DAT used for the diagnosis of VL.
416
E. Oliveira et al. / Transactions of the Royal Society of Tropical Medicine and Hygiene 105 (2011) 414–416
Authors’ contributions: EO and AR conceived and designed the study; SWS and CFA carried out the immunoassays and analysis and interpretation of the data; SWS, CFA and EO drafted the manuscript; AR and EO revised the manuscript critically for intellectual content. All authors read and approved the final manuscript. AR and EO are guarantors of the paper.
MG, Brazil, approved the use of stored serum samples (CEPSH/CPqRR no. 13/2003), in agreement with Resolution 357/05 of the National Health Council of the Ministry of Health, which regulates research involving human subjects in Brazil.
Acknowledgements: The authors are grateful to Amanda de Morais Prates for technical support.
1. Pedras MJ, de Gouvêa Viana LG, de Oliveira EJ, Rabello A. Comparative evaluation of direct agglutination test, rK39 and soluble antigen ELISA with IFAT for the diagnosis of visceral leishmaniasis. Trans R Soc Trop Med Hyg 2008;102:172–8. 2. Oliveira E, Pedras MJ, de Assis IEM, Rabello A. Improvement of direct agglutination test (DAT) for laboratory diagnosis of visceral leishmaniasis in Brazil. Trans R Soc Med Trop Hyg 2009;103: 1279–81. 3. de Assis TS, Braga ASC, Pedras MJ, Oliveira E, Barral A, de Siqueira IC, et al. Multi-centric prospective evaluation of rk39 rapid test and direct agglutination test for the diagnosis of visceral leishmaniasis in Brazil. Trans R Soc Med Trop Hyg 2011;105: 81–5. 4. WHO Expert Committee on the Control of the Leishmaniases. Control of the leishmaniases: report of a meeting of the WHO Expert Committee (No. 949) on the Control of Leishmaniases, Geneva, 22–26 March 2010. Geneva: World Health Organization, 2011. Technical Report Series No. 949.
Funding: Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq), Brasília, DF, Brazil (grants 350594/1995-3 and 311641/2009-1); Fundac¸ão de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), Belo Horizonte, MG, Brazil (CDS: 55013/02); and FIOCRUZ, Rio de Janeiro, RJ, Brazil (PDTIS RID06). Conflicts of interest: None declared. Ethical approval: The Ethical Review Board of Centro de Pesquisas René Rachou (CPqRR)—FIOCRUZ, Belo Horizonte,
References
Contents lists available at ScienceDirect
Transactions of the Royal Society of Tropical Medicine and Hygiene journal homepage: http://www.elsevier.com/locate/trstmh
Short Communication
Direct agglutination test (DAT): improvement of biosafety for laboratory diagnosis of visceral leishmaniasis Edward Oliveira, Soraya Wilke Saliba, Camila Filizzola de Andrade, Ana Rabello ∗ Clinical Research Laboratory, Centro de Pesquisas René Rachou–FIOCRUZ, Av. Augusto de Lima 1715, 30190-002 Belo Horizonte, Minas Gerais, Brazil
a r t i c l e
i n f o
Article history: Received 23 November 2010 Received in revised form 13 April 2011 Accepted 13 April 2011 Available online 25 May 2011 Keywords: Visceral leishmaniasis Serological diagnosis DAT Kaolin suspension NAC
a b s t r a c t In this study, the direct agglutination test (DAT), using 2-mercaptoethanol (2-ME), kaolin or N-acetylcysteine (NAC) as sample diluents, was used to assay 89 samples from visceral leishmaniasis (VL) patients and 130 samples from patients with other diseases and healthy individuals. Maintaining a cut-off of 1:100, the DAT assays with 2-ME, kaolin or NAC presented sensitivities of 94.4%, 95.5% and 100% (P = 0.09) and specificities of 99.2%, 100% and 97.7% (P = 0.17), respectively. Based on these results, we suggest that NAC can be used as a replacement for 2-ME in the DAT, increasing biosafety in the diagnosis of VL. © 2011 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.
1. Introduction We recently standardised, optimised and evaluated the direct agglutination test (DAT) using Leishmania chagasi promastigotes for diagnosis of visceral leishmaniasis (VL) in Brazil.1–3 Use of the DAT and rK39 tests has been indicated as the best option for VL diagnosis.4 However, the DAT requires use of 2-mercaptoethanol (2-ME) as a sample diluent to eliminate non-specific agglutination. 2-ME can injure the eyes, respiratory tract and nervous system, thus manipulation of 2-ME requires the use of a cabinet fume hood and personal protection equipment in order to prevent the release of toxic gas into the laboratory environment. To eliminate the use of 2-ME in the DAT assay, we have conducted experiments with alternative reagents such as kaolin and N-acetylcysteine (NAC) for the treatment of serum samples; both are non-toxic, odourless and nonharmful. NAC is used as an antioxidant and for fluidising of
∗ Corresponding author. Tel.: +55 31 3349 7782; fax: +55 31 3295 3115. E-mail address: ana@cpqrr.fiocruz.br (A. Rabello).
