303
DIAGN MICROBIOLINFECTDIS 1990;13:303-306
VIROLOGY
Evaluation of Latex Agglutination and Dot Immunobinding Assay for the Detection of Cytomegalovirus Antibody Maria T. Wilson, Robert L. Sautter, William D. LeBar, and Timothy R. Mervak
The Virogen CMV Antibody Test and Difco CMV-Cube were compared with three other available methods, enzyme immunoassay, immunofluorescence and latex agglutination, for the detection of cytomegalovirus (CMV) antibody in 126 random sera submitted to the clinical laboratory. Based on concordance
of three or more methods, 72% of the samples were positive for CMV antibody. The sensitivities and specificities of the assays were Virogen, 98% and 97%, and CMV-Cube 97% and 97%, respectively.
INTRODUCTION
bury, NJ), a rapid LA test, and the CMV-Cube (Difco Laboratories, Ann Arbor, MI), a dot immunobinding EIA; as compared to three other procedures for the detection of CMV antibody.
Accurate screening for cytomegalovirus (CMV) antibody status can be essential for identification of seronegative donors and recipients who may benefit from seronegative blood products (Ringden et al., 1984; Bowden et al., 1986; Tegtmeier, 1988). A variety of methods, including latex agglutination (LA), enzyme immunoassay (EIA), indirect immunofluorescence (IFA), anticomplement immunofluorescence, indirect hemagglutination, and immunoblotting have been used for the detection of CMV antibodies in patient sera (Beckwith et al., 1985; McHugh et al., 1985; Hursh et al., 1989; Leland et al., 1989; Miller et al., 1989). This report describes the performance of two new assays, the Virogen CMV Antibody Test (Wampole Laboratories, CranFrom the MicrobiologyLaboratory,Harrisburg Hospital (M.T.W., R.L.S.), Harrisburg, Pennsylvania;and Departments of Pathologyand Microbiology(W.D.L., T.R.M.), Providence Hospital, Southfield,Michigan. Address reprint requests to: WilliamLeBar, Departmentof Pathology, ProvidenceHospital, 16001 West Nine Mile Road, Southfield, MI 48037. Received January25, 1990; revised and accepted March 20, 1990. © 1990 ElsevierScience PublishingCo., Inc. 655 Avenues of the Americas, New York, NY 10010 0732-8893/90/$3.50
MATERIALS A N D METHODS Serum Specimens In this study, 126 randomly selected serum samples were sent to the clinical laboratory for testing. All sera were stored at -20°C until use.
Virogen CMV Antibody Test The Virogen test was performed as follows. Undiluted serum (0.025 ml) was mixed with one drop of Virogen CMV latex on a disposable card slide. The slide was rotated for 10 min at 100 rpm and examined for agglutination under a light held above the slide. All reactions were compared against the negative control for determination of a positive or negative result.
CMV-Cube All sera were diluted 1:5 in CMV-Cube specimen diluent prior to testing. Three drops of diluted spec-
304
TABLE 1.
M.T. Wilson et al.
C o m p a r i s o n of CMV A n t i b o d y Assays
Assay
TABLE 2.
Performance Characteristics of CMV A n t i b o d y Assays
Result Performance (%)
Virogen CMV Scan CMV-Cube Abbott EIA IFA
+ + + + +
+ + + + -
+ + +
+ +
Totals
84
2
3
1
. .
13
. .
. . . . + + 20
1
. + + +
+ + + +
1
1
imen were applied to the center of the CMV-Cube membrane and allowed to pass through. Three drops of wash reagent w e r e applied to the m e m b r a n e a n d allowed to pass. Three d r o p s of a n t i - h u m a n alkaline p h o s p h a t a s e conjugate were t h e n applied to the membrane, allowed to pass, and incubated for I min. The m e m b r a n e surface was w a s h e d with five d r o p s of wash solution, and three d r o p s of c h r o m o g e n reagent were a d d e d . After 2 min, the reaction was s t o p p e d by the addition of a stopping reagent and the m e m b r a n e was o b s e r v e d for color d e v e l o p m e n t . A h o m o g e n e o u s blue dot in the center of the m e m brane is c o n s i d e r e d positive for CMV antibody; the absence of the blue dot is negative for CMV antibody.
CMV Scan The CMV Scan (BBL Microbiology Systems, Cockeysville, MD) was p e r f o r m e d according to manufacturer's instructions and as described previously (Beckwith et al., 1985; M c H u g h et al., 1985; Leland et al., 1989). ALl sera were screened undiluted.
EIA The Abbott CMV Total AB EIA (Abbott Laboratories, Chicago, IL) was p e r f o r m e d by using a 1:21 dilution of serum per m a n u f a c t u r e r ' s instructions, as described previously (Beckwith et al., 1985; M c H u g h et al., 1985). Absorbance was read on a Q u a n t u m II s p e c t r o p h o t o m e t e r (Abbott), and a cutoff value was calculated b y a d d i n g 0.075 to the m e a n absorbance of the negative controls. Samples with absorbance >10% above the cutoff were positive.
Assay
Sensitivity
Specificity
PVPa
PVN ~'
Virogen CMV CMV-Cube CMV Scan IFA Abbott EIA
98 97 99 98 97
97 97 97 43 100
99 98 99 75 100
94 92 97 88 92
aPVP, positive predictive value. ~PVN, negative predictive value.
Analysis of Results C o n c o r d a n c e of three or more m e t h o d s was necessary for a sample to be c o n s i d e r e d positive for CMV antibody (Beckwith et al., 1985). This criterion was u s e d to calculate sensitivity, specificity, and positive and negative predictive values.
RESULTS A total of 126 s e r u m specimens were tested by five different m e t h o d s for the detection of CMV antibody. Based on concordance of three or more of the five m e t h o d s , 91 (72%) of the 126 sera tested were positive for antibody to CMV. The test p e r f o r m a n c e for each m e t h o d is detailed in Tables I a n d 2. Results for samples yielding discordant Virogen or CMVCube results are detailed in Table 3. The false-positive Virogen test, as well as one of the two false-negative tests, agreed with the CMV Scan test results. The two latex tests agreed 99% (125/126) of the time. The discrepant negative Virogen test was positive by all of the other methods. All three discordant negative CMV-Cube results were negative in the Abbott EIA assay. The CMVCube a n d the Abbott EIA also d e m o n s t r a t e d 99% (125/126) agreement. The discrepant positive CMVCube result was negative by the other four methods.
DISCUSSION IFA The CMV A n t i b o d y IFA (Electro-Nucleonics, Columbia, MD) was p e r f o r m e d b y using a 1:16 dilution of serum per m a n u f a c t u r e r ' s instructions. Fluorescence was read on a Leitz epifluorescence microscope. The presence of fluorescent intranuclear inclusions was considered positive for CMV antibody.
Currently, there are m a n y reliable m e t h o d s available for the detection of antibody to CMV. CMV antibody testing is i m p o r t a n t in the screening of blood and blood p r o d u c t s and in d e t e r m i n i n g the serological status in b o n e m a r r o w transplant candidates and recipients, i m m u n o c o m p r o m i s e d patients, and n e o n a t e s (Feng et al., 1986; H u r s h et
Assay Comparisons for Detection of CMV
TABLE
3.
