Quantification of human cytomegalovirus viremia by using monoclonal antibodies to different viral proteins

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Abstract

Human cytomegalovirus (HCMV) viremia in peripheral blood polymorphonuclear leukocytes (PMNLs) from 187 immunosuppressed patients (79 heart transplant recipients and 108 patients with acquired immunodeficiency syndrome [AIDS]) was investigated. Five mouse monoclonal antibodies (MAbs), one specifically reactive to HCMV immediate-early antigen (IEA) in PMNLs, two specifically reactive to IEA in infected cell cultures, and two specifically reactive to late antigens, were used in immunofluorescence and/or immunoperoxidase test systems for (i) detection of HCMV IEA in human embryonic lung fibroblast (HELF) cell cultures inoculated with PMNL samples, (ii) direct detection of HCMV IEA in PMNL nuclei of cytospin preparations, and (iii) identification of HCMV isolates from PMNL samples in HELF cells. Quantification of viremia was achieved by counting the number of infected PMNLs per 2 x 105 cells examined directly on cytospin preparations as well as by counting the number of IEA-positive HELF cells inoculated with 2 x 105 PMNLs. A significant correlation was found between the number of infected PMNLs and the number of infected HELF cells. When the number of infected PMNLs per 2 x 105 cells was > 10, both methods (PMNL IEA and HELF IEA) gave concordant results; when it was > 80/2 x 105 cells, clinical symptoms were consistently associated with HCMV viremia. Ten patients with heart transplants and three patients with AIDS who had high or increasing levels of HCMV viremia underwent antiviral treatment with ganciclovir. Treatment was discontinued only after disappearance of IEA-positive PMNLs from blood (the last marker of infection to become negative). On the other hand, in the presence of low levels of viremia (<10 infected PMNLs per 2 x 105 cells), different methods often provided discordant results and overt clinical symptoms were never observed. IEA-negative results with PMNL samples or HELF cells in the presence of positive virus isolation could never be attributed to the inability of IEA MAbs to recognize individual HCMV strains, since all of the relevant viral isolates were recognized by IEA MAbs. In addition, all five MAbs used in the study were capable of identifying all 135 conventional HCMV isolates obtained from the study population. It was concluded that (i) maximal sensitivity in diagnosing HCMV viremia may be achieved by combining different techniques; (ii) direct detection of HCMV IEA in PMNLs, yielding results on the day of blood collection, is a very rapid and sensitive technique, and thus one can rely on it for clinical management of HCMV infections; and (iii) low levels of viremia are clinically irrelevant, whereas high levels are associated with clinical symptoms.

Original languageEnglish
Pages (from-to)2681-2688
Number of pages8
JournalJournal of Clinical Microbiology
Volume28
Issue number12
Publication statusPublished - 1990

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Viremia
Viral Proteins
Cytomegalovirus
Monoclonal Antibodies
Neutrophils
Antigens
Fibroblasts
Lung
Acquired Immunodeficiency Syndrome
Cell Culture Techniques
Forensic Anthropology
Ganciclovir
Cytomegalovirus Infections
Leukocyte Count

ASJC Scopus subject areas

  • Microbiology
  • Microbiology (medical)

Cite this

@article{63ae5ac1d638413f95eca29c3772f0a4,
title = "Quantification of human cytomegalovirus viremia by using monoclonal antibodies to different viral proteins",
abstract = "Human cytomegalovirus (HCMV) viremia in peripheral blood polymorphonuclear leukocytes (PMNLs) from 187 immunosuppressed patients (79 heart transplant recipients and 108 patients with acquired immunodeficiency syndrome [AIDS]) was investigated. Five mouse monoclonal antibodies (MAbs), one specifically reactive to HCMV immediate-early antigen (IEA) in PMNLs, two specifically reactive to IEA in infected cell cultures, and two specifically reactive to late antigens, were used in immunofluorescence and/or immunoperoxidase test systems for (i) detection of HCMV IEA in human embryonic lung fibroblast (HELF) cell cultures inoculated with PMNL samples, (ii) direct detection of HCMV IEA in PMNL nuclei of cytospin preparations, and (iii) identification of HCMV isolates from PMNL samples in HELF cells. Quantification of viremia was achieved by counting the number of infected PMNLs per 2 x 105 cells examined directly on cytospin preparations as well as by counting the number of IEA-positive HELF cells inoculated with 2 x 105 PMNLs. A significant correlation was found between the number of infected PMNLs and the number of infected HELF cells. When the number of infected PMNLs per 2 x 105 cells was > 10, both methods (PMNL IEA and HELF IEA) gave concordant results; when it was > 80/2 x 105 cells, clinical symptoms were consistently associated with HCMV viremia. Ten patients with heart transplants and three patients with AIDS who had high or increasing levels of HCMV viremia underwent antiviral treatment with ganciclovir. Treatment was discontinued only after disappearance of IEA-positive PMNLs from blood (the last marker of infection to become negative). On the other hand, in the presence of low levels of viremia (<10 infected PMNLs per 2 x 105 cells), different methods often provided discordant results and overt clinical symptoms were never observed. IEA-negative results with PMNL samples or HELF cells in the presence of positive virus isolation could never be attributed to the inability of IEA MAbs to recognize individual HCMV strains, since all of the relevant viral isolates were recognized by IEA MAbs. In addition, all five MAbs used in the study were capable of identifying all 135 conventional HCMV isolates obtained from the study population. It was concluded that (i) maximal sensitivity in diagnosing HCMV viremia may be achieved by combining different techniques; (ii) direct detection of HCMV IEA in PMNLs, yielding results on the day of blood collection, is a very rapid and sensitive technique, and thus one can rely on it for clinical management of HCMV infections; and (iii) low levels of viremia are clinically irrelevant, whereas high levels are associated with clinical symptoms.",
author = "G. Gerna and Revello, {M. G.} and E. Percivalle and M. Zavattoni and M. Parea and M. Battaglia",
year = "1990",
language = "English",
volume = "28",
pages = "2681--2688",
journal = "Journal of Clinical Microbiology",
issn = "0095-1137",
publisher = "American Society for Microbiology",
number = "12",

}

TY - JOUR

T1 - Quantification of human cytomegalovirus viremia by using monoclonal antibodies to different viral proteins

AU - Gerna, G.