mucus in pharmaceutical formulations. On the other hand, kaolin is an aluminium silicate used in making paper, plastics, rubber, paints and many other products. Here we present the results of DAT assays in which 0.15 M 2-ME, 10% kaolin and 5 mM NAC were used as sample diluents. 2. Materials and methods 2.1. Antigen preparation The antigen was prepared from L. chagasi promastigotes recovered (MHOM/BR/2002/LPC-RPV) after 4 days of cultivation in biphasic NNN-LIT medium containing 20% heat-inactivated fetal bovine serum (FBS), following the modifications proposed previously.2 Initially, the suspension was centrifuged for 10 min at 25 ◦ C at 10 × g and was then incubated for 30 min in a biological oxygen demand incubator at 26 ◦ C (FANEM, São Paulo, Brazil) to separate dead from viable parasites. Next, the supernatant was carefully transferred into Falcon tubes and was washed three times with 0.01 M PBS (pH 7.4) supplemented with 2% bovine serum albumin (PBS-BSA). Then the parasites
0035-9203/$ – see front matter © 2011 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.trstmh.2011.04.010
E. Oliveira et al. / Transactions of the Royal Society of Tropical Medicine and Hygiene 105 (2011) 414–416
415
Table 1 Diagnostic validity of the direct agglutination test (DAT) using three sample diluents for patients with visceral leishmaniasis (VL) (n = 89) and for the control group (comprising other diseases, other infectious diseases and healthy individuals) (n = 130) DAT
Sensitivity (n = 89) [% (95% CI)]a
Specificity (n = 130) [% (95% CI)]b
PPV (n = 219) [% (95% CI)]c
NPV (n = 219) [% (95% CI)]d
DAT-Mod DAT-Kaolin DAT-NAC
94.4 (86.8–97.9) 95.5 (83.3–98.6) 100 (94.8–100)
99.2 (95.2–100) 100 (96.4–100) 97.7 (92.9–99.4)
98.8 (92.7–99.9) 100 (94.6–100) 96.7 (90.1–99.2)
96.3 (91.1–98.6) 97.0 (92.1–99.0) 100 (96.3–100)
PPV: positive predictive value; NPV: negative predictive value; DAT-Mod/Kaolin/NAC: direct agglutination test with 2-mercaptoethanol, kaolin and Nacetylcysteine, respectively. a Difference between sensitivity rates, P = 0.09. b Difference between specificity rates, P = 0.17. c Difference between PPVs: DAT-Mod vs. DAT-Kaolin, P = 0.25; DAT-Mod vs. DAT-NAC, P = 0.2; and DAT-Kaolin vs. DAT-NAC, P = 0.02. d Difference between NPVs: DAT-Mod vs. DAT-Kaolin, P = 0.79; DAT-Mod vs. DAT-NAC, P = 0.01; and DAT-Kaolin vs. DAT-NAC, P = 0.02.
were treated with 1.2% 2-ME (Sigma Chemical Co., St Louis, MO, USA) and fixed with 2% formaldehyde diluted in PBS (w/v) at 37 ◦ C under constant agitation. Next, the promastigotes were washed with PBS and stained with 0.1% Coomassie Brilliant Blue R-250 diluted in PBS. The concentration of promastigotes was adjusted to an optical density at 590 nm (OD590 ) of 0.500, which corresponds to a concentration of 2 × 108 parasites/ml. The antigen was stored in a refrigerator (4–8 ◦ C) and was used in DAT assays referred to as DAT-Mod, DAT-Kaolin and DAT-NAC as detailed below. 2.2. Performance of test 2.2.1. Direct agglutination test with 2-mercaptoethanol (DAT-Mod) Sera were diluted in PBS containing 1% heat-inactivated FBS and 0.15 M 2-ME solution and a two-fold dilution series was made from 1:100 to 1:102 400. Then, 50 l of DAT antigen suspension was added to each well of a Vshaped microtitre plate (Sarstedt Inc., Newton, NC, USA) containing 50 l of diluted serum. After 18 h of incubation at room temperature, the end titre was read as the dilution corresponding to the well immediately before the first well with a clear sharp-edged blue spot identical in size to the reaction-negative control. Appropriate negative and positive control samples with known DAT titres were included. The cut-off for interpretation of positive and negative results was 1:100, as determined previously.2 2.2.2. Direct agglutination test with kaolin (DAT-Kaolin) Sera were diluted 1:100 in PBS containing 10% kaolin, homogenised, incubated for 15 min at room temperature and centrifuged for 15 min at 1500 × g. Then, a two-fold dilution series in PBS containing 1% FBS was made from 1:100 to 1:102 400, considering the supernatant to be 1:100. Next, 50 l of DAT antigen suspension was added to each well of a V-shaped microtitre plate containing 50 l of diluted serum. The reading was taken after 18 h of incubation following the same criteria used for the DAT-Mod assay. 2.2.3. Direct agglutination test with N-acetylcysteine (DAT-NAC) Sera were diluted in PBS containing 1% FBS and 5 mM NAC and a two-fold dilution series was made from 1:100
