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Serological diagnosis of cytomegalovirus infections: comparison of six commercial methods of ELISA
Scrodiclgnosis and Immunotherapy
in Infectious Disease (1988) 2, 42343
I
Serological diagnosis of cytomegalovirus infections: comparison of six commercial methods of ELISA
Fernando de Ory*, Pilar Perez-Breiia and Jo& M. Echevarria
Servicio de Virologia. Centro National de Microhiologia, Virologia Sanitarias, Majadahonda, 28220 Madrid, Spain
e lnmunologia
Six commercial enzyme-linked immunosorbent assays (ELISA) (three indirect and three u chain capture methods, two of them with enzyme-labelled antigen, ELA) for detection of cytomegalovirus (CMV) specific IgM were compared, using sera from patients with CMV infection, from recent Epstein-Barr virus (EBV) infections. and sera containing rheumatoid factor (RF) and CMV specific IgG. The six methods showed similar sensitivities. In sera from congenital infections the most sensitive
assayswere the p chain capture methods.In two indirect methods,false positive reactionsappearedwhen assayingserawith RF and specificIgG. The sameindirect methods and both ELA assays gave some CMV analysing sera from recent EBV infections. Ke~;words: cytomegalovirus, antigen
specific IgM,
IgM
positive
enzyme immunoassay,
reactions
when
enzyme-labelled
Introduction Cytomegalovirus (CMV) is recognized as an important human pathogen, capable of causing serious diseasein immunocompromised patients and in neonates infected in utero. CMV is also responsible for casesof acute hepatitis, infectious mononucleosis, and other pathologies. The best diagnostic method is the isolation of CMV from urine or throat washes.However, virus isolation is limited by the availability of cell cultures, and requires experienced personnel. Serological diagnosis of CMV infections is usually made by the complement fixation test (CFT). However, early diagnosis cannot be obtained by CFT, since sequential serum samplesare required. Rapid diagnosis of CMV infections can be obtained in some casesby detection of specific IgM antibodies. In recent years, a number of methods to detect specific IgM have been developed, including indirect immunofluorescence (IIF)‘, radioimmunoassay (RIA)’ and enzyme immunoassay (ELBA)“. All these tests show lower sensitivity than isolation when assaying cord sera or sera from congenitally infected children4.‘. Some false-positive results can be obtained by IIF in sera from normal blood donor@. RIA and ELBA show similar sensitivity when used for the detection of specific IgM in postnatal infections7, including pregnant women with primary infections*. *To whom correspondence should be addressed. 0888-0786/88/060423
+ 09 $03.00/O
423
(0 1988 Academic
Press Limited
424
F. de Ory et al.
ELISA offers some practical advantages over RIA since it uses stable and safe reagents. Thus, ELISA is applicable in the majority of clinical virology laboratories. In the present paper we compare the characteristics of commercial ELISA kits for detection of CMV specific IgM, including both indirect and antibody capture tests. Material and methods Sera A total of 88 serum sampleswere tested by all ELISA methods. They were grouped into four panels. Panel 1 68 serum samplesfrom 57 patients with evidence of CMV infection and with CMV specific IgM detected by the ELISA that we are currently using in our laboratory (Enzygnost CMV). Ten sera were from eight congenitally or perinatally infected babies (~4 months); one serum from a pregnant woman; seven sera from patients with hepatitis; 14 sera from 12 organ recipients, including bone marrow (4) kidney (4) heart (3) and liver (I); 22 sera from 15 patients with infectious mononucleosis like illness; and 14 sera from patients with fever and/or adenopathies. In four of the congenital or perinatal infections and in one cardiac recipient CMV was isolated from urine. Panel 2 Six serum samples from congenitally or perinatally infected babies (< 4 months), diagnosed by CMV isolation from urine, but negative in Enzygnost CMV for specific IgM. Panel 3 Ten sera from patients with infectious mononucleosis who were positive for specific IgM against the viral capsid antigen (VCA) of Epstein-Barr virus (EBV) and heterophil antibodies. Panel 4
Four sera containing rheumatoid factor (RF) and CMV specific IgG.
