Journal of Microbiological Methods 112 (2015) 1–2
Contents lists available at ScienceDirect
Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth
Note
Development and evaluation of a latex agglutination test for the rapid serodiagnosis of tularemia Waldemar Rastawicki ⁎, Natalia Rokosz-Chudziak, Anna Chróst, Rafał Gierczyński Department of Bacteriology, National Institute of Public Health‐National Institute of Hygiene, Warsaw, Poland
a r t i c l e
i n f o
Article history: Received 11 February 2015 Received in revised form 25 February 2015 Accepted 25 February 2015 Available online 26 February 2015
a b s t r a c t A latex agglutination test (LAT) was developed for a rapid detection of antibodies against Francisella tularensis. The assay is performed by mixing serum with antigen-coated latex beads and read within 5 min. Developed LAT has been proved to be a specific, sensitive, fast, easy-to-perform and cost-efficient tool for the routine diagnosis of tularemia. © 2015 Elsevier B.V. All rights reserved.
Keywords: Latex agglutination test F. tularensis Tularemia Serodiagnosis
Tularemia is a highly infectious disease caused by an intracellular Gram-negative bacterium, Francisella tularensis. The clinical presentation in humans depends on the route of infection and varies from relatively mild skin lesions and lymphadenopathy to severe pneumonia and septicemia (Eliasson et al., 2006; Rimawi et al., 2014). The laboratory diagnosis of tularemia is based on bacteriological, molecular and serological investigations (Harik, 2013; Hepburn and Simpson, 2008). The commonly utilized serological assays for tularemia are enzymelinked immunosorbent assay (ELISA) and tube agglutination test. Indirect ELISA is particularly appropriate for seroepidemiological studies because it is sensitive and specific but is expensive, laborious and requires enzyme-conjugated secondary antibodies against immunoglobulin of respective animal species (Porsch-Ozcurumez et al., 2004). Tube agglutination test seems to be an appropriate assay for routine serodiagnosis of tularemia because it is cheap, easy to perform and applicable to human and various animal species. However, results of tube agglutination test are usually possible to obtain after 18 h of incubation (Ransmeier and Ewing, 1941). In the present study, we developed the latex agglutination test for the rapid detection of antibodies against F. tularensis and evaluated this test by comparing it with tube agglutination test, in-house ELISA and commercial Serion ELISA. A total number of 113 sera obtained from patients suspected in clinical investigations for tularemia were tested. Seventy seven (68.1%) out of them were positive and 36 (31.9%) were negative in routine serodiagnosis of tularemia conducted in our laboratory by standard tube agglutination test or/and by in-house ELISA. Fifty six of the sera were also tested by commercial Serion ELISA classic Francisella tularensis ⁎ Corresponding author. E-mail address: [email protected] (W. Rastawicki).
http://dx.doi.org/10.1016/j.mimet.2015.02.012 0167-7012/© 2015 Elsevier B.V. All rights reserved.
