RIGA STRADIN'S UNIVERSITY
Inese Folkmane
INFLUENCE OF IMMUNOSUPPRESSIVE THERAPY ON
- HERPESVIRUSES INFECTION IN RENAL
TRANSPLANT RECIPIENTS
(SPECIALITY-NEPHROLOGY)
A Summary of Doctoral Thesis
Supervisors of the research study:
Habilitated Doctor of Medicine, Professor Rafail Rosental
Habilitated Doctor of Biology Svetlana Chapenko
Riga - 2004
Introduction
In the last decade several new potent immunosuppressive drugs have been
introduced in renal transplantation. Although these drugs work more selectively on
the immune system, opportunistic infections continue to produce serious complications.
The herpesviruses involved in infectious complications of the posttransplant
period represent an important cause of morbidity and even mortality after renal
transplantation [Gerna, 2002]. The human -herpesviruses comprise the cytomegalovirus (CMV), and the closely related human herpesviruses 6 and 7
(HHV-6, HHV-7). The viruses are ubiquitous and can be reactivated from latency
in a period of immunosuppression. Assessment of the contribution of each virus in
posttransplant complications is difficult due to their concurrent infection as well as
their possible simultaneous reactivation after organ transplantation. CMV is the
known pathogen in posttransplant complications with well-documented clinical
spectrum of the active infection. The role of HHV-6 and HHV-7 in the pathogenesis of posttransplant complications was evaluated differently [Osman et al, 1996;
Griffits et al. 1997]. The additional investigations are necessary to evaluate the
role of new immunosuppressive drugs (and combined drug regimens) on the activation of each virus as well as on the interaction between the viruses in order to
make early and exact diagnosis of -herpesvirus infections and to improve the
patient and graft survival.
The research has been elaborated at the Laboratory of Transplantation, Riga
Stradin's University; at the Latvian Transplantation Centre, P. Stradin's Clinical
University hospital and August Kirchenstein Institute of Microbiology and Virology,
Department of Oncovirology, University of Latvia, within the period of 1999 to
2003. Supervisors of the research study: Habilitated Doctor of Medicine, Professor
l
Rafail Rosental - Head of the Latvian Transplantation Centre, P. Stradin's Clinical
University Hospital, Head of Laboratory of Transplantology, Riga Stradin's University and Habilitated Doctor of Biology, Svetlana Chapenko - senior research
worker at the August Kirchenstein Institute of Microbiology and Virology, Department of Oncovirology, University of Latvia.
Significance and urgency of the study
Renal transplantation (RT) with appropriate immunosuppression is a choice
method in the treatment of patients with end-stage renal failure. The clinical development of new immunosuppressive agents represents one of the most significant
events in the field of organ transplantation today. Up-to-date immunosuppression
has reduced the number and severity of acute rejections and improved the early
patients' and graft survival. However, as well as offering many advantages, the
potential risk of drug-related over-immunosuppression (immunodeficiency), in the
daily practice of IS therapy, continues to be of significant clinical concern. Infections are and still remain the most common complications after RT.
Today, serious infectious complications develop in 15-44% of RT recipients
within the first year of surgery [Soulilou et al, 2001]. Bacterial infections are no
longer the common mortal problem they once were. Rarely is a patient lost from
opportunistic bacterial infection, unless the patient has a viral pneumonia with
superinfection or has been given excessive antibody or bone marrow toxic immunosuppression. The immunomodulating viruses particularly CMV, but also Epstein-Barr virus (EBV), other human herpes viruses, hepatitis B and C viruses
(HBV and HCV), and human immunodeficiency virus (HIV), if present, are still
major problems [R.A. Sells, 1998].
CMV has major implications on the outcome of RT due to so called "direct"
tissue injury and clinical disease as well as "indirect" effects - as additional opportunistic infections, increased graft rejection, decreased long-term patient survival
and finally, increased costs of transplantation [Paya, 2001]. Less is known about
the role of the more recently identified HHV-6 and HHV-7 on the posttransplant
viral infection complications and the role of these viruses as helper viruses in the
development of CMV disease [Kidd et al, 2000; DesJardin et al, 1998 ].
Different forms of immunosuppression used in organ transplantation affect
different aspects of viral infection; antilymphocyte antibodies as well as pulse steroids enhance viral activation from latency, whereas corticosteroids, calcineurin
inhibitors and rapamycin promote the persistence and spread of virus by suppressing the host's antiviral immune responses [Rubin, 2001; Jamil et al, 2000].
There have been widely varying results with regard to -herpesvirus infections in subjects given mycophenolate mofetil (MMF) and little information about
new anti-interleukin-2 receptor (IL-2R) antibodies basiliximab (chimeric anti-CD25specific murine antibody) and dclizumab (human lgG1 monoclonal antibody consisting of 90% human and 10% murine amino acid sequences).
Latvian Transplantation Centre performs about 60-65 kidney transplantations
per year. Postoperative therapy includes almost all combinations of immunosuppressants. One year patient survival is 92% and one year graft survival is 85%.
The most patients (54%) experience no rejection episodes; whereas, side-effects
and toxicity are common, and the risk of infections, particularly CMV infection
(15%), increases with all standard immunosuppressive regimens, -herpesvirus
infections are very urgent in Latvia. It has been shown before that latent CMV,
HHV-6 and HHV-7 infections are highly prevalent among the healthy blood donors
in Latvia [Kozireva et al, 2001]. At the same time the prevalence, activation as well
as the influence of -herpesviruses infection on renal transplant patients are
studied insufficiently and the observed effects of viral activation against an immunosuppression background differ between the transplantation centres. The additional investigations are necessary to evaluate the role of new immunosuppressive drugs (and combined drug regimens) on the activation of each virus as well
as on the interaction between the viruses in order to make early and exact diagnosis of (3-herpesviruses infection and to improve the patient and graft survival.
