Pediatr Transplantation 2014: 18: 47–51
© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Pediatric Transplantation DOI: 10.1111/petr.12176
Clinical consequences of human herpesvirus-6 DNAemia in peripheral blood in pediatric liver transplant recipients Al Fawaz T, Ng V, Richardson SE, Barton M, Allen U. Clinical consequences of human herpesvirus-6 DNAemia in peripheral blood in pediatric liver transplant recipients.
Tariq Al Fawaz1,*, Vicky Ng2,3, Susan E. Richardson4, Michelle Barton1,† and Upton Allen1,3 1
Abstract: The significance of HHV6 DNAemia after solid organ transplantation has not been fully determined. Our objectives were to determine the prevalence of HHV6 DNAemia in pediatric liver transplant recipients and to describe the associated clinical characteristics and outcomes. This was a retrospective case–control study. Eligible liver transplant patients aged ≤ 18 yr with HHV6 DNAemia were matched with two subjects without HHV6 DNAemia. Matching was by age 6 months. Among 154 subjects, 25 patients (16%) had HHV6 DNAemia detected by PCR in whole blood or plasma (M:F ratio = 0.9:1). While 28% of subjects with DNAemia (7/25) had symptoms consistent with HHV6 infection, active infection was detected in only four subjects (2.6% of liver transplant patients). The major symptoms/signs were fever, vomiting, lethargy, splenomegaly, bone marrow suppression, and elevated transaminases. The prevalence of DNAemia due to other herpesviruses in cases vs. controls was EBV 56% vs. 60%, CMV 12% vs. 12%, HHV7 20% vs. 12%; p value is not significant for all pairwise comparisons. HHV6 DNAemia in pediatric liver transplant patients is not an uncommon entity. While the clinical relevance is still not entirely established, active HHV6 infection and attributable symptoms are relatively rare.
Division of Infectious Diseases, Hospital for Sick Children, University of Toronto, Toronto, ON, Canada, 2Division of Gastroenterology, Hepatology and Nutrition, Hospital for Sick Children, University of Toronto, Toronto, ON, Canada, 3Transplant Centre, Department of Pediatrics, Hospital for Sick Children, University of Toronto, Toronto, ON, Canada, 4The Division of Microbiology, Department of Pediatric Laboratory Medicine, Hospital for Sick Children, University of Toronto, Toronto, ON, Canada Key words: herpesvirus – HHV6 – pediatric transplantation – liver transplant Upton Allen, Division of Infectious Diseases, Hospital for Sick Children, 555 University Avenue, Toronto, ON M5G 1X8, Canada Tel.: 416 813 8129 Fax: 416 813 8404 E-mail: [email protected] *Present affiliation: King Fahad Medical City, Riyadh, Saudi Arabia † Present affiliation: Division of Microbiology, Hospital for Sick Children, Toronto, ON, Canada Accepted for publication 16 September 2013
HHV6 is a ubiquitous virus, which is widespread throughout the world. It belongs to the b-herpes group family to which CMV and HHV7 also belong. As with other herpesviruses, it has the ability to remain latent and can be reactivated with immunosuppression (1, 2). In this context, HHV6 is increasingly being recognized as a significant opportunistic pathogen in solid organ as well as stem cell transplant recipients (3–11). In the normal host, HHV6 is the causative agent of the self-limited illness, roseola (exanAbbreviations: CMV, cytomegalovirus; EBV, Epstein Barr virus; HHV6, human herpesvirus 6; HHV7, human herpesvirus 7; MMF, mycophenolate mofetil; PCR, polymerase chain reaction; PTLD, post-transplant lymphoproliferative disorder.
thema subitum). Seroconversion usually occurs by the age of two yr, and healthy individuals typically encounter no adverse consequences of infection. However, in liver transplant patients, HHV6 has the potential to cause severe illnesses. These illnesses include, but are not limited to hepatitis, interstitial pneumonitis, encephalitis, and bone marrow suppression (10) – clinical entities that are not unlike what could be attributable to CMV. This makes it imperative to rule out CMV disease in any situation where a diagnosis of HHV6 disease is being considered. While there are several reports summarizing the clinical features of HHV6 infection in liver transplant patients (3–7), the true burden and clinical significance of this infection after solid organ transplantation are yet to be fully 47
Al Fawaz et al.
determined. In this regard, this study was conducted to determine the prevalence of HHV6 DNAemia in a cohort of pediatric liver transplant recipients and to describe the clinical characteristics and outcomes in this population of patients. This information was intended to inform our approach to herpesvirus surveillance in the posttransplant period, with specific reference to the relative merits of routine HHV6 surveillance.
Antiviral prophylaxis
Methods
Patients were identified through the Hospital’s Medical Records, and cross-verification was performed using the transplant database. Relevant clinical and laboratory data were extracted and compared among cases and controls. Data were summarized using descriptive statistics (e.g., medians and ranges), and wherever appropriate, comparisons were made using conditional statistics, given the matched case–control design. The 2:1 matching of controls to cases was intended to provide sufficient power for meaningful comparisons.
Setting This study was conducted at the Hospital for Sick Children, Toronto, a quaternary pediatric institution where transplantation of solid organs is performed. The study was approved by our institution’s Research Ethics Board.
Study design This was a retrospective case–control study. Cases were matched by age (6 months) with controls using a 1:2 ratio of cases-to-controls.
Eligibility criteria Cases were liver or combined liver–small bowel or combined liver–kidney transplant recipients aged ≤ 18 yr old and who were transplanted over a 10-yr period, between January 1997 and January 2007. During this period, 154 liver transplants were performed. Routine surveillance testing for CMV and EBV was performed every 1–2 wk for the first three months after transplantation (6–12 samples per patient). Beyond three months, testing was carried out approximately every four wk for the first six months post-transplantation; then every 4–8 wk thereafter in relation to clinic visits. The PCR testing approach enabled real-time concurrent screening for HHV6 on all samples that were tested for CMV. In addition, additional testing for HHV6 was also based on the clinicians’ decisions to evaluate clinical syndromes by testing of blood or biopsy samples. Controls were identified from among subjects who did not have positive HHV6 DNA test results and who were transplanted during the above period.
PCR assay and definitions HHV6 DNA was detected using PCR as previously described and is used in other studies from our center (11, 12). This was a qualitative assay with a detection threshold of 2–20 copies and with primers as previously published (12). DNAemia was defined as the presence of a positive HHV6 DNA in blood or plasma. CMV, EBV, and HHV7 DNAemia were similarly defined. In this regard, not all cases of DNAemia are consistent with active infection. For these viruses, the detection of DNA does not necessarily indicate active infection and may represent latent infection. However, it has been previously determined that the detection of HHV6 DNA in plasma is consistent with active infection (13). Thus, active HHV6 infection was defined as a positive plasma HHV6 DNA PCR. A positive tissue PCR by itself was not regarded as being indicative of active infection (in the absence of compatible histopathology), given the poor utility of herpesvirus tissue PCR in distinguishing active infection from latent infection.
48
Our liver transplant patient population received ganciclovir for the first three months after transplantation, if they were at risk of CMV infection (donor-seropositive, recipientseronegative; recipient-seropositive regardless of donor status). Ganciclovir was administered at a dose of 10 mg/kg/ day divided twice daily for two wk, followed by 5 mg/kg once daily to complete 12 wk.