Serological methods ELISA. Both indirect ELISA and p chain capture methods were tested. The indirect methods were Enzygnost CMV (Behring Institute, Marburg. F.R.G.). CMV Stat M (Whittaker M.A. Bioproducts, Walkersville, MD, U.S.A.), and Abbott CMV M (Abbott Laboratories, North Chicago, IL, U.S.A.) The u chain capture methods were Vironostika anti-CMV IgM (Organon Teknika, Turnhout, Belgium) and two enzymelabelled antifen (ELA) methods: CMV IgM ELA (Medac, Hamburg, F.R.G.) and Cytotek M ELA ‘(Flow Laboratories, Irvine, Scotland). Sera were tested at a single dilution in each test as follows: 1 : 40, 1 : 80, 1 : 560, 1 : 100, 1 : 100 and 1 : 100, respectively. In two indirect methods (Enzygnost CMV and CMV Stat M) IgG was removed from the serum samplesprior to testing, using anti-human IgG antisera. Both Enzygnost CMV and CMV Stat M usescontrol antigen consisting of not infected cell cultures. With Cytotek M ELA three different methods were used: short (30 min), medium (60 min) or long (I 8 h) incubation periods with labelled antigen.
Serological diagnosis of cytomegalovirus infections
425
Other serological methods Heterophil antibodies were detected using the TM Absorption kit (Mercia Diagnostics, Shalford, U.K.). IgM to VCA-EBV was detected with a commercial IIF (Gull Laboratories, Salt L.ake City, UT, U.S.A.). RF was determined by a latex agglutination test (RapiTex RF, Behring Institute). CMV specific IgG was determined with a commercial ELISA (Enzygnost CMV). I;irzis
kolutiori
llrine samples were inoculated on cultures of human diploid fibroblasts and incubated at 37°C until cytopathic effect became apparent’. The isolated virus was identified as CMV by IIF using monoclonal antibodies (Monofluokit CMV. Diagnostics Pasteur, Marnes la Coquette. France). Results All ELISA methods showed agreement with 60 of the 68 serum samplesfrom Panel I (88.2%). Discrepant results are listed in Table I. The serum 1462~was the second serum sample from a congenitally or perinatally infected child, and it was positive only by Vironostika anti-CMV IgM. The first sample of this case (7%) was doubtful in CMV Stat M and Abbott CMV M. A serum sample (1366~) from another congenital or perinatal case, with a previous sample positive by all methods, was negative by Enzygnost CMV and doubtful by Abbott CMV M. The latter test gave equivocal results with two additional samplesfrom congenitally or perinatally infected babies. In the u chain capture methods two samples from cardiac recipients gave discrepant results. In one of these patients (1167i) CMV was grown from urine. A second sample from this patient, taken 2 weeks later, was positive in all methods including CMV IgM ELA. Finally, a serum sample from a patient with fever of unknown origin was negative by both ELAs (139Of). Results corresponding to samplesof Panel 2 are shown in Table 2. Vironostika antiCMV IgM. CMV IgM ELA, Cytotek M ELA, CMV Stat M and Abbott CMV M detected as positive 4, 4, 3, 2 and I samplesrespectively. One additional doubtful result was obtained by CMV Stat M. When we tested serum samplesfrom casesof IM caused by EBV (Panel 3) only five were negative by all methods. False--positive reactions are listed in Table 3. No falsepositive results were obtained by Enzygnost CMV or Vironostika anti-CMV IgM. CMV IgM ELA detected as positive one case (12%); CMV Stat M detected two as positive (2517 and 12%) and two doubtful (88f and 121f); and Abbott CMV M detected two as positive (2517f and 88f). In Cytotek M ELA the results were different depending on the procedure used. Incubation with labelled antigen for 18 h yielded two positive results. These false positive results disappeared when procedures with short incubation periods were used. Finally. results corresponding to Panel 4 are shown in Table 4. All the four sera tested were negative in Enzygnost CMV and in all u chain capture methods. In CMV Stat M and Abbott M CMV, however, we obtained positive results with two and three serum samples respectively.
Congenital Congenital Congenital Congenital Congenital H.R.11 H.R. Fever
Enzygnost CMV .~ ~~~___ + + + + + + * f + + + + + +
Stat M
of life from
CMV
*RF: rheumatoid factor tHA: heterophil antibodies. fSecond sample of 75~. @econd serum sample taken at the third month /IRecipient of a heart transplant.
7% 1462c$ 1366~s 828~ 3029~ 1167i 1705i 1390f
Sample No. Patient
M
with specific
* zt + It f + + +
CMV
IgM
by all ELISA
+ + + + + -
IgM ELA
methods
+ + + + + -
when
Cytotek
old.
t + + + + + +
___~ Vironostika anti - CMV IgM
he was 2 months
M ELA
p Chain capture methods
specific IgM (ELBA)
on serum samples from Panel 1
CMV
CMV
results obtained
Abbott
a child
~. ~~~ Indirect methods
Table 1. Discrepant
+1/5 +1/5 -
RF*
-
HA-f
4 2 P
0
.F %k
62c 661c 702~ 22oc 952~ 1121c
Sample No.
