- Email: [email protected]
Comparison of hemagglutination and hemadsorption tests for influenza detection
DIAGN MICROBIOLINFECTDIS 1992;15:363-365
363
Comparison of Hemagglutination and Hemadsorption Tests for Influenza Detection Sheryl L.G. Johnston, Kathleen Wellens, and Charles Siegel
Hemagglutination (HA) testing of the supernatant of primary rhesus monkey kidney cell culture tubes inoculated with clinical specimens was compared with hemadsorption (HAd) of the same tube for identifying the presence of hemagglutinin activity due to influenza. A total of 476 respiratory specimens were screened for influenza by this method. The 127 influenza or
parainfluenza positive cultures showed 100% concordance between the two tests. All negative cultures were both also negative by HA and HAd. In contrast to the HAd method, HA testing was performed with less tube manipulation, less handson time, and less potential for cell culture tube contamination.
Conventional recommendations for influenza isolation and identification employ the use of conventional cell culture tubes and hemadsorption (HAd) to identify the presence of hemagglutinin activity (Mills et al., 1989; Munnich and Ray, 1987; Pothier et al., 1986; Stokes et al., 1988; Wails et al., 1986). Hemagglutinin activity is typically present before the advent of cytopathic effect (CPE) and earlier than 7 days after inoculation in the case of the influenza virus (Chanock, 1979; Dowdle et al., 1979; Greenberg and Krilov, 1986). Although the HAd test is sensitive and readily available, it requires the use of additional cell culture tubes, which is costly, or washing the tubes free of red blood cells (RBC) after testing is complete, which is cumbersome and can lead to contamination. To circumvent either problem, hemagglutinin activity can be detected outside the cell culture tube through the use of the hemagglutination test (HA) (Hsiung, 1982), although the sensitivity of this test compared with the HAd test has not been established.
In our laboratory, we used the HA test in microtiter plates to conveniently and rapidly screen conventional primary rhesus monkey kidney (RMK) culture tubes inoculated with clinical specimens for the presence of hemagglutinin activity. This method was compared with typical HAd testing. The presence of hemagglutinin evidenced by positive HA and/or HAd testing was confirmed by the staining of infected cells with type-specific monoclonal antibodies to influenza A and B and parainfluenza 1, 2, and 3. A total of 476 respiratory specimens submitted for influenza screening were inoculated into confluent RMK (Viromed Laboratories, Minnetonka, MN) cells and concurrently tested for hemagglutinin activity by HA and HAd. Each specimen was inoculated (0.50 ml) into two RMK cell culture tubes that had previously been washed twice with serumfree medium. This medium contained antibiotics in Eagle's minimum essential medium. The tubes were incubated at 35°C on roller drums for up to 14 days. Blind passages to a third RMK tube were made around day 7 after inoculation. HA and HAd testing was performed at least three times on all tubes other than those already positive for a hemagglutinating virus. This testing was done after inoculation at 3- to 5-day intervals. The inconsistency was due to laboratory staffing. The HAd test was performed as described by Chanock (1979). Approximately 0.20 ml of a fresh
From the Department of LaboratoryMedicine, BellinMemorial Hospital, Green Bay, Wisconsin, USA. Address reprint requests to Dr. S.L.G.Johnston, VirologyLaboratory, St. VincentHospital, 835 South Van Buren Street, Green Bay, WI 54305, USA. Received 28 March 1991; revised and accepted 1 July 1991. © 1992 ElsevierScience Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010 0732-8893/92/$5.00
364
0.50% solution of guinea pig red blood cells (RBCs) (Kroy Medical, Stillwater, MN) in isotonic saline were added to culture tubes containing specimen inoculum in 1-1.5 ml of serum-free medium. These tubes were then incubated horizontally for 30 min. The culture tube was gently tipped to resuspend settled RBCs and read microscopically for adsorption. The HA test was performed with the same RBC suspension as in the HAd test. A 96-well, U-bottom microtiter plate was used. With a multichannel pipette, 100 ~I of sterile, physiologic saline were added to each well of the plate, and - 2 5 ~1 of cell culture supernatant were aseptically added to the appropriately labeled well of the plate, followed by 50 ~1 of the RBC suspension. The plate was covered, gently tapped to mix, and incubated at room temperature for 2 hr before being read. A magnifying mirror was tipped so that the plate could be read from the underside. Wells displaying partial or complete hemagglutination, as evidenced by the lack of a RBC "button" in the bottom of the well, were considered positive for