Detection of Epidemiologically Significant Organisms from Surveillance Specimensusing Pooled eSwabs Compared to Swabs in Amies Transport Medium

B.M. WILLEY, P. GNANASUNTHARAM, G. NGUYEN, B. BORGUNDVAAG, W. LEUNG, M. LOFTUS, R. THANABALAN, V. PORTER, G. SMALL

I. EDWARDS, P. LO, K. WONG, Y. WEI, G. MAHLI, A.J. MCGEER, S.M. POUTANENUniversity Health Network/Mount Sinai Hospital, University of Toronto, Toronto, Ontario

Results cont’dMethicillin-Resistant Staphylococcus aureus (MRSA)

Of the 475 patients who consented to parallel study specimens, MRSA were identified in 7 (1.5%; Table

2). Of these, 5 (1.1%) isolates were detected from nasal ATM, 4 (0.8%) from rectal ATM, with overall 6

(1.3%) cases being identified when the results from the paired nasal/rectal ATM swabs from the same

patient were combined. Similarly, MRSA were identified from 6 (1.3%) pooled eSwab specimens. When

combining culture data from both swab types there were a total of 7 MRSA cases identified.

Growth of non-S. aureus on MSSA and MRSA selective agars

The amount of breakthrough other than MSSA or MRSA growing from ATM rectal and nasal swabs was

compared to that growing from pooled eSwabs. This was done to assess the difference in bacterial load

and its potential effect on the recovery of ESO (Table 4).

Conclusions and DiscussionFor staphylococci and R-GNB, WASP-planted pooled eSwabs, containing nasal and rectal samples,

identified a statistically equivalent number of patients with ESO as did nasal and rectal specimens

collected and planted separately using mattress ATM swabs. Due to the lack of positives in the patient

population screened, this study was unable to determine whether this equivalence held true for VRE.

Although overgrowth of ESO by breakthrough commensal flora is possible due to pooling rectal with

nasal and the higher sample volume being tested from eSwabs (30uL versus direct rolling of the ATM

swab surface), the data obtained in this study was not conclusive in this regard.

In conclusion, the data from this study indicates that the ESO detection rate will not be negatively

affected by switching from separately obtained mattress swabs in ATM to pooled rectal and nasal

eSwabs planted by the WASP automated system.

AbstractObjectives: A pilot study comparing 254 single eSwabs to mattress swabs in Amies Transport Medium (ATM) for detection of methicillin-resistant and methicillin–susceptible S. aureus (MRSA, MSSA) from nares and axilla indicated that yield from eSwabs fell in between ATM with direct plating and ATM broth enrichment culture. This study compared

the yield from pooled nasal (NAS) and rectal (REC) eSwabs (PeS) to individual swabs in ATM.

Methods: From Aug-Dec 2010, with ethics approval and informed consent, single NAS and REC swabs in ATM were

collected in parallel to PeS from Emergency Room and Inflammatory Bowel Disease Clinic patients. The ATM swabs were plated manually as per laboratory protocol to selective agars for MRSA, MSSA, resistant (R) Gram-negative bacilli (R-GNB; defined as acquired 3rd generation cephalosporin-R plus fluoroquinolones and/or aminoglycoside resistance) and

vancomycin-resistant enterococci (VRE). The automated WASP instrument (Copan) plated 30uL of liquid from each PeS to the same four media. Incubation and identification methods were standardized.

Results: In total, 475 patients consented to parallel specimens. MRSA were identified from 7 patients, of which 5 (1.1%) were detected from NAS-ATM, 4 (0.8%) from REC-ATM, 6 (1.3%) from either NAS-ATM or REC-ATM, and 6 (1.3%) from PeS. Due to the low MRSA prevalence among this population, comparison of swab types was performed using MSSA

detection as a proxy. MSSA was identified from 102 (21.5%) NAS-ATM, 37 (7.9%) REC-ATM, 112 (23.6%) from either NAS-ATM or REC-ATM, while 107 (22.5%) were detected by the PeS, from a total 118 MSSA cases. The difference in overall number of MSSA patients detected between the combined ATM and the PeS was not statistically significant (107 PeS

MSSA+ versus 112 ATM MSSA+: Fisher’s exact test/two tailed P value=0.7079). Similarly, there was no statistical difference between detection for R-GNB by the 2 swab systems: REC-ATM and PeS both detected 49 R-GNB, respectively, from 51 positive patients. Of the 51 patients with R-GNB (1 C. freundii, 42 E. coli, 4 K. pneumoniae, 4 P. mirabilis), 47 were identified from both REC-ATM and PeS, while E. coli from 4 remaining patients were identified from

REC-ATM (2 isolates) or PeS (2 isolates) only. No VRE were identified from any swab.

Conclusions: This study found the WASP planted PeS (nasal plus rectal) to have an equivalent yield of ESO compared to NAS-ATM and REC-ATM planted separately.

IntroductionDue to escalating workloads associated with surveillance screening for epidemiologically significant

organisms (ESO) coupled with the fiscal constraints faced by most clinical microbiology laboratories,

there has been pressure to automate specimen management. Furthermore, cost cutting has led many

institutions to pooling surveillance swabs in order to minimize costs, albeit no study comparing

recovery of ESO from single versus pooled specimens has been done to support this practice.

To meet these needs, various automated specimen plating and streaking instruments have been

devised enabling staff to be freed up for other tasks. However, implementation of systems such as the

WASP (Copan Diagnostics) require that laboratories switch from mattress swabs in semi-solid Amies

Transport Medium (ATM) to flocked eSwabs comprising nylon fiber tips transported in liquid Amies in

standardized screw-capped tubes.

While previous studies have shown flocked swabs to be comparable to mattress swabs for most

common organisms, prospective studies specifically investigating their efficacy in the recovery of

MRO from surveillance swabs have not been performed. A proof-of-principle pilot study performed in

Summer 2010 in a high-prevalence population, prospectively compared 254 single eSwabs collected in

parallel to single mattress ATM swabs (StarSwab II, StarPlex Scientific) from nares and axillae to

determine the eSwab efficacy for detecting methicillin-susceptible and -resistant Staphylococcus

aureus (MSSA, MRSA). The results from this pilot sampling indicated that the yield of MRSA and

MSSA from eSwabs fell in between ATM with direct plating and ATM with broth enrichment culture

(data not shown).

Since reliable recovery of ESO is integral to patient safety and infection control, this study aimed to

compare the yield from pooled flocked swabs processed automatically to that of single mattress ATM

swabs processed manually for recovery of ESO.

Materials and MethodsSource and type of specimensWith Mount Sinai Hospital and University of Toronto Ethics Review Boards’ approval and informed

consent from patients in the Emergency Room and Inflammatory Bowel Disease Clinics, single nasal

and single rectal swabs were collected using mattress ATM swabs (StarSwab II, StarPlex) in parallel to

pooled nasal and rectal eSwabs placed into the single standard tube containing 1 mL modified liquid

Amies (Copan Dual MRSA ESWAB KIT, see Figure 1 for inoculation sequence).

ECCMID 2011

Figure 1. Using the Copan Dual eSwab kit for ESO screening: 1) after swabbing the rectum

(pink) the flocked swab is inoculated into the liquid Amies medium and discarded; after

swabbing the nares (white) the flocked swab is placed into the tube, broken at the notch, and

the lid is screwed tightly onto the tube to ensure no leakage. The pooled eSwab is labelled and

sent to the laboratory for processing on the WASP instrument.

Figure 2. On laboratory receipt of the inoculated

Copan dual eSwabs, the WASP instrument

automatically labels appropriate media as ordered by

test information embedded into the patient barcode

on the tube. The WASP unscrews the lid, removes

the swab, plates a 30uL loop-full of liquid modified

Amies medium containing the eluted material from

the pooled sample to each medium, and streaks the

inoculum to obtain single colonies. In this study, the

WASP sequentially plated eluted samples to 4

selective agars for isolating MSSA, MRSA, R-GNB

and VRE, respectively.

Table 1: Description of surveillance swabs from 475 patients and plating order

9504751425Total specimens

MSSA>MRSA>ESBL>VRE373Rectal

MSSA>MRSA373373

NasalInflammatory Bowel Disease

Clinic

MSSA>MRSA>ESBL>VRE102Rectal

MSSA>MRSA102102

NasalEmergency Department

Plating order indicated by

organism type

Mattress

ATM swabs

Pooled

eSwab

Swab

site

Source of surveillance swabs

Materials and Methods cont’dSpecimen processingThe ATM swabs were plated manually to Oxoid Chromogenic Staphylococcus agar for MSSA, Oxoid Denim Blue agar

for MRSA, Oxoid ESBL agar for resistant R-GNB; defined as acquired 3rd generation cephalosporin-R plus

fluoroquinolone or aminoglycoside R), and Difco mEnterococcus Vancomycin agar prepared in-house for

vancomycin-resistant enterococci (VRE).

Manual plating entailed the all sides of each swab tip being smeared over a circular area ~3cm in diameter on each

plate surface. Selective agars were inoculated in the precise order shown in Table 1 above. This was followed by

automated streaking of each plate on the IsoPlater Instrument.

The pooled eSwabs were placed into the feed tray on the automated WASP instrument for plating (Figure 2.). A total

of 30uL of liquid from each eSwab was inoculated to the same media in sequence. The inoculation order of the WASP

was irrelevant as the loop of the WASP was sterilized between each plate inoculation, and before and after each

streaking step.

Incubation of streaked plates in ambient conditions at 37oC from both study arms was simultaneous and methods

used to work-up positive cultures from all media were standardized and in accordance with laboratory protocols.

ResultsOverall numbers of ESO detected from the 1425 specimens screened in this study are summarized in Table 2.

Table 2: Summary of ESO results obtained 475 patients using the two different swab types (No.)

0/950*98/950*16/1425246/14251425Total ESO-positive swabs /Total swabs tested

*Nasal swabs were not processed for R-GNB or VRE

0517118475

Total ESO patient cases identified from all swab types

combined (i.e. Total patient cases)

**6112475

Total MSSA/MRSA patient cases identified from either

Nasal or Rectal Mattress ATM swabs

**5102475

Nasal samples collected via the StarPlex II Mattress

ATM swabs

049437475

Rectal samples collected via the StarPlex II Mattress

ATM swabs

0496107475

Pooled nasal/rectal samples collected via the Copan

Dual eSwab Kit

VRE+R-GNB+MRSA+MSSA+Total no.Swab type

Resistant Gram-negative bacilli (R-GNB)Table 5 describes the R-GNB diversity and the discrepancies between the rectal ATM and the pooled

eSwabs identified from the 475 patient screens. The same 49 distinct R-GNB were isolated from the

parallel specimens obtained from 47 patients, while 4 other R-GNB were detected by one swab type

only: 2 from ATM and 2 from pooled eSwabs, resulting in 51 (10.7%) patients with R-GNB overall.

Similar to the staphylococcal screens, no statistical difference was found in detection capabilities of the

2 swab types for R-GNB screens, but what was notable was the quantity of growth between the 2 swab

types. Yield was equivalent in 26/51 positives, 1+ greater from eSwabs from 14 patients, 2+ more from

eSwabs for 5 patients, while ATM only producing 1+ more growth in 4 and 2+ heavier growth from 1

specimen. In all 4 discrepant specimens, growth quantity for the positive was less than 1+.

Table 3: Comparison of growth quantification of MRSA identified from both swab types in the study

5 colony+++6

*The nasal and rectal swabs taken using the mattress ATM from a single case were automatically planted using broth enrichment as the patient was a known MRSA carrier. The direct plating of the mattress and eSwabs from this patient failed to grow MRSA, however, the enriched mattress nasal swab was positive.

1 colony+/-+++7

+++0++5

+1 colony+/-4

0++/-3

0++++++2

00; +++ broth enrichment*01

Amies Rectal SwabsAmies Nasal SwabsPooled eSwabsMRSA No.

39 (4.1)615 (64.7)950Either Mattress ATM swabs

6 (1.3)270 (56.8)475Nasal Mattress ATM swabs

Table 4: Summary of breakthrough of non-S. aureus* from ATM versus pooled eSwabs (No. %).

*Includes colonies of colours other than Denim blue (both media) that may/ may not require work and may/may not overgrow ESO

33 (7)345 (72.6)475Rectal Mattress ATM swabs

75 (15.8)352 (74.1)475Pooled rectal and nasal eSwabs

Growth of other organisms

on MRSA Selective agar

Growth of other organisms

on MSSA Selective agar

Total

no.Swab type

Study period and number of parallel specimensSpecimens were collected from August through December 2010 from 475 Emergency and Inflammatory

Bowel Diseases Clinic patients. These included 475 pooled eSwabs containing rectal and nasