Cepacia and TX Article Part 1

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Journal of Clinical Microbiology, April 2002, p. 1188-1193, Vol. 40, No.

4 Copyright © 2002, American Society for Microbiology. All Rights

Reserved.

Six-Year Molecular Analysis of Burkholderia cepacia Complex Isolates

among Cystic Fibrosis Patients at a Referral Center for Lung

Transplantation

G. Heath,1, Kathy Hohneker,2 Charlene Carriker,3 ,1

Routh,1 J. LiPuma,4 M. Aris,2 Weber,2,3,5 and

H. Gilligan1,6*  Clinical Microbiology-Immunology Laboratories,1

Department of Hospital

Epidemiology, University of North Carolina Hospitals,3

Microbiology-Immunology,6 Medicine,2 Pediatrics, The University of North

Carolina at Chapel Hill, Chapel Hill, North Carolina,5 Department of

Pediatrics and Communicable Diseases, The University of Michigan, Ann

Arbor, Michigan4  Received 28 June 2001/ Returned for modification 26

October 2001/

Accepted 24 December 2001

Over a 6-year period, Burkholderia cepacia complex species were isolated

from cystic fibrosis (CF) patients receiving care at The University of

North Carolina Hospitals (clinic CF patients) and from those referred

from other treatment centers. Fifty-six isolates collected from 30

referred patients and 26 clinic CF patients were characterized by

pulsed-field gel electrophoresis (PFGE) and were assayed by PCR to detect

the cable pilin gene, cblA. PFGE results indicated that six separate

clusters (clusters A to F) were present among the 56 isolates and that

three clusters (clusters A, B, and E) consisted only of isolates from

referred patients infected with B. cepacia complex isolates prior to

referral. However, one cluster (cluster C) consisted of isolates from

four CF patients, and hospital records indicate that this cluster began

with an isolate that came from a referred patient and that spread to

three clinic CF patients. Cluster D consisted of two isolates from clinic

CF patients, and hospitalization records are consistent with nosocomial,

patient-to-patient spread. cblA was present in only 4 of the 56 isolates

and included isolates in cluster E from the referred patients. Our

results indicate a lack of spread of a previously characterized,

transmissible clone from referred patients to our clinic CF population.

Only two instances of nosocomial, patient-to-patient spread could be

documented over the 6-year period. An additional spread of an isolate

(cluster F) from a referred patient to a clinic patient could not be

documented as nosocomial and may have been the result of spread in a

nonhospitalized setting. The majority (36 of 56) of our B. cepacia

complex-infected CF patients harbor isolates with unique genotypes,

indicating that a diversity of sources account for infection. These data

suggest that CF patients infected with B. cepacia complex and referred

for lung transplantation evaluation were not a major source of B. cepacia

complex strains that infected our resident CF clinic population.

The Burkholderia cepacia complex is composed of at least seven closely

related species or genomovars consisting of B. cepacia (genomovar I), B.

multivorans (genomovar II), B. stabilis (genomovar IV), B. vietnamiensis

(genomovar V), and B. ambifaria (genomovar VII), and genomovars III and

VI, with species designations for genomovars III and VI still pending (4,

8).

Nearly 4% of cystic fibrosis (CF) patients in the United States are

infected with a member of the B. cepacia complex (13). Of these patients,

some will develop the cepacia syndrome, a rapidly fatal necrotizing

pneumonia with bacteremia (9, 10, 26). Previous studies have shown that

transmissible clones of B. cepacia exist, and subsequent infection with

these clones of both healthy uninfected CF patients and CF patients

seeking lung transplantation can be devastating (9, 11, 12, 22, 25).

Therefore, screening of CF patients for the detection of B. cepacia

complex and management of patients once infection is documented are

extremely important. Furthermore, CF patients infected with B. cepacia

complex isolates are stigmatized, being segregated from the general CF

patient population in terms of clinical care and social interaction. In

many North American transplant centers, infection with B. cepacia complex

is a strict contraindication for lung transplantation in CF patients

(14).  There are more than 120 lung transplantation centers in North

America.  The University of North Carolina (UNC) Hospitals is one of the

few CF centers in North America that will perform lung transplantation

for CF patients infected with B. cepacia complex. For this reason, about

14% of adult CF patients (i.e., roughly three times the national

percentage) referred to the UNC Hospitals for double lung transplantation

are infected with B. cepacia complex.  Some transmissible clones of B.

cepacia complex exist, such as the cable

pilin-positive (cblA+) electropherotype (ET) ET 12 clone responsible for

epidemic transmission in both Canada and the United Kingdom (7, 9, 11,

21, 22, 25). Therefore, we believed that it was important to study the CF

patient population at the UNC Hospitals by asking three questions. First,

have referred patients brought a previously characterized, transmissible

clone into our (the UNC Hospitals) center, and has it been transmitted to

our clinic CF patient population? Second, are any new, previously

uncharacterized, transmissible clones evident among our referred

patients, and has transmission from referred patients to our local clinic

CF population occurred? Third, how much intracenter spread of B. cepacia

complex has occurred?