305
Samples Yielding Discordant Virogen or CMV-Cube Result Assay Result
Sample
Virogen
17 27
CMV-Cube
Consensus
Comment
+ +
+ -
CMV Scan positive Abbott EIA negative Abbott EIA positive CMV Scan positive Abbott EIA negative Abbott EIA negative CMV Scan negative
67 89 93
+ + +
-
-
+ +
94
+
-
+
+
+
123
al., 1989; Leland et al., 1989). Requirements for a test to screen both patients and blood products should include a high sensitivity for detection of positive donors and a high specificity and negative predictive value for the selection of blood components (Beckwith et al., 1985). Each method evaluated in the present study, with the exception of IFA, fulfilled these requirements. The increased number of false-positive results using IFA is undoubtedly due to the occurrence of nonspecific Fc receptor staining near the perinudear region (Gallo, 1986). Unlike Fc receptor staining in herpes simplex virus (HSV)-infected cells, CMV-infected cells lose both specific and nonspecific reactivity when treated with glacial acetic acid to eliminate Fc receptor sites (Gallo, 1986; LeBar et al., 1989); however, this problem may be overcome by the use of
anticomplement immunofluorescence (Kettering et al., 1977; Gallo, 1986). The low sensitivity and positive predictive value for IFA has been reported previously (Beckwith et al., 1985). The Virogen CMV Antibody Test and Difco CMV-Cube are both rapid and sensitive tests for the detection of CMV antibody. The performance characteristics of these assays are comparable to those of other currently marketed CMV antibody assays. The results of this study indicate that both the Virogen CMV Antibody Test and The CMV-Cube may be useful in the determination of CMV antibody status. We are grateful to Ms. Ann Ralph for her skillful technical assistance. This study was supported by Wampole Laboratories (Cranbury, NJ).
REFERENCES
Beckwith DG, Halstead DC, Alpaugh K, Schweder A, Blount-Fronefield DA, Toth K (1985) Comparison of a latex agglutination test with five other methods for determining the presence of antibody against cytomegalovirus. J Clin Microbiol 21:328-331. Bowden RA, Sayers M, Flournoy N, Newton B, Banajii M, Thomas ED, Myers JD (1986) Cytomegalovirus immune globulin and seronegative blood products to prevent primary cytomegalovirus infection after marrow transplantation. N Engl J Med 314:1006-1010. Feng CS, William IB, Gohd RS, Causey AP (1986) A comparison of four commercial kits for detection of cytomegalovirus antibodies in blood donors. Transfusion 26:203-204. Gallo D (1986) Elimination of Fc receptor binding of human immunoglobulin G in immunofluorescence assays for herpes simplex virus antibodies. J Clin Microbio124:672674. Hursh DA, Abbott AD, Sun R, Iltis JP, Rice DH, Gleaves CA (1989) Evaluation of a latex particle agglutination
assay for the detection of cytomegalovirus antibody in patient serum. J Clin Microbiol 27:2878-2879. Kettering JD, Schmidt NJ, Gallo D, Lennette EH (1977) Anticomplement immunofluorescence test for antibodies to human cytomegalovirus. ] Clin Microbiol 6:627632. LeBar WD, Resek CM, Crist AE Jr, Sautter RL (1989) Comparison of a rapid latex agglutination assay and a fluorescent-antibody technique for the detection of herpes simplex antibody. Diagn Microbiol Infect Dis 11:21-24. Leland DS, Barth KA, Cunningham EB, Jansen J, Tricot GJ, French MLV (1989) Evaluation of four methods for cytomegalovirus antibody detection for use by a bone marrow transplantation service. J Clin Microbio127:176178. McHugh TM, Casavant CH, Wilber JC, Stites DP (1985) Comparison of six methods for detection of antibody to cytomegalovirus. J Clin MicrobioI 22:1014-1019. Miller H, McCulloch B, Landini MP, Rossier E (1989) Comparison of irnmunoblotting with other serological meth-
306
ods and virus isolation for early detection of primary cytoinegalovirus infection in allograft recipients. J Clin Microbiol 27:2672-2677. Ringden O, Paulin T, Pihlstedt P, Lonnqvist B, Wahren B (1984) Cytomegalovirus antibody screening of blood
M.T. Wilson et al.
donors for immunocompromised seronegative patients. Lancet 2:1044. Tegtmeier GE (1988) The use of cytomegalovirus screened blood in neonates. Transfusion 28:201-203.
