Materials and methods

Between July 1999 and November 2002, we obtained weekly surveillance stool cultures for VRE from all hospitalized pediatric SCT recipients. Cultures were obtained during the initial transplant admission and in all subsequent inpatient stays.

The pediatric SCT unit at Vanderbilt Children's Hospital consists of five hepa-filtered rooms. Patients stay in these rooms from the time of admission until the time of discharge. Strict hand washing is enforced before entry to the rooms for all health-care workers and family members. All patients had double lumen Hickman-type central venous catheter at the time of admission. Empiric broad-spectrum antibiotics, generally a third-generation cephalosporin, were started when the absolute neutrophil count (ANC) was less than 0.5 10⁹/l. Vancomycin was started if the patient had a positive blood culture with Gram-positive cocci, had persistent fever, or upon the discretion of the attending physician. Blood cultures were obtained if the patients developed fever or any signs or symptoms of bacteremia or sepsis. Surveillance blood cultures were obtained twice weekly from all patients receiving steroids at a dose of greater than 1 mg/kg/day.

When the stool surveillance cultures were obtained, a plate containing bile esculin with azide base with 10 g/ml of vancomycin was streaked. In addition, a colisitin nalidixic agar plate and a 5% sheep's blood agar plate (from Bectin-Dickinson Biological Laboratories) were streaked. These VRE screening plates were examined at 24, 48, and 72 h of incubation at 37°C. When suspicious colonies were identified via Gram stain appearance, a second blood plate was cultured, and checked for catalase and pyroglutamate aminopeptidase. Then, a vancomycin/gentamycin/ampicillin/streptomycin quad plate was streaked. If enterococcus was identified, and this was the patient's first documented VRE, a 4-h rapid identification for streptococci test (RapID STR Panel Test by Remel, Inc.) was carried out to further speciate the isolate. In addition, a sensitivity plate was inoculated to identify the antibiogram.

When VRE was identified, the patients were placed on strict contact isolation. The isolation consisted of gloves, gown, hand washing on entrance and exit to a room, and a dedicated stethoscope. For patients who tested positive, weekly cultures were obtained in the outpatient department until the clearance of VRE colonization. In the outpatient clinic, a dedicated room and bathroom were assigned for VRE colonized patients. These rooms were thoroughly cleaned after each visit. Clearance of VRE colonization was defined as three consecutive negative weekly cultures.

Wilcoxon's rank-sum test and Fisher's exact test were used to evaluate risk factors of VRE. Survival differences were evaluated via Kaplan–Meier curves and the log-rank test. All tests were conducted at the 0.05 significance level. The study was approved by the Institutional Review Board of Vanderbilt University Medical Center.

Results

Between July 1999 and November 2002, 61 patients underwent SCT at Vanderbilt Children's Hospital and had weekly surveillance stool cultures for VRE. Of these, 15 patients (24.6%) had a positive stool culture for VRE. The median age for those who tested positive for VRE was 3.6 years (range 0.7–17.9 years) compared to 5.6 years (range 0.6–20.5 years) for those who remained negative. Of the 15, 13 (87%) VRE-positive and 32 of 46 (69.5%) VRE-negative patients received allogeneic SCT. The most common diagnosis in both groups was acute leukemia (Table 1). Neither patient age (P=0.89), sex (P=0.83), type of transplant (P=0.31), nor time to engraftment (P=0.34) were found to be significantly different between the two groups. Analysis of survival data (median follow-up 271 days) did not indicate a relationship between VRE colonization and survival (P=0.78). Underlying diagnosis for the VRE-positive patients included acute myelogenous leukemia (n=6), acute lymphocytic leukemia (n=4), Ewing's sarcoma, myelodysplastic syndrome, Hodgkin's disease, hypophosphatasia, and Wiskott–Aldrich syndrome (n=1 each) (Table 2). Of the 15, 11 (73%) had received vancomycin therapy prior to SCT. All patients were receiving broad-spectrum antibiotics (third- or fourth-generation cephalosporin) at the time of colonization. Three patients had VRE detected on the first culture after admission, and 12 developed VRE colonization at a median of 3.5 days (range 1–227 days) post transplant. Species of VRE were E. faecium (n=14) and E. faecalis (n=1).

Table 1 - Patients characteristics.

Full table

Table 2 - Clinical feature and outcome of patients with VRE colonization.

Full table

Five of the 15 (33%) colonized patients developed VRE bacteremia; two at the time of first colonization and three at days 20, 32, and 34 following the first positive stool culture. Only two of the five patients who developed bacteremia had an ANC of <0.5 10⁹/l. All but one of the VRE isolates (patient number 12) was sensitive to quinupristin/dalfopristin and linezolid. The isolate from patient number 12 showed intermediate sensitivity to quinupristin/dalfopristin and linezolid. The bacteremia resolved without significant morbidity in all five patients after therapy with quinupristin/dalfopristin. Central line removal occurred in three of the five patients due to persistent positive blood cultures 48 h after the initiation of quinupristin/dalfopristin therapy. Therapy with quinupristin/dalfopristin was continued for a total of 10 days after the first negative blood cultures.

Four patients died (38–153 days post-SCT) due to relapse (patients 4 and 14) or multisystem organ failure as a complication of the transplant (patients 1 and 8). However, all remained colonized with VRE at the time of death. None of the deaths was directly caused by VRE infection. Of the 11 surviving patients, seven cleared the VRE at a median of 144 days (range 61–198 days) postcolonization. Negative VRE status was defined by three negative sequential weekly cultures. Two patients (patient numbers 2 and 5) who received autologous SCT cleared the colonization on days 80 and 61, respectively. The five patients who received allogeneic SCT and cleared the VRE colonization all did so while on continued immune suppression. Four patients remained colonized at 68–702 days after the first positive culture. All those patients received allogeneic SCT and are receiving therapy for acute (patient 12) or clinically extensive chronic graft-versus-host disease (GVHD) (patients 3, 6, and 9).