Characterization of UNC Hospitals CF center and infection control. All B.

cepacia complex isolates were recovered from CF patients between 1

January 1995 and 1 March 2001. We studied two distinct patient

populations from which B. cepacia complex organisms were isolated. One

consisted of 30 CF patients who were referred to the UNC Hospitals, often

for lung transplantation evaluation, already infected with B. cepacia

complex. The second population was of 26 CF patients who received their

routine care at the UNC Hospitals and who became newly infected during a

study period from 1 January 1997 to March 2001. For these patients to be

considered " newly infected, " prior cultures of samples from these

patients at our institution had to be negative for B. cepacia and the

organism had to be isolated during the study period.  For the year 2000,

451 CF patients received care at the UNC Hospitals, with 251 of these

patients being pediatric patients, while 200 were adult patients.

Nineteen (9.5%) of the 200 adult patients and 8 (3.2%) of the 251

pediatric patients were infected with B. cepacia complex. A total of 107

CF patients are awaiting lung transplantation at the UNC Hospitals, with

15 (14%) of these patients infected with B. cepacia complex. As part

of the evaluation of possible nosocomial transmission among CF patients

whose isolates were in clusters C, D, and F, all patient charts were

reviewed by a nurse trained in infection control. The review included

production of a time line of all hospital admissions including hospital

location, clinic visits, physical and occupational therapy visits,

pulmonary rehabilitation visits, and evaluations in specialty clinics

(e.g., pulmonary function laboratory, radiology, and transplantation

clinics). Patients with known B. cepacia complex colonization requiring

inpatient hospitalization were admitted to private rooms and placed on

contact precautions. When pediatric patients with B. cepacia complex were

seen in the outpatient clinic, they either were scheduled to be seen on a

different day than other CF patients or were scheduled to be seen at the

end of the day, after all patients not infected or colonized with B.

cepacia had left the facility. Until 1998, adult CF patients were seen in

the general pulmonary clinics in order to minimize contact with each

other. In 1998, the formation of a dedicated adult CF clinic prompted the

institution of several infection control measures, including patient

education, masking of all patients upon arrival at the clinic and in all

common areas, strict adherence to hand-washing procedures, and

disinfection of rooms between patients. The UNC Hospitals Lung Transplant

Clinic adopted similar infection control guidelines in 2000.  B. cepacia

isolation, PFGE, and cblA analysis. All strains were isolated

on either Pseudomonas cepacia agar or B. cepacia selective agar prepared

in-house and were further characterized biochemically and by PCR

genomovar analysis as described previously (6, 16, 19). Once a member of

the B. cepacia complex was isolated from a CF patient, a subculture was

prepared from a single colony and stocks were prepared in skim milk and

frozen at -70°C.  Pulsed-field gel electrophoresis (PFGE) was conducted

as described previously, with the following revisions (7). All cultures

were grown

overnight in tryptic soy broth and adjusted to an optical density at 600

nm of 1. One milliliter was removed and centrifuged for 5 min to pellet

the cells. The cells were then resuspended in formalin (3.8% [vol/vol])

(5) and allowed to sit on ice for 1 h. The cells were pelleted and rinsed

three times in TE (10 mM Tris-HCl, 10 mM EDTA [pH 8.0]) before being

placed in agarose blocks. Genomic DNA was restricted with SpeI (New

England Biolabs); and electrophoresis was conducted for 24 h in 0.5x TBE

(Tris-borate, EDTA), with initial and final pulse times of 1.2 and 54 s,

respectively, by using a CHEF Mapper system (Bio-Rad). An ET 12 strain of

B. cepacia complex was kindly provided by R. Goldstein, Boston University

School of Medicine (25). Gels were analyzed visually according to the

criteria of Tenover et al. (27).   All isolates of B. cepacia complex

were tested by PCR for the presence of the cable pilin gene, cblA, as

described previously (22).

Fifty-six B. cepacia complex isolates cultured from 56 CF patients were

characterized for their PFGE patterns (genotypes) after digestion with

SpeI (Table 1). Genotypes were considered related if they differed by

three or fewer bands (27). In total, 42 distinct genotypes were present,

which likely indicates a wide diversity in the sources of infection.

There were 26 genomovar II (B.  ultivorans) isolates, 25 genomovar III

isolates, two genomovar I (B. cepacia) isolates, and three genomovar V

(B. vietnamiensis) isolates. Our PFGE analysis revealed that six clusters

of PFGE patterns were present within the CF population at the UNC

Hospitals during this study period (Fig. 1). Three clusters consisted of

strains from multiple patients (clusters A to C), while clusters D to F

consisted of isolates from only two patients each.

Becki

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