-
-
Enzygnost
CMV
CMV
Indirect
f -
+ +
Stat M
methods Abbott + -
M
CMV + + + + -
IgM ELA
specific IgM (ELISA)
on samples from Panel 2
CMV
CMV
Table 2. Results obtained
Cytotek + + + -
methods
M ELA
p Chain capture
+ + + + -
Vironostika anti-CMV IgM
% 4 a 3 4 EL 3. 2 ti
0% a 8.
*Results
2517f 2525f 88f 121f 128f
obtained
Sample’ No.
by using
short
-
Enzygnost
(Mm).
CMV
medium
+ I!c * +
Stat M
methods Abbott
--
+ + -
on samples from Panel 3
periods
M
with
CMV
labelled
+
antigen.
-/-I-i-i+ -1-l-t -l-i-l-l-
p Chain capture methods -IgM ELA Cytotek M ELA*
specific IgM (ELISA)
CMV
CMV
results obtained
(60 m) and long (18 h) incubation
CMV
Indirect
Table 3. Discrepant
-
Vironostika anti - CMV IgM --
F W 0 G 2 %
RF titre
I:10 1:40 I:20 I:5
Sample No.
4286 5 4 2
-
Enzygnost CMV
CMV
methods
+ +
Stat M
Indirect Abbott
Table 4. Results obtained
+ + +
CMV
CMV
M
CMV
IgM ELA
specific IgM (ELISA)
on samples from Panel 4
Cytotek
-
M ELA
p Chain capture methods Vironostika anti - CMV IgM
vl 0
430
F. de Ory et al. Discussion
The diagnosis of CMV infections has been hampered for several years in most clinical laboratories by the difficulties of virus isolation and by the lack of specific, sensitive and easy methods for detection of specific IgM antibodies. Since the development of ELISA methods for the serological diagnosis of viral infections, several commercial kits for the detection of CMV specific IgM are available to diagnostic laboratories. A number of different kinds of ELISA methods are now available, including indirect and u chain capture methods. In this study, a total of 74 serum samples from 63 patients with evidence of CMV infection (Panels 1 and 2) were tested. Out of these, 16 sera were from I4 congenitally or perinatally infected babies. None of the methods were able to detect specific IgM in all cases, as previously reported using ELISA and RIAs and IIF5. However, u chain capture methods showed a higher sensitivity, since Vironostika anti-CMV IgM detected 14 sera, CMV IgM ELA 13 sera and Cytotek M ELA 12 sera as positive. CMV Stat M and Abbott CMV M detected 10 and six positive sera respectively. Of the remaining 58 serum samples from 49 CMV cases (all in Panel l), only u chain capture methods failed to detect specific IgM in three cases (in two recipients of heart transplantation and one in a patient with fever). The discrepant results obtained could be caused by CMV strain heterogeneity, as has been described when measuring CMV total antibodies by CFT and indirect hemagglutination’“. However, these discrepancies were not detected when different CMV strains were processed in the same way to obtain CMV antigens to be used in ELISA for detection of specific IgG”. Unfortunately, we have no data about the specific strains used in the commercial kits compared in this paper for preparation of antigens, nor about the procedure followed to obtain the antigen in each case. Thus, we can not provide a satisfactory explanation for our discrepant results. CMV and EBV can in some instances cause similar clinical pictures (infectious mononucleosis, hepatitis, fever, adenopathies). The differential diagnosis of CMV and EBV infections is hampered by crossreaction between the two viruses. This cross reactivity becomes apparent when assaying specific IgM class antibodies, and it has been reported with IIF6, indirect ELISA”, RIA’ and ELA13. In the present study, Enzygnost and Vironostika anti-CMV IgM showed the highest specificity, since they detected as negative all the sera from acute EBV infections tested. For several years, rheumatoid factor has been recognized as a source of false positive results when assaying IgM specific antibodies, especially by indirect immunoassays. This problem can be avoided by absorption of sera with IgG coated latex beads. aggregated human IgG, Staph&coccus aureu~ protein A, or anti-human IgG for removal of RF or IgG prior to assay. These methods were recently compared for their ability to eliminate non-specific CMV TgM reactivity14. The treatment of serum samples with anti-human IgG was found to be the best method for eliminating such interference. Two of the indirect methods tested in this study involve the elimination of IgG from the samples prior to assay (Enzygnost CMV and CMV Stat M); however, only the first method was found to be free of non-specific reactions, while the other identified two RF positive sera as positive. This probably could be due to an insufficient amount of anti-human IgG in the antiserum supplied with the CMV Stat M. The remaining indirect method (Abbott CMV M) does not use any pre-treatment for eliminating such interference and three sera containing RF were identified as positive by this kit. All u chain capture methods were free of interferences due to the presence of specific IgG and RF.