DIAGN MICROBIOLINFECTDIS 1990;13:303-306
VIROLOGY
Evaluation of Latex Agglutination and Dot Immunobinding Assay for the Detection of Cytomegalovirus Antibody Maria T. Wilson, Robert L. Sautter, William D. LeBar, and Timothy R. Mervak
The Virogen CMV Antibody Test and Difco CMV-Cube were compared with three other available methods, enzyme immunoassay, immunofluorescence and latex agglutination, for the detection of cytomegalovirus (CMV) antibody in 126 random sera submitted to the clinical laboratory. Based on concordance
of three or more methods, 72% of the samples were positive for CMV antibody. The sensitivities and specificities of the assays were Virogen, 98% and 97%, and CMV-Cube 97% and 97%, respectively.
INTRODUCTION
bury, NJ), a rapid LA test, and the CMV-Cube (Difco Laboratories, Ann Arbor, MI), a dot immunobinding EIA; as compared to three other procedures for the detection of CMV antibody.
Accurate screening for cytomegalovirus (CMV) antibody status can be essential for identification of seronegative donors and recipients who may benefit from seronegative blood products (Ringden et al., 1984; Bowden et al., 1986; Tegtmeier, 1988). A variety of methods, including latex agglutination (LA), enzyme immunoassay (EIA), indirect immunofluorescence (IFA), anticomplement immunofluorescence, indirect hemagglutination, and immunoblotting have been used for the detection of CMV antibodies in patient sera (Beckwith et al., 1985; McHugh et al., 1985; Hursh et al., 1989; Leland et al., 1989; Miller et al., 1989). This report describes the performance of two new assays, the Virogen CMV Antibody Test (Wampole Laboratories, CranFrom the MicrobiologyLaboratory,Harrisburg Hospital (M.T.W., R.L.S.), Harrisburg, Pennsylvania;and Departments of Pathologyand Microbiology(W.D.L., T.R.M.), Providence Hospital, Southfield,Michigan. Address reprint requests to: WilliamLeBar, Departmentof Pathology, ProvidenceHospital, 16001 West Nine Mile Road, Southfield, MI 48037. Received January25, 1990; revised and accepted March 20, 1990. © 1990 ElsevierScience PublishingCo., Inc. 655 Avenues of the Americas, New York, NY 10010 0732-8893/90/$3.50
MATERIALS A N D METHODS Serum Specimens In this study, 126 randomly selected serum samples were sent to the clinical laboratory for testing. All sera were stored at -20°C until use.
Virogen CMV Antibody Test The Virogen test was performed as follows. Undiluted serum (0.025 ml) was mixed with one drop of Virogen CMV latex on a disposable card slide. The slide was rotated for 10 min at 100 rpm and examined for agglutination under a light held above the slide. All reactions were compared against the negative control for determination of a positive or negative result.
CMV-Cube All sera were diluted 1:5 in CMV-Cube specimen diluent prior to testing. Three drops of diluted spec-
304
TABLE 1.
M.T. Wilson et al.
C o m p a r i s o n of CMV A n t i b o d y Assays
Assay
TABLE 2.
Performance Characteristics of CMV A n t i b o d y Assays
Result Performance (%)
Virogen CMV Scan CMV-Cube Abbott EIA IFA
+ + + + +
+ + + + -
+ + +
+ +
Totals
84
2
3
1
. .
13
. .
. . . . + + 20
1
. + + +
+ + + +
1
1
imen were applied to the center of the CMV-Cube membrane and allowed to pass through. Three drops of wash reagent w e r e applied to the m e m b r a n e a n d allowed to pass. Three d r o p s of a n t i - h u m a n alkaline p h o s p h a t a s e conjugate were t h e n applied to the membrane, allowed to pass, and incubated for I min. The m e m b r a n e surface was w a s h e d with five d r o p s of wash solution, and three d r o p s of c h r o m o g e n reagent were a d d e d . After 2 min, the reaction was s t o p p e d by the addition of a stopping reagent and the m e m b r a n e was o b s e r v e d for color d e v e l o p m e n t . A h o m o g e n e o u s blue dot in the center of the m e m brane is c o n s i d e r e d positive for CMV antibody; the absence of the blue dot is negative for CMV antibody.