hemagglutinin activity. HA- or HAd-positive tubes were scraped and stained with a monoclonal antibody (Bartels, Issaquah, WA) for typing and confirmation of the presence of influenza A or B, or parainfluenza 1, 2, or 3. A portion of the supernatant was saved for reinoculation into cell culture if the stained smear results were negative. HAd-positive tubes were rinsed free of RBCs before being scraped as the presence of these cells made accurate staining of the cultured cell difficult. In the advent of distinctive CPE, the smears were appropriately stained with type-specific monoclonal antibodies to influenza or parainfluenza. If CPE was indistinctive or absent, the smear was stained with antibody to all five of the aforementioned viruses. The inherent presence of endogenous simian paramyxovirus (SV5), which occurs in RMK cells on occasion, can make HAd interpretation subjective based on correlation of nonspecific hemadsorption of a negative control. Therefore, the use of controls was extremely important to both procedures. Negative RMK tubes from each of the 16 lots of inoculated tubes tested were incorporated into both the HA and HAd tests. The difficulty of nonspecific simian virus hemagglutinin activity was not observed during HA testing. In all lots of RMK-negative control cells, HA testing at dilution above 1:5 produced no positive results. In three of these negative lots of uninoculated cultures, nonspecific hemadsorption was interpreted as due to inherent simian virus. These tubes displayed a foamy-virus CPE and, when scraped and stained with monoclonal antibodies to
S.L.G. Johnston et al.
influenza A and B and parainfluenza 1, 2, and 3, no fluorescence was observed. Of the 476 specimens tested, 349 were negative for hemagglutinin activity by both the HA and HAd tests, and they lacked any detectable CPE. In all cultures, observable influenza/parainfluenza CPE was confirmed by both positive HA and HAd testing. There were 127 HA- and HAd-positive cultures; 92 influenza A, 20 influenza B, seven parainfluenza 1, one parainfluenza 2, and seven parainfluenza 3. In all cases, the results of the HA and HAd tests agreed 100% on the same day, postinoculation, of testing. Because testing was not performed on a daily basis, no interpretation can be made as to which method would produce more rapid results. The purpose of this investigation was to demonstrate an efficient means of screening a large number of viral specimens for hemagglutinating activity, not to demonstrate that one method was more rapid. Based on the data gathered in our laboratory, HA testing by the described method appears to be a useful alternative to HAd testing in screening for the presence of viral hemagglutinin produced by influenza. HA testing may be useful for detecting parainfluenza and mumps, but more data are necessary. The HAd test was invasive into the cell culture tube and posed the potential risk of tube contamination. To keep the testing cost effective, negative tubes less than 14 days after inoculation were washed of RBCs after HAd testing and returned for incubation rather than discarded. HAd testing required manipulation of the culture tube at least twice during the procedure, first to introduce the RBCs and then again to read the tube under the microscope for adsorption. A third handling was necessary if a wash was made before returning the tube for incubation. This wash is recommended (Chanock, 1979) because incubation with these RBCs present can result in nonspecific hemadsorption in subsequent testing. In contrast, manipulation of the culture tube for HA testing was minimal. The tube was entered only once by a sterile pipette and returned for incubation. Once the HA plate was completely set up, further manipulation was not necessary for reading. All 96 wells could be read with a magnifying mirror, which was used to hold the plate while the HA test was in progress. A multichannel pipette used to introduce saline and RBCs into the HA plate made setup efficient by saving time and manipulation. The intact cell culture tube was subsequently free of RBC and available for the detection of other viruses such as adenovirus and respiratory syncytial virus. In our laboratory, all hemagglutinin-producing viruses detected by HAd testing were also de-
Note
tected b y H A testing. All cultures negative by the HA test w e r e also negative by the H A d test, and none of these cultures p r o d u c e d detectable influenza or parainfluenza CPE. In c o m p a r i s o n to the H A d test, the H A test was m o r e efficient, as sensitive, posed less contamination potential, and was
365
less subject to misinterpretation caused by the presence of simian virus. Further investigations m u s t be m a d e to d e t e r m i n e the usefulness of this H A test in c o m p a r i s o n to H A d for the detection of parainfluenza and m u m p s .
