Journal qf’ Virological Methods, 40 (1992) 2 19-224 ic 1992 Elsevier Science Publishers B.V. / All rights
VIRMET
219 reserved
/ 0166-0934/92/$05.00
01410
Detection of cytomegalovirus by a rapid culture system: a comparison of monoclonal antibodies in a clinical setting Deenan
Pillay, Heather
Division of’ Communicable
Charman,
Jennie Lok and Paul D. Griffiths
Diseases, Royal Free Hospital and School (Accepted
14 June
of Medicine, London (UK)
1992)
Summary A monoclonal antibody cocktail, directed against immediate early antigens of cytomegalovirus, was assessed in a rapid culture system for detection of CMV (DEAFF) in clinical samples. The antibody cocktail was shown to be equivalent in sensitivity and specificity to our currently used monoclonal antibody. A comparison of DEAFF (using either monoclonal preparation) and conventional cell culture shows that DEAFF detects a smaller proportion of CMV positive samples from those receiving antiviral chemotherapy. The reasons for the discrepancies observed between these techniques are discussed. Cytomegalovirus; therapy
Rapid culture system; Monoclonal
antibody;
Antiviral chemo-
Introduction Cytomegalovirus infection is a major cause of morbidity and mortality in immunocompromised individuals (Griffiths, 1990). The availability of licensed drugs for treatment of CMV disease (Balfour, 1990) has heightened the requirements for rapid diagnostic tests which are both sensitive and specific and can easily be introduced into the diagnostic laboratory (Pillay et al., 1992). The detection of CMV from clinical samples received by our laboratory is routinely undertaken by immunofluorescent staining of CMV immediate early antigens Correspondence to: D. Pillay. Department of Virology. Rowland Hill Street. London NW3 2PF. UK.
Royal
Free Hospital
and School
of Medicine.
in previously inoculated cell monolayers (Griffiths et al.. 1984). We now report a comparison of a cocktail of two monoclonal antibodies prepared by A.D.I. Diagnostics (Toronto, Canada) with our currently used monoclonal antibody (~63327) in the processing of 1558 consecutive clinical samples obtained from multiple sites. In addition, a subgroup of these was also assessed for viral infection by conventional cell culture. and the results related to clinical illness and antiviral therapy.
Materials and Methods A total of 1558 consecutive clinical samples received in the laboratory from 330 individuals (52 HIV/AIDS, 50 bone marrow transplant, 80 renal transplant, 25 liver transplant and 123 others) were assessed in this study. These samples comprised urine (41X), whole blood (23%), saliva (19X), bronchoalveolar lavage fluid (5%) and others (13%) including tissue biopsies. Specimens were tested either as part of protocol surveillance or as an investigation of symptoms. The presence or absence of CMV disease and antiviral therapy (acyclovir or ganciclovir) in these patients at the time of sampling was documented. Disease was defined as characteristic signs and symptoms. in association with detection of CMV from the relevant tissue, except in the case of retinitis, which was diagnosed by an experienced ophthalmologist on the basis of typical clinical features. Specimens for routine cell culture were inoculated in duplicate onto monolayers of human embryonic lung fibroblasts and incubated for a minimum of 3 weeks. The monolayers were observed at least twice weekly for signs of a characteristic cytopathic effect. For the rapid culture system, detection of early antigenic fluorescent foci (DEAFF), eight-well Labtec slides. containing monolayers of human embryo lung fibroblasts, were inoculated with four clinical specimens each in duplicate. 16624 h later, the cells were fixed and monoclonal ~63-27 added to the upper row of cells, whereas to the lower row of cells the A.D.I. monoclonal cocktail was added in a dilution of 1:’1000 of the original material. The slides were processed subsequently by the addition of an affinity-purified goat anti-mouse IgG serum conjugated with fluorescein isothiocyanate. Positive and negative controls were run with each batch of slides. A positive result was scored where at least one fluorescent focus was observed (Griftiths et al., 1984). The monoclonal antibody ~63-27 was kindly provided by Dr. W. Britt (Birmingham, Alabama) and is used routinely in the DEAFF test used by our laboratory. This replaced the pool of monoclonal antibodies previously used in our DEAFF test (Stirk and Griffiths, 1987). when these became unavailable. The AD1 cocktail consisted of two monoclonal antibodies prepared and Canada). Both preparations of supplied by A.D.I. Diagnostics (Ontario. monoclonals were directed against immediate early antigens of human cytomegalovirus.