AU - Revello, M. G.

AU - Percivalle, E.

AU - Zavattoni, M.

AU - Parea, M.

AU - Battaglia, M.

PY - 1990

Y1 - 1990

N2 - Human cytomegalovirus (HCMV) viremia in peripheral blood polymorphonuclear leukocytes (PMNLs) from 187 immunosuppressed patients (79 heart transplant recipients and 108 patients with acquired immunodeficiency syndrome [AIDS]) was investigated. Five mouse monoclonal antibodies (MAbs), one specifically reactive to HCMV immediate-early antigen (IEA) in PMNLs, two specifically reactive to IEA in infected cell cultures, and two specifically reactive to late antigens, were used in immunofluorescence and/or immunoperoxidase test systems for (i) detection of HCMV IEA in human embryonic lung fibroblast (HELF) cell cultures inoculated with PMNL samples, (ii) direct detection of HCMV IEA in PMNL nuclei of cytospin preparations, and (iii) identification of HCMV isolates from PMNL samples in HELF cells. Quantification of viremia was achieved by counting the number of infected PMNLs per 2 x 105 cells examined directly on cytospin preparations as well as by counting the number of IEA-positive HELF cells inoculated with 2 x 105 PMNLs. A significant correlation was found between the number of infected PMNLs and the number of infected HELF cells. When the number of infected PMNLs per 2 x 105 cells was > 10, both methods (PMNL IEA and HELF IEA) gave concordant results; when it was > 80/2 x 105 cells, clinical symptoms were consistently associated with HCMV viremia. Ten patients with heart transplants and three patients with AIDS who had high or increasing levels of HCMV viremia underwent antiviral treatment with ganciclovir. Treatment was discontinued only after disappearance of IEA-positive PMNLs from blood (the last marker of infection to become negative). On the other hand, in the presence of low levels of viremia (<10 infected PMNLs per 2 x 105 cells), different methods often provided discordant results and overt clinical symptoms were never observed. IEA-negative results with PMNL samples or HELF cells in the presence of positive virus isolation could never be attributed to the inability of IEA MAbs to recognize individual HCMV strains, since all of the relevant viral isolates were recognized by IEA MAbs. In addition, all five MAbs used in the study were capable of identifying all 135 conventional HCMV isolates obtained from the study population. It was concluded that (i) maximal sensitivity in diagnosing HCMV viremia may be achieved by combining different techniques; (ii) direct detection of HCMV IEA in PMNLs, yielding results on the day of blood collection, is a very rapid and sensitive technique, and thus one can rely on it for clinical management of HCMV infections; and (iii) low levels of viremia are clinically irrelevant, whereas high levels are associated with clinical symptoms.

AB - Human cytomegalovirus (HCMV) viremia in peripheral blood polymorphonuclear leukocytes (PMNLs) from 187 immunosuppressed patients (79 heart transplant recipients and 108 patients with acquired immunodeficiency syndrome [AIDS]) was investigated. Five mouse monoclonal antibodies (MAbs), one specifically reactive to HCMV immediate-early antigen (IEA) in PMNLs, two specifically reactive to IEA in infected cell cultures, and two specifically reactive to late antigens, were used in immunofluorescence and/or immunoperoxidase test systems for (i) detection of HCMV IEA in human embryonic lung fibroblast (HELF) cell cultures inoculated with PMNL samples, (ii) direct detection of HCMV IEA in PMNL nuclei of cytospin preparations, and (iii) identification of HCMV isolates from PMNL samples in HELF cells. Quantification of viremia was achieved by counting the number of infected PMNLs per 2 x 105 cells examined directly on cytospin preparations as well as by counting the number of IEA-positive HELF cells inoculated with 2 x 105 PMNLs. A significant correlation was found between the number of infected PMNLs and the number of infected HELF cells. When the number of infected PMNLs per 2 x 105 cells was > 10, both methods (PMNL IEA and HELF IEA) gave concordant results; when it was > 80/2 x 105 cells, clinical symptoms were consistently associated with HCMV viremia. Ten patients with heart transplants and three patients with AIDS who had high or increasing levels of HCMV viremia underwent antiviral treatment with ganciclovir. Treatment was discontinued only after disappearance of IEA-positive PMNLs from blood (the last marker of infection to become negative). On the other hand, in the presence of low levels of viremia (<10 infected PMNLs per 2 x 105 cells), different methods often provided discordant results and overt clinical symptoms were never observed. IEA-negative results with PMNL samples or HELF cells in the presence of positive virus isolation could never be attributed to the inability of IEA MAbs to recognize individual HCMV strains, since all of the relevant viral isolates were recognized by IEA MAbs. In addition, all five MAbs used in the study were capable of identifying all 135 conventional HCMV isolates obtained from the study population. It was concluded that (i) maximal sensitivity in diagnosing HCMV viremia may be achieved by combining different techniques; (ii) direct detection of HCMV IEA in PMNLs, yielding results on the day of blood collection, is a very rapid and sensitive technique, and thus one can rely on it for clinical management of HCMV infections; and (iii) low levels of viremia are clinically irrelevant, whereas high levels are associated with clinical symptoms.

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