Aim of the study
The research aim is to evaluate the influence of different immunosuppressive
therapy protocols on -herpesviruses activation in renal transplant recipients in
order to improve the outcome of renal transplantation.
Tasks of the study:
1.
To investigate donors and recipients before and after renal transplantation for
6-herpesvirus infection prevalence.
2.
To evaluate the role of latent infection as well as donor factor for the develop
ment of CMV disease.
3.
To analyze -herpesvirus infection upon different immunosuppressive proto
cols accentuating new immunosuppressive drugs like mycophenolate mofetil,
basiliximab, daclizumab.
4.
To determine the activation and interaction of -herpesviruses upon antirejection therapy.
5.
To analyze the influence of -herpesviruses infection on renal transplant func
tion as well as on short -term graft survival.
Novelty, scientific and practical importance of the study
1.
The first time the activation, interaction and role of recently discovered herpesviruses (HHV-6 and HHV-7) in the development of post-transplant
complications were assessed.
2.
The first time the activation of -herpesviruses as well as severity of viral
disease were studied with regard to application of new immunosuppressive
drugs like mycophenolate mofetil, basiliximab, daclizumab.
3.
The new and very sensitive molecular method - nested polymerase chain
reaction (n PCR) was used for detection of latent and active CMV, HHV-6 and
HHV-7.
4.
The obtained results will permit to elaborate optimal immunosuppressive pro
tocols, decreasing the rate and severity of viral disease as well as increasing
the patient and graft survival thereby improving quality of life.
Presentation of the study results
The study results were reported: at the European Society for Clinical Virology
Winter Meeting in Rotterdam, the Netherlands (1999), International Symposium
"MMF in renal transplantation" in Riga, Latvia (1999), XI European Congress on
Clinical Microbiology and Infectious Diseases in Istanbul, Turkey (2001), V Congress of the Polish Transplantation Society in Mikolajki, Poland (2001), II World
Latvian Scientists' Congress in Riga, Latvia (2001), International Congress on
Transplantology - "A Transplant Odyssey" in Istanbul, Turkey (2001), Conference
of Tutors and Researchers of the Riga Stradin's University in Riga, Latvia (2002),
Rapamune Baltic Meeting in Vienna, Austria (2002), XXI Congress of the Scandinavian Transplantation Society in Reykjavik, Iceland (2002), Russian Congress on
Transplantolagy and artificial organs in Moscow, Russia (2002), The International
Rapamune Expierence Meeting in Riga, Latvia (2003). Posters on the subject
were demonstrated: at the IV International Conference on New Trends in Clinical
and Experimental Immunosuppression in Geneva, Switzerland (2000), XX Congress of the Scandinavian Transplantation Society in Helsinki, Finland (2000), XII
International Congress of Virology, Paris, France (2002), Conference of Tutors
and Researchers of the Riga Stradin's University in Riga, Latvia (2003), VI International Conference on New Trends in Immunosuppression in Salzburg, Austria
(2004).
The Thesis volume is 82 pages computer text, containing introduction, description of the topic importance, formulation of the work aim and tasks, literature
review, description of material, methods and results, discussion and references.
The thesis contains 15 tables and 11 figures.
Subjects and Methods
A total of 71 patients of both genders who underwent their first or second
cadaveric RT in the Transplantation Centre of Latvia during 1997 - 2000 were
studied. Mean age of recipients was 43.4±14.2 years (range 15 to 70 years).
Cohorts were established with the approval of the local Ethics Committee and,
prior to their examination, all participants provided informed consent.
EDTA anticoagulated peripheral blood and serum samples from the patients
were collected before transplantation and weekly thereafter for a minimum of 12
weeks. Plasma/serum and peripheral blood mononuclear cells (PBMC) were processed on the day of collection and then stored at - 70° C.
Further the characterization of subjects for each clinical study will be given
separately.
1. Primary CMV infection and dual (CMV and HHV-7) -herpesviruses
infection as an increased risk factor for CMV disease in patients undergoing renal transplantation
The study included 49 patients who underwent first RT. Each patient received
an intravenous injection of methylprednisolone (Pharmacia&Upjon) up to 500 mg
on the day of transplantation (day 0), which was tapered gradually within the four
days postoperatively as well as maintenance conventional triple immunosuppressive therapy with cyclosporine A (CsA), azathioprine (Aza) and prednisolon (P).
Oral CsA (Sandimun Neoral, Novartis) was initiated immediately after the transplant operation and adjusted based on target trough levels (at 150-350 ng/ml during the first four weeks and at 150 to 250 ng/ml for the remainder of the study).
Oral steroids (Prednisolon, Gedeon Richter) were started on day 1 at 0.5 mg/kg/
per day and tapered to a minimal dose of 10-5 mg/day until month 12. The maintenance dosage of orally administered azathioprine (Imuran, Glaxo Wellcome)
was 1-2 mg/kg/ per day. Acute rejection (AR) episodes were treated with intavenous injection of 500 mg of methylprednisolone for three to five days followed by a
return to the preepisode dose of steroids. Steroid-resistant and severe cellular or
vascular AR episodes were subsequently treated with policlonal antibodies - anithymocyte globulin (ATG; Fresenius) 4 mg/kg/ per day for 10 to 14 days.