CMV Scan The CMV Scan (BBL Microbiology Systems, Cockeysville, MD) was p e r f o r m e d according to manufacturer's instructions and as described previously (Beckwith et al., 1985; M c H u g h et al., 1985; Leland et al., 1989). ALl sera were screened undiluted.
EIA The Abbott CMV Total AB EIA (Abbott Laboratories, Chicago, IL) was p e r f o r m e d by using a 1:21 dilution of serum per m a n u f a c t u r e r ' s instructions, as described previously (Beckwith et al., 1985; M c H u g h et al., 1985). Absorbance was read on a Q u a n t u m II s p e c t r o p h o t o m e t e r (Abbott), and a cutoff value was calculated b y a d d i n g 0.075 to the m e a n absorbance of the negative controls. Samples with absorbance >10% above the cutoff were positive.
Assay
Sensitivity
Specificity
PVPa
PVN ~'
Virogen CMV CMV-Cube CMV Scan IFA Abbott EIA
98 97 99 98 97
97 97 97 43 100
99 98 99 75 100
94 92 97 88 92
aPVP, positive predictive value. ~PVN, negative predictive value.
Analysis of Results C o n c o r d a n c e of three or more m e t h o d s was necessary for a sample to be c o n s i d e r e d positive for CMV antibody (Beckwith et al., 1985). This criterion was u s e d to calculate sensitivity, specificity, and positive and negative predictive values.
RESULTS A total of 126 s e r u m specimens were tested by five different m e t h o d s for the detection of CMV antibody. Based on concordance of three or more of the five m e t h o d s , 91 (72%) of the 126 sera tested were positive for antibody to CMV. The test p e r f o r m a n c e for each m e t h o d is detailed in Tables I a n d 2. Results for samples yielding discordant Virogen or CMVCube results are detailed in Table 3. The false-positive Virogen test, as well as one of the two false-negative tests, agreed with the CMV Scan test results. The two latex tests agreed 99% (125/126) of the time. The discrepant negative Virogen test was positive by all of the other methods. All three discordant negative CMV-Cube results were negative in the Abbott EIA assay. The CMVCube a n d the Abbott EIA also d e m o n s t r a t e d 99% (125/126) agreement. The discrepant positive CMVCube result was negative by the other four methods.
DISCUSSION IFA The CMV A n t i b o d y IFA (Electro-Nucleonics, Columbia, MD) was p e r f o r m e d b y using a 1:16 dilution of serum per m a n u f a c t u r e r ' s instructions. Fluorescence was read on a Leitz epifluorescence microscope. The presence of fluorescent intranuclear inclusions was considered positive for CMV antibody.
Currently, there are m a n y reliable m e t h o d s available for the detection of antibody to CMV. CMV antibody testing is i m p o r t a n t in the screening of blood and blood p r o d u c t s and in d e t e r m i n i n g the serological status in b o n e m a r r o w transplant candidates and recipients, i m m u n o c o m p r o m i s e d patients, and n e o n a t e s (Feng et al., 1986; H u r s h et
Assay Comparisons for Detection of CMV
TABLE
3.
