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Cytomegalovirus infection in kidney transplant recipients: early diagnosis and monitoring of antiviral therapy by the antigenemia assay
Cytomegalovirus Infection in Kidney Transplant Recipients: Early Diagnosis and Monitoring of Antiviral Therapy by the Antigenemia Assay T. Said, M.R.N. Nampoory, A.S. Pacsa, K.V. Johny, M.P. Nair, M. Abdel-Haleem, M. Samhan, and M. Al-Mousawi
C
YTOMEGALOVIRUS (CMV) is one of the most frequent infections in renal transplant recipients. After primary infection the virus remains dormant in various cells/organs for years. There are many factors that may reactivate virus replication; among them, organ transplantation certainly is the most important one, often causing serious disease1 and often associated with decreased graft survival.2 Though treatment is available, its timing and dosage should be tailored according to the ongoing CMV disease.3,4 There are different approaches for the diagnosis of CMV infections. The “gold standard” is cell culture in which the virus can be isolated.5 However, it is a timeconsuming and expensive procedure. Though combining virus isolation with immunofluorescence technique reduces the time from weeks to days, it is still not enough to meet the demand of a rapid diagnosis.6 Polymerase chain reaction (PCR) may have a strong impact on the diagnosis of CMV infection, but there is a need for further standardization and simplification.7 Many of the kidney transplant recipients fail to produce antibodies due to immunosuppressive therapy,5 making serological diagnosis undependable in CMV infections. Recently, detection of a specific CMV replication-related antigen (CMV antigen pp65) directly in leukocytes of patients has shown a good correlation with the clinical condition of the patient. This, CMV antigenemia assay (AA) seems to provide not only a reliable, specific, and sensitive diagnostic tool, but also facilitates monitoring the effectiveness of antiviral therapy.8 –13 PATIENTS AND METHODS This prospective study was conducted in 102 consecutive kidney transplant recipients. They included 63 males and 39 females with a mean age of 38 (⫾28) years. Kidney donors were living related in Table 1. Presence of CMV pp65 Antigen in Leukocytes of Kidney Recipients No of patients
With Symptoms (%) 34/102 (33)
Without Symptoms (%) 68/102 (67)
Total 102
AA-positive AA-negative
28/34 (82) 6/34 (18)
7/68 (10) 61/68 (90)
35 67
Table 2. Correlation Between AA Positivity and Clinical Presentation No of AA-Positive Cells Clinical Presentation
Total No of Patients
No of AA-Positive Patients
Range
Average
Asymptomatic Prolonged fever Fever ⫹ leukopenia Systemic
68 11 13 10
7 8 10 10
1–9 5–25 7–266 10 –500
3 12 80 147
52, cadaver in 20, and living unrelated in 30 transplants, of which 25 were done abroad. Immunosuppression included cyclosporin, azathioprin, and prednisolone in 70 and cyclosporin, mycophenolate mofetil (MMF), and prednisolone in 32 recipients. Induction immunosuppression with antilymphocytic globulin (ATG) was used in immunologically high-risk recipients. Mild to moderate acute rejections (Banff I and II) were treated with methylprednisolone pulse therapy, while patients with severe (Banff III) or steroidresistant episodes received either OKT3 alone or OKT3 and plasmapharesis. Blood samples (n ⫽ 773) were collected in EDTAcontaining tubes from these recipients at 2 to 20 months posttransplantation during their regular outpatient visits. All the samples were processed for antigen detection on the same day of collection. Clinical diagnosis of CMV disease was suspected from the presence of prolonged fever, leucopenia, and/or systemic involvement. Patients with CMV disease were treated with intravenous ganciclovir (Cymevene) in a dose titrated according to body weight and renal function for 2 to 3 weeks. Some of those with systemic involvement required treatment for more than 3 weeks. At the time of blood extraction, 5 mL of blood was taken from each patient and processed according to the standard technique for CMV antigenemia assay (CMV-vue, Inestar, USA). The number of cells containing the CMV-specific pp65 antigen/50,000 cells was counted under light microscope. The test is read as follows: negative (less than 5 cells), low positive (from 6 to 10 cells), high positive (from 11 to 20 cells), and strong positive (more than 20 cells). For
From Hamed Al-Essa Organ Transplant Centre (T.S., M.R.N.N., M.P.N., M.A.-H., M.S., M.A.-M,), Ministry of Health, Kuwait, and Departments of Microbiology and Medicine (A.S.P., K.V.J.), Faculty of Medicine, University of Kuwait, Kuwait. Address reprint requests to Dr Tarek Said, PO Box 2498 Ardheya, Code 92400, Kuwait.