2. Activation of dual -herpesviruses (CMV and HHV-7) after kidney transplantation with MMF-based maintenance immunosuppression
The study included 48 patients who underwent first cadaveric kidney transplantation. Kidney recipients were randomized and compared in 2 different maintenance immunosuppressive therapy protocols. The group I (n=24) received
conventional triple immunosuppressive therapy (Aza+P+CsA), the group II (n=24)
received MMF-based triple therapy (MMF+P+CsA). MMF (CellCept; Hoffman-La
Roche) medication was fixed doses of 0.75 -1 g b.i.d. Rejections were treated by
i.v. methylprednisolone 5mg/kg for 5 days. No induction therapy with policlonal or
monoclonal antibodies was applied. No prophylaxis with ganciclovir was applied
for "CMV at risk" recipients.
3. Concurrent -herpesviruses (CMV, HHV-6, HHV-7) infection and antirejection therapy: role in viral disease development in renal transplant
recipients
50 patients of both genders who underwent their first cadaveric RT were reviewed retrospectively. To analyze viral infection complications upon antirejection
treatment and with regard to type of viral infection (concurrent viral infection or
viral infection alone) the recipients were grouped in three groups and compared to
receive either conventional therapy (group I, n=20) or MMF-based triple therapy
(group II, n=21) or besides maintenance triple immunosuppressive therapy received high doses of steroids ("pulse" steroids) and polyclonal antibodies - ATG
(group III, n=9) due to AR or as induction therapy. The group I was also control
group. Patient demographic characteristics showed no significant differences between the groups.
4. Results of renal transplantation with different immunosuppressive
regimens (MMF- based triple therapy and induction with IL-2R
monoclonal antibodies)
The study included 71 patients of both genders who underwent first or second
cadaveric RT. Patients were divided into three groups according treatment after
RT. The group I (n=25) received conventional triple CsA-based therapy (CsA,
AzA.P), the group II (n=23) received MMF-based triple therapy (CsA, MMF, P)
and the group III (n=23) received induction with basiliximab (Simulect; Novartis) or
daclizumab (Zenapax; Roche) and background triple immunosuppressive therapy
(CsA, P, Aza). The first of the two 20 mg doses of basiliximab was performed immediately before transplant surgery and the second dose was administered on
day 4 after RT. The first dose of daclizumab was administered at a dosage 2mg/
kg/d i.v. immediately before transplant surgery and the second dose (1mg/kg/d) at
2-week interval. All the rest immunosuppressants were applied accordingly the
above mentioned studies.
Methods
Hematologic parameters (RBC count and hemoglobin concentration; hematokrit;
erythrocyte qualitative parameters) were determined on the hematologic analyser
Cell-Dyn 1700 (Abbot Laboratories, USA).
Blood biochemical tests (plasma creatinine; plasma urea; plasma phosphorus;
plasma potassium; plasma sodium; plasma calcium) was used biochemical analyzer Abbot Spectrum Series II (Abbot Laboratories, USA), where to quantitatively
estimate concentrations of various substances in plasma, determination of light
absorption level by different length waves was done.
The Cyclosporine A through blood level was determined on AxSYM autoanalyzer (Abbot Laboratories, USA) using immune fluorescence polarization
technique.
The diagnosis of the latent CMV, HHV-6, and HHV-7 infection was based on
detection of sequences in peripheral blood leukocytes (PBL) by nPCR. Latent
CMV infection was affirmed by microparticle enzyme immunoassay (MEIA) for
anti-CMV IgG and anti-CMV IgM (Imx, Abbot) in serum. The diagnosis of an
active CMV infection was based on CMV serology (seroconvertion or 4-fold
increase of IgG titer), on the detection of viral DNA sequence in blood plasma by
nPCR and identification of the virus in blood by inoculation of PBL onto monolayer
MRC-5 cell culture (cytopathic effect [CPE], the presence of CMV antigens
detected by indirect immunofluorescence assay [IFA], the presence of specific
DNA in cell culture fractions), as well as on clinical signs. The active HHV-6 and
HHV-7 infections were determined by the presence of viral DNA in blood
plasma using nPCR and by the identification of viruses in cell cocultures of
PBMC from recipients with umbilical cord blood mononuclear cells (CPE, IFA,
nPCR).
Acute rejection episodes were diagnosed on the basis of an otherwise unexplained rise in serum creatinine greater than 25% from baseline together with clinical evidence and improvement within 3 days after the initiation of empiric antirejection therapy. In the vast majority of patients the diagnosis was confirmed on
graft biopsy. Histological examination and classification of a core biopsy was done
according to the Banff 1993 scheme [Solez K, et al. 1993]. CMV infection and
disease were defined as outlined by previous international CMV workshops
[Proceedings from the 5th International Cytomegalovirus Conference. 1995].
10
Statistics
The results were expressed as mean ± standard deviation (SD) or percentage. Paired and unpaired Student's f-test for comparisons between and within
groups or Fisher's exact test for qualitative variables respectively, were used. Results of Cox proportional analyses were expressed as relative risk and 95% confidence intervals. The threshold of statistical significance was set at P<0.05.
Results and discussion
1. Primary CMV infection and dual (CMV and HHV-7) -herpesviruses
infection as an increased risk factor for CMV disease in patients
undergoing renal transplantation
Before RT latent -herpesviruses infection was detected in 44 of 49 (89.8%)
patients and from them CMV infection in 26.5%, HHV-7 - 12.2% and dual (CMV
and HHV-7) infection in 51% of patients (Table 1).
Tablei. Latent -herpesviruses infection before and at 6 months after RT
Only 5 of 49 (10.2%) patients were free of -herpesviruses infection. As
shown in Table 1, the latent dual infection was prevalent in patients before RT.
