Alcaligenes xylosoxidans Bloodstream Infections in Outpatient Oncology Office

Gaps in infection control led to biofilm production in central venous catheters and resultant bloodstream infection.

To confi rm the presence of an outbreak, ACDC conducted a telephone survey, which asked the microbiology laboratories of Hospital A and 4 surrounding hospitals for a list of all patients who had had positive blood cultures for A. xylosoxidans in the past 3 months. One laboratory identifi ed 3 such patients, and Hospital A laboratory identifi ed 7; all 9 patients (1 case-patient had positive blood CME ACTIVITY Medscape, LLC is pleased to provide online continuing medical education (CME) for this journal article, allowing clinicians the opportunity to earn CME credit. Medscape, LLC is accredited by the Accreditation Council for Continuing Medical Education (ACCME) to provide CME for physicians. Medscape, LLC designates this educational activity for a maximum of 0.5 AMA PRA Category 1 Credits™. Physicians should only claim credit commensurate with the extent of their participation in the activity. All other clinicians completing this activity will be issued a certificate of participation. To participate in this journal CME activity: (1) review the learning objectives and author disclosures; (2) study the education content; (3) take the post-test and/or complete the evaluation at http://www.medscape.com/cme/eid; (4) view/print certifi cate.

Learning Objectives
Upon completion of this activity, participants will be able to: Identify properties of cultures reported from both laboratories) were associated with a single outpatient oncology offi ce, Offi ce B. The other 3 hospitals reported that they had not identifi ed any A. xylosoxidans bloodstream infections in the past 3 months.
To identify the source of the outbreak and risk factors for infection and to implement control measures, ACDC then initiated an outbreak investigation.

Methods
The outbreak investigation focused on Offi ce B. To identify all patients associated with Offi ce B who had had a positive A. xylosoxidans culture, we requested a review of medical records and laboratory reports.

Matched Case-Control Study
A matched case-control study was performed to determine risk factors for infection. A case-patient was defi ned as a patient of Offi ce B who had a positive A. xylosoxidans blood culture from November 2001 through January 2002. Controls were defi ned as patients who had no symptoms or signs of bloodstream infection (fever, chills, rigors, myalgias, nausea, vomiting, weakness, or hypotension). For each casepatient, 5-7 controls were randomly selected and matched by the closest date of their visits to Offi ce B to the casepatient's date of visit. Variables included age, sex, underlying diagnosis, intravenous medications received, peripheral white blood cell counts, presence and type of central venous catheter (CVC), clinic visits, hospitalization dates, symptoms, and types of invasive procedures. Data were collected on standardized forms and analyzed by using Epi Info 2000 version 1.1.2 (Centers for Disease Control and Prevention [CDC], Atlanta, GA, USA); odds ratios were used to estimate risk, t tests were performed for continuous variables, and p<0.05 indicated statistical signifi cance.

Prospective Cohort Study
To identify possible A. xylosoxidans bloodstream infection, ACDC conducted prospective blood culture surveillance. On February 15, 2002, all patients with a CVC who had visited Offi ce B since November 2001 were sent a letter requesting them to have a culture performed on blood drawn through the CVC. CVCs were removed from those whose culture results were positive.

Environmental Investigation
On January 17,2002, numerous open containers (multidose 30-mL vials of heparin; 100-mL and 150-mL bottles of saline; and containers of alcohol, hydrogen peroxide, betadine, and iodine) were collected and sent to the Los Angeles County Public Health Laboratory for analysis. On February 15, 2002, environmental samples and swabs were collected for culture from work surfaces (e.g., countertops, sinks, hoods, kitchens) and from tap water and hands of healthcare workers who accessed CVCs, collected blood, prepared fl ushes, or administered chemotherapy.

Infection Control
During January and February 2002, we made several site visits to Offi ce B to observe procedures, review medical records, and interview the offi ce staff. Specifi cally, we observed procedures for preparation and administration of intravenous medications and asked offi ce staff about level of technical training, experience, and license status.

Molecular Studies
Blood isolates of A. xylosoxidans from case-patients were obtained from Hospital A's laboratory. For comparison, all A. xylosoxidans isolated from patients from Los Angeles County in the past 6 months were obtained from a large local reference laboratory. Pulsed-fi eld gel electrophoresis (PFGE) was performed at the Los Angeles County Public Health Laboratory by using standard methods (18) for Salmonella spp. with the exception that isolates were digested with XbaI and SpeI. PFGE pattern comparisons were performed visually by using criteria established by Tenover et al. (19).