Statistical
analysis of groups was undertaken
by the chi-squared
test
Results Of all the samples processed, 62 (4%) were found positive by the use of either monoclonal preparation. Of these, 13% were positive only with the A.D.I. monoclonal cocktail, 10% were positive only with monoclonal ~63-27, and 77% were positive with both (Table 1). All the samples found positive were obtained from individuals who were CMV seropositive, or who had received organs from seropositive donors, and we therefore had no evidence of false positive results. Accordingly, reactivity with either monoclonal was scored as positive. The overall sensitivity of the A.D.I. cocktail was then calculated to be 90% vs. 87% for mAb ~63-27. Subjectively, the fluorescent pattern given by the A.D.I. cocktail was noticeably brighter, and, therefore, easier to read. There were no major differences in sensitivities of detection between samples from different sites or different patient groups, in cases where sufficient positive results were obtained. A plot of number of foci observed between monoclonals gave a good correlation, and the samples missed by one preparation had only one or two foci per field with the other. In addition to tests in DEAFF with both sets of monoclonals, 812 of the above samples were inoculated concurrently into routine cell culture. CMV was detected in 82 samples by any method, of which 95% were positive by conventional cell culture compared to 43% by DEAFF. Patients whose samples were DEAFF positive. culture negative, and on whom previous surveillance had been undertaken by our laboratory. had all excreted CMV at some other time, providing further evidence for the specificity of DEAFF. The sensitivity of DEAFF is lower than that previously reported (Stirk and Griffiths, 1987). Since the sensitivity is related to the amount of virus inoculum into the assay system, the results were evaluated in the context of antiviral therapy at the time of sampling. DEAFF detected a smaller proportion of CMV positive samples from those receiving anti-CMV prophylaxis or therapy (acyclovir or ganciclovir). than from those who did not (28% vs. 47%) but this TABLE I A comparison DEAFF
of monoclonal
antibodies
in the analysis of 1558 consecutive
mAb 6327 (no. samples)
A.D.I. mAbs (no. samples) + Total
clinical specimens by Total
+
_
48 6
8 1496
I502
54
I.504
1558
56
777 ___
TABLE
2
DEAFF antiviral
results in 82 clinical therapy
Therapy
DEAFF
samples
(No.
positive
by any method
Negative
Total
Yes No
5 30
I3 34
IX 64
Total
35
47
81
analysis
TABLE
3
DEAFF disease
results
was undertaken
in X1 culture
DEAFF
Disease
positive
(No.
to compare
clinical
DEAFF
specimens
taken
m relation
to
from
Yes No
I’ 23
13 34
15 51
Total
35
47
x1
to compare
between
individuals
groups.
in relation
to CMV
Y2 Total
was undertaken
samples
samples) Negative
analysis
individuals
2.09
positive
Positive
Statistical
from
samples)
Positive
Statistical
taken
DEAFF
positive
II.41
results
between
groups
difference did not reach statistical significance (Table 2). We also investigated the relationship of CMV disease at time of sampling to the DEAFF results. since this may assist in clinical management decisions. The proportion of such samples detected by DEAFF from individuals with CMV disease compared to those without were similar (48% vs. 40%) (Table 3). Culture positive. DEAFF negative samples appeared to be spread evenly among those from whom multiple specimens were taken. rather than there being a preponderance of such results in samples from particular individuals.