305
Samples Yielding Discordant Virogen or CMV-Cube Result Assay Result
Sample
Virogen
17 27
CMV-Cube
Consensus
Comment
+ +
+ -
CMV Scan positive Abbott EIA negative Abbott EIA positive CMV Scan positive Abbott EIA negative Abbott EIA negative CMV Scan negative
67 89 93
+ + +
-
-
+ +
94
+
-
+
+
+
123
al., 1989; Leland et al., 1989). Requirements for a test to screen both patients and blood products should include a high sensitivity for detection of positive donors and a high specificity and negative predictive value for the selection of blood components (Beckwith et al., 1985). Each method evaluated in the present study, with the exception of IFA, fulfilled these requirements. The increased number of false-positive results using IFA is undoubtedly due to the occurrence of nonspecific Fc receptor staining near the perinudear region (Gallo, 1986). Unlike Fc receptor staining in herpes simplex virus (HSV)-infected cells, CMV-infected cells lose both specific and nonspecific reactivity when treated with glacial acetic acid to eliminate Fc receptor sites (Gallo, 1986; LeBar et al., 1989); however, this problem may be overcome by the use of
anticomplement immunofluorescence (Kettering et al., 1977; Gallo, 1986). The low sensitivity and positive predictive value for IFA has been reported previously (Beckwith et al., 1985). The Virogen CMV Antibody Test and Difco CMV-Cube are both rapid and sensitive tests for the detection of CMV antibody. The performance characteristics of these assays are comparable to those of other currently marketed CMV antibody assays. The results of this study indicate that both the Virogen CMV Antibody Test and The CMV-Cube may be useful in the determination of CMV antibody status. We are grateful to Ms. Ann Ralph for her skillful technical assistance. This study was supported by Wampole Laboratories (Cranbury, NJ).
REFERENCES
Beckwith DG, Halstead DC, Alpaugh K, Schweder A, Blount-Fronefield DA, Toth K (1985) Comparison of a latex agglutination test with five other methods for determining the presence of antibody against cytomegalovirus. J Clin Microbiol 21:328-331. Bowden RA, Sayers M, Flournoy N, Newton B, Banajii M, Thomas ED, Myers JD (1986) Cytomegalovirus immune globulin and seronegative blood products to prevent primary cytomegalovirus infection after marrow transplantation. N Engl J Med 314:1006-1010. Feng CS, William IB, Gohd RS, Causey AP (1986) A comparison of four commercial kits for detection of cytomegalovirus antibodies in blood donors. Transfusion 26:203-204. Gallo D (1986) Elimination of Fc receptor binding of human immunoglobulin G in immunofluorescence assays for herpes simplex virus antibodies. J Clin Microbio124:672674. Hursh DA, Abbott AD, Sun R, Iltis JP, Rice DH, Gleaves CA (1989) Evaluation of a latex particle agglutination
assay for the detection of cytomegalovirus antibody in patient serum. J Clin Microbiol 27:2878-2879. Kettering JD, Schmidt NJ, Gallo D, Lennette EH (1977) Anticomplement immunofluorescence test for antibodies to human cytomegalovirus. ] Clin Microbiol 6:627632. LeBar WD, Resek CM, Crist AE Jr, Sautter RL (1989) Comparison of a rapid latex agglutination assay and a fluorescent-antibody technique for the detection of herpes simplex antibody. Diagn Microbiol Infect Dis 11:21-24. Leland DS, Barth KA, Cunningham EB, Jansen J, Tricot GJ, French MLV (1989) Evaluation of four methods for cytomegalovirus antibody detection for use by a bone marrow transplantation service. J Clin Microbio127:176178. McHugh TM, Casavant CH, Wilber JC, Stites DP (1985) Comparison of six methods for detection of antibody to cytomegalovirus. J Clin MicrobioI 22:1014-1019. Miller H, McCulloch B, Landini MP, Rossier E (1989) Comparison of irnmunoblotting with other serological meth-
306
ods and virus isolation for early detection of primary cytoinegalovirus infection in allograft recipients. J Clin Microbiol 27:2672-2677. Ringden O, Paulin T, Pihlstedt P, Lonnqvist B, Wahren B (1984) Cytomegalovirus antibody screening of blood
M.T. Wilson et al.
donors for immunocompromised seronegative patients. Lancet 2:1044. Tegtmeier GE (1988) The use of cytomegalovirus screened blood in neonates. Transfusion 28:201-203.