Examination of CVC for Biofi lm
A CVC (PASport, a single-lumen, 6 French catheter with an under-the-skin titanium port; SIMS Deltec, Inc., St. Paul, MN, USA) was surgically removed from an asymptomatic patient identifi ed in the prospective cohort study. Aseptic methods were used. The distal lumen opening was clamped with a sterile hemostat to retain the liquid within the lumen, and the catheter was placed in a sealed, sterile container and shipped overnight to CDC in Atlanta for processing within 24 hours of collection. At CDC, the CVC was placed into a Class II Biological Safety Cabinet, and a 1-cm segment was removed with a sterile scalpel. This segment was cut lengthwise to expose the lumen, and the individual pieces were placed into 5% glutaraldehyde (Ted Pella, Redding, CA, USA) in 0.67 M cacodylate buffer, pH 6.2, and processed for scanning electron microscopy (20). Samples were examined by using a Philips XL 20 Scanning Electron Microscope (FEI Company, a subsidiary of Philips, Hillsboro, OR, USA). The remaining catheter attached to the titanium port was clamped, and the outer surface was cleaned with a 70% alcohol wipe, disinfected, and processed to recover biofi lm organisms (20). The recovered organisms were plated on trypticase soy agar containing 5% sheep blood (blood agar; Becton, Dickinson, Sparks, MD, USA). Plates were incubated for up to 72 h at 35°C and then examined. Colonies were spread onto blood agar for subculture and identifi ed by using standard clinical microbiologic methods (21). Biofi lm isolates were also characterized by PFGE (methods described above).

Results
A total of 12 patients with A. xylosoxidans bloodstream infection were found: 9 from the retrospective case-control study and 3 from the prospective study (Table). All 12 were immunocompromised. Their ages ranged from 41 to 79 years (mean 65.8 years), and 10 (83.3%) were female. Case-patients had differing underlying diagnoses and chemotherapy regimens. Casepatients had had fevers, chills, and/or rigors within minutes to days after an infusion through their CVC. Several case-patients had multiple episodes of fever and chills during and immediately after visits to Offi ce B when their CVC was accessed for blood collection, chemotherapy, or routine fl ushes. For some, these symptoms were attributed to possible side effects of chemotherapy. All casepatients had visited Offi ce B from November 12 through December 18, 2001. Case-patient 1 was hospitalized from October 12 through November 10, 2001, and had visited Offi ce B for daily collection to monitor neutropenia from November 13 through 19,2001. Patients with A. xylosoxidans bloodstream infection were treated with antimicrobial drugs and CVC removal. Available records showed case-patients were treated with piperacillin/tazobactam; 1 case-patient who was allergic to penicillin was treated with aztreonam. One patient died from underlying malignancy (end-stage pancreatic cancer).

Matched Case-Control Study
Of the 9 case-patients identifi ed, 7 who had clear onset date of bloodstream infection symptoms were selected for the case-control study. Case-patients were younger than controls (mean age 63.5 years [range 41-73 years] and mean age 73.2 years [range 35-89 years], respectively; p = 0.047). Case-patients were signifi cantly more likely to have a CVC than controls. Matched case-control analysis showed that all 7 case-patients versus 4 of 47 control patients had a CVC at the time of illness onset (p<0.0001). The 2 other case-patients not included in the case-control study also had CVCs. Patients with CVCs received heparin and saline fl ushes before and after the CVC was used for blood collection or infusions. No records documented when each of the Offi ce B nurses accessed the CVCs. Patients without CVCs who needed only blood collection for testing did not receive any fl ushes; however, those without CVCs who needed blood tests before receiving an infusion received a heparin and saline fl ush after a peripheral intravenous line was placed. Case-patients and controls did not have statistically signifi cant differences in peripheral leukocyte counts, intravenous medications administered, types of chemotherapy received, or underlying diseases.

Prospective Cohort Study
In February 2002, 29 patients with CVCs had blood collected for culture. Of the 3 (10%) who had positive cul-

Environmental Investigation
Cultures from available open solutions in the oncology offi ce, collected 6 weeks after the initial cluster of A. xylosoxidans-positive blood cultures, and environmental cultures did not grow A. xylosoxidans. A sample from a sterile saline bottle that was open in the infusion room was positive for Bacillus circulans, and a tap water sample was positive for Moraxella spp.