Serological diagnosis of cytomegalovirus infections
431
The levels of sensitivity obtained with all methods make them useful for the diagnosis of CMV infections. However, some false positive results can be obtained, specially in CMV Stat and Abbott CMV M. For this reason, the positive resuls obtained with these kits should be interpreted with caution when sera from patients with RF or from casesof suspected acute EBV infections are analysed. Acknowledgements The authors are grateful to Mrs T. Minguito and Mrs M. A. Bustillo for their excellent technical assistance,and Mrs P. Sagties for typing the manuscript. References I. Hanshaw JB, Steinfeld HJ, White CJ. Fluorescent antibody test for cytomegalovirus macroglobulin. New Engl J Med 1968; 279: 56670. 2. Kangro HO. Evaluation of a radioimmunoassay for IgM class antibodies against cytomegalovirus. Br J exp Path01 1980; 61: 512-20. 3. Schmitz H, Doerr HW, Kampa D, Vogt, A. Solid phaseenzymeimmunoassayfor immunoglobulin M antibodies to cytomegalovirus. J Clin Microbial 1977; 5: 629-34. 4. Ahlfors K, Forsgreen M, Griffiths P, Mijller Nielsen C. Comparison of four serological tests for the detection of specificimmunoglobulinM in cord seraof infants congenitally infected with cytomegalovirus. Stand J Infect Dis 1987; 19: 303-8. 5. Thomson RB, Benedict ML, Congeni BL. Evaluation of a commercially available cytomegalovirus specific IgM test for the diagnosis of cytomegalovirus infection in the symptomatic newborn. Diagn Microbial Infect Dis 1987; 7: 2114. 6. Hekker AC, Brand-Saathof B. Vis J, Miejers RC. Indirect immunofluorescence test for detection of IgM antibodies to cytomegalovirus. J Infect Dis 1979; 140: 596600. 7. Demmler GJ, Six HR, Hurst SM, Yow MD. Enzyme linked immunosorbent assay for the detection of IgM class antibodies to cytomegalovirus. J Infect Dis 1986; 153: 1152-5. 8. Stagno S, Tinker MK, Elrod C, Fuccillo DA, Cloud G, O’Beirne AJ. Immunoglobulin M antibodies detected by enzyme linked immunosorbent assay and radioimmunoassay in the diagnosis of cytomegalovirus infections in pregnant women and newborn infants. J Clin Microbial 1985; 21: 93&5. 9. Reynolds DW, Stagno S, Alford CA. Laboratory diagnosis of cytomegalovirus infections. In: Lennette EH, Schmidt NJ, eds. Diagnostic procedures for viral rickettsial and chlamydial infections, 5th edn. Washington: American Public Health Association, 1979: 3999439. IO. Faix RG. Cytomegalovirus antigenic heterogeneity can cause false negative results in indirect hemagglutination and complement fixation antibody assays. J Clin Microbial 1985; 22: 768 71. 11. Adler SP, McVoy M. Detection of cytomegalovirus antibody enzyme immunoassay and lack of evidence for an effect resulting from strain heterogeneity. J Clin Microbial 1986; 24: 870-2. 12. Krishna RV, Meurman OH, Ziegler T, Krech UH. Solid phase enzyme immunoassay for determination of antibodies to cytomegalovirus. J Clin Microbial 1980; 12: 46-51. 13. Ory F de, Echevarria JM, Leon P, Rodriguez M, Najera R. Diagnostic0 diferencial de mononucleosis infecciosa por virus Epstein-Barr y citomegalovirus mediante detection de IgM especifica. Enf Infec Microbial Clin 1988; 6: 19-23. 14. Joassin L, Reginster M. Elimination of non specific cytomegalovirus immunoglobulin M activities in the enzyme linked immunosorbent assay by using anti-human immunoglobulin G. J Clin Microbial 1986; 23: 57681. (Mamucripr
accepted
Ist July
1988)
in Infectious Disease (1988) 2, 42343
I
Serological diagnosis of cytomegalovirus infections: comparison of six commercial methods of ELISA
Fernando de Ory*, Pilar Perez-Breiia and Jo& M. Echevarria
Servicio de Virologia. Centro National de Microhiologia, Virologia Sanitarias, Majadahonda, 28220 Madrid, Spain
e lnmunologia
