Adenoviruses are non-enveloped double-stranded DNA viruses. Fifty-one serotypes of human ADV have been identified and are classified into six subgroups (A-F) (1). The viruses are endemic in the general pediatric population with the peak incidence of infection occurring between the ages of 6 months and 5 years (2). In normal, healthy children, ADV causes a mild self-limiting illness affecting the respiratory tract, gastrointestinal system, eyes and skin. In contrast, in immunocompromised patients, ADV infections have been associated with significant morbidity and mortality. A case fatality rate as high as 60% has been reported among pediatric and young adult HSCT patients (3). Clinical manifestations of ADV infection in HSCT recipients can range from asymptomatic infection to disseminated disease resulting in multi-organ failure. Four distinct clinical syndromes are reported in HSCT recipients: gastrointestinal infections with diarrhea, hemorrhagic colitis or hepatitis; pulmonary infections, resulting in potentially fatal pneumonia; urinary tract infections, associated with hemorrhagic cystitis and possible renal failure; and asymptomatic nasopharyngeal infections (4).
Several studies have identified risk factors for the development of ADV infection and for progression from infection to invasive disease following HSCT. Children appear to be almost three times more likely to develop ADV infection than adults (5, 6). Recipients of HSCT from matched unrelated donors and/or of T-cell depleted grafts and patients with GVHD are at increased risk of developing ADV infection (7–9). Isolation of the virus from more than one site and detection of ADV DNA in peripheral blood correlate significantly with progression to disseminated disease and fatal outcome (9, 11–13). The highest risk of death is reported in those patients who develop hepatitis or pneumonitis (3, 14, 15).
ADV is traditionally identified in clinical specimens using culture-based methods. While these methods are effective in detecting end organ infection in symptomatic patients, they lack sensitivity for detecting low levels of circulating virus in peripheral blood samples. In addition, while some positives can be detected in 1–2 days using rapid shell-vial techniques, conventional tube culture methods can take weeks to produce a definitive result. PCR-based assays are now increasingly used in the detection of human ADV from blood, body fluids and tissue specimens (7, 8, 16). Recently developed multiplex real-time PCR-assays are rapid (24 hr turnaround time) and can detect all common ADV serotypes. These assays provide a quantitative result (viral load) which may be useful for the clinical assessment of therapeutic response and the prediction of disease outcome (17, 18).
There are few effective therapeutic options for the treatment of ADV infection. Regimens that have been described for treating ADV infection in immunocompromised patients include the use of ribavirin, ganciclovir, vidarabine, intravenous immunoglobulin and adoptive immunotherapy with leukocyte transfusions. These attempts at treatment have been described primarily in case reports and small series with variable results (10, 19–24). Cidofovir (CDV) is a nucleotide analog with broad antiviral activity against DNA viruses including ADV (8, 25–28). Some case reports have demonstrated success in using CDV to treat disseminated ADV in immunocompromised patients, including HSCT recipients. While encouraging in its antiviral effects, some reports have shown limited utility due to dose-limiting nephrotoxicity when using conventional dosing of 5 mg/kg once a week. Others have suggested alternative dosing and adequate prehydration can mitigate this concern (25, 27, 28). To date, most studies published with CDV have involved adult patients with only small numbers of pediatric patients described. No large pediatric series have been published, and none has evaluated the safety and effectiveness of this agent in a large pediatric stem cell transplant population, the population most at risk from this virus.