After RT, CMV infection alone was determined in 15 out of 49 (30.6%) recipients
and 2 of them had primary infection (Table 1). The number of recipients with HHV7 infection alone (12.2%) and with dual infection (51,0%) remained unchanged
after RT. Three recipients, who were negative for CMV and HHV-7 infection before and after RT, had no post-transplantation complications. Viral disease was
clinically diagnosed in 18 out of 49 (36.7%) recipients.
The risk of the viral disease in recipients was calculated depending on the
type of latent viral infection (Table 2). The results showed that the risk of disease
for recipients with dual infection was 3.6 and 2.2-fold higher in comparison with
CMV infection and HHV-7 infection alone, respectively.
Table 2. The risk of the viral disease depending on the type of latent viral
infection
*P< 0,01, the risk of the viral disease in recipients with dual -herpesviruses infection vs. CMV and HHV-7 infection alone.
The data, indicating that dual infection is an increased risk factor for the development of viral disease, were confirmed by the results of the investigation of 18
recipients with viral disease after RT. CMV infection alone was revealed in 3
(16.7%), HHV-7 infection alone in 2 (11.1%) and dual infection in 13 (72.2%) recipients (Table 3). Frequency of dual infection in recipients with viral disease was
feasibly higher than CMV and HHV-7 infection alone (P<0.025).
To determine the time of reactivation of each virus and the association of their
reactivation with the development of the disease, 18 recipients with viral disease
were divided into 3 groups (A, B, C) depending on the type of the viral infection
(CMV, HHV-7, CMV+HHV-7). The results of virological and serological examination for the recipients with viral disease after RT are presented in Table 3.
Primary CMV infection and reinfection with donor virus are known predictors
for the development of CMV disease in transplant recipients [Chou S, 1986;
Grundy et al, 1988]. In the present study, the severe CMV disease had developed
in 2 recipients with CMV primary infection and viral syndrome in 1 recipient with
latent CMV infection (group A), although these recipients were not treated with
ATG. Activation of CMV was detected in all recipients prior to onset of the
disease.
13
Table 3. The results of virological and serological examination for the recipients
with viral disease after RT
D, donor; R, recipient; PBL, peripheral blood leukocytes; VS, viral syndrome; "+",
positive result;"-", negative result; +*, 4-fold rise of specific CMV IgG antibody titer
increase; ND, not detected
14
Anti-rejection therapy with monoclonal (OKT3) and polyclonal (ATG) antibodies is now recognized as a major risk factor for CMV reactivation and development
of CMV disease in the CMV-seropositive recipients [Morris, 1996]. Four recipients
treated with ATG had reactivation of -herpesviruses (HHV-7 - in the case of
HHV-7 infection alone; CMV and HHV-7 - in the case of dual infection). Reactivation of viruses preceded the development of viral disease, which was diagnosed in
all 4 recipients.
The significance of HHV-7 in the development of post-transplantation infectious complications is not determined so far. A relationship between identification
of HHV-7 DNA in plasma and CMV disease has been shown [Tong et al, 2000]
and evidence suggests that this herpesvirus have indirect effect such as contributing to the risk of opportunistic infections. Our study showed the correlation between reactivation of latent HHV-7 after RT and development of febrile syndrome
in both recipients with HHV-7 infection alone (group B). The appearance and duration of febrile syndrome coincided with the time when viral DNA was detectable
in blood plasma.
Thus, our study showed the association of virus reactivation with viral disease
in the cases of CMV and HHV-7 infections alone.
A total of 51.0% of patients had latent dual (CMV+HHV-7) infection before
RT. Our investigation demonstrated that, after RT, the risk of viral disease was 3.6
and 2.2-fold higher in patients with latent dual infection in comparison with latent
CMV or latent HHV-7 infection alone, respectively (P<0.025). The virulogical monitoring revealed reactivation of both viruses in 100% of recipients with dual infection and development of viral disease after RT. However, HHV-7 reactivation preceded CMV reactivation in 77.0% cases and was not related to the treatment by
ATG. These data coincide with the results of Osman et al [Osman et al, 1996 ],
15
who also noted that HHV-7 reactivation preceded the CMV reactivation in the
post-transplantation period. After reactivation, HHV-7 might enhance the state of
immunodeficiency due to its selective tropism to CD4+ T lymphocytes [Secchiero
et al, 1997], or might alter cytokine profile [Ongradi et al, 1999] and thus create
conditions for CMV reactivation.
2. Activation of dual - herpesviruses (CMV and HHV-7) after kidney
transplantation with MMF-based maintenance immunosuppression
The study included 48 patients. The patients were randomized and compared
in 2 different maintenance immunosuppressive therapy protocols. The group I
(n=24) received conventional triple immunosuppressive therapy - control group,
the group II (n=24) received MMF-based triple immunosuppressive therapy. The 2
groups were matched for age, sex, donor and recipient pretransplant CMV serological status and treatment of steroid-pulse therapy.
Before RT the presence of CMV, HHV-7 and CMV+HHV-7 DNA sequences
were detected in PBL, but not in the plasma samples of 50.0%, 29.1% and 37.5%
of group I patients and of 58.3%, 33.3% and 29.1% of group II patients, respectively, that indicates the presence of latent, but not active virus infection in these
patients. After RT number of recipients with latent -herpesviruses infection were
higher than before one. It was made 58.3% for CMV infection alone and 41.6% for
dual (CMV+HHV-7) infection in group I and 66.6% and 33.3% in group II, respectively. During the 6 - months observation period after RT viral activation as well as
the risk of viral activation upon different immunosuppressive protocols was
analyzed (Table 4).
