Use of multilocus variable number of tandem repeats analysis genotyping to determine the role of asymptomatic carriers in Clostridium difficile transmission

Abstract

Background: Previous studies have suggested that asymptomatic carriers of toxigenic Clostridium difficile are a source of hospital-associated (HA) infections. Multilocus variable number of tandem repeats analysis (MLVA) is a highly discriminatory molecular subtyping tool that helps to determine possible transmission sources.

Methods: Clostridium difficile isolates were recovered from perirectal swabs collected for vancomycin-resistant Enterococcus (VRE) surveillance as well as from clinical C. difficile toxin-positive stool samples from July to November 2009 at the University of Pittsburgh Medical Center Presbyterian (UPMC). MLVA was performed to determine the genetic relationships between isolates from asymptomatic carriers and patients with HA C. difficile infection (HA-CDI). Asymptomatic carriage and HA-CDI isolates were considered to be associated if the carriage isolate was collected before the HA-CDI isolate and if the MLVA genotypes had a summed tandem-repeat difference of ≤ 2.

Results: Of 3006 patients screened, 314 (10.4%) were positive for toxigenic C. difficile, of whom 226 (7.5%) were detected only by VRE surveillance cultures. Of 56 incident cases of CDI classified as HA at UPMC during the study with available isolates, 17 (30%) cases were associated with CDI patients, whereas 16 (29%) cases were associated with carriers. Transmission events from prior bed occupants with CDI (n = 2) or carriers (n = 2) were identified in 4 of 56 cases.

Conclusions: In our hospital with an established infection control program designed to contain transmission from symptomatic CDI patients, asymptomatic carriers appear to have played an important role in transmission. Identification and isolation of carriers may be necessary to further reduce transmission of C. difficile in such settings.

Keywords: Clostridium difficile; MLVA; genotyping; screening; transmission.

Figure 1.

Minimum spanning tree depicting the relationships between 739 isolates in 524 genotypes by multilocus variable number of tandem repeats analysis (MLVA) recovered at the University of Pittsburgh Medical Center (UPMC) July–November 2009. Duplicate isolates from the same patient are included. Single circles represent 1 MLVA genotype; double circles represent complexes of highly related isolates by MLVA (summed tandem-repeat distances of ≤2), each labeled by capital letter designations conforming to those in Table 1. The numbers within each complex/genotype represent the tcdC genotype. Circle/complex size is proportional to the number of isolates included. Complexes labeled by capital letters contain cases of Clostridium difficile infection (CDI) acquired at UPMC according to epidemiologic criteria occurring within the screening period +10 days; complexes with hospital-associated (HA) UPMC case isolates only from the 30 days before and after this period remain unlabeled. Complexes and genotypes are color-coded according to epidemiologic classifications: red = HA-CDI at UPMC; yellow = colonization detected by toxin testing; blue = C. difficile colonization detected on screening tests; white = environmental isolates; green = all other C. difficile infections detected on toxin testing (recurrent CDI, community-acquired CDI, and HA-CDI not acquired at UPMC).

Figure 2.

Minimum spanning tree depicting the detailed relationships between multilocus variable number of tandem repeats analysis (MLVA) genotypes in complexes M and N (Figure 1). Each circle represents 1 MLVA genotype scaled in size to the number of isolates comprising it. Numbers between genotypes indicate the summed tandem-repeat difference between them. The color coding of MLVA genotypes conforms to that in Figure 1. Numbers within genotypes represent the study day(s) on which each isolate within the genotype was collected. Isolates with capital letters (A–F) indicate isolates from patients with HA C. difficile infection (HA-CDI) listed in Table 1; the source isolates for these cases based on epidemiological analysis are labeled by corresponding lower-case letters (a–f). One isolate labeled e/E is both a hospital-associated CDI case isolate as well as a source for a subsequent case. The MLVA genotype indicated by the arrow shows an example of a C. difficile genotype recovered by screening tests only that was essential to the formation of a complex.