IgG/IgM (Institut Virion/Serion GmbH, Würzburg, Germany, cat. number ESR 142G/M). In order to assess potential cross-reactivity, 273 sera of humans without known history of tularemia were tested by LAT, including 134 sera from blood donors and 139 sera from a control group of patients with diagnosed other than tularemia bacterial infections (Yersinia spp. 86 patients; Salmonella spp. 13 patients; VTEC 8 patients; Borrelia burgdorferi 11 patients; Legionella pneumophila 11 patients and Campylobacter jejuni 10 patients). The antigen for the latex agglutination test was prepared as described previously for the in-house ELISA F. tularensis (Rastawicki et al., 2005). Briefly, the formalin-killed F. tularensis suspension, purchased from Serum and Vaccine Manufactory BIOMED S.A. Cracow, Poland was sonicated for 10 min at 18–20 KC in a MSE Ultrasonic Disintegration machine and centrifuged at 30,000 ×g for 30 min. The pellet was discarded, and the supernatant was then diluted to the titer of activity (6 μg/ml) in glycine-buffered saline (pH 8.2), added to an equal volume of 1% suspension of 0.81 μm latex particles, and incubated for 2 h at 37 °C. After incubation, sensitized latex particles were recovered by centrifugation, washed twice with glycine-buffered saline and finally diluted to 1% with glycine-buffered saline (pH 8.2) containing 0.1% sodium azide and 0.3% of BSA. Latex particles sensitized with antigens of F. tularensis and particles sensitized with albumin, bovine (Sigma Chemical Co., USA) — control latex reagent, were mixed for 5 min with the same volume (25 μl) of sera diluted in phosphate-buffered saline (PBS) from 1: 12.5 to 1: 1600 on a glass plate. Agglutination titers were expressed as reciprocals of the highest serum dilution showing agglutination of at least 50% of the latex particles. Titers of 25 or higher were considered LAT positive. Tube agglutination was performed according to the classical Widal reaction as described previously (Ransmeier and Ewing, 1941). To serial
2
W. Rastawicki et al. / Journal of Microbiological Methods 112 (2015) 1–2
Table 1 Results of the latex agglutination test (LAT) against different serum samples. Patients/number of tested sera
Number of positive results by latex agglutination test (titers)
Suspected for tularemia, positive by tube agglutination test or/and in-house ELISA N = 77 Suspected for tularemia, negative by tube agglutination test or/and ELISA N = 36 Clinically healthy blood donors N = 134 Patients positive for different bacterial infections N = 139
twofold dilutions of serum in PBS, equal amounts (50 μl) of a standardized suspension of formalin-killed F. tularensis (Bioveta, Czech Republic), were added. Agglutination was recorded after incubation for 18 h at 37 °C. Titers were expressed as reciprocals of the last serum dilution giving visible agglutination. A titer of 1/25 or above was regarded as a positive result. The in-house ELISA was carried out using 96-well Maxisorp polystyrene microtiter plates (Nunc, Denmark) as described previously (Rastawicki et al., 2005). The secondary antibody was a horseradish peroxidase-conjugated goat anti-human IgM/IgG/ IgA antibodies (Rockland, USA). The commercial Serion ELISA classic Francisella tularensis IgG/IgM was performed and interpreted according to the manufacturer's instruction. Results of the developed latex agglutination test with respectively titration of serum samples for antibody against F. tularensis are presented in Table 1. Among the 77 serum samples obtained from patients with tularemia, confirmed by tube agglutination test and/or in-house ELISA, 76 (98.7%) were positive by latex agglutination test. No positive reaction was observed by the LAT in the 36 sera of patients suspected for tularemia but negative by routine serodiagnosis, 134 sera from blood donors and 139 sera obtained from the control group of patients. None of these control samples reacted even at a titer 12.5. In the present study there was also no positive reaction with control latex reagent. Comparison between the results of LAT, tube agglutination test, inhouse ELISA and commercial Serion ELISA to detect antibodies against F. tularensis showed the 100% specificity and very high sensitivity (100%, 98.3% and 97.4%, respectively) of the developed rapid latex assay (Table 2). In all of the cases of positive results of ELISA both IgM and IgG classes of antibodies were observed in a significant diagnostic level. Only one serum sample, that was positive by in-house ELISA and Serion ELISA, gave negative result by the LAT. This sample was also negative by tube agglutination test. Detailed case analysis showed that the LAT-negative serum sample was collected from a patient in the convalescent phase of tularemia and the results of ELISA's were, especially in the IgM class of antibody, low positive. Although serology is frequently preferred in laboratory diagnosis of tularemia some patients may have cross-reacting antibodies due to infections with other bacteria, such as Yersinia, Brucella, Salmonella and Legionella (Foley and Nieto, 2010). However, in the presented study we did not observe positive latex agglutination reactions when tested