REFERENCES Chanock RM (1979) Parainfluenza viruses. In Diagnostic
procedures for Viral, Rickettsial and Chlamydial Infections, 5th ed. Eds, EH Lennette and NJ Schmidt. Washington, DC: American Public Health Association, pp 611-632. Dowdle WA, Kendal AP, Noble GR (1979) Influenza viruses. In Diagnostic Procedures for Viral, Rickettsial and Chlamydial Infections, 5th ed. Eds, EH Lennette and NJ Schmidt. Washington, DC: American Public Health Association, pp 585-609. Greenberg, SB, Krilov LR (1986) Cumitech 21, Laboratory Diagnosis of Viral Respiratory Disease. Coord eds, WL Drew and SJ Rubin. Washington, DC: American Society for Microbiology. Hsiung GD (1982) Myxoviridae. In Diagnostic Virology Ed. GD Hsiung. New Haven: Yale University Press, pp 3541 and 138-150. Mills RD, Cain KJ, Woods GL (1989) Detection of influenza
virus by centrifugal inoculation of MDCK cells and staining with monoclonal antibodies. ] Clin Microbiol 27:2505-2508. Minnich LL, Ray CG (1987) Early testing of cell cultures for detection of hemadsorbing viruses. J Clin Microbiol 25:421-422. Pothier P, Denoyel, GA, Ghim, S, Prudhomme de Saint Maur G, Freymuth F (1986) Use of monoclonal antibodies for rapid detection of influenza A virus in nasopharyngeal secretions. Eur J Clin Microbiol 5:336-339. Stokes CE, Bernstein JM, Kyger SA, Hayden FG (1988) Rapid diagnosis of influenza A and B by 24-h fluorescent focus assays. J Clin Microbiol 26:1263-1266. Walls HH, Harmon MW, Slagle JJ, Stocksdale C, Kendal, AP (1986) Characterization and evaluation of monoclonal antibodies developed for typing influenza A and influenza B viruses. J Clin Microbiol 23:240-245.
363
Comparison of Hemagglutination and Hemadsorption Tests for Influenza Detection Sheryl L.G. Johnston, Kathleen Wellens, and Charles Siegel
Hemagglutination (HA) testing of the supernatant of primary rhesus monkey kidney cell culture tubes inoculated with clinical specimens was compared with hemadsorption (HAd) of the same tube for identifying the presence of hemagglutinin activity due to influenza. A total of 476 respiratory specimens were screened for influenza by this method. The 127 influenza or
parainfluenza positive cultures showed 100% concordance between the two tests. All negative cultures were both also negative by HA and HAd. In contrast to the HAd method, HA testing was performed with less tube manipulation, less handson time, and less potential for cell culture tube contamination.
Conventional recommendations for influenza isolation and identification employ the use of conventional cell culture tubes and hemadsorption (HAd) to identify the presence of hemagglutinin activity (Mills et al., 1989; Munnich and Ray, 1987; Pothier et al., 1986; Stokes et al., 1988; Wails et al., 1986). Hemagglutinin activity is typically present before the advent of cytopathic effect (CPE) and earlier than 7 days after inoculation in the case of the influenza virus (Chanock, 1979; Dowdle et al., 1979; Greenberg and Krilov, 1986). Although the HAd test is sensitive and readily available, it requires the use of additional cell culture tubes, which is costly, or washing the tubes free of red blood cells (RBC) after testing is complete, which is cumbersome and can lead to contamination. To circumvent either problem, hemagglutinin activity can be detected outside the cell culture tube through the use of the hemagglutination test (HA) (Hsiung, 1982), although the sensitivity of this test compared with the HAd test has not been established.