PATIENTS AND METHODS
Following Institutional Review Board approval, a retrospective review of medical records for all consecutive patients undergoing myeloablative autologous (n=8) or allogeneic (n=169) HSCT at St. Jude Children’s Research Hospital during a 39 month-period (January 2001–March 2004) was performed. Fifty-seven (57) of these patients tested positive for ADV (see viral detection methods, below), and were included in this study. Data collected on the latter patients included information on demographics, clinical presentation and hospital course, pertinent laboratory results, treatment, toxicities and clinical outcome. Patients undergoing autologous HSCT received a disease-specific conditioning regimen without total body irradiation (TBI). Eighteen patients received 1200 cGy TBI in 8 fractions as part of their conditioning regimen; 3 patients received 2 cGy TBI before a non-myeloablative HSCT for solid tumors. GVHD prophylaxis consisted of cyclosporin (CSP) and mycophenolate mophetil (MMF) in matched sibling donor recipients, CSP and methotrexate in matched unrelated donor recipients(MUD), and MMF in recipients of HLA-mismatched family members (MMFM). T-cell depletion was performed on unrelated donor grafts using the CD34 antibody on the CliniMACS system [Miltenyi Biotec, Germany] and adding back 5×105 CD34+ cells /kg to the final graft. For mismatched family member grafts, T-cell depletion was performed either by using the CD34 antibody or the murine monoclonal anti-CD3 antibody OKT3 (Muronomab) [Miltenyi Biotec, Germany]. In addition, all patients received rabbit antithymocyte globulin (ATG) [Genzyme Transplant, Fremont, CA] from day – 3 to day -1 and recipients of mismatched related donor grafts received OKT3 and methylprednisolone on days 1 to 18 postHSCT.
Adenovirus disease was defined according to published criteria (14). Patients who were culture or PCR positive for ADV with corresponding clinical signs and symptoms were considered to have ADV infection involving those site(s). Disseminated ADV disease was defined by clinical evidence of ADV infection involving two or more sites. Transient ADV viremia occurred when blood was PCR-positive for no more than two consecutive weeks. Compromised renal function was defined as elevated serum creatinine (>1.5 mg/dl), low creatinine clearance (<90 ml/min per 1.73m2) and proteinuria (>2+). Acute and chronic GVHD were graded according to published criteria (29, 30). Regimen-related toxicity was scored by the National Cancer Institute Common Toxicity Criteria (NCI-CTC), version 2.0.
Peripheral blood was prospectively screened weekly for ADV by quantitative real-time PCR, for the first 100 days post-HSCT or longer in patients with persistent symptoms of ADV infection, recipients of T-cell depleted grafts and patients with active GVHD or on immunosuppressive therapy. Cultures for viral pathogens were collected when clinically indicated from sites including the throat, nasopharynx, trachea, conjunctiva, urine, stool and rectum. Tissue biopsies from the gut, liver or lungs and autopsy specimens were routinely sent for histologic examination and for viral identification by culture and PCR. All patients who were culture-positive for ADV had subsequent weekly cultures taken from the site of involvement until culture-negative. Renal function was assessed before and after CDV administration by checking serum chemistry and urinalysis.
Creatinine clearance was monitored prior to each dose of CDV; serum creatinine and urine protein were measured before and after each dose of CDV.
Detection of Adenovirus
Specimens were cultured for ADV using standard shell-vial and tube-culture methodologies (31). Cell lines used for viral culture included A549, RMK (Viromed-Labcorp., Inc, St. Paul, MN) and R-Mix™ (Shell vials only, Diagnostic Hybrids, Inc., Athens, OH). Tube cultures were held for 3 weeks (21 days) prior to discarding and reporting as negative. Shell vials were inoculated in duplicate, held for 48 hr, with fluorescent antibody (FA) staining at 24 and 48 hr. Tube cultures that showed cytopathic effect consistent with ADV were confirmed by FA staining.
Blood samples were tested for ADV using a previously described, quantitative real-time PCR method (17) that detects all common serotypes of human ADV. PCR was performed on isolated peripheral blood mononuclear cells (PBMC’s) and reported as viral copies per ml of blood. Tissue specimens were examined histopathologically. Suspected involvement by ADV was confirmed using immunohistochemical methods.
Treatment with CDV was initiated immediately upon detection of ADV by PCR, culture or histopathology. Five milligrams per kilogram (5 mg/kg) of intravenous CDV was administered weekly, for 2 consecutive weeks, then every 2 weeks until 3 consecutive ADV-negative samples. (PCR or culture) were documented from all previously involved sites. Patients were hydrated with normal saline before and after the infusion of CDV. Probenecid, 1g/m2 of body surface area was given orally 3 hr before CDV infusion and then 500 mg/m2 both 2 and 8 hr after completion of CDV infusion. When possible, the concomitant use of cidofovir with other nephrotoxins such as cyclosporine, aminoglycosides, pentamidine or foscarnet was avoided. However, in situations where the use of these nephrotoxins was unavoidable, dose adjustments were made to achieve a low therapeutic level. When indicated for anti-fungal therapy, liposomal amphotericin B was used in all cases, instead of amphotericin B deoxycholate. Patients were monitored for other previously described CDV-related toxicities and adverse clinical reactions by checking daily white blood counts and clinical exams for skin or ocular changes. In patients with compromised renal function (see “Definitions” section, above) the dose and schedule of CDV was changed to 1 mg/kg given on 3 alternate days weekly for 2 consecutive weeks, the same dose was repeated every other week until 3 consecutive ADV-negative specimens were documented from all previously involved sites. Patients with recurrent ADV were retreated using an identical regimen as with first infection.
All patients received antibacterial, antiviral and antifungal prophylaxis according to the standard of practice at the time of transplant. Intravenous immunoglobulin (IVIG) was administered weekly to recipients of allogeneic grafts until 100 days post-HSCT. Patients with Cytomegalovirus (CMV) reactivation by PCR assay received primary preemptive therapy with ganciclovir and, in case of persistent viremia or bone marrow toxicity, secondary preemptive treatment with foscarnet, until 3 consecutive negative results were obtained.
The comparison between the two groups (symptomatic and asymptomatic patients) for continuous outcome measure was done using exact (or Monte Carlo estimates of p-value based on 17000 simulations) Wilcoxon Rank Sum test or two sample t test as implanted in statistical software package SAS. The comparison for the binary outcome measure between the two groups was done using Fisher’s Exact-test as implemented in SAS. A p-value of <0.05 was considered significant.
Patient Risk Factors and Clinical Presentations
Fifty-seven patients diagnosed with ADV infection following HSCT were included in this study, out of 177 patients who received HSCT during the study period (32% rate of infection). Their demographic and clinical characteristics are shown in Table 1. Fifty-two (91%) of the 57 patients were children, 16 years or younger, and the remaining 5 patients were between the ages of 18–26 years. Thirty-five (61%) patients received T-cell depleted grafts, 26 from mismatched family members and 9 from matched unrelated donors. Nine patients received unmanipulated grafts from matched unrelated donors. Sixteen patients had preexisting GVHD, 10 of whom were on immunosuppressive therapy for GVHD at the time of ADV infection. Four patients presented with exacerbation of ongoing GVHD. Clinical presentations at the time of ADV detection included diarrhea (n=30), febrile illness (n=12), hemorrhagic cystitis (n=7), pneumonitis (n=6), and hepatitis (n=1). Fourteen patients (25%) were asymptomatic at the time of initial ADV detection [Table 2].
Detection of Virus
ADV was first detected at a median of 53 days (range 6–319 days) after HSCT. Sites of viral detection were the following: stool (n=30), peripheral blood (n=37), urine (n=7), nasopharyngeal washings (n=5), bronchoalveolar lavage (n=2), endotracheal aspirate (n=2) and cerebrospinal fluid (n=1). Among 37 patients in whom peripheral blood was positive for ADV, 14 patients were asymptomatic at initial presentation, 23 patients had clinical evidence of ADV infection concurrent with viremia, and eight of these symptomatic patients had transient viremia. Twenty-three (62%) of 37 patients with viremia had antecedent diarrhea as their presenting symptom. Conversely, a total of 30 patients presented with diarrhea, 77% progressing to viremia. Fifteen patients (26%) had ADV detected in 2 or more sites simultaneously. Eight (14%) of 57 patients had disseminated disease manifested by: pneumonia and enteritis (n=4); hepatitis, pneumonia and colitis (n=1) and hemorrhagic cystitis and enteritis (n=3). All eight of these patients initially presented with diarrhea (stool positive for ADV with negative blood PCR) and subsequently became positive at other sites.