Conclusions There is increasing demand for rapid viral diagnostic techniques as a wider array of effective antiviral therapies become available. Many of these techniques utilise monoclonal antibodies in order to detect viral proteins. Although highly specific, the use of such antibodies demands that they recognise an epitope which is highly conserved between strains of virus. The introduction of a new monoclonal antibody into an existing assay requires therefore full validation against previously used reagents. The results obtained with the A.D.I. monoclonal cocktail accord well with our existing monoclonal
223
antibody, ~63-27, when introduced into a rapid culture system to diagnose CMV infection in a variety of patient groups. The fact that samples missed by one monoclonal preparation had only one or two foci per field with the other, supports our original proposition that DEAFF is operating at a level where low quantities of virus are missed. Since there is a stochastic probability of detecting a single focus per field, it is concluded that the two monoclonal preparations are equivalent. The sensitivity of DEAFF, using either monoclonal set, was low compared to conventional cell culture. Analysis suggests that this sensitivity is reduced further, although not significantly, when the technique is used on samples from individuals receiving antiviral treatment and presumably reflects reduced viral shedding into the specimen. Alternatively, the drug may itself be inoculated, especially when the specimen is urine from an individual receiving acyclovir. On the other hand, samples from ill patients, in whom an increased viral excretion might be expected, were not more likely to be positive in DEAFF than samples from those with asymptomatic CMV infection. Finally, since specimens showing discrepant culture and DEAFF results did not all tend to be drawn from the same few individuals, it is unlikely that our findings are explained by the existence of virus strains (Colimon et al., 1985) which escape detection by the monoclonal antibodies used in our study. Such a lack of variation at the antigenic site recognised by mAb ~63-27 has recently been confirmed in an assessment of a rapid detection system for CMV in neonatal urine samples (Boppana et al.. 1992). These results are surprising, since those obtained by a previous comparison of diagnostic techniques, in which the sensitivity of DEAFF was calculated to be 77% (Stirk et al., 1987). A number of differences may have contributed to this discrepancy, such as the monoclonal antibodies and cell lines used in the assay, the patient groups from which samples were obtained and the use of antiviral chemotherapy. It also illustrates that the performance of assays may vary and must be kept under constant review. The factors contributing to such performance may lie outside laboratory control, and show the importance of clinico-pathological liaison. Further, our results underline the importance of maintaining cell culture as a ‘gold standard’ for CMV diagnosis. Clearly, improvements in rapid diagnostic tests for CMV are required. The polymerase chain reaction (PCR) may provide an advantage in terms of speed and sensitivity (Desselberger et al., 1990) and has been used for CMV in a research setting (Demmler et al., 1987). We are currently evaluating this technique, compared to DEAFF and conventional cell culture. for diagnosing clinical CMV infection in a routine laboratory setting.
References Balfour, H.H. 12 (Suppl.
(1990) Management 7). S849--S860.
of cytomegalovirus
disease
with antiviral
drugs.
Rev. Infect.
Dis.
1’4 Boppana, S.B.. Smith, R.J.. Stagno. S. and Britt. W.J. (1993) Evaluation of a microtitre plate fluorescent-antibody assay for rapid detection of human cytomegalovirus infection. J. Clin. Micro. 30, 721 7X. Colimon, R.. Somogyi. T.. Bertrand, C. and Michelson. S. (1985) Demonstration of the colincarity of human cytomegalovirus genomes and construction of restriction maps of unknown isolates using cloned subgenomic fragments. J. Gen. Virol. 66. 2183 2198. Demmler. G.J., Buffone G.J.. Schimbor. C.M. and May, R.A. (1988) Detection of cytomegalovirus in urine from newborns by using polymcrase chain reaction DNA amplification. J. Infect. Dis. 158. II77 1184. Desselberger. U. and Collingham. K. (1990) Molecular techniques in the diagnosis of human infectious diseases. Genitourin. Med. 66. 3 I3 323. Griffiths. P.D. (1990) ‘Cytomegalovirus‘. In: A.J. Zuckerman. J.E. Banatvala. and J.R. Pattison (Eds). Principles and Practice of Clinical Virology, 2nd edn. 699101. Wiley. London. Griftiths. P.D.. Panjwani, D.D.. Stirk. P.R. et al. (1984) Rapid detection of cytomegalovirua infection in immunocompromised patients by detection of early antigcnic fluorescent foci. Lancet ii, 1242--l 245. Pillay. D. and Griffiths, P.D. (1991) Diagnosis of cytomegalovirus infection: a review. Genitnurin. Med. 6X. 1833188. Stirk. P.R. and Griffiths. P.D. (1987) Use uf monoclonal antibodies for the diagnosis 01 cytomegalovirus infection by the detection of early antigenic fluorescent POCI (DEAFF) in ccl1 culture. J. Med. Virol. 21. 329 337.