Data extraction and statistical analyses
Results
Among 154 children and adolescents who underwent liver transplantation and were tested for HHV6, 25 were documented to have HHV6 DNA in blood or plasma after transplantation, for a prevalence of 16.2%. There were 12 males and 13 females with a male/female ratio of 0.9:1. The median age at the time of transplantation was 0.75 yr, with a range of 0.25–14.5 yr. The underlying diseases and descriptive characteristics of study subjects and their HHV6 pretransplant serostatus are shown in Table 1. There were 50 age-matched control subjects identified who were transplanted during the same period. Seventeen (34%) of these subjects were HHV6 seropositive pretransplant, 16 (32%) were seronegative, and the serostatus was unknown in 17 (34%) patients. The pretransplant CMV and EBV serostatus for cases vs. controls were statistically comparable, as shown in Table 1. Among the 25 cases, the breakdown of positive HHV6 DNA PCR results according to the sources of samples was as follows: all 25 were positive in whole blood, three with additional plasma-positive PCR tests (12%), two with additional liver-positive PCR tests (8%), and one with liver- and plasma-positive PCR tests (4%). Thus, based on our definition of active infection (positive plasma PCR tests), four of the 25 (16% of cases) with positive DNA PCR tests had active infection. HHV6 PCR became positive during the first six wk post-transplantation in 14 patients (56% of cases). The time of initial detection ranged from 11 days to six yr, according to the clinical indications for testing. Later detections (e.g.,
HHV6 in liver transplant patients Table 1. Characteristics of HHV6 patients with HHV6 DNAemia Cases
Variable
Number
Controls Percentage Percentage Number or number
0.75 n/a Age at transplantation (0.25–14.5) (yr), median (range) Gender (female/ 13:12 52:48 male) Organ transplanted Liver 21 84 Liver and small 3 12 bowel Liver and kidney 1 4 Liver, small bowel, 0 0 pancreas Underlying disease Biliary atresia 12 48 TPN cholestasis 6 24 Hepatoblastoma 0 0 Pediatric acute liver 3 12 failure (undetermined etiology) Autoimmune 1 4 hepatitis Metabolic liver 2 8 disease Alagille syndrome 1 4 HHV6 serology pretransplantation Seropositive 11 44 Seronegative 7 28 Unknown 7 28 EBV serology pretransplantation* Seropositive 16 64 Seronegative 8 32 CMV serology pretransplantation* Seropositive 16 64 Seronegative 8 32 Immunosuppressive drugs† Corticosteroids 25 100 Tacrolimus 20 80 Sirolimus 1 4 Thymoglobulin 4 16 MMF 5 20 Time of HHV6 DNAemia First six wk post14 56 transplant Median duration of 6 (0.7–120) n/a HHV6 DNAemia, weeks (range) Recurrence of 7 28 positive PCR Median period 3.9 (3–32) – after resolution, weeks (range) Clinical manifestations Asymptomatic 18 72 Symptomatic 7 28
1.16
0.83–15
30:20
60:40
46 3
92 6
0 1
0 2
28 5 4 7
56 10 8 14
1
2
5
10
0
0
17 16 17
34 32 34
30 20
60 40
21 28
42 56
50 47 5 3 4
100 94 10 6 8
n/a
n/a
n/a
n/a
years post-transplantation) were invariably as a result of diagnostic evaluations as opposed to surveillance. The median duration of a positive PCR in whole blood or plasma was six wk, range five days to 30 months; however, for seven patients, repeat PCR testing was not carried out in a manner that allowed us to document the precise date of resolution of positive PCR results. Eighteen patients had no recurrence of positive PCRs after an initial positive result followed by clearance, while seven patients had recurrence within a median of 27 days (range 21 days to eight months) after resolution. Eighteen patients (72% of cases) were asymptomatic, while seven patients (28% of cases) were symptomatic with symptoms that were initially presumed to be due to HHV6. However, based on positive DNA PCR tests in plasma, only four of these were regarded as having HHV6-attributable symptoms (two liver and two liver–small bowel transplants). The major symptoms and signs were fever in seven patients and vomiting, lethargy, splenomegaly, bone marrow suppression in one patient each, respectively. At the time of the initial positive PCR tests, 10 patients (40% of cases) with HHV6 DNAemia had rising transaminases with median AST and ALT values of 59 (range 30–507 U/L) and 81 (range 22–565 U/L), respectively. Herpesvirus co-infections
The relationship between the presence of a positive HHV6 DNA test and DNA positivity (DNAemia) for other herpesviruses was examined. Table 2 shows that when cases were compared with controls, there were no significant differences in the frequency of DNAemia with CMV, EBV and HHV7. The proportion of PTLD diagnoses were comparable, with two (8%) in the study group and five (10%) in the control group.
Table 2. Co-infections between HHV6 and other herpesviruses among cases and controls* n/a
n/a
n/a
n/a
n/a n/a
n/a n/a
*Serostatus unknown or indeterminate for some subjects. † Other agents received included azathioprine and cyclosporine A in
© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Pediatric Transplantation DOI: 10.1111/petr.12176
Clinical consequences of human herpesvirus-6 DNAemia in peripheral blood in pediatric liver transplant recipients Al Fawaz T, Ng V, Richardson SE, Barton M, Allen U. Clinical consequences of human herpesvirus-6 DNAemia in peripheral blood in pediatric liver transplant recipients.
Tariq Al Fawaz1,*, Vicky Ng2,3, Susan E. Richardson4, Michelle Barton1,† and Upton Allen1,3 1
Abstract: The significance of HHV6 DNAemia after solid organ transplantation has not been fully determined. Our objectives were to determine the prevalence of HHV6 DNAemia in pediatric liver transplant recipients and to describe the associated clinical characteristics and outcomes. This was a retrospective case–control study. Eligible liver transplant patients aged ≤ 18 yr with HHV6 DNAemia were matched with two subjects without HHV6 DNAemia. Matching was by age 6 months. Among 154 subjects, 25 patients (16%) had HHV6 DNAemia detected by PCR in whole blood or plasma (M:F ratio = 0.9:1). While 28% of subjects with DNAemia (7/25) had symptoms consistent with HHV6 infection, active infection was detected in only four subjects (2.6% of liver transplant patients). The major symptoms/signs were fever, vomiting, lethargy, splenomegaly, bone marrow suppression, and elevated transaminases. The prevalence of DNAemia due to other herpesviruses in cases vs. controls was EBV 56% vs. 60%, CMV 12% vs. 12%, HHV7 20% vs. 12%; p value is not significant for all pairwise comparisons. HHV6 DNAemia in pediatric liver transplant patients is not an uncommon entity. While the clinical relevance is still not entirely established, active HHV6 infection and attributable symptoms are relatively rare.
Division of Infectious Diseases, Hospital for Sick Children, University of Toronto, Toronto, ON, Canada, 2Division of Gastroenterology, Hepatology and Nutrition, Hospital for Sick Children, University of Toronto, Toronto, ON, Canada, 3Transplant Centre, Department of Pediatrics, Hospital for Sick Children, University of Toronto, Toronto, ON, Canada, 4The Division of Microbiology, Department of Pediatric Laboratory Medicine, Hospital for Sick Children, University of Toronto, Toronto, ON, Canada Key words: herpesvirus – HHV6 – pediatric transplantation – liver transplant Upton Allen, Division of Infectious Diseases, Hospital for Sick Children, 555 University Avenue, Toronto, ON M5G 1X8, Canada Tel.: 416 813 8129 Fax: 416 813 8404 E-mail: [email protected] *Present affiliation: King Fahad Medical City, Riyadh, Saudi Arabia † Present affiliation: Division of Microbiology, Hospital for Sick Children, Toronto, ON, Canada Accepted for publication 16 September 2013
HHV6 is a ubiquitous virus, which is widespread throughout the world. It belongs to the b-herpes group family to which CMV and HHV7 also belong. As with other herpesviruses, it has the ability to remain latent and can be reactivated with immunosuppression (1, 2). In this context, HHV6 is increasingly being recognized as a significant opportunistic pathogen in solid organ as well as stem cell transplant recipients (3–11). In the normal host, HHV6 is the causative agent of the self-limited illness, roseola (exanAbbreviations: CMV, cytomegalovirus; EBV, Epstein Barr virus; HHV6, human herpesvirus 6; HHV7, human herpesvirus 7; MMF, mycophenolate mofetil; PCR, polymerase chain reaction; PTLD, post-transplant lymphoproliferative disorder.
thema subitum). Seroconversion usually occurs by the age of two yr, and healthy individuals typically encounter no adverse consequences of infection. However, in liver transplant patients, HHV6 has the potential to cause severe illnesses. These illnesses include, but are not limited to hepatitis, interstitial pneumonitis, encephalitis, and bone marrow suppression (10) – clinical entities that are not unlike what could be attributable to CMV. This makes it imperative to rule out CMV disease in any situation where a diagnosis of HHV6 disease is being considered. While there are several reports summarizing the clinical features of HHV6 infection in liver transplant patients (3–7), the true burden and clinical significance of this infection after solid organ transplantation are yet to be fully 47
Al Fawaz et al.
determined. In this regard, this study was conducted to determine the prevalence of HHV6 DNAemia in a cohort of pediatric liver transplant recipients and to describe the clinical characteristics and outcomes in this population of patients. This information was intended to inform our approach to herpesvirus surveillance in the posttransplant period, with specific reference to the relative merits of routine HHV6 surveillance.
Antiviral prophylaxis
Methods
Patients were identified through the Hospital’s Medical Records, and cross-verification was performed using the transplant database. Relevant clinical and laboratory data were extracted and compared among cases and controls. Data were summarized using descriptive statistics (e.g., medians and ranges), and wherever appropriate, comparisons were made using conditional statistics, given the matched case–control design. The 2:1 matching of controls to cases was intended to provide sufficient power for meaningful comparisons.
Setting This study was conducted at the Hospital for Sick Children, Toronto, a quaternary pediatric institution where transplantation of solid organs is performed. The study was approved by our institution’s Research Ethics Board.
Study design This was a retrospective case–control study. Cases were matched by age (6 months) with controls using a 1:2 ratio of cases-to-controls.
Eligibility criteria Cases were liver or combined liver–small bowel or combined liver–kidney transplant recipients aged ≤ 18 yr old and who were transplanted over a 10-yr period, between January 1997 and January 2007. During this period, 154 liver transplants were performed. Routine surveillance testing for CMV and EBV was performed every 1–2 wk for the first three months after transplantation (6–12 samples per patient). Beyond three months, testing was carried out approximately every four wk for the first six months post-transplantation; then every 4–8 wk thereafter in relation to clinic visits. The PCR testing approach enabled real-time concurrent screening for HHV6 on all samples that were tested for CMV. In addition, additional testing for HHV6 was also based on the clinicians’ decisions to evaluate clinical syndromes by testing of blood or biopsy samples. Controls were identified from among subjects who did not have positive HHV6 DNA test results and who were transplanted during the above period.
PCR assay and definitions HHV6 DNA was detected using PCR as previously described and is used in other studies from our center (11, 12). This was a qualitative assay with a detection threshold of 2–20 copies and with primers as previously published (12). DNAemia was defined as the presence of a positive HHV6 DNA in blood or plasma. CMV, EBV, and HHV7 DNAemia were similarly defined. In this regard, not all cases of DNAemia are consistent with active infection. For these viruses, the detection of DNA does not necessarily indicate active infection and may represent latent infection. However, it has been previously determined that the detection of HHV6 DNA in plasma is consistent with active infection (13). Thus, active HHV6 infection was defined as a positive plasma HHV6 DNA PCR. A positive tissue PCR by itself was not regarded as being indicative of active infection (in the absence of compatible histopathology), given the poor utility of herpesvirus tissue PCR in distinguishing active infection from latent infection.
48
Our liver transplant patient population received ganciclovir for the first three months after transplantation, if they were at risk of CMV infection (donor-seropositive, recipientseronegative; recipient-seropositive regardless of donor status). Ganciclovir was administered at a dose of 10 mg/kg/ day divided twice daily for two wk, followed by 5 mg/kg once daily to complete 12 wk.