Asymptomatic patients tended to have significantly lower median viral load than the symptomatic patients, as shown in Figure 1 (p≤0.005). The eight patients with disseminated disease appeared to have a higher viral load at presentation (P=0.0031, based on the exact Wilcoxon Rank Sum test) with a median of 2.6×104 viral copies/ml (range, 0.1×103– 5.6×107) compared to those who did not have disseminated disease with a median of 0.4×103 viral copies/ml (range, 0.1×103–1.4×105). The one patient that died from ADV disease had a presenting viral load of 1.7×106 copies/ml.
The median duration of therapy with CDV was 60 days (range 1–270), with a median of 5 doses given (range, 1–22). Four patients with compromised renal function before the diagnosis of ADV infection were treated with a modified dosing regimen of CDV (see Methods section). Eight patients needed to be re-treated with CDV after completing their initial course of therapy (see “Therapeutic Response”, below). No cases of dose-limiting nephrotoxicity were observed in association with CDV therapy even in those patients with previously compromised renal function. All 4 patients with preexisting reduced renal function cleared the virus without further rise of creatinine, worsening of creatinine clearance, proteinuria or reduction in urinary output. Furthermore, none of the other previously reported side effects attributable to CDV were observed, the latter including bone marrow suppression, nausea, vomiting or ocular complications.
Therapeutic Response and Risk of Recurrence
Cidofovir treatment was associated with resolution of diarrhea, hemorrhagic cystitis, fever, hepatitis and pneumonia, with the virus becoming undetectable in 56/57 (98%) patients. Peripheral blood viral load in all but one patient showed an immediate and progressive decrease, following initiation of therapy. Patients that were asymptomatic cleared the virus more quickly than those that were symptomatic (Fig. 2). The patient who died had an initial response then exhibited rising peripheral blood viral load and died of pneumonitis while on therapy with CDV.
Eight patients needed to be re-treated with CDV after completing their initial course of therapy. The median time to recurrence of ADV infection was 16 days and the median duration of re-treatment in these patients was 5 months. No recurrences were seen among the 14 asymptomatic patients with viremia, while 8 of 23 symptomatic patients with viremia recurred (35% recurrence rate). Symptomatic infection in viremic patients was thus predictive of recurrence (P=0.0148, by Fisher’s exact test). Five of the 8 patients who recurred were recipients of T-cell depleted grafts and 3 were receiving immunosuppressive therapy for GVHD. All patients had ADV detected in their blood at the time of recurrence, 4 had diarrhea, 2 had hemorrhagic cystitis and 1 had hepatitis. Seven of the 8 patients that recurred initially had disseminated disease and had a higher viral load compared to those patients that did not recur (see data on viral load in patients with disseminated disease, above)Table 3.
The case fatality rate in this series was 2% with the one ADV-related death due to pneumonitis and ARDS. This patient was an infant (age 18 months) who received a T-cell depleted graft from a mismatched family member. At autopsy, lung tissue was positive for ADV by both culture and PCR. Overall survival in the cohort was 28/57 (49%) patients with a median follow up of 18 months (range 5–43). Causes of death for all 29 patients are shown in Table 4.
This study demonstrates the safe and effective use of CDV for the treatment of ADV infection in the immunocompromised pediatric HSCT patients. The results obtained (case fatality rate of 2%) reflect a striking reduction in morbidity and mortality, compared to previous series which demonstrated case-fatality rates of up to 60% in HCST patients positive for ADV. Additional data gathered during the course of this study support the use of such therapy as an aggressive preemptive measure that can be used most effectively in combination with frequent monitoring of patients for ADV infection, using both culture and molecular amplification techniques.