VIRMET
219 reserved
/ 0166-0934/92/$05.00
01410
Detection of cytomegalovirus by a rapid culture system: a comparison of monoclonal antibodies in a clinical setting Deenan
Pillay, Heather
Division of’ Communicable
Charman,
Jennie Lok and Paul D. Griffiths
Diseases, Royal Free Hospital and School (Accepted
14 June
of Medicine, London (UK)
1992)
Summary A monoclonal antibody cocktail, directed against immediate early antigens of cytomegalovirus, was assessed in a rapid culture system for detection of CMV (DEAFF) in clinical samples. The antibody cocktail was shown to be equivalent in sensitivity and specificity to our currently used monoclonal antibody. A comparison of DEAFF (using either monoclonal preparation) and conventional cell culture shows that DEAFF detects a smaller proportion of CMV positive samples from those receiving antiviral chemotherapy. The reasons for the discrepancies observed between these techniques are discussed. Cytomegalovirus; therapy
Rapid culture system; Monoclonal
antibody;
Antiviral chemo-
Introduction Cytomegalovirus infection is a major cause of morbidity and mortality in immunocompromised individuals (Griffiths, 1990). The availability of licensed drugs for treatment of CMV disease (Balfour, 1990) has heightened the requirements for rapid diagnostic tests which are both sensitive and specific and can easily be introduced into the diagnostic laboratory (Pillay et al., 1992). The detection of CMV from clinical samples received by our laboratory is routinely undertaken by immunofluorescent staining of CMV immediate early antigens Correspondence to: D. Pillay. Department of Virology. Rowland Hill Street. London NW3 2PF. UK.
Royal
Free Hospital
and School
of Medicine.
in previously inoculated cell monolayers (Griffiths et al.. 1984). We now report a comparison of a cocktail of two monoclonal antibodies prepared by A.D.I. Diagnostics (Toronto, Canada) with our currently used monoclonal antibody (~63327) in the processing of 1558 consecutive clinical samples obtained from multiple sites. In addition, a subgroup of these was also assessed for viral infection by conventional cell culture. and the results related to clinical illness and antiviral therapy.
Materials and Methods A total of 1558 consecutive clinical samples received in the laboratory from 330 individuals (52 HIV/AIDS, 50 bone marrow transplant, 80 renal transplant, 25 liver transplant and 123 others) were assessed in this study. These samples comprised urine (41X), whole blood (23%), saliva (19X), bronchoalveolar lavage fluid (5%) and others (13%) including tissue biopsies. Specimens were tested either as part of protocol surveillance or as an investigation of symptoms. The presence or absence of CMV disease and antiviral therapy (acyclovir or ganciclovir) in these patients at the time of sampling was documented. Disease was defined as characteristic signs and symptoms. in association with detection of CMV from the relevant tissue, except in the case of retinitis, which was diagnosed by an experienced ophthalmologist on the basis of typical clinical features. Specimens for routine cell culture were inoculated in duplicate onto monolayers of human embryonic lung fibroblasts and incubated for a minimum of 3 weeks. The monolayers were observed at least twice weekly for signs of a characteristic cytopathic effect. For the rapid culture system, detection of early antigenic fluorescent foci (DEAFF), eight-well Labtec slides. containing monolayers of human embryo lung fibroblasts, were inoculated with four clinical specimens each in duplicate. 16624 h later, the cells were fixed and monoclonal ~63-27 added to the upper row of cells, whereas to the lower row of cells the A.D.I. monoclonal cocktail was added in a dilution of 1:’1000 of the original material. The slides were processed subsequently by the addition of an affinity-purified goat anti-mouse IgG serum conjugated with fluorescein isothiocyanate. Positive and negative controls were run with each batch of slides. A positive result was scored where at least one fluorescent focus was observed (Griftiths et al., 1984). The monoclonal antibody ~63-27 was kindly provided by Dr. W. Britt (Birmingham, Alabama) and is used routinely in the DEAFF test used by our laboratory. This replaced the pool of monoclonal antibodies previously used in our DEAFF test (Stirk and Griffiths, 1987). when these became unavailable. The AD1 cocktail consisted of two monoclonal antibodies prepared and Canada). Both preparations of supplied by A.D.I. Diagnostics (Ontario. monoclonals were directed against immediate early antigens of human cytomegalovirus.