16
Table 4. Activation of CMV and dual -herpesviruses (CMV and HHV-7) as well
as risk of virus activation in recipients with different immunosuppression at 6
months after RT
A considerably higher incidence of CMV and dual infection activation was
observed in recipients with MMF-based therapy (62.5% and 75% vs. 28.5% and
30%, P<0.05). The risk of CMV and dual b- herpesviruses activation was 4.0 and
7.5-fold higher, respectively, in group II as compare to group I (P<0.05). The patients were also evaluated in terms of the incidence and severity of viral disease in
recipients with CMV infection alone and with dual infection in both groups
(Figure 1).
17
Figure 1. The characteristics of viral disease in recipients with CMV infection
alone and with dual (CMV+HHV-7) b- herpesviruses infection depending on immunosuppressive therapy. *P<0,01, the frequency of CMV tissue-invasive disease
in recipients with CMV infection alone in group II vs. control group; **P<0,001, the
frequency of CMV tissue- invasive disease in recipients with dual b- herpesviruses infection in group II vs. control group.
Viral disease was clinically diagnosed in 4 of 14 (28.5%) recipients with CMV
infection alone and in 3 of 10 (30%) recipients with dual b- herpesviruses infection
in control group (Figure 1). Accordingly, viral disease was clinically diagnosed in
10 of 16 (62.5%) recipients with CMV infection alone and in 6 of 8 (75%) recipients with dual (3-herpesviruses infection in MMF-based group. Also the clinical
signs of viral disease tended to be more severe in patients treated with MMFbased immunosuppression (incidence of tissue-invasive disease in 37% of
18
recipients with CMV infection alone and in 50% of recipients with dual b- herpesviruses infection ), whereas, in the control group, CMV tissue-invasive disease
was not diagnosed at all (P<0.001).
There have been widely varying results with regard to viral infectious complications in subjects given MMF. One study [Tricontinental MMF Renal Study
group, 1996] showed that a patient group receiving 2 mg/d MMF had no increased
incidence of tissue-invasive CMV infection. Our results showed statistically significant increasing of active of CMV infection alone and dual B-herpesviruses infection (62.5% and 75%) in recipients with MMF-based immunosuppression as compare to conventional treatment group (28.5% and 30%), as well as more severe
clinical signs of viral disease (tissue invasive disease in 37.5% and in 50% recipients, respectively) in recipients with MMF-based immunosuppression as compare
to conventional treatment group (P<0.001). The similar results have receantly
been reported by others [Land, 1999; Meulen et al, 2000]. However, more frequently tissue-invasive CMV disease is associated with dose of 3g/d MMF
[ Moreso et al, 1998], we found, that even dose of 2g/d of MMF creates conditions for activation of -herpesviruses and promotes recipients to more severe
clinical signs of viral disease. A fixed-dose regimen of 2 g/d MMF may be excessive.
3. Concurrent -herpesviruses (CMV, HHV-6, HHV-7) infection and antirejection therapy: role in viral disease development in renal transplant
recipients
50 patients of both genders who underwent their first cadaveric RT were reviewed retrospectively. Before RT latent/persistent -herpesviruses infection was
disclosed in 44 of 50 (88%) patients (Figure 2). The concurrent latent/persistent
19
-herpesviruses infection was diagnosed in 29 of 50 (58%) patients (dual CMV
+HHV-6 infection - 2/50 , 4%, dual CMV +HHV-7 infection - 13/50, 26%, dual
HHV-6+HHV-7 infection - 2/50, 4% and triple CMV+HHV-6+HHV-7 infection 12/50, 24%). The concurrent latent/persistent -herpesviruses infection was more
prevalent (58%) in comparison with viral infection alone, that was diagnosed in 15
of 50 (30%) patients (CMV -11/50, 22%; HHV-7 - 4/50 8%; HHV-6 - 0/50, 0%)
(P=0.008) before RT (Figure 2).
Figure 2. Prevalence of latent/persistent 6-herpesviruses infection in the patients
before and after RT (nPCR results), *P= 0,008, prevalence of concurrent pherpesviruses infection vs. viral infection alone before RT; #P= 0,0005, prevalence
of concurrent -herpesviruses infection vs. viral infection alone after RT.
After RT the percent of recipients with latent/persistent -herpesviruses infection increased to 92% (46/50) (Figure 2). Four recipients (8%), who remained free
from -herpesviruses infection, the viral disease complications did not develop
within the first 6 months after RT. The concurrent latent/persistent -herpesviruses
20
infection was significantly prevalent also after RT as compared to viral infection
alone (64% vs. 28%, P=0.005).
Viral disease was clinically diagnosed in 22 of 50 recipients (44%). In seventeen recipients with viral disease, concurrent (-herpesviruses infection was diagnosed, in 3 - CMV infection alone and in 2 recipients - HHV-7 alone.
The risk of the viral disease was calculated depending on the type of viral
infection (Table 5). Our results showed that the risk of viral disease for the recipients with concurrent -herpesviruses infection was feasibly higher, 4.46 (95% Cl:
0.87 - 6.95), in comparison with CMV infection alone, 0.45 (95% Cl: 0.16 -1.27)
(P=0.045).
Table 5. Risk of the development of viral disease in recipients with either viral infection alone or concurrent -herpesviruses infection
*P=0,045, the risk of the development of viral disease in recipients with concurrent
-herpesviruses infection in comparison with CMV infection alone
To analyze viral disease complications upon antirejection treatment the recipients were compared to receive either conventional therapy (group I) or MMFbased triple therapy (group II) or besides maintenance triple immunosuppressive
21
therapy received high doses of steroids and polyclonal antibodies - ATG (group
III) due to AR or as induction therapy. The analysis of post-transplant complications (Table 6) depending on the type of immunosuppressive treatment showed
that frequency of viral disease was considerably higher in group II (13/21, 62%)
and in group III (7/9, 78%) patients, who received more potent immunosuppressive drugs - MMF and ATG in comparison to patients who received conventional
therapy (4/20, 20%) (P=0.01). Also frequency of viral disease complications was
3-fold higher in patients with concurrent -herpesviruses infection (3/4, 75%) in
comparison to patients with -herpesviruses infection alone (1/4, 25%) in group I.