The clinical manifestations of ADV infection seen in this study mirrored those previously reported in immunocompromised patients, with a broad spectrum of disease, ranging from asymptomatic infection to fatal, disseminated disease (3, 5, 6, 15). Asymptomatic patients were detected often, possibly due in part to the use of frequent surveillance for ADV. This may explain the higher incidence of ADV seen here in comparison to a previously reported study (15). Fourteen (25%) of all patients in the current study were asymptomatic. Among those who had symptoms, diarrhea was the most common clinical presentation, with stool being the most frequent site of initial viral isolation (52% compared with 83% in a previous study) (5). The most severe manifestations of invasive ADV are usually reported as due to respiratory and hepatic disease (3, 15). In this report, one patient had evidence of hepatitis with elevated liver function tests, associated with colitis and pneumonia. In the latter patient, ADV was isolated in the stool, liver tissue, and lung, with a very high peripheral blood viral load of 4.2×109 copies/ml. Six patients had clinical and radiological evidence of pneumonitis and one suffered from Acute Respiratory Distress Syndrome (ARDS) which contributed to his demise.
Several risk factors have been identified for the development of ADV infection following HSCT. These include young age, GVHD and its therapy, the use of T-cell depleted grafts and grafts from unrelated donors (5, 6, 8, 33). In this report, 32 patients (61%) received a T-cell depleted graft with all of these having low CD4 count at the time of ADV isolation. This is comparable to that reported in previous studies (14, 25, 27, 28). Ten patients (18%) were receiving immunosuppressive therapy for GVHD, and the 3 patients that received an autologous graft all had been pretreated with a myeloablative conditioning regimen.
Isolation of ADV from more than two sites, detection of ADV DNA in peripheral blood, and high viral load in peripheral blood have been shown by others to correlate with progression to disseminated disease and fatal outcome, whereas detection of ADV from any single site (excluding blood) has not (9, 11–13). In the series presented here, 37 (65%) had detectable ADV DNA in peripheral blood and 15 (26%) patients had ADV isolated from two or more sites. The incidence of patients with ADV isolated from multiple sites is same as the 39% reported by Shields et al. (33) but higher than 19% reported by Howard et al. (6) The higher numbers seen here may be explained, at least in part, by the policy of prospective surveillance together with an increased number of high risk and pediatric patients. The high incidence of ADV viremia seen in this series may be related both to the use of routine screening using sensitive molecular diagnostic methods and to the rate of gastrointestinal (GI) infection. GI adenovirus infection can present with diarrhea and frequently progresses to involve other sites (35, 13). Seventy-seven percent (77%) of patients in the present series who initially presented with ADV positive diarrhea went on to develop viremia with eight progressing to disseminated disease. It is noteworthy that despite the higher percentage of patients with ADV viremia and frequency of multiple-site involvement, only 8 (14%) patients developed disseminated disease. This contrasts with a recent report in which 73% of patients with ADV in peripheral blood developed disseminated disease (12). The marked reduction in disseminated disease rate seen in this series may be due to the policy of early preemptive use of CDV therapy for any ADV-positive result, irrespective of site, symptoms, or viral load.
We noted a correlation between the viral load in the peripheral blood with the presence and severity of disease. In symptomatic patients, a rise in viral copy number corresponded temporally with the onset or worsening of symptoms. As viral load decreased, clinical signs and symptoms of infection diminished. Additionally, asymptomatic patients tended to have relatively lower viral load than the symptomatic patients. All patients in whom ADV infection recurred had a high viral load at the time of initial therapy with CDV.
The timing of ADV infection following HSCT in this study was variable and did not appear to predict clinical course or responsiveness to therapy nor was it related to viral load at initial ADV detection. The median time to onset of ADV infection was 53 days with 6 patients developing the infection within the first 30 days postHSCT. Our data is consistent with a previous report from this center, (15) but contrasts with a study that found 50% of ADV infection in pediatric HSCT recipients occurred in the first 30 days post-HSCT (35).
Treatment options for ADV disease are limited. There have been case reports showing variable outcomes with ribavirin and ganciclovir in ADV infection, (19–23) but no prospective randomized trials have been conducted with either of these agents. Cidofovir has shown promise as an effective agent against ADV infection, with evaluations limited to very small groups of patients (less than 60 patients in all reported studies) (25–28). Many have been reluctant to use CDV because of early reports of dose-limiting toxicity (21, 26, 27, 37). The toxicity profile of CDV includes nephrotoxicity and bone marrow suppression. Such adverse affects were not seen in our series, with no instances of dose-limiting toxicity or reduced renal function seen in 57 treated patients. These findings may be attributed to a number of steps taken to reduce the risk of nephrotoxicity, cited most prominently as a risk of this medication. Preventative measures included a modified dosing regimen in patients with evidence of preexisting renal impairment and aggressive prehydration of patients. The concomitant use of other potentially nephrotoxic medications was also minimized in these patients. Beyond these factors, it is also possible that children tolerate CDV better than adult patients, in whom most experience with CDV use and toxicity has been reported.
CDV appears to demonstrate a high degree of efficacy in this study. 56/57 (98%) of patients had clinical resolution of ADV infection, with clearance of the virus documented by culture and quantitative PCR. Clearance occurred irrespective of peak viral load, with one patient recovering from a level of 4.2×109 viral copies/ ml of blood, with concomitant involvement of liver, lungs and colon. While the level of viremia or number of sites involved did correlate with the length of time it took to clear the virus and with the risk of recurrence, it had no impact on overall mortality again, supporting the overall effectiveness of this treatment.
Although others have demonstrated the potential utility of CDV in treating ADV infection in immunocompromised pediatric patients (25–28), this is the first large series to support such therapy, the first to demonstrate the safety of such treatment in the pediatric population, and the first to demonstrate such a high rate of resolution of ADV infection. This result is particularly remarkable considering the high risk status of these patients. The apparent increased effectiveness seen here can, at least in part, be attributed to the aggressive screening of these patients for ADV infection, using a combination of culture-based and molecular techniques, with a strategy of preemptive therapy even in asymptomatic patients and regardless of viral load, a strategy not previously evaluated in earlier studies. The patients in this series showed a case-fatality rate of 2%, far lower than those reported by other groups, which have reported up to a 60% mortality rate in similar patients (82% mortality in patients with disseminated ADV) (12, 13).
This study was limited by the fact that it was a retrospective, single-arm study, with a relatively small number of treated patients. As in all studies in such critically ill patients, it is difficult to fully evaluate risk factors and confounding co-morbidities. The number of patients with high viral load or disseminated disease in this study was small. This may be a reflection of the efficacy of CDV therapy in controlling viral replication, evidenced by the high cure rate among even the most severely affected patients. Finally, when comparing the results seen here with those from other centers, variability in absolute viral load data may be seen. What will likely remain are the relative differences in viral quantity seen between prognostic groups and the relative changes seen in individual patients during disease onset and therapeutic response.
Prospective surveillance for ADV by quantitative PCR assay and culture can be highly effective in early diagnosis of ADV infection in pediatric patients after HSCT. Early use of CDV offers the hope of dramatically reducing morbidity and mortality from ADV infection and disease, currently a major risk in this population of severely immunocompromised patients. The risk of drug-related toxicities in this population can be substantially mitigated through the use of alternative dosing regimens in patients with preexisting renal dysfunction, aggressive hydration, and avoidance of other potentially nephrotoxic agents during the period of CDV treatment. A large, controlled prospective trial is warranted to confirm the encouraging results in this report.
We are indebted to Imella Herrinton and Deanna Combs for their valuable assistance in the preparation of this manuscript.
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