Statistical
analysis of groups was undertaken
by the chi-squared
test
Results Of all the samples processed, 62 (4%) were found positive by the use of either monoclonal preparation. Of these, 13% were positive only with the A.D.I. monoclonal cocktail, 10% were positive only with monoclonal ~63-27, and 77% were positive with both (Table 1). All the samples found positive were obtained from individuals who were CMV seropositive, or who had received organs from seropositive donors, and we therefore had no evidence of false positive results. Accordingly, reactivity with either monoclonal was scored as positive. The overall sensitivity of the A.D.I. cocktail was then calculated to be 90% vs. 87% for mAb ~63-27. Subjectively, the fluorescent pattern given by the A.D.I. cocktail was noticeably brighter, and, therefore, easier to read. There were no major differences in sensitivities of detection between samples from different sites or different patient groups, in cases where sufficient positive results were obtained. A plot of number of foci observed between monoclonals gave a good correlation, and the samples missed by one preparation had only one or two foci per field with the other. In addition to tests in DEAFF with both sets of monoclonals, 812 of the above samples were inoculated concurrently into routine cell culture. CMV was detected in 82 samples by any method, of which 95% were positive by conventional cell culture compared to 43% by DEAFF. Patients whose samples were DEAFF positive. culture negative, and on whom previous surveillance had been undertaken by our laboratory. had all excreted CMV at some other time, providing further evidence for the specificity of DEAFF. The sensitivity of DEAFF is lower than that previously reported (Stirk and Griffiths, 1987). Since the sensitivity is related to the amount of virus inoculum into the assay system, the results were evaluated in the context of antiviral therapy at the time of sampling. DEAFF detected a smaller proportion of CMV positive samples from those receiving anti-CMV prophylaxis or therapy (acyclovir or ganciclovir). than from those who did not (28% vs. 47%) but this TABLE I A comparison DEAFF
of monoclonal
antibodies
in the analysis of 1558 consecutive
mAb 6327 (no. samples)
A.D.I. mAbs (no. samples) + Total
clinical specimens by Total
+
_
48 6
8 1496
I502
54
I.504
1558
56
777 ___
TABLE
2
DEAFF antiviral
results in 82 clinical therapy
Therapy
DEAFF
samples
(No.
positive
by any method
Negative
Total
Yes No
5 30
I3 34
IX 64
Total
35
47
81
analysis
TABLE
3
DEAFF disease
results
was undertaken
in X1 culture
DEAFF
Disease
positive
(No.
to compare
clinical
DEAFF
specimens
taken
m relation
to
from
Yes No
I’ 23
13 34
15 51
Total
35
47
x1
to compare
between
individuals
groups.
in relation
to CMV
Y2 Total
was undertaken
samples
samples) Negative
analysis
individuals
2.09
positive
Positive
Statistical
from
samples)
Positive
Statistical
taken
DEAFF
positive
II.41
results
between
groups
difference did not reach statistical significance (Table 2). We also investigated the relationship of CMV disease at time of sampling to the DEAFF results. since this may assist in clinical management decisions. The proportion of such samples detected by DEAFF from individuals with CMV disease compared to those without were similar (48% vs. 40%) (Table 3). Culture positive. DEAFF negative samples appeared to be spread evenly among those from whom multiple specimens were taken. rather than there being a preponderance of such results in samples from particular individuals.