Statistically significantly higher rate of viral disease complications was disclosed in
group II patients with concurrent -herpesviruses infection as compare to viral
infections alone (85% vs. 15%, P=0.001) (Table 6).
22
Table 6. The viral disease complications depending on the type of (herpesviruses infection (concurrent -herpesviruses infection or viral infection
alone) and immunosuppressive treatment
*P=0,01, the frequency of viral disease complications in group II and III
patients vs. control group; **P=0,001, the frequency of viral disease
complications in group II recipients with concurrent (-herpesviruses infection vs.
viral infection alone.
The similar tendency was observed in group III where viral disease
complications developed in 71.4% of recipients with concurrent herpesviruses infection vs. 28.6% of recipients with viral infection alone (Table
6).
CMV is the known pathogen in posttransplant complications with welldocumented clinical spectrum of the active infection. The role of HHV-6 and
HHV-7 in the pathogenesis of posttransplant complications was evaluated
differently
23
[Paya, 2001; Dockrell et al, 2001]. Our study showed that concurrent herpesviruses infection was prevalent in patients before (58%) and after (64%)
RT. The risk of development of viral disease was significantly higher by concurrent
infection (4.46) in comparison with CMV infection alone (0.45) (P=0.045). Also
higher rate of viral disease complications was disclosed in MMF-treated recipients
with concurrent -herpesviruses infection as compared to viral infections alone
(85% vs. 15%, P=0.001). Taking into account that concurrent -herpesviruses
infection is an increased risk factor for the development of complications in recipients after RT, screening diagnosis should include also testing for CMV, HHV-6,
and HHV-7 infections in recipients before and after RT as well as in donors.
4. Results of renal transplantation with different immunosuppressive regimens
(MMF- based triple therapy and induction with IL-2R monoclonal antibodies)
The study included 71 patients of both genders who underwent their first or
second cadaveric RT. To assess the efficacy (graft survival, the incidence of acute
rejection and CMV disease rate) of three different immunosuppressive regimens
up to 12 months post RT, the patients were divided into three groups according
immunosuppressive treatment (group I - conventional therapy, control; group II MMF- based therapy and group III - induction therapy). There was no significant
difference in baseline characteristics between treatment groups with the exception
of mean age of patients of the control group - it was 45.1±13.0 and significantly
higher than the mean age of MMF and induction therapy groups (40.6±13.2 and
39.8±10.4;P=0.05).
The comparative criteria of the efficacy of 3 immunosuppressive treatment
regimens are presented in Figure 3. The functional survival rates of the renal
24
allograft up to 12 months after transplantation for 3 treatment regimens groups
were similar - 88% in group 1,91% - in group II and III, respectively.
Figure 3. The comparative criteria of the efficacy (graft survival, the incidence of
acute rejection and CMV disease rate) of three different immunosuppressive regimens up to 12 months post RT. *P<0,05, the incidence of acute rejection in group
III vs. control group; #P<0,05, the incidence of CMV disease in group II vs. control
group and group III.
The incidence of AR within 12 months after RT was 8 of 25 (32%) in the control group; 5 of 23 (21.7%) in the group II and 4 of 23 (17.3%) in the group III, respectively. There were significantly fewer AR episodes in group III than in control
group (17.3% vs. 32%, P=0.05) whereas the difference between AR episodes in
group II and III was not significant (Figure 3).
Further, we concentrate on the CMV results because the rate of CMV
infection may be a more sensitive marker for overimmunosuppression than the
overall rate of infections. The effect of two immunosuppressive regimens against a
25
background of conventional immunosuppressive therapy on the incidence and
nature of CMV infections is depicted in Figure 4.
Figure 4. The effect of two immunosuppressive regimens against a background of
conventional immunosuppressive therapy on the incidence and nature of CMV
infections. *P<0,05, the incidence of CMV disease in group II vs. control group
and group III; #P<0,05, tissue invasive CMV disease in group II vs. control group
and group III.
In MMF group, there were more frequent incidence of CMV disease as well
as more severe clinical signs of CMV disease: five (21.7%) cases of tissue invasive CMV disease vs. one (4.3%) case of tissue invasive CMV disease in control
group and induction therapy group (P<0.05). CMV syndrome was clinically diagnosed in 2 of 25 patients in control group, in 4 of 23 patients in group II and in
group III. There was no significant difference among the groups.
The effect of CMV disease on graft function during the early period as well as
at 12 months after RT was assessed, as well. The mean serum creatinine of
26
recipients with CMV disease in three immunosuppressive therapy groups at 1, 3,
6,9, and 12 months after RT is given in Figure 5.
Figure 5. The mean serum creatinine of recipients with CMV disease in three
immunosuppressive therapy groups at 1, 3, 6, 9, and 12 months after RT.
*P<0,05, the mean serum creatinine in control group vs. group II and III.
The mean serum creatinine level in patients with CMV disease during the first
6 months after RT was significantly higher (0,22±0,08 mmol/l) in control group as
compared to group II (0,14±0,03 mmol/l) and group III (0,1510,06 mmol/l) patients, P<0.05. At 12 months after RT, no further deterioration of graft function was
noted in patients with CMV disease and the mean serum creatinine level was
similar in all three groups (group I - 0,17±0,06 mmol/l, group II - 0,11±0,05 mmol/l,
group III - 0,13±0,06 mmol/l). Differences in creatinine values among the study
groups most likely reflect the fact that the control group experienced more episodes of AR compared to group II and group III.