Six commercial enzyme-linked immunosorbent assays (ELISA) (three indirect and three u chain capture methods, two of them with enzyme-labelled antigen, ELA) for detection of cytomegalovirus (CMV) specific IgM were compared, using sera from patients with CMV infection, from recent Epstein-Barr virus (EBV) infections. and sera containing rheumatoid factor (RF) and CMV specific IgG. The six methods showed similar sensitivities. In sera from congenital infections the most sensitive
assayswere the p chain capture methods.In two indirect methods,false positive reactionsappearedwhen assayingserawith RF and specificIgG. The sameindirect methods and both ELA assays gave some CMV analysing sera from recent EBV infections. Ke~;words: cytomegalovirus, antigen
specific IgM,
IgM
positive
enzyme immunoassay,
reactions
when
enzyme-labelled
Introduction Cytomegalovirus (CMV) is recognized as an important human pathogen, capable of causing serious diseasein immunocompromised patients and in neonates infected in utero. CMV is also responsible for casesof acute hepatitis, infectious mononucleosis, and other pathologies. The best diagnostic method is the isolation of CMV from urine or throat washes.However, virus isolation is limited by the availability of cell cultures, and requires experienced personnel. Serological diagnosis of CMV infections is usually made by the complement fixation test (CFT). However, early diagnosis cannot be obtained by CFT, since sequential serum samplesare required. Rapid diagnosis of CMV infections can be obtained in some casesby detection of specific IgM antibodies. In recent years, a number of methods to detect specific IgM have been developed, including indirect immunofluorescence (IIF)‘, radioimmunoassay (RIA)’ and enzyme immunoassay (ELBA)“. All these tests show lower sensitivity than isolation when assaying cord sera or sera from congenitally infected children4.‘. Some false-positive results can be obtained by IIF in sera from normal blood donor@. RIA and ELBA show similar sensitivity when used for the detection of specific IgM in postnatal infections7, including pregnant women with primary infections*. *To whom correspondence should be addressed. 0888-0786/88/060423
+ 09 $03.00/O
423
(0 1988 Academic
Press Limited
424
F. de Ory et al.
ELISA offers some practical advantages over RIA since it uses stable and safe reagents. Thus, ELISA is applicable in the majority of clinical virology laboratories. In the present paper we compare the characteristics of commercial ELISA kits for detection of CMV specific IgM, including both indirect and antibody capture tests. Material and methods Sera A total of 88 serum sampleswere tested by all ELISA methods. They were grouped into four panels. Panel 1 68 serum samplesfrom 57 patients with evidence of CMV infection and with CMV specific IgM detected by the ELISA that we are currently using in our laboratory (Enzygnost CMV). Ten sera were from eight congenitally or perinatally infected babies (~4 months); one serum from a pregnant woman; seven sera from patients with hepatitis; 14 sera from 12 organ recipients, including bone marrow (4) kidney (4) heart (3) and liver (I); 22 sera from 15 patients with infectious mononucleosis like illness; and 14 sera from patients with fever and/or adenopathies. In four of the congenital or perinatal infections and in one cardiac recipient CMV was isolated from urine. Panel 2 Six serum samples from congenitally or perinatally infected babies (< 4 months), diagnosed by CMV isolation from urine, but negative in Enzygnost CMV for specific IgM. Panel 3 Ten sera from patients with infectious mononucleosis who were positive for specific IgM against the viral capsid antigen (VCA) of Epstein-Barr virus (EBV) and heterophil antibodies. Panel 4
Four sera containing rheumatoid factor (RF) and CMV specific IgG.
Serological methods ELISA. Both indirect ELISA and p chain capture methods were tested. The indirect methods were Enzygnost CMV (Behring Institute, Marburg. F.R.G.). CMV Stat M (Whittaker M.A. Bioproducts, Walkersville, MD, U.S.A.), and Abbott CMV M (Abbott Laboratories, North Chicago, IL, U.S.A.) The u chain capture methods were Vironostika anti-CMV IgM (Organon Teknika, Turnhout, Belgium) and two enzymelabelled antifen (ELA) methods: CMV IgM ELA (Medac, Hamburg, F.R.G.) and Cytotek M ELA ‘(Flow Laboratories, Irvine, Scotland). Sera were tested at a single dilution in each test as follows: 1 : 40, 1 : 80, 1 : 560, 1 : 100, 1 : 100 and 1 : 100, respectively. In two indirect methods (Enzygnost CMV and CMV Stat M) IgG was removed from the serum samplesprior to testing, using anti-human IgG antisera. Both Enzygnost CMV and CMV Stat M usescontrol antigen consisting of not infected cell cultures. With Cytotek M ELA three different methods were used: short (30 min), medium (60 min) or long (I 8 h) incubation periods with labelled antigen.
Serological diagnosis of cytomegalovirus infections
425
Other serological methods Heterophil antibodies were detected using the TM Absorption kit (Mercia Diagnostics, Shalford, U.K.). IgM to VCA-EBV was detected with a commercial IIF (Gull Laboratories, Salt L.ake City, UT, U.S.A.). RF was determined by a latex agglutination test (RapiTex RF, Behring Institute). CMV specific IgG was determined with a commercial ELISA (Enzygnost CMV). I;irzis
kolutiori
llrine samples were inoculated on cultures of human diploid fibroblasts and incubated at 37°C until cytopathic effect became apparent’. The isolated virus was identified as CMV by IIF using monoclonal antibodies (Monofluokit CMV. Diagnostics Pasteur, Marnes la Coquette. France). Results All ELISA methods showed agreement with 60 of the 68 serum samplesfrom Panel I (88.2%). Discrepant results are listed in Table I. The serum 1462~was the second serum sample from a congenitally or perinatally infected child, and it was positive only by Vironostika anti-CMV IgM. The first sample of this case (7%) was doubtful in CMV Stat M and Abbott CMV M. A serum sample (1366~) from another congenital or perinatal case, with a previous sample positive by all methods, was negative by Enzygnost CMV and doubtful by Abbott CMV M. The latter test gave equivocal results with two additional samplesfrom congenitally or perinatally infected babies. In the u chain capture methods two samples from cardiac recipients gave discrepant results. In one of these patients (1167i) CMV was grown from urine. A second sample from this patient, taken 2 weeks later, was positive in all methods including CMV IgM ELA. Finally, a serum sample from a patient with fever of unknown origin was negative by both ELAs (139Of). Results corresponding to samplesof Panel 2 are shown in Table 2. Vironostika antiCMV IgM. CMV IgM ELA, Cytotek M ELA, CMV Stat M and Abbott CMV M detected as positive 4, 4, 3, 2 and I samplesrespectively. One additional doubtful result was obtained by CMV Stat M. When we tested serum samplesfrom casesof IM caused by EBV (Panel 3) only five were negative by all methods. False--positive reactions are listed in Table 3. No falsepositive results were obtained by Enzygnost CMV or Vironostika anti-CMV IgM. CMV IgM ELA detected as positive one case (12%); CMV Stat M detected two as positive (2517 and 12%) and two doubtful (88f and 121f); and Abbott CMV M detected two as positive (2517f and 88f). In Cytotek M ELA the results were different depending on the procedure used. Incubation with labelled antigen for 18 h yielded two positive results. These false positive results disappeared when procedures with short incubation periods were used. Finally. results corresponding to Panel 4 are shown in Table 4. All the four sera tested were negative in Enzygnost CMV and in all u chain capture methods. In CMV Stat M and Abbott M CMV, however, we obtained positive results with two and three serum samples respectively.
Congenital Congenital Congenital Congenital Congenital H.R.11 H.R. Fever
Enzygnost CMV .~ ~~~___ + + + + + + * f + + + + + +
Stat M
of life from
CMV
*RF: rheumatoid factor tHA: heterophil antibodies. fSecond sample of 75~. @econd serum sample taken at the third month /IRecipient of a heart transplant.
7% 1462c$ 1366~s 828~ 3029~ 1167i 1705i 1390f
Sample No. Patient
M
with specific
* zt + It f + + +
CMV
IgM
by all ELISA
+ + + + + -
IgM ELA
methods
+ + + + + -
when
Cytotek
old.
t + + + + + +
___~ Vironostika anti - CMV IgM
he was 2 months
M ELA
p Chain capture methods
specific IgM (ELBA)
on serum samples from Panel 1
CMV
CMV
results obtained
Abbott
a child
~. ~~~ Indirect methods
Table 1. Discrepant
+1/5 +1/5 -
RF*
-
HA-f
4 2 P
0
.F %k
62c 661c 702~ 22oc 952~ 1121c
Sample No.
-
-
Enzygnost
CMV
CMV
Indirect
f -
+ +
Stat M
methods Abbott + -
M
CMV + + + + -
IgM ELA
specific IgM (ELISA)
on samples from Panel 2
CMV
CMV
Table 2. Results obtained
Cytotek + + + -
methods
M ELA
p Chain capture
+ + + + -
Vironostika anti-CMV IgM
% 4 a 3 4 EL 3. 2 ti
0% a 8.
*Results
2517f 2525f 88f 121f 128f
obtained
Sample’ No.
by using
short
-
Enzygnost
(Mm).
CMV
medium
+ I!c * +
Stat M
methods Abbott
--
+ + -
on samples from Panel 3
periods
M
with
CMV
labelled
+
antigen.
-/-I-i-i+ -1-l-t -l-i-l-l-
p Chain capture methods -IgM ELA Cytotek M ELA*
specific IgM (ELISA)
CMV
CMV
results obtained
(60 m) and long (18 h) incubation
CMV
Indirect
Table 3. Discrepant
-
Vironostika anti - CMV IgM --
F W 0 G 2 %
RF titre
I:10 1:40 I:20 I:5
Sample No.
4286 5 4 2
-
Enzygnost CMV
CMV
methods
+ +
Stat M
Indirect Abbott
Table 4. Results obtained
+ + +
CMV
CMV
M
CMV
IgM ELA
specific IgM (ELISA)
on samples from Panel 4
Cytotek
-
M ELA
p Chain capture methods Vironostika anti - CMV IgM
vl 0
430
F. de Ory et al. Discussion
The diagnosis of CMV infections has been hampered for several years in most clinical laboratories by the difficulties of virus isolation and by the lack of specific, sensitive and easy methods for detection of specific IgM antibodies. Since the development of ELISA methods for the serological diagnosis of viral infections, several commercial kits for the detection of CMV specific IgM are available to diagnostic laboratories. A number of different kinds of ELISA methods are now available, including indirect and u chain capture methods. In this study, a total of 74 serum samples from 63 patients with evidence of CMV infection (Panels 1 and 2) were tested. Out of these, 16 sera were from I4 congenitally or perinatally infected babies. None of the methods were able to detect specific IgM in all cases, as previously reported using ELISA and RIAs and IIF5. However, u chain capture methods showed a higher sensitivity, since Vironostika anti-CMV IgM detected 14 sera, CMV IgM ELA 13 sera and Cytotek M ELA 12 sera as positive. CMV Stat M and Abbott CMV M detected 10 and six positive sera respectively. Of the remaining 58 serum samples from 49 CMV cases (all in Panel l), only u chain capture methods failed to detect specific IgM in three cases (in two recipients of heart transplantation and one in a patient with fever). The discrepant results obtained could be caused by CMV strain heterogeneity, as has been described when measuring CMV total antibodies by CFT and indirect hemagglutination’“. However, these discrepancies were not detected when different CMV strains were processed in the same way to obtain CMV antigens to be used in ELISA for detection of specific IgG”. Unfortunately, we have no data about the specific strains used in the commercial kits compared in this paper for preparation of antigens, nor about the procedure followed to obtain the antigen in each case. Thus, we can not provide a satisfactory explanation for our discrepant results. CMV and EBV can in some instances cause similar clinical pictures (infectious mononucleosis, hepatitis, fever, adenopathies). The differential diagnosis of CMV and EBV infections is hampered by crossreaction between the two viruses. This cross reactivity becomes apparent when assaying specific IgM class antibodies, and it has been reported with IIF6, indirect ELISA”, RIA’ and ELA13. In the present study, Enzygnost and Vironostika anti-CMV IgM showed the highest specificity, since they detected as negative all the sera from acute EBV infections tested. For several years, rheumatoid factor has been recognized as a source of false positive results when assaying IgM specific antibodies, especially by indirect immunoassays. This problem can be avoided by absorption of sera with IgG coated latex beads. aggregated human IgG, Staph&coccus aureu~ protein A, or anti-human IgG for removal of RF or IgG prior to assay. These methods were recently compared for their ability to eliminate non-specific CMV TgM reactivity14. The treatment of serum samples with anti-human IgG was found to be the best method for eliminating such interference. Two of the indirect methods tested in this study involve the elimination of IgG from the samples prior to assay (Enzygnost CMV and CMV Stat M); however, only the first method was found to be free of non-specific reactions, while the other identified two RF positive sera as positive. This probably could be due to an insufficient amount of anti-human IgG in the antiserum supplied with the CMV Stat M. The remaining indirect method (Abbott CMV M) does not use any pre-treatment for eliminating such interference and three sera containing RF were identified as positive by this kit. All u chain capture methods were free of interferences due to the presence of specific IgG and RF.