Table 2 Comparison between the results of the latex agglutination test (LAT), tube agglutination test, in-house ELISA and commercial Serion ELISA to detect antibodies against F. tularensis. Test
LAT + LAT− Total Sensitivity Specificity
Tube agglutination test
In-house ELISA
Serion ELISA
(+)
(−)
(+)
(−)
(+)
(−)
62 – 62 100% 100%
– 36 36
58 1 59 98.3% 100%
– 18 18
37 1 38 97.4% 100%
– 18 18
12,5
25
50
100
200
400
800
1600
Total
–
3
3
25
19
19
4
3
76
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
the sera from the control group of patients with bacterial infections other than tularemia as well as clinically healthy blood donors. It is worth noting that the cross-reactions were not observed also in our previous study when serum samples from patients with yersiniosis were tested by tube agglutination test and in-house ELISA (Rastawicki and Wolaniuk, 2013). Moreover, our results also showed a high sensitivity of the latex agglutination test for tularemia detection, even when compared to ELISA, that is generally considered as much more sensitive test than agglutination methods. For observed in our study a high usefulness of LAT in serodiagnosis of tularemia may be responsible a strong, immunospecific and long-lasting humoral immunity during F. tularensis infection (Koskela and Salminen, 1985; Tärnvik, 1989). In tularemia, antibodies usually appear within first two weeks after the onset of the symptoms and in a significant proportion of patients, persist in detectable quantities for many months (Eliasson et al., 2008; Syrjälä et al., 1986). From a clinical point of view the early recognition of the disease is very important for initiating appropriate treatment to avoid severe complications. The clear advantage of the latex agglutination test is that it gives a result within 5 min. compared with a few hours for ELISA and 18 h for tube agglutination test. In conclusion, the latex agglutination test developed in our laboratory is a specific, sensitive, fast, easy-to-perform and cost-efficient tool for the detection of antibodies to F. tularensis. It may be used as a screening test in the routine diagnosis of tularemia, particularly useful in a small, mobile laboratories. References Eliasson, H., Broman, T., Forsman, M., Bäck, E., 2006. Tularemia: current epidemiology and disease management. Infect. Dis. Clin. N. Am. 20, 289–311. Eliasson, H., Olcén, P., Sjöstedt, A., Jurstrand, M., Bäck, E., Andersson, S., 2008. Kinetics of the immune response associated with tularemia: comparison of an enzyme-linked immunosorbent assay, a tube agglutination test, and a novel whole-blood lymphocyte stimulation test. Clin. Vaccine Immunol. 15, 1238–1243. Foley, J.E., Nieto, N.C., 2010. Tularemia. Vet. Microbiol. 140, 332–338. Harik, N.S., 2013. Tularemia: epidemiology, diagnosis, and treatment. Pediatr. Ann. 42, 288–292. Hepburn, M.J., Simpson, A.J., 2008. Tularemia: current diagnosis and treatment options. Expert. Rev. Anti Infect. Ther. 6, 231–240. Koskela, P., Salminen, A., 1985. Humoral immunity against Francisella tularensis after natural infection. J. Clin. Microbiol. 22, 973–979. Porsch-Ozcurumez, M., Kischel, N., Priebe, H., Splettstosser, W., Finke, E.J., Grunow, R., 2004. Comparison of enzyme-linked immunosorbent assay, Western blotting, microagglutination, indirect immunofluorescence assay, and flow cytometry for serological diagnosis of tularemia. Clin. Diagn. Lab. Immunol. 11, 1008–1015. Ransmeier, J.C., Ewing, C.L., 1941. The agglutination reaction in tularemia. J. Infect. 69, 193–205. Rastawicki, W., Wolaniuk, N., 2013. Comparison of usefulness of commercial ELISA Virion/ Serion, home-made ELISA and tube agglutination test in serodiagnosis of tularemia. Med. Dosw. Mikrobiol. 65, 255–261. Rastawicki, W., Jagielski, M., Gierczyński, R., 2005. Evaluation of the enzyme-linked immunosorbent assay for the serodiagnosis of tularemia. Med. Dosw. Mikrobiol. 57, 425–429. Rimawi, R.H., Shah, K.B., Chowdhary, R.A., Cook, P.P., 2014. Hunting for tularemia — a review of cases in North Carolina. http://dx.doi.org/10.1111/zph.12114. Syrjälä, H., Koskela, P., Ripatti, T., Salminen, A., Herva, E., 1986. Agglutination and ELISA Methods in the Diagnosis of Tularemia in Different Clinical Forms and Severities of the Disease. 153, 142–145. Tärnvik, A., 1989. Nature of protective immunity to Francisella tularensis. Rev. Infect. Dis. 11, 440–451.