In our laboratory, we used the HA test in microtiter plates to conveniently and rapidly screen conventional primary rhesus monkey kidney (RMK) culture tubes inoculated with clinical specimens for the presence of hemagglutinin activity. This method was compared with typical HAd testing. The presence of hemagglutinin evidenced by positive HA and/or HAd testing was confirmed by the staining of infected cells with type-specific monoclonal antibodies to influenza A and B and parainfluenza 1, 2, and 3. A total of 476 respiratory specimens submitted for influenza screening were inoculated into confluent RMK (Viromed Laboratories, Minnetonka, MN) cells and concurrently tested for hemagglutinin activity by HA and HAd. Each specimen was inoculated (0.50 ml) into two RMK cell culture tubes that had previously been washed twice with serumfree medium. This medium contained antibiotics in Eagle's minimum essential medium. The tubes were incubated at 35°C on roller drums for up to 14 days. Blind passages to a third RMK tube were made around day 7 after inoculation. HA and HAd testing was performed at least three times on all tubes other than those already positive for a hemagglutinating virus. This testing was done after inoculation at 3- to 5-day intervals. The inconsistency was due to laboratory staffing. The HAd test was performed as described by Chanock (1979). Approximately 0.20 ml of a fresh
From the Department of LaboratoryMedicine, BellinMemorial Hospital, Green Bay, Wisconsin, USA. Address reprint requests to Dr. S.L.G.Johnston, VirologyLaboratory, St. VincentHospital, 835 South Van Buren Street, Green Bay, WI 54305, USA. Received 28 March 1991; revised and accepted 1 July 1991. © 1992 ElsevierScience Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010 0732-8893/92/$5.00
364
0.50% solution of guinea pig red blood cells (RBCs) (Kroy Medical, Stillwater, MN) in isotonic saline were added to culture tubes containing specimen inoculum in 1-1.5 ml of serum-free medium. These tubes were then incubated horizontally for 30 min. The culture tube was gently tipped to resuspend settled RBCs and read microscopically for adsorption. The HA test was performed with the same RBC suspension as in the HAd test. A 96-well, U-bottom microtiter plate was used. With a multichannel pipette, 100 ~I of sterile, physiologic saline were added to each well of the plate, and - 2 5 ~1 of cell culture supernatant were aseptically added to the appropriately labeled well of the plate, followed by 50 ~1 of the RBC suspension. The plate was covered, gently tapped to mix, and incubated at room temperature for 2 hr before being read. A magnifying mirror was tipped so that the plate could be read from the underside. Wells displaying partial or complete hemagglutination, as evidenced by the lack of a RBC "button" in the bottom of the well, were considered positive for hemagglutinin activity. HA- or HAd-positive tubes were scraped and stained with a monoclonal antibody (Bartels, Issaquah, WA) for typing and confirmation of the presence of influenza A or B, or parainfluenza 1, 2, or 3. A portion of the supernatant was saved for reinoculation into cell culture if the stained smear results were negative. HAd-positive tubes were rinsed free of RBCs before being scraped as the presence of these cells made accurate staining of the cultured cell difficult. In the advent of distinctive CPE, the smears were appropriately stained with type-specific monoclonal antibodies to influenza or parainfluenza. If CPE was indistinctive or absent, the smear was stained with antibody to all five of the aforementioned viruses. The inherent presence of endogenous simian paramyxovirus (SV5), which occurs in RMK cells on occasion, can make HAd interpretation subjective based on correlation of nonspecific hemadsorption of a negative control. Therefore, the use of controls was extremely important to both procedures. Negative RMK tubes from each of the 16 lots of inoculated tubes tested were incorporated into both the HA and HAd tests. The difficulty of nonspecific simian virus hemagglutinin activity was not observed during HA testing. In all lots of RMK-negative control cells, HA testing at dilution above 1:5 produced no positive results. In three of these negative lots of uninoculated cultures, nonspecific hemadsorption was interpreted as due to inherent simian virus. These tubes displayed a foamy-virus CPE and, when scraped and stained with monoclonal antibodies to
S.L.G. Johnston et al.