Data extraction and statistical analyses
Results
Among 154 children and adolescents who underwent liver transplantation and were tested for HHV6, 25 were documented to have HHV6 DNA in blood or plasma after transplantation, for a prevalence of 16.2%. There were 12 males and 13 females with a male/female ratio of 0.9:1. The median age at the time of transplantation was 0.75 yr, with a range of 0.25–14.5 yr. The underlying diseases and descriptive characteristics of study subjects and their HHV6 pretransplant serostatus are shown in Table 1. There were 50 age-matched control subjects identified who were transplanted during the same period. Seventeen (34%) of these subjects were HHV6 seropositive pretransplant, 16 (32%) were seronegative, and the serostatus was unknown in 17 (34%) patients. The pretransplant CMV and EBV serostatus for cases vs. controls were statistically comparable, as shown in Table 1. Among the 25 cases, the breakdown of positive HHV6 DNA PCR results according to the sources of samples was as follows: all 25 were positive in whole blood, three with additional plasma-positive PCR tests (12%), two with additional liver-positive PCR tests (8%), and one with liver- and plasma-positive PCR tests (4%). Thus, based on our definition of active infection (positive plasma PCR tests), four of the 25 (16% of cases) with positive DNA PCR tests had active infection. HHV6 PCR became positive during the first six wk post-transplantation in 14 patients (56% of cases). The time of initial detection ranged from 11 days to six yr, according to the clinical indications for testing. Later detections (e.g.,
HHV6 in liver transplant patients Table 1. Characteristics of HHV6 patients with HHV6 DNAemia Cases
Variable
Number
Controls Percentage Percentage Number or number
0.75 n/a Age at transplantation (0.25–14.5) (yr), median (range) Gender (female/ 13:12 52:48 male) Organ transplanted Liver 21 84 Liver and small 3 12 bowel Liver and kidney 1 4 Liver, small bowel, 0 0 pancreas Underlying disease Biliary atresia 12 48 TPN cholestasis 6 24 Hepatoblastoma 0 0 Pediatric acute liver 3 12 failure (undetermined etiology) Autoimmune 1 4 hepatitis Metabolic liver 2 8 disease Alagille syndrome 1 4 HHV6 serology pretransplantation Seropositive 11 44 Seronegative 7 28 Unknown 7 28 EBV serology pretransplantation* Seropositive 16 64 Seronegative 8 32 CMV serology pretransplantation* Seropositive 16 64 Seronegative 8 32 Immunosuppressive drugs† Corticosteroids 25 100 Tacrolimus 20 80 Sirolimus 1 4 Thymoglobulin 4 16 MMF 5 20 Time of HHV6 DNAemia First six wk post14 56 transplant Median duration of 6 (0.7–120) n/a HHV6 DNAemia, weeks (range) Recurrence of 7 28 positive PCR Median period 3.9 (3–32) – after resolution, weeks (range) Clinical manifestations Asymptomatic 18 72 Symptomatic 7 28
1.16
0.83–15
30:20
60:40
46 3
92 6
0 1
0 2
28 5 4 7
56 10 8 14
1
2
5
10
0
0
17 16 17
34 32 34
30 20
60 40
21 28
42 56
50 47 5 3 4
100 94 10 6 8
n/a
n/a
n/a
n/a
years post-transplantation) were invariably as a result of diagnostic evaluations as opposed to surveillance. The median duration of a positive PCR in whole blood or plasma was six wk, range five days to 30 months; however, for seven patients, repeat PCR testing was not carried out in a manner that allowed us to document the precise date of resolution of positive PCR results. Eighteen patients had no recurrence of positive PCRs after an initial positive result followed by clearance, while seven patients had recurrence within a median of 27 days (range 21 days to eight months) after resolution. Eighteen patients (72% of cases) were asymptomatic, while seven patients (28% of cases) were symptomatic with symptoms that were initially presumed to be due to HHV6. However, based on positive DNA PCR tests in plasma, only four of these were regarded as having HHV6-attributable symptoms (two liver and two liver–small bowel transplants). The major symptoms and signs were fever in seven patients and vomiting, lethargy, splenomegaly, bone marrow suppression in one patient each, respectively. At the time of the initial positive PCR tests, 10 patients (40% of cases) with HHV6 DNAemia had rising transaminases with median AST and ALT values of 59 (range 30–507 U/L) and 81 (range 22–565 U/L), respectively. Herpesvirus co-infections
The relationship between the presence of a positive HHV6 DNA test and DNA positivity (DNAemia) for other herpesviruses was examined. Table 2 shows that when cases were compared with controls, there were no significant differences in the frequency of DNAemia with CMV, EBV and HHV7. The proportion of PTLD diagnoses were comparable, with two (8%) in the study group and five (10%) in the control group.
Table 2. Co-infections between HHV6 and other herpesviruses among cases and controls* n/a
n/a
n/a
n/a
n/a n/a
n/a n/a
*Serostatus unknown or indeterminate for some subjects. † Other agents received included azathioprine and cyclosporine A in