Contents lists available at ScienceDirect
Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth
Note
Development and evaluation of a latex agglutination test for the rapid serodiagnosis of tularemia Waldemar Rastawicki ⁎, Natalia Rokosz-Chudziak, Anna Chróst, Rafał Gierczyński Department of Bacteriology, National Institute of Public Health‐National Institute of Hygiene, Warsaw, Poland
a r t i c l e
i n f o
Article history: Received 11 February 2015 Received in revised form 25 February 2015 Accepted 25 February 2015 Available online 26 February 2015
a b s t r a c t A latex agglutination test (LAT) was developed for a rapid detection of antibodies against Francisella tularensis. The assay is performed by mixing serum with antigen-coated latex beads and read within 5 min. Developed LAT has been proved to be a specific, sensitive, fast, easy-to-perform and cost-efficient tool for the routine diagnosis of tularemia. © 2015 Elsevier B.V. All rights reserved.
Keywords: Latex agglutination test F. tularensis Tularemia Serodiagnosis
Tularemia is a highly infectious disease caused by an intracellular Gram-negative bacterium, Francisella tularensis. The clinical presentation in humans depends on the route of infection and varies from relatively mild skin lesions and lymphadenopathy to severe pneumonia and septicemia (Eliasson et al., 2006; Rimawi et al., 2014). The laboratory diagnosis of tularemia is based on bacteriological, molecular and serological investigations (Harik, 2013; Hepburn and Simpson, 2008). The commonly utilized serological assays for tularemia are enzymelinked immunosorbent assay (ELISA) and tube agglutination test. Indirect ELISA is particularly appropriate for seroepidemiological studies because it is sensitive and specific but is expensive, laborious and requires enzyme-conjugated secondary antibodies against immunoglobulin of respective animal species (Porsch-Ozcurumez et al., 2004). Tube agglutination test seems to be an appropriate assay for routine serodiagnosis of tularemia because it is cheap, easy to perform and applicable to human and various animal species. However, results of tube agglutination test are usually possible to obtain after 18 h of incubation (Ransmeier and Ewing, 1941). In the present study, we developed the latex agglutination test for the rapid detection of antibodies against F. tularensis and evaluated this test by comparing it with tube agglutination test, in-house ELISA and commercial Serion ELISA. A total number of 113 sera obtained from patients suspected in clinical investigations for tularemia were tested. Seventy seven (68.1%) out of them were positive and 36 (31.9%) were negative in routine serodiagnosis of tularemia conducted in our laboratory by standard tube agglutination test or/and by in-house ELISA. Fifty six of the sera were also tested by commercial Serion ELISA classic Francisella tularensis ⁎ Corresponding author. E-mail address: [email protected] (W. Rastawicki).
http://dx.doi.org/10.1016/j.mimet.2015.02.012 0167-7012/© 2015 Elsevier B.V. All rights reserved.
IgG/IgM (Institut Virion/Serion GmbH, Würzburg, Germany, cat. number ESR 142G/M). In order to assess potential cross-reactivity, 273 sera of humans without known history of tularemia were tested by LAT, including 134 sera from blood donors and 139 sera from a control group of patients with diagnosed other than tularemia bacterial infections (Yersinia spp. 86 patients; Salmonella spp. 13 patients; VTEC 8 patients; Borrelia burgdorferi 11 patients; Legionella pneumophila 11 patients and Campylobacter jejuni 10 patients). The antigen for the latex agglutination test was prepared as described previously for the in-house ELISA F. tularensis (Rastawicki et al., 2005). Briefly, the formalin-killed F. tularensis suspension, purchased from Serum and Vaccine Manufactory BIOMED S.A. Cracow, Poland was sonicated for 10 min at 18–20 KC in a MSE Ultrasonic Disintegration machine and centrifuged at 30,000 ×g for 30 min. The pellet was discarded, and the supernatant was then diluted to the titer of activity (6 μg/ml) in glycine-buffered saline (pH 8.2), added to an equal volume of 1% suspension of 0.81 μm latex particles, and incubated for 2 h at 37 °C. After incubation, sensitized latex particles were recovered by centrifugation, washed twice with glycine-buffered saline and finally diluted to 1% with glycine-buffered saline (pH 8.2) containing 0.1% sodium azide and 0.3% of BSA. Latex particles sensitized with antigens of F. tularensis and particles sensitized with albumin, bovine (Sigma Chemical Co., USA) — control latex reagent, were mixed for 5 min with the same volume (25 μl) of sera diluted in phosphate-buffered saline (PBS) from 1: 12.5 to 1: 1600 on a glass plate. Agglutination titers were expressed as reciprocals of the highest serum dilution showing agglutination of at least 50% of the latex particles. Titers of 25 or higher were considered LAT positive. Tube agglutination was performed according to the classical Widal reaction as described previously (Ransmeier and Ewing, 1941). To serial
2
W. Rastawicki et al. / Journal of Microbiological Methods 112 (2015) 1–2
Table 1 Results of the latex agglutination test (LAT) against different serum samples. Patients/number of tested sera
Number of positive results by latex agglutination test (titers)
Suspected for tularemia, positive by tube agglutination test or/and in-house ELISA N = 77 Suspected for tularemia, negative by tube agglutination test or/and ELISA N = 36 Clinically healthy blood donors N = 134 Patients positive for different bacterial infections N = 139
twofold dilutions of serum in PBS, equal amounts (50 μl) of a standardized suspension of formalin-killed F. tularensis (Bioveta, Czech Republic), were added. Agglutination was recorded after incubation for 18 h at 37 °C. Titers were expressed as reciprocals of the last serum dilution giving visible agglutination. A titer of 1/25 or above was regarded as a positive result. The in-house ELISA was carried out using 96-well Maxisorp polystyrene microtiter plates (Nunc, Denmark) as described previously (Rastawicki et al., 2005). The secondary antibody was a horseradish peroxidase-conjugated goat anti-human IgM/IgG/ IgA antibodies (Rockland, USA). The commercial Serion ELISA classic Francisella tularensis IgG/IgM was performed and interpreted according to the manufacturer's instruction. Results of the developed latex agglutination test with respectively titration of serum samples for antibody against F. tularensis are presented in Table 1. Among the 77 serum samples obtained from patients with tularemia, confirmed by tube agglutination test and/or in-house ELISA, 76 (98.7%) were positive by latex agglutination test. No positive reaction was observed by the LAT in the 36 sera of patients suspected for tularemia but negative by routine serodiagnosis, 134 sera from blood donors and 139 sera obtained from the control group of patients. None of these control samples reacted even at a titer 12.5. In the present study there was also no positive reaction with control latex reagent. Comparison between the results of LAT, tube agglutination test, inhouse ELISA and commercial Serion ELISA to detect antibodies against F. tularensis showed the 100% specificity and very high sensitivity (100%, 98.3% and 97.4%, respectively) of the developed rapid latex assay (Table 2). In all of the cases of positive results of ELISA both IgM and IgG classes of antibodies were observed in a significant diagnostic level. Only one serum sample, that was positive by in-house ELISA and Serion ELISA, gave negative result by the LAT. This sample was also negative by tube agglutination test. Detailed case analysis showed that the LAT-negative serum sample was collected from a patient in the convalescent phase of tularemia and the results of ELISA's were, especially in the IgM class of antibody, low positive. Although serology is frequently preferred in laboratory diagnosis of tularemia some patients may have cross-reacting antibodies due to infections with other bacteria, such as Yersinia, Brucella, Salmonella and Legionella (Foley and Nieto, 2010). However, in the presented study we did not observe positive latex agglutination reactions when tested
Table 2 Comparison between the results of the latex agglutination test (LAT), tube agglutination test, in-house ELISA and commercial Serion ELISA to detect antibodies against F. tularensis. Test