influenza A and B and parainfluenza 1, 2, and 3, no fluorescence was observed. Of the 476 specimens tested, 349 were negative for hemagglutinin activity by both the HA and HAd tests, and they lacked any detectable CPE. In all cultures, observable influenza/parainfluenza CPE was confirmed by both positive HA and HAd testing. There were 127 HA- and HAd-positive cultures; 92 influenza A, 20 influenza B, seven parainfluenza 1, one parainfluenza 2, and seven parainfluenza 3. In all cases, the results of the HA and HAd tests agreed 100% on the same day, postinoculation, of testing. Because testing was not performed on a daily basis, no interpretation can be made as to which method would produce more rapid results. The purpose of this investigation was to demonstrate an efficient means of screening a large number of viral specimens for hemagglutinating activity, not to demonstrate that one method was more rapid. Based on the data gathered in our laboratory, HA testing by the described method appears to be a useful alternative to HAd testing in screening for the presence of viral hemagglutinin produced by influenza. HA testing may be useful for detecting parainfluenza and mumps, but more data are necessary. The HAd test was invasive into the cell culture tube and posed the potential risk of tube contamination. To keep the testing cost effective, negative tubes less than 14 days after inoculation were washed of RBCs after HAd testing and returned for incubation rather than discarded. HAd testing required manipulation of the culture tube at least twice during the procedure, first to introduce the RBCs and then again to read the tube under the microscope for adsorption. A third handling was necessary if a wash was made before returning the tube for incubation. This wash is recommended (Chanock, 1979) because incubation with these RBCs present can result in nonspecific hemadsorption in subsequent testing. In contrast, manipulation of the culture tube for HA testing was minimal. The tube was entered only once by a sterile pipette and returned for incubation. Once the HA plate was completely set up, further manipulation was not necessary for reading. All 96 wells could be read with a magnifying mirror, which was used to hold the plate while the HA test was in progress. A multichannel pipette used to introduce saline and RBCs into the HA plate made setup efficient by saving time and manipulation. The intact cell culture tube was subsequently free of RBC and available for the detection of other viruses such as adenovirus and respiratory syncytial virus. In our laboratory, all hemagglutinin-producing viruses detected by HAd testing were also de-
Note
tected b y H A testing. All cultures negative by the HA test w e r e also negative by the H A d test, and none of these cultures p r o d u c e d detectable influenza or parainfluenza CPE. In c o m p a r i s o n to the H A d test, the H A test was m o r e efficient, as sensitive, posed less contamination potential, and was
365
less subject to misinterpretation caused by the presence of simian virus. Further investigations m u s t be m a d e to d e t e r m i n e the usefulness of this H A test in c o m p a r i s o n to H A d for the detection of parainfluenza and m u m p s .
REFERENCES Chanock RM (1979) Parainfluenza viruses. In Diagnostic
procedures for Viral, Rickettsial and Chlamydial Infections, 5th ed. Eds, EH Lennette and NJ Schmidt. Washington, DC: American Public Health Association, pp 611-632. Dowdle WA, Kendal AP, Noble GR (1979) Influenza viruses. In Diagnostic Procedures for Viral, Rickettsial and Chlamydial Infections, 5th ed. Eds, EH Lennette and NJ Schmidt. Washington, DC: American Public Health Association, pp 585-609. Greenberg, SB, Krilov LR (1986) Cumitech 21, Laboratory Diagnosis of Viral Respiratory Disease. Coord eds, WL Drew and SJ Rubin. Washington, DC: American Society for Microbiology. Hsiung GD (1982) Myxoviridae. In Diagnostic Virology Ed. GD Hsiung. New Haven: Yale University Press, pp 3541 and 138-150. Mills RD, Cain KJ, Woods GL (1989) Detection of influenza
virus by centrifugal inoculation of MDCK cells and staining with monoclonal antibodies. ] Clin Microbiol 27:2505-2508. Minnich LL, Ray CG (1987) Early testing of cell cultures for detection of hemadsorbing viruses. J Clin Microbiol 25:421-422. Pothier P, Denoyel, GA, Ghim, S, Prudhomme de Saint Maur G, Freymuth F (1986) Use of monoclonal antibodies for rapid detection of influenza A virus in nasopharyngeal secretions. Eur J Clin Microbiol 5:336-339. Stokes CE, Bernstein JM, Kyger SA, Hayden FG (1988) Rapid diagnosis of influenza A and B by 24-h fluorescent focus assays. J Clin Microbiol 26:1263-1266. Walls HH, Harmon MW, Slagle JJ, Stocksdale C, Kendal, AP (1986) Characterization and evaluation of monoclonal antibodies developed for typing influenza A and influenza B viruses. J Clin Microbiol 23:240-245.