to 1:102 400 following the same procedure used for the DAT-Mod assay. 2.3. Diagnostic validity The internal diagnostic validity of the DAT, using the three reagents as sample diluents, was evaluated using serum samples from the following individuals: 89 VL patients with a clinically and parasitologically confirmed diagnosis of VL; and 130 control serum samples, comprising 24 patients with a firm diagnosis of another disease such as neoplasia (41.7%), fungal, viral or bacterial infection (20.8%), autoimmune disease (16.7%), blood disease (12.5%) or hepatic disease (8.3%), 12 patients with schistosomiasis, 21 patients with Chagas disease, 30 patients with malaria, 10 patients with tegumentary leishmaniasis and 33 healthy individuals. Epi Info 6.04 software (CDC, Atlanta, GA, USA) was used to calculate the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV). The 2 test was used for comparison of the sensitivities, specificities, PPVs and NPVs and P-values of <0.05 were considered significant. 3. Results and discussion The DAT using 2-ME, kaolin or NAC as sample diluents did not show any statistically significant differences in sensitivity and specificity (P = 0.09 and P = 0.17, respectively). The DAT-Kaolin assay presented a higher PPV than DATNAC (100% vs. 96.7%; P = 0.02). DAT-NAC demonstrated a higher NPV than DAT-Mod (100% vs. 96.3%; P = 0.01) and DAT-Kaolin (100% vs. 97.0%; P = 0.02) (Table 1). In addition, dithiothreitol (DTT) was tested at a concentration of 0.15 M as a sample diluent but the results were not as good and the smell of the reagent was strong and unpleasant. Treatment of the samples with 10% kaolin suspension requires incubation and centrifugation steps before dilution of the supernatant, which makes its use less practical than the use of 2-ME or NAC. In relation to the cost, it was observed that 2-ME, kaolin and NAC are cheap and there are no differences in the costs associated with the use of any of these reagents. Thus, based on practicality and the results obtained, we suggest substitution of 2-ME by NAC as a sample diluent. This substitution can increase the biosafety of the DAT used for the diagnosis of VL.
416
E. Oliveira et al. / Transactions of the Royal Society of Tropical Medicine and Hygiene 105 (2011) 414–416
Authors’ contributions: EO and AR conceived and designed the study; SWS and CFA carried out the immunoassays and analysis and interpretation of the data; SWS, CFA and EO drafted the manuscript; AR and EO revised the manuscript critically for intellectual content. All authors read and approved the final manuscript. AR and EO are guarantors of the paper.
MG, Brazil, approved the use of stored serum samples (CEPSH/CPqRR no. 13/2003), in agreement with Resolution 357/05 of the National Health Council of the Ministry of Health, which regulates research involving human subjects in Brazil.
Acknowledgements: The authors are grateful to Amanda de Morais Prates for technical support.
1. Pedras MJ, de Gouvêa Viana LG, de Oliveira EJ, Rabello A. Comparative evaluation of direct agglutination test, rK39 and soluble antigen ELISA with IFAT for the diagnosis of visceral leishmaniasis. Trans R Soc Trop Med Hyg 2008;102:172–8. 2. Oliveira E, Pedras MJ, de Assis IEM, Rabello A. Improvement of direct agglutination test (DAT) for laboratory diagnosis of visceral leishmaniasis in Brazil. Trans R Soc Med Trop Hyg 2009;103: 1279–81. 3. de Assis TS, Braga ASC, Pedras MJ, Oliveira E, Barral A, de Siqueira IC, et al. Multi-centric prospective evaluation of rk39 rapid test and direct agglutination test for the diagnosis of visceral leishmaniasis in Brazil. Trans R Soc Med Trop Hyg 2011;105: 81–5. 4. WHO Expert Committee on the Control of the Leishmaniases. Control of the leishmaniases: report of a meeting of the WHO Expert Committee (No. 949) on the Control of Leishmaniases, Geneva, 22–26 March 2010. Geneva: World Health Organization, 2011. Technical Report Series No. 949.
Funding: Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq), Brasília, DF, Brazil (grants 350594/1995-3 and 311641/2009-1); Fundac¸ão de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), Belo Horizonte, MG, Brazil (CDS: 55013/02); and FIOCRUZ, Rio de Janeiro, RJ, Brazil (PDTIS RID06). Conflicts of interest: None declared. Ethical approval: The Ethical Review Board of Centro de Pesquisas René Rachou (CPqRR)—FIOCRUZ, Belo Horizonte,
References