LAT + LAT− Total Sensitivity Specificity
Tube agglutination test
In-house ELISA
Serion ELISA
(+)
(−)
(+)
(−)
(+)
(−)
62 – 62 100% 100%
– 36 36
58 1 59 98.3% 100%
– 18 18
37 1 38 97.4% 100%
– 18 18
12,5
25
50
100
200
400
800
1600
Total
–
3
3
25
19
19
4
3
76
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
the sera from the control group of patients with bacterial infections other than tularemia as well as clinically healthy blood donors. It is worth noting that the cross-reactions were not observed also in our previous study when serum samples from patients with yersiniosis were tested by tube agglutination test and in-house ELISA (Rastawicki and Wolaniuk, 2013). Moreover, our results also showed a high sensitivity of the latex agglutination test for tularemia detection, even when compared to ELISA, that is generally considered as much more sensitive test than agglutination methods. For observed in our study a high usefulness of LAT in serodiagnosis of tularemia may be responsible a strong, immunospecific and long-lasting humoral immunity during F. tularensis infection (Koskela and Salminen, 1985; Tärnvik, 1989). In tularemia, antibodies usually appear within first two weeks after the onset of the symptoms and in a significant proportion of patients, persist in detectable quantities for many months (Eliasson et al., 2008; Syrjälä et al., 1986). From a clinical point of view the early recognition of the disease is very important for initiating appropriate treatment to avoid severe complications. The clear advantage of the latex agglutination test is that it gives a result within 5 min. compared with a few hours for ELISA and 18 h for tube agglutination test. In conclusion, the latex agglutination test developed in our laboratory is a specific, sensitive, fast, easy-to-perform and cost-efficient tool for the detection of antibodies to F. tularensis. It may be used as a screening test in the routine diagnosis of tularemia, particularly useful in a small, mobile laboratories. References Eliasson, H., Broman, T., Forsman, M., Bäck, E., 2006. Tularemia: current epidemiology and disease management. Infect. Dis. Clin. N. Am. 20, 289–311. Eliasson, H., Olcén, P., Sjöstedt, A., Jurstrand, M., Bäck, E., Andersson, S., 2008. Kinetics of the immune response associated with tularemia: comparison of an enzyme-linked immunosorbent assay, a tube agglutination test, and a novel whole-blood lymphocyte stimulation test. Clin. Vaccine Immunol. 15, 1238–1243. Foley, J.E., Nieto, N.C., 2010. Tularemia. Vet. Microbiol. 140, 332–338. Harik, N.S., 2013. Tularemia: epidemiology, diagnosis, and treatment. Pediatr. Ann. 42, 288–292. Hepburn, M.J., Simpson, A.J., 2008. Tularemia: current diagnosis and treatment options. Expert. Rev. Anti Infect. Ther. 6, 231–240. Koskela, P., Salminen, A., 1985. Humoral immunity against Francisella tularensis after natural infection. J. Clin. Microbiol. 22, 973–979. Porsch-Ozcurumez, M., Kischel, N., Priebe, H., Splettstosser, W., Finke, E.J., Grunow, R., 2004. Comparison of enzyme-linked immunosorbent assay, Western blotting, microagglutination, indirect immunofluorescence assay, and flow cytometry for serological diagnosis of tularemia. Clin. Diagn. Lab. Immunol. 11, 1008–1015. Ransmeier, J.C., Ewing, C.L., 1941. The agglutination reaction in tularemia. J. Infect. 69, 193–205. Rastawicki, W., Wolaniuk, N., 2013. Comparison of usefulness of commercial ELISA Virion/ Serion, home-made ELISA and tube agglutination test in serodiagnosis of tularemia. Med. Dosw. Mikrobiol. 65, 255–261. Rastawicki, W., Jagielski, M., Gierczyński, R., 2005. Evaluation of the enzyme-linked immunosorbent assay for the serodiagnosis of tularemia. Med. Dosw. Mikrobiol. 57, 425–429. Rimawi, R.H., Shah, K.B., Chowdhary, R.A., Cook, P.P., 2014. Hunting for tularemia — a review of cases in North Carolina. http://dx.doi.org/10.1111/zph.12114. Syrjälä, H., Koskela, P., Ripatti, T., Salminen, A., Herva, E., 1986. Agglutination and ELISA Methods in the Diagnosis of Tularemia in Different Clinical Forms and Severities of the Disease. 153, 142–145. Tärnvik, A., 1989. Nature of protective immunity to Francisella tularensis. Rev. Infect. Dis. 11, 440–451.