© 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
0041-1345/01/$–see front matter PII S0041-1345(01)02195-9
Transplantation Proceedings, 33, 2799–2801 (2001)
2799
2800
SAID, NAMPOORY, PACSA ET AL
Table 3. Antigenemia Assay Positivity in Relation to the Type of Kidney Donor Living Related
Living Unrelated
Cadaver
Total
52 12 23
30 13 43
20 10 50
102 35 34
Number AA positives %
calculating the positive predictive value (Pos.PV) and the negative predictive value (Neg.PV) the following formulas were used: Pos.PV ⫽ (number of patients with positive test results and CMV-related disease/total number of patients with positive test result) ⫻ 100; Neg.PV ⫽ (number of patients with negative test result and without CMV-related symptoms/total number of patients with negative test result) ⫻ 100.
RESULTS
During the period of the study, 34 patients (33%) developed symptoms compatible with CMV infection. Of these patients, 28 (82%) had the antigen in their leukocytes, while only 7 of the 68 symptom-free patients (10%) were antigenpositive (Table 1). Though the number of antigen-positive cells varied from zero to hundreds, it showed a strict correlation with the severity of clinical symptoms. The average number of AA-positive cells increased steadily according to the clinical condition of the patients (Table 2). Among the asymptomatic group, only seven patients were positive for the AA but their average number of AApositive cells was six times lower than that detected in the eight AA-positive patients with prolonged fever. The group of patients with fever and leukopenia had a relatively higher number of positive cells, while most of those with systemic CMV disease had a very high number. When the number of AA-positive patients was correlated with the type of donor, a higher percentage (50%) was found in the group of recipients who received cadaveric renal transplant (Table 3). Among the 28 symptomatic patients with AA-positive cells, 19 were diagnosed to have active CMV disease and were treated with ganciclovir. Presentation was prolonged fever in one, fever and leucopenia in eight, and systemic involvement in 10 patients (hepatitis in nine, gastritis in two, colitis in one, cystitis in one, and retinitis in one). The number of AA-positive cells was proportional to the severity of the disease, and with treatment their number decreased substantially (Table 4). Most of the treated patients received ganciclovir for 2 to 3 weeks and they became both antigenemia-negative and symptom-free within 4 weeks of treatment. Only two patients with systemic involvement required 3 months of treatment to become AA-negative.