Conclusions There is increasing demand for rapid viral diagnostic techniques as a wider array of effective antiviral therapies become available. Many of these techniques utilise monoclonal antibodies in order to detect viral proteins. Although highly specific, the use of such antibodies demands that they recognise an epitope which is highly conserved between strains of virus. The introduction of a new monoclonal antibody into an existing assay requires therefore full validation against previously used reagents. The results obtained with the A.D.I. monoclonal cocktail accord well with our existing monoclonal
223
antibody, ~63-27, when introduced into a rapid culture system to diagnose CMV infection in a variety of patient groups. The fact that samples missed by one monoclonal preparation had only one or two foci per field with the other, supports our original proposition that DEAFF is operating at a level where low quantities of virus are missed. Since there is a stochastic probability of detecting a single focus per field, it is concluded that the two monoclonal preparations are equivalent. The sensitivity of DEAFF, using either monoclonal set, was low compared to conventional cell culture. Analysis suggests that this sensitivity is reduced further, although not significantly, when the technique is used on samples from individuals receiving antiviral treatment and presumably reflects reduced viral shedding into the specimen. Alternatively, the drug may itself be inoculated, especially when the specimen is urine from an individual receiving acyclovir. On the other hand, samples from ill patients, in whom an increased viral excretion might be expected, were not more likely to be positive in DEAFF than samples from those with asymptomatic CMV infection. Finally, since specimens showing discrepant culture and DEAFF results did not all tend to be drawn from the same few individuals, it is unlikely that our findings are explained by the existence of virus strains (Colimon et al., 1985) which escape detection by the monoclonal antibodies used in our study. Such a lack of variation at the antigenic site recognised by mAb ~63-27 has recently been confirmed in an assessment of a rapid detection system for CMV in neonatal urine samples (Boppana et al.. 1992). These results are surprising, since those obtained by a previous comparison of diagnostic techniques, in which the sensitivity of DEAFF was calculated to be 77% (Stirk et al., 1987). A number of differences may have contributed to this discrepancy, such as the monoclonal antibodies and cell lines used in the assay, the patient groups from which samples were obtained and the use of antiviral chemotherapy. It also illustrates that the performance of assays may vary and must be kept under constant review. The factors contributing to such performance may lie outside laboratory control, and show the importance of clinico-pathological liaison. Further, our results underline the importance of maintaining cell culture as a ‘gold standard’ for CMV diagnosis. Clearly, improvements in rapid diagnostic tests for CMV are required. The polymerase chain reaction (PCR) may provide an advantage in terms of speed and sensitivity (Desselberger et al., 1990) and has been used for CMV in a research setting (Demmler et al., 1987). We are currently evaluating this technique, compared to DEAFF and conventional cell culture. for diagnosing clinical CMV infection in a routine laboratory setting.
References Balfour, H.H. 12 (Suppl.
(1990) Management 7). S849--S860.
of cytomegalovirus
disease
with antiviral
drugs.
Rev. Infect.
Dis.
1’4 Boppana, S.B.. Smith, R.J.. Stagno. S. and Britt. W.J. (1993) Evaluation of a microtitre plate fluorescent-antibody assay for rapid detection of human cytomegalovirus infection. J. Clin. Micro. 30, 721 7X. Colimon, R.. Somogyi. T.. Bertrand, C. and Michelson. S. (1985) Demonstration of the colincarity of human cytomegalovirus genomes and construction of restriction maps of unknown isolates using cloned subgenomic fragments. J. Gen. Virol. 66. 2183 2198. Demmler. G.J., Buffone G.J.. Schimbor. C.M. and May, R.A. (1988) Detection of cytomegalovirus in urine from newborns by using polymcrase chain reaction DNA amplification. J. Infect. Dis. 158. II77 1184. Desselberger. U. and Collingham. K. (1990) Molecular techniques in the diagnosis of human infectious diseases. Genitourin. Med. 66. 3 I3 323. Griffiths. P.D. (1990) ‘Cytomegalovirus‘. In: A.J. Zuckerman. J.E. Banatvala. and J.R. Pattison (Eds). Principles and Practice of Clinical Virology, 2nd edn. 699101. Wiley. London. Griftiths. P.D.. Panjwani, D.D.. Stirk. P.R. et al. (1984) Rapid detection of cytomegalovirua infection in immunocompromised patients by detection of early antigcnic fluorescent foci. Lancet ii, 1242--l 245. Pillay. D. and Griffiths, P.D. (1991) Diagnosis of cytomegalovirus infection: a review. Genitnurin. Med. 6X. 1833188. Stirk. P.R. and Griffiths. P.D. (1987) Use uf monoclonal antibodies for the diagnosis 01 cytomegalovirus infection by the detection of early antigenic fluorescent POCI (DEAFF) in ccl1 culture. J. Med. Virol. 21. 329 337.