Infection Control Practices and Procedures
Offi ce B had 10 patient examination rooms and a separate, large, open infusion room where several patients could receive chemotherapy. The infusion room contained a hood and sink for preparation of intravenous medication. Of the 4 staff members at Offi ce B who regularly accessed CVCs; inserted peripheral intravenous catheters; collected blood; and prepared or administered chemotherapy, fl ushes, or intravenous medications, only 1 was a registered nurse who had a California state license. The 3 nonlicensed staff members were reported to have received nursing training in their native country but did not have documented formal training or education. One nurse wore artifi cial fi ngernails but had removed them before hand culture samples were collected; thus, the fi ngernails were unavailable for culture. The following breaches in infection control were noted: intravenous catheters were inserted by persons not wearing gloves; unlabeled, prefi lled syringes were stored in the hood; no documentation of hood cleaning was found; open, multidose heparin vials and saline bottles, some undated, were found throughout the facility; nonhygienic material was stored in the chemotherapy medication preparation hood; and failure to wash hands between patients was noted. No pharmacists were employed at Offi ce B. No documentation of staff training and evaluation for chemotherapy preparation or infection control competency was available.

CVC Biofi lm Studies
Scanning electron microscopy of the CVC showed a biofi lm that contained rod-shaped bacteria in association with fi brinlike material on the catheter surface (Figure 1). A pure bacterial culture recovered from the CVC lumen was identifi ed as A. xylosoxidans.

Molecular Studies
A. xylosoxidans blood culture isolates from case-patients were indistinguishable by PFGE analysis (Figure 2); in contrast, 3 A. xylosoxidans isolates from a local reference laboratory had different PFGE patterns. The isolate from the CVC biofi lm matched the A. xylosoxidans bloodstream infection outbreak strain.