DISCUSSION
Detection of pp65 antigen has become an increasingly important assay for the diagnosis of CMV infection/disease in transplant recipients.14,15 This AA gives rapid results and correlates well with the patient’s condition. Furthermore, it is useful for monitoring the effectiveness of antiviral therapy16 as we have shown in our earlier report on monitoring antiviral therapy.17 The assay is rather easy to perform and inexpensive when compared to other methodologies like cell culture and PCR.18 Using a cutoff point of 5 AApositive leukocytes/50,000 as an indicator of CMV infection/disease, the assay gave both highly positive (80%) and negative (91%) predictive values. Among the 68 asymptomatic patients there were only seven (10%) with positive AA and the number of antigen-positive cells remained under 10. Therefore, using a cutoff point of 10 positive cells, none of the asymptomatic patients will fall into the positive range. Based on the number of AA-positive cells quantification seems possible19,20 and important in relation to patient management.21 Indeed, the number of positive cells increased according to the severity of clinical condition and showed more than 10-fold increase from prolonged fever to systemic involvement. Similar data were obtained by using CMV PCR for measuring the DNA levels.22 The use of antilymphocyte immunoglobulins (ATG or OKT3) as induction immunosuppression or antirejection therapy is well known to be associated with increased incidence of CMV disease.23,24 This can explain the higher incidence of CMV disease in our cadaveric renal transplant group. CONCLUSION
In kidney transplant recipients, CMV antigenemia assay is a useful method for both diagnosis of CMV disease and monitoring the effectiveness of antiviral therapy. The number of antigen-positive leukocytes correlates well with the severity of disease and the response to treatment. Cadaver renal transplant recipients have a higher incidence of CMV disease, most probably due to the use of antilymphocyte immunoglobulins. ACKNOWLEDGMENTS We greatly acknowledge the office of the Vice-Rector for Research, Kuwait University, who supported this study (grant no. MI 096, 112). We also acknowledge the excellent technical help rendered by Mrs A. El-Shazly (virology lab). We appreciate the effort done by the outpatient nursing staff of Hamed Al-Essa Organ Transplant Centre in collecting the blood samples regularly.
Table 4. Effect of Ganciclovir Treatment on Antigen Positivity and Clinical Manifestation No of Patients with Positive Cells After Treatment Clinical Presentation
No of Patients (19)
No of AA-Positive Cells at Start of Treatment
Week 3
Week 4
Month 2
Month 3
Prolonged fever Fever and leukopenia Systemic
1 8 10
25 31–100 (52) 10 –500 (140)
0 3 4
0 2 2
0 1 2
0 0 0
CYTOMEGALOVIRUS INFECTION IN KIDNEY RECIPIENT
REFERENCES 1. Jeffrey DJ: J Antimicrob Chemother 23(suppl E):1, 1989 2. Fujinami RS, Nelson JA, Walker L, et al: J Virol 62:100, 1980 3. Erice A, Jordan MC, Chace KBA, et al: JAMA 257:3082, 1987 4. Dunn DL, Mayoral JL, Gillingham KJ, et al: Transplantation 51:98, 1991 5. Landini MP: Progr Med Virol 40:157, 1993 6. Ravello MG, Percivalle E, Zavattoni M, et al: J Med Virol 29:88, 1989 7. Persing DH: J Cli Microbiol 29:1281, 1991 8. Erice A, Holm MA, Gill PC, et al: J Clin Microbiol 28:2681, 1992 9. Tanaba K, Takahashi K, Kotoma I, et al: Transpl Proc 28:1508, 1996 10. Degre M, Buhholm G, Holter E, et al: Eur J Clin Microbiol Inf Dis 13:668, 1994 11. The TH, Van der Bij W, Van der Berg AP, et al: Rev Inf Dis 12:737, 1990 12. Perez L, Mariscal D, Tubau F, et al: Diagn Microbiol Inf Dis 19:15, 1994
2801 13. Egan JJ, Barber L, Lomax J, et al: Thorax 50:9, 1995 14. Van der Bij W, Torensma R, Van Son WJ, et al: J Med Virol 25:179, 1988 15. Van der Berg AP, Van der Bij W, Van Son J, et al: Transplantation 48:991, 1989 16. Gerna G, Zipeto D, Parea M, et al: J Inf Dis 164:488, 1991 17. Pacsa AS, El-Shazly A, Gupta RK, et al: Med Princ Pract 8:85, 1999 18. Van der Meer JTM, Drew WL, Bowden RA, et al: Antiviral Res 32:119, 1996 19. Van den Berg AP, Klompmaker IJ, Haagsma EB, et al: J Inf Dis 164:265, 1991 20. Niubo J, Perez JL, Martinez-Lacasa JT, et al: Diagn Microbiol Infect Dis 24:19, 1996 21. Meyer-Konig U, Serr A, Von Laer D, et al: J Inf Dis 171:705, 1994 22. Toyoda M, Carlos JB, Galera OA, et al: Transplantation 63:957, 1997 23. Hibberd PL, Tolkoff-Rubin NE, Cosimi AB, et al: Transplantation 53:68, 1992 24. Bailey TC, Powderly WG, Storch GA, et al: Am J Kidney Dis 21:196, 1993