The mean serum creatinine level for patients without previous CMV disease
is given in Figure 6. At one month after RT, the mean serum creatinine for patients
27
without previous CMV disease was significantly lawer, particularly in contol group,
(0.22±0.07 mmol/l) than those with CMV disease (0.3±0.09 mmol/l). At the same
time, the mean serum creatinine for both patients (with CMV disease and without
CMV disease) within three treatment groups did not differ at 12 months after RT
(Figure 5 and Figure 6).
Figure 6. The mean serum creatinine for recipients without CMV disease in three
immunosuppressive therapy groups at 1, 3, 6, 9, and 12 months after RT.
* P<0,05, the mean serum creatinine in control group vs. group II and III.
Our data demonstrates that the no deterioration of graft function was observed in patients with CMV disease and the mean serum creatinine was not significantly different from those without CMV disease at 12 months after RT. This
suggests that the major effect of CMV disease on graft function occurred during
the early period after RT, however our data cannot distinguish between AR increasing the likelihood of subsequent infection, or CMV infection increasing the
risk of AR. None of the patients in this study was lost due to CMV disease complications.
28
The balance between AR and infection after transplantation continues to be
of significant clinical concern, especially during the early posttransplantation period [Nashan et al, 1999]. The recent introduction of several new and effective
immunosuppressive agents may allow the utilization of immunosuppressive regimens that are effective and less broadly toxic. In our study we compared two potent immunosuppressive regimens (MMF-based triple therapy and quadruple induction protocol with new monoclonal antibodies - basiliximab or daclizumab) with
conventional triple therapy. The one-year graft survival rate was excellent in all
treatment groups. The incidence of AR episodes was significantly lower in group
II! recipients than in control group (17% vs. 32%, P<0.05). None of the patients in
groups II and III experienced severe, grade III AR or steroid-resistant AR,
whereas, 3 vascular type AR episodes occurred in control group. Another situation
was with regard to CMV infection complications under different immunosuppressive protocols. Significantly frequent and severe CMV disease complications
(P<0.05) were observed in MMF group (39.1%) as compared to the control group
(12%) and the induction therapy group (17.3%).This is consistent with a recent
studies of association of MMF-based immunosuppressive protocols with higher
rate of CMV disease [Kuypers et al, 2002].
29
Conclusions
1.
The concurrent latent -herpesviruses infection is prevalent in patients
before and after RT.
2.
The clinical signs of the viral disease are more severe in recipients with
primary CMV infection (D+/R-) than in recipients with the reactivation of
CMV.
3.
In recipients with concurrent dual -herpesviruses infection (CMV+HHV7), the HHV-7 reactivation precedes CMV reactivation and this reactivation take place before the onset of the clinical symptoms of the disease.
4.
The risk of viral disease for the recipients with concurrent herpesviruses infection is 10-fold higher (4.46), in comparison with CMV
infection alone (0.45).
5.
The immunosuppressive protocols based on MMF and concurrent 0herpesviruses infection are associated with increased incidence of CMV
disease as well as with more severe clinical signs of CMV disease in
recipients after RT.
6.
Addition of MMF to cyclosporine and prednisolone as well as induction
therapy protocols with IL-2R monoclonal antibodies can provide excellent
1-year graft survivals and a low incidence of AR episodes.
7.
The combination of cyclosporine, azathioprine, and steroids with IL-2R
monoclonal antibodies is a highly effective immunosuppressive protocol
that greatly reduces the incidence of AR and does not associate with an
increased risk of CMV disease within 12 months after RT.
8.
The presence of CMV disease within 6 months after RT does not
associate with poor graft function and patients survival at 12 months.
30
Practical recommendations
1.
Taking into account that concurrent -herpesviruses infection is an in
creased risk factor for the development of complications in recipients
after RT, screening diagnosis should include also testing for CMV,
HHV-6, and HHV-7 infections in recipients before and after RT as well as
in donors.
2.
In recipients with latent concurrent -herpesviruses infection prophylactic
antiviral therapy with ganciclovir would be advisable, and an immunosuppressive drug regimen that is less prone to viral disease (induction
therapy protocols with IL-2R monoclonal antibodies or MMF-based triple
immunosuppression with reduced MMF dose) would be chosen.
3.
Quadruple induction immunosuppressive protocol with IL-2R monoclonal
antibodies (in combination with cyclosporine, azathioprine, and steroids)
would be applied for older recipients, and for CMV high risk recipients
(D+/R-)
31
The list of publications
Original articles
1. I. Folkmane, J. Bicāns, B. Bērziņa, L. Salaka, R. Rozentāls. Post-transplant
diabetes melllitus after kidney transplantation. Acta medica Lituanica. 1999.
Suppl. 4, pp. 27-29. Vilnius ACADEMIA 1999
2. S.Chapenko, I.Folkmane,V.Tomsone, D.Amerika, M. Murovska, R. Rozental.
Co-infection of two betta- herpesviruses (CMV and HHV-7) as a risk factor for
CMV disease in patients undergoing renal transplantation. Clinical
Transplantation 2000:14:486-492
3. I.Folkmane, S. Chapenko, D. Amerika, J. Bicans, M. Murovska, R. Rosentals.
(-herpesvirus activation after kidney transplantation with mycophenolate
mofetil - based maintenance immunosuppression. Transplantation
Proceedings, Vol 33, No 3, 2001, p. 2384-2385
4. S. Chapenko, I. Folkmane, V. Tomsone, S. Kozireva, J. Bicans, D. Amerika, R.
Rozentals, M. Murovska. Infection of p-herpesviruses (CMV, HHV-6, HHV-7):
role in post-renal transplantation complications. Transplantation Proceedings,
Vol 33, No 4, 2001, p. 2463-2464
5. I.Folkmane, S. Chapenko, M. Murovska, R. Rosentals. Low rate of acute
rejection and cytomegalovirus infection in renal transplant recipients with
basiliximab. Transplantation Proceedings, Vol 33, No 7-8,2001, p. 3209-3210.