Discussion
This large outbreak (N = 12) of A. xylosoxidans bloodstream infections was caused by 1 strain, which was also isolated from CVC biofi lm. Symptoms of bloodstream infection probably occurred when fl ushes detached bacteria from the CVC biofi lm. The prospective study found that 3 (10%) of 29 patients had A. xylosoxidans-positive blood cultures. Our case-control and prospective studies support the association of A. xylosoxidans bloodstream infection and CVCs, and our molecular biologic studies confi rm A. xylosoxidans biofi lm of the same outbreak strain on a CVC. A. xylosoxidans outbreaks reported to date have been associated with healthcare Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 14, No. 7, July 2008  and contamination of hospital products (1,2,5,(12)(13)(14), but none occurred in an outpatient setting.
The cause of this outbreak most likely was the use of contaminated multidose vials of heparin or saline fl ushes, leading to the formation of an A. xylosoxidans biofi lm on CVCs. Case-patient 1 had been hospitalized from late October through early November at Hospital A. During November [13][14][15][16][17][18][19]2001, this case-patient had had blood collected and her CVC line fl ushed numerous times at Offi ce B; on November 19, culture result indicated A. xylosoxidans infection, which was successfully treated. We observed multiple breaches in infection control at Offi ce B: use of unlabeled, prefi lled syringes, poor hand hygiene, and lack of glove use. The CVC of case-patient 1 may have been fl ushed by using the same syringe and needle inserted into multidose vials, causing contamination of the vials. Another possible route of contamination is through artifi cial fi ngernails. A cluster of Serratia marcescens and A. xylosoxidans bacteremia cases linked to multidose heparin vials contaminated by a nurse with artifi cial fi ngernails has been reported (22); however, the artifi cial fi ngernails from the nurse at Offi ce B were unavailable for testing. We suspect that multidose vials were contaminated with A. xylosoxidans and subsequently used on other patients from November 12 through December 18, 2001, when all case-patients had overlapping visits at Offi ce B and received CVC fl ushes. A culture from an open, supposedly sterile saline bottle grew B. circulans, which suggests possible breaches in infection control. Multidose heparin and saline vials have been reported as the cause of outbreaks of hepatitis C (23,24), S. marcescens (25), and Pseudomonas aeruginosa (26) infections.
Although the heparin and the saline vials could have been contaminated from case-patient 1 in November, casepatients who subsequently received fl ushes from these vials may not have become immediately ill with symptoms of bloodstream infection. A. xylosoxidans biofi lm could have developed on their CVCs and intermittently caused clinical illness when the CVCs were manipulated; i.e., fl ushing dislodged the biofi lm and caused symptomatic bacteremia. Although indwelling catheters are frequently colonized with biofi lm shortly after insertion (27), colonization does not necessarily lead to infection; bloodstream infection symptoms from an organism in contaminated intravenous solutions have been delayed for as long as 421 days after exposure (28). The fi nding of an asymptomatic patient with a CVC with A. xylosoxidans biofi lm supports this. A number of variables may be associated with detachment of microbial cells from a biofi lm (29), resulting in erosion or sloughing. Flushing, which could mechanically shear the biofi lm, could result in detachment of cells or aggregates that could in turn colonize the bloodstream and cause signs and symptoms of bacteremia. This phenomenon has been recently reported (28).
The case-patients in this outbreak had their CVCs removed and were treated with antimicrobial agents. The presence of A. xylosoxidans biofi lm and the mechanism of bloodstream infection after disruption by catheter fl ushing suggests that eradication of infection would require catheter removal, as reported by others (4,9). Previously, recurrent A. xylosoxidans bacteremia has been reported in those patients whose indwelling catheters were not removed (11). Formation of A. xylosoxidans biofi lm provides an explanation for the organism most commonly causing bacteremia in patients with CVCs (7,10).
The California Code of Regulations, Title 17, Section 2500 (30), requires all healthcare professionals to immediately report outbreaks of any cause; however, this outbreak was not recognized early on. The initial cluster of patients at Offi ce B had symptoms and positive blood cultures growing this uncommon organism for 6 weeks before the cases were reported to the Los Angeles County Department of Public Health. Because outpatient settings may lack surveillance systems, outbreaks may not be recognized immediately, thus potentially exposing more patients. In addition, some of the symptoms of bloodstream infection were initially attributed to side effects of chemotherapy. Because 10% of patients in our prospective cohort study had blood cultures positive for A. xylosoxidans, further studies are needed to determine whether active surveillance for patients with CVCs would help recognize infections.
Because we noted not only infection control breaches but also that unlicensed offi ce personnel were manipulating the CVCs, line fl ushes, infusions, and blood collection through CVCs, we reported the situation to the California Medical Board and the California Department of Health Services. Although no California state regulations for infection control in outpatient physician's offi ces exist, the California Department of Health Services and Los Angeles County Department of Public Health recommended that the oncology offi ce improve infection control standards; handling, storage, exposure, and access to pharmaceuticals; and improve medical record documentation. New infection control policies were established, and the offi ce subsequently hired new, properly licensed registered nurses and nurse practitioners to handle insertion of intravenous catheters and administration of intravenous medications and chemotherapy. After proper education of the oncology offi ce staff and removal of multidose vials of heparin and saline, no more A. xylosoxidans bloodstream infections were reported from Offi ce B.
Our investigation has limitations. We did not culture A. xylosoxidans from the multidose vials. The original vials, used when the outbreak began, had already been discarded and were not available for testing by the time we were notifi ed in January 2002. Our investigation was also limited by the absence of medical records indicating when nursing staff accessed the CVCs. Although the contamination of multidose vials remains suggestive, we suspect that they were the most likely source. The outbreak ended after discontinuing their use and enacting improved infection control practices. The organism could have been introduced into multidose vials by a needle or syringe used on an infected patient or by the artifi cial fi ngernails of the nurse, through gaps in infection control.
For patients who received infusion therapy at home, receipt of therapy in an outpatient clinic or physician's offi ce was an independent risk factor for bloodstream infection (31). Therefore, clinicians need to be vigilant because minor breaches in infection control can lead to large outbreaks with uncommon human pathogens, especially in patients with CVCs. Clinicians also need to ensure that appropriate infection control practices are adhered to consistently, especially in outpatient care settings, where oversight of infection control procedures may be absent. Unlike standards that exist for nursing homes or hospitals, no written standards regarding infection control in outpatient settings exist from the California Department of Health Services or the California Medical Board. However, routine monitoring and adherence to infection control could prevent outbreaks. Clinicians providing care in outpatient settings should review appropriate infection control standards and consider establishing written policies to ensure that standards are met. As healthcare delivery continues to move toward outpatient care (32), the lack of formal infection control procedures and accountability in the outpatient offi ce setting can lead to large disease outbreaks (33,34); the need for oversight in this setting should be considered.
Our investigation helps characterize the mechanisms of A. xylosoxidans bloodstream infection in immunocompromised patients with CVCs. It provides a better understanding of how biofi lm formation in a CVC can result in a clinical infectious disease process with this uncommon organism. Substantial illness and death can occur in outpatient settings that lack formal oversight. This outbreak highlights an unaddressed infection control problem in the outpatient setting for regulating agencies to further review.