Serological diagnosis of cytomegalovirus infections
431
The levels of sensitivity obtained with all methods make them useful for the diagnosis of CMV infections. However, some false positive results can be obtained, specially in CMV Stat and Abbott CMV M. For this reason, the positive resuls obtained with these kits should be interpreted with caution when sera from patients with RF or from casesof suspected acute EBV infections are analysed. Acknowledgements The authors are grateful to Mrs T. Minguito and Mrs M. A. Bustillo for their excellent technical assistance,and Mrs P. Sagties for typing the manuscript. References I. Hanshaw JB, Steinfeld HJ, White CJ. Fluorescent antibody test for cytomegalovirus macroglobulin. New Engl J Med 1968; 279: 56670. 2. Kangro HO. Evaluation of a radioimmunoassay for IgM class antibodies against cytomegalovirus. Br J exp Path01 1980; 61: 512-20. 3. Schmitz H, Doerr HW, Kampa D, Vogt, A. Solid phaseenzymeimmunoassayfor immunoglobulin M antibodies to cytomegalovirus. J Clin Microbial 1977; 5: 629-34. 4. Ahlfors K, Forsgreen M, Griffiths P, Mijller Nielsen C. Comparison of four serological tests for the detection of specificimmunoglobulinM in cord seraof infants congenitally infected with cytomegalovirus. Stand J Infect Dis 1987; 19: 303-8. 5. Thomson RB, Benedict ML, Congeni BL. Evaluation of a commercially available cytomegalovirus specific IgM test for the diagnosis of cytomegalovirus infection in the symptomatic newborn. Diagn Microbial Infect Dis 1987; 7: 2114. 6. Hekker AC, Brand-Saathof B. Vis J, Miejers RC. Indirect immunofluorescence test for detection of IgM antibodies to cytomegalovirus. J Infect Dis 1979; 140: 596600. 7. Demmler GJ, Six HR, Hurst SM, Yow MD. Enzyme linked immunosorbent assay for the detection of IgM class antibodies to cytomegalovirus. J Infect Dis 1986; 153: 1152-5. 8. Stagno S, Tinker MK, Elrod C, Fuccillo DA, Cloud G, O’Beirne AJ. Immunoglobulin M antibodies detected by enzyme linked immunosorbent assay and radioimmunoassay in the diagnosis of cytomegalovirus infections in pregnant women and newborn infants. J Clin Microbial 1985; 21: 93&5. 9. Reynolds DW, Stagno S, Alford CA. Laboratory diagnosis of cytomegalovirus infections. In: Lennette EH, Schmidt NJ, eds. Diagnostic procedures for viral rickettsial and chlamydial infections, 5th edn. Washington: American Public Health Association, 1979: 3999439. IO. Faix RG. Cytomegalovirus antigenic heterogeneity can cause false negative results in indirect hemagglutination and complement fixation antibody assays. J Clin Microbial 1985; 22: 768 71. 11. Adler SP, McVoy M. Detection of cytomegalovirus antibody enzyme immunoassay and lack of evidence for an effect resulting from strain heterogeneity. J Clin Microbial 1986; 24: 870-2. 12. Krishna RV, Meurman OH, Ziegler T, Krech UH. Solid phase enzyme immunoassay for determination of antibodies to cytomegalovirus. J Clin Microbial 1980; 12: 46-51. 13. Ory F de, Echevarria JM, Leon P, Rodriguez M, Najera R. Diagnostic0 diferencial de mononucleosis infecciosa por virus Epstein-Barr y citomegalovirus mediante detection de IgM especifica. Enf Infec Microbial Clin 1988; 6: 19-23. 14. Joassin L, Reginster M. Elimination of non specific cytomegalovirus immunoglobulin M activities in the enzyme linked immunosorbent assay by using anti-human immunoglobulin G. J Clin Microbial 1986; 23: 57681. (Mamucripr
accepted
Ist July
1988)