C
YTOMEGALOVIRUS (CMV) is one of the most frequent infections in renal transplant recipients. After primary infection the virus remains dormant in various cells/organs for years. There are many factors that may reactivate virus replication; among them, organ transplantation certainly is the most important one, often causing serious disease1 and often associated with decreased graft survival.2 Though treatment is available, its timing and dosage should be tailored according to the ongoing CMV disease.3,4 There are different approaches for the diagnosis of CMV infections. The “gold standard” is cell culture in which the virus can be isolated.5 However, it is a timeconsuming and expensive procedure. Though combining virus isolation with immunofluorescence technique reduces the time from weeks to days, it is still not enough to meet the demand of a rapid diagnosis.6 Polymerase chain reaction (PCR) may have a strong impact on the diagnosis of CMV infection, but there is a need for further standardization and simplification.7 Many of the kidney transplant recipients fail to produce antibodies due to immunosuppressive therapy,5 making serological diagnosis undependable in CMV infections. Recently, detection of a specific CMV replication-related antigen (CMV antigen pp65) directly in leukocytes of patients has shown a good correlation with the clinical condition of the patient. This, CMV antigenemia assay (AA) seems to provide not only a reliable, specific, and sensitive diagnostic tool, but also facilitates monitoring the effectiveness of antiviral therapy.8 –13 PATIENTS AND METHODS This prospective study was conducted in 102 consecutive kidney transplant recipients. They included 63 males and 39 females with a mean age of 38 (⫾28) years. Kidney donors were living related in Table 1. Presence of CMV pp65 Antigen in Leukocytes of Kidney Recipients No of patients
With Symptoms (%) 34/102 (33)
Without Symptoms (%) 68/102 (67)
Total 102
AA-positive AA-negative
28/34 (82) 6/34 (18)
7/68 (10) 61/68 (90)
35 67
Table 2. Correlation Between AA Positivity and Clinical Presentation No of AA-Positive Cells Clinical Presentation
Total No of Patients
No of AA-Positive Patients
Range
Average
Asymptomatic Prolonged fever Fever ⫹ leukopenia Systemic
68 11 13 10
7 8 10 10
1–9 5–25 7–266 10 –500
3 12 80 147
52, cadaver in 20, and living unrelated in 30 transplants, of which 25 were done abroad. Immunosuppression included cyclosporin, azathioprin, and prednisolone in 70 and cyclosporin, mycophenolate mofetil (MMF), and prednisolone in 32 recipients. Induction immunosuppression with antilymphocytic globulin (ATG) was used in immunologically high-risk recipients. Mild to moderate acute rejections (Banff I and II) were treated with methylprednisolone pulse therapy, while patients with severe (Banff III) or steroidresistant episodes received either OKT3 alone or OKT3 and plasmapharesis. Blood samples (n ⫽ 773) were collected in EDTAcontaining tubes from these recipients at 2 to 20 months posttransplantation during their regular outpatient visits. All the samples were processed for antigen detection on the same day of collection. Clinical diagnosis of CMV disease was suspected from the presence of prolonged fever, leucopenia, and/or systemic involvement. Patients with CMV disease were treated with intravenous ganciclovir (Cymevene) in a dose titrated according to body weight and renal function for 2 to 3 weeks. Some of those with systemic involvement required treatment for more than 3 weeks. At the time of blood extraction, 5 mL of blood was taken from each patient and processed according to the standard technique for CMV antigenemia assay (CMV-vue, Inestar, USA). The number of cells containing the CMV-specific pp65 antigen/50,000 cells was counted under light microscope. The test is read as follows: negative (less than 5 cells), low positive (from 6 to 10 cells), high positive (from 11 to 20 cells), and strong positive (more than 20 cells). For
From Hamed Al-Essa Organ Transplant Centre (T.S., M.R.N.N., M.P.N., M.A.-H., M.S., M.A.-M,), Ministry of Health, Kuwait, and Departments of Microbiology and Medicine (A.S.P., K.V.J.), Faculty of Medicine, University of Kuwait, Kuwait. Address reprint requests to Dr Tarek Said, PO Box 2498 Ardheya, Code 92400, Kuwait.
© 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
0041-1345/01/$–see front matter PII S0041-1345(01)02195-9
Transplantation Proceedings, 33, 2799–2801 (2001)
2799
2800
SAID, NAMPOORY, PACSA ET AL
Table 3. Antigenemia Assay Positivity in Relation to the Type of Kidney Donor Living Related
Living Unrelated
Cadaver
Total
52 12 23
30 13 43
20 10 50
102 35 34
Number AA positives %
calculating the positive predictive value (Pos.PV) and the negative predictive value (Neg.PV) the following formulas were used: Pos.PV ⫽ (number of patients with positive test results and CMV-related disease/total number of patients with positive test result) ⫻ 100; Neg.PV ⫽ (number of patients with negative test result and without CMV-related symptoms/total number of patients with negative test result) ⫻ 100.
RESULTS
During the period of the study, 34 patients (33%) developed symptoms compatible with CMV infection. Of these patients, 28 (82%) had the antigen in their leukocytes, while only 7 of the 68 symptom-free patients (10%) were antigenpositive (Table 1). Though the number of antigen-positive cells varied from zero to hundreds, it showed a strict correlation with the severity of clinical symptoms. The average number of AA-positive cells increased steadily according to the clinical condition of the patients (Table 2). Among the asymptomatic group, only seven patients were positive for the AA but their average number of AApositive cells was six times lower than that detected in the eight AA-positive patients with prolonged fever. The group of patients with fever and leukopenia had a relatively higher number of positive cells, while most of those with systemic CMV disease had a very high number. When the number of AA-positive patients was correlated with the type of donor, a higher percentage (50%) was found in the group of recipients who received cadaveric renal transplant (Table 3). Among the 28 symptomatic patients with AA-positive cells, 19 were diagnosed to have active CMV disease and were treated with ganciclovir. Presentation was prolonged fever in one, fever and leucopenia in eight, and systemic involvement in 10 patients (hepatitis in nine, gastritis in two, colitis in one, cystitis in one, and retinitis in one). The number of AA-positive cells was proportional to the severity of the disease, and with treatment their number decreased substantially (Table 4). Most of the treated patients received ganciclovir for 2 to 3 weeks and they became both antigenemia-negative and symptom-free within 4 weeks of treatment. Only two patients with systemic involvement required 3 months of treatment to become AA-negative.
DISCUSSION
Detection of pp65 antigen has become an increasingly important assay for the diagnosis of CMV infection/disease in transplant recipients.14,15 This AA gives rapid results and correlates well with the patient’s condition. Furthermore, it is useful for monitoring the effectiveness of antiviral therapy16 as we have shown in our earlier report on monitoring antiviral therapy.17 The assay is rather easy to perform and inexpensive when compared to other methodologies like cell culture and PCR.18 Using a cutoff point of 5 AApositive leukocytes/50,000 as an indicator of CMV infection/disease, the assay gave both highly positive (80%) and negative (91%) predictive values. Among the 68 asymptomatic patients there were only seven (10%) with positive AA and the number of antigen-positive cells remained under 10. Therefore, using a cutoff point of 10 positive cells, none of the asymptomatic patients will fall into the positive range. Based on the number of AA-positive cells quantification seems possible19,20 and important in relation to patient management.21 Indeed, the number of positive cells increased according to the severity of clinical condition and showed more than 10-fold increase from prolonged fever to systemic involvement. Similar data were obtained by using CMV PCR for measuring the DNA levels.22 The use of antilymphocyte immunoglobulins (ATG or OKT3) as induction immunosuppression or antirejection therapy is well known to be associated with increased incidence of CMV disease.23,24 This can explain the higher incidence of CMV disease in our cadaveric renal transplant group. CONCLUSION
In kidney transplant recipients, CMV antigenemia assay is a useful method for both diagnosis of CMV disease and monitoring the effectiveness of antiviral therapy. The number of antigen-positive leukocytes correlates well with the severity of disease and the response to treatment. Cadaver renal transplant recipients have a higher incidence of CMV disease, most probably due to the use of antilymphocyte immunoglobulins. ACKNOWLEDGMENTS We greatly acknowledge the office of the Vice-Rector for Research, Kuwait University, who supported this study (grant no. MI 096, 112). We also acknowledge the excellent technical help rendered by Mrs A. El-Shazly (virology lab). We appreciate the effort done by the outpatient nursing staff of Hamed Al-Essa Organ Transplant Centre in collecting the blood samples regularly.
Table 4. Effect of Ganciclovir Treatment on Antigen Positivity and Clinical Manifestation No of Patients with Positive Cells After Treatment Clinical Presentation
No of Patients (19)
No of AA-Positive Cells at Start of Treatment
Week 3
Week 4
Month 2
Month 3
Prolonged fever Fever and leukopenia Systemic
1 8 10
25 31–100 (52) 10 –500 (140)
0 3 4
0 2 2
0 1 2
0 0 0
CYTOMEGALOVIRUS INFECTION IN KIDNEY RECIPIENT
REFERENCES 1. Jeffrey DJ: J Antimicrob Chemother 23(suppl E):1, 1989 2. Fujinami RS, Nelson JA, Walker L, et al: J Virol 62:100, 1980 3. Erice A, Jordan MC, Chace KBA, et al: JAMA 257:3082, 1987 4. Dunn DL, Mayoral JL, Gillingham KJ, et al: Transplantation 51:98, 1991 5. Landini MP: Progr Med Virol 40:157, 1993 6. Ravello MG, Percivalle E, Zavattoni M, et al: J Med Virol 29:88, 1989 7. Persing DH: J Cli Microbiol 29:1281, 1991 8. Erice A, Holm MA, Gill PC, et al: J Clin Microbiol 28:2681, 1992 9. Tanaba K, Takahashi K, Kotoma I, et al: Transpl Proc 28:1508, 1996 10. Degre M, Buhholm G, Holter E, et al: Eur J Clin Microbiol Inf Dis 13:668, 1994 11. The TH, Van der Bij W, Van der Berg AP, et al: Rev Inf Dis 12:737, 1990 12. Perez L, Mariscal D, Tubau F, et al: Diagn Microbiol Inf Dis 19:15, 1994
2801 13. Egan JJ, Barber L, Lomax J, et al: Thorax 50:9, 1995 14. Van der Bij W, Torensma R, Van Son WJ, et al: J Med Virol 25:179, 1988 15. Van der Berg AP, Van der Bij W, Van Son J, et al: Transplantation 48:991, 1989 16. Gerna G, Zipeto D, Parea M, et al: J Inf Dis 164:488, 1991 17. Pacsa AS, El-Shazly A, Gupta RK, et al: Med Princ Pract 8:85, 1999 18. Van der Meer JTM, Drew WL, Bowden RA, et al: Antiviral Res 32:119, 1996 19. Van den Berg AP, Klompmaker IJ, Haagsma EB, et al: J Inf Dis 164:265, 1991 20. Niubo J, Perez JL, Martinez-Lacasa JT, et al: Diagn Microbiol Infect Dis 24:19, 1996 21. Meyer-Konig U, Serr A, Von Laer D, et al: J Inf Dis 171:705, 1994 22. Toyoda M, Carlos JB, Galera OA, et al: Transplantation 63:957, 1997 23. Hibberd PL, Tolkoff-Rubin NE, Cosimi AB, et al: Transplantation 53:68, 1992 24. Bailey TC, Powderly WG, Storch GA, et al: Am J Kidney Dis 21:196, 1993