6.1.Folkmane, J. Bicans, S. Chapenko, M. Murovska, R. Rozentals. The results of
kidney transplantation with different immunosuppressive regimens.
Transplantation Proceedings, 34,2002, p. 558-559.
7.1.Folkmane, K. Bernarde, J. Bicans, D. Amerika, R. Rozentals. Benefitial effects
of low-toxicity regimens with MMF in renal recipients with late allograft
dysfunction.Transplantation Proceedings, 35,2003, p. 789-790
8. M. Mihailova, J. Jansons, I. Sominska, *l. Folkmane, *R. Rozentals, P.
Pumpens.Structural features of hepatitis B virus from long-term
immunosuppressed patients in Latvia. Proceedings of the Latvian Academy of
Sciences. Section B, Vol. 57 (2003). No.5, p. 158-163
32
Abstracts
1. I.Folkmane1, D.Amerika1, LSamoshenkova2, S.Chapenko3, J.Bicans1,
R.Rozentals1. "Cytomegaloviais infection in patients after kidney transplantation".
International Meeting "Blood borne virus infections". Radisson SAS Hotel, Riga,
Latvia, May 26-27,1998. Abstract, p. 18-19.
2. S.Chapenko1, V.Tomsons1, D.Amerika2, I.Folkmane2, R.Rosentals,
M.Murovska1. "A prospective study of human -herpesviruses infection in renal
transplantation". International Meeting "Blood borne virus infections". Radisson
SAS Hotel, Riga, Latvia, May 26-27,1998. Abstract, p. 17
3. S. Chapenko1, V. Tomsons1, I. Folkmane2, D. Amerika2, R. Rozentals2, M.
Murovska1. "Activation of CMV and HHV-7 in Patients After Renal
Transplantation" . ESCV Winter Meeting 1999, Rotterdam the Netherlands, 7-9
January. Abstract, p.51.
4. I. Folkmane, J. Bicans, B. Berzina, L. Salaka, R. Rozentals. Department of
Transplantation, Latvian Medical Academy, Riga, Latvia. "Post-transplant diabetes
melllitus after kidney transplantation". 3-rd Baltic meeting on nephrology.
23-25 April 1999, Vilnius, Lithuania. Abstract, p. 6
5. I.Folkmane1, S. Chapenko2, D. Amerika1, J. Bicans1, M. Murovska2, R.
Rozental1. "-herpesviruses activation after kidney transplantation with MMFbased maintenance immunosuppression". 4th International Conference on new
trends in clinical and experimental immunosuppression. Geneva, Switzerland,
February 17-20,2000. Abstract, p. 194.
6. Chapenko S.1, Folkmane I.2, Tomsone V. \ Kozireva S.1,2Bicans J.2, Amerika
D.2, Rozentals R.2, Murovska M.1 "Infection of p-herpesviruses (CMV, HHV-6,
HHV-7): role in post-renal transplantation complications". Scandinavian
Transplantation Society XX Congress. May 11-12,2000, Marina Congress Center,
Helsinki, Finland. Abstract p.51.
7. S. Chapenko1, I. Folkmane2, D. Amerika2, R. Rozental2, M. Murovska1.
"Importance of concurrent beta-herpesvirus infection in the development of
postrenal-transplantation complications upon antirejection therapy". 11 th European
Congress of Clinical Mirobiology and Infectious Diseases. Istanbul, Turky, 1-4
April, 2001. Abstract No.0115. Abstract publication in Clinical Microbiology and
Infection, Volume 7, Supplement 1, 2001
33
8. I. Folkmane1, S. Chapenko2, M. Murovska2, R. Rosentals1. "Low rate of acute
rejection and cytomegalovirus infection in renal transplant recipients with
basiliximab". A Transplant Odyssey. The scientific and educational congress.
Istanbul, Turky, 20-23 August, 2001, Abstract, p.153.
9. I.Folkmane, K. Bemarde, J. Bicans, D. Amerika, R. Rozentals. "Benefitial
effects of low-toxicity regimens with MMF in renal recipients with late allograft
dysfunction".The Scandinavian Transplantation society XXI congress, May 22-24,
2002, Reykjavik, Iceland. Abstract p. 9.
10. Z. Nora1, I. Folkmane2, A. Fostiropolo1, R. Rozentals2, S. Chapenko1, M.
Murovska 1 . "Human herpesvirus 5, 6, 7 and 8 as the risk factors for
posttransplant complications". XII th International congress of Virology, 27th to 1st
August 2002, Paris, France. Abstract, p.208
11. I.Sominskaya1, J. Jansons1, M. Mikhailova1, U. Dumpis1, I. Folkmane2, R.
Rozentals2, P. Pumpens1. "Structural features of HBV and HCV infection in a
patients from kidney transplantation centre". XII th International congress of
Virology, 27th to 1st August 2002, Paris, France. Abstract, p.200.
12. I. Folkmane1, J. Bicans1, S. Chapenko2, M. Murovska2, K. Bernarde1, R.
Rosentals1. "Influence of sirolimus-based immunosuppressive protocols on graft
function and CMV infection in renal transplant patients: 12 months results".
6th international conference on new trends in immunosuppression. Salzburg,
Austria, February 5-8,2004. Abstract, p.152.
34