Discussion

Over the past decade, significant improvement in supportive care in SCT units has led to better prevention and treatment of infections in transplant recipients. However, the use of prophylactic broad-spectrum antibiotics has raised the concern for increasing incidence of resistant organisms. VRE has emerged as an important pathogen in liver, kidney, and SCT units in the United States.^4,5,6,7,8,20 Colonization with VRE has been reported in as many as 50% of pediatric oncology patients.¹⁷ At present, there are very limited data with regard to the incidence and clinical consequences of VRE in pediatric SCT patients.

Koc et al¹¹ reported on two adult cases of fatal VRE infection following unrelated donor SCT in 1998.¹¹ Kapur et al performed surveillance cultures on 29 recipients of autologous SCT and found that 15 were colonized. Of these, 10 patients developed bacteremia one of whom died as a direct result of the VRE bacteremia.¹⁰ In 1999, another outbreak of VRE was reported in five patients at Johns Hopkins Hospital. All patients received prophylactic vancomycin therapy prior to the colonization. One of these patients developed bacteremia with no significant associated morbidity or mortality.²⁰ Carretto et al reported on three children who developed five episodes of VRE bacteremia following unrelated SCT. All the children presented with high spiking fever and hemorrhagic enteritis. The bacteremia resolved in all of these children with quinupristin/dalfopristin and teicoplanin therapy, in addition to central line removal. Information about the duration of intestinal colonization was not reported.¹⁵

Our study represents the largest prospective screening of pediatric SCT patients. The VRE incidence of 24.6% in our study is similar to the outcomes in the previous studies of pediatric oncology patients. Of the 15 patients, 13 (93 %) were colonized in the first 3 weeks post-SCT. Three were positive on the first culture following admission to the hospital. Only one patient (patient number 6) developed colonization at day 227 post transplant. That particular patient had multiple cycles of broad-spectrum antibiotics as well as extensive chronic GVHD. His colonization was noted during one of his many admissions for GVHD flare up. Since none of these patients had VRE screening prior to the SCT admission, this early colonization might represent VRE colonization before their SCT admission.

Four of five patients who developed bacteremia had significant gastrointestinal pathology with either transplant-related mucositis or gut GVHD. This is similar to what has been observed by others in the setting of SCT.^15,16 In contrast to other reports, our patients did not develop any significant morbidity or mortality directly related to VRE bacteremia. All five cleared the bacteremia with quinupristin/dalfopristin therapy as well as central line removal in three patients. Two of our patients cleared the bacteremia without the need to remove the central line. Central line removal was performed when patients had at least one positive blood culture despite therapy with quinupristin/dalfopristin. All of our isolates except one were sensitive to quinupristin/dalfopristin. Although one patient (patient number 12) had an isolate that showed intermediate sensitivity to quinupristin/dalfopristin, he successfully cleared the bacteremia after central line removal. All five patients continued to have intestinal colonization after completing therapy with quinupristin/dalfopristin. This observation is similar to other reports in which therapy with antibiotics cleared the bacteremia but had no effect on the intestinal colonization.^15,21

All four patients who died in our study remained colonized with VRE, but none of the deaths was directly related to VRE. The overall survival, transplant-related mortality TRM and relapse were very comparable in patients who were VRE positive as compared to those who were not colonized (Table 1). This was in contrast to the observation reported by Gregory et al. They reported increased TRM and graft failure in patients who had VRE colonization and bacteremia.¹⁶

Seven of 11 surviving patients cleared the VRE colonization. They did so in a minimum of 60 days after the first colonization with four patients requiring more than 4 months to clear VRE colonization. Of note, the two patients who received autologous SCT cleared the infection more rapidly (days 61 and 81) as compared to the 5 patients who received allogeneic SCT (days 64, 198, 198, 149, and 144). None of the patients who cleared the VRE colonization had recolonization during the study period. One limitation of our study is that patients who met the criteria for clearing VRE colonization did not have further screening unless they had a complication necessitating hospitalization. Four patients (27%) remain colonized with VRE from 68 to 702 days post first colonization. While all of those who remain colonized had active GVHD requiring immune suppression, only two of the five who received allogeneic SCT and cleared the colonization had active GVHD. Despite the small numbers, recipients of allogeneic SCT and those with active GVHD tend to have longer period of colonization.

Although our study did not detect any differences between patients with and without VRE colonization, the analysis was limited by the small sample size. Perhaps a larger multicenter study is needed to be able to identify specific risk factors predisposing SCT patients to VRE colonization.

The current report is the first to ascertain the incidence and clinical outcome of VRE colonization in pediatric SCT recipients. It is not surprising that VRE colonization is common and can last for prolonged periods. In a recent study, Wong et al²² reported that therapy with ramoplanin, a glycolipodepsipeptide, significantly suppresses VRE gastrointestinal colonization. There are no studies to show that therapy with ramoplanin will decrease the incidence of bacteremia. The efficacy and safety of ramoplanin has not been established in SCT recipients. Since the gastrointestinal tract can remain colonized without clinically apparent disease, screening and identification of colonized patients is critical. To prevent nosocomial infections, contact isolation and precautions directed specifically against VRE contamination remain the only effective measure.²³ We continue to perform weekly surveillance stool cultures on all hospitalized pediatric SCT patients and apply strict contact isolation to all those identified with intestinal colonization of VRE.

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Acknowledgements

We gratefully acknowledge the excellent care provided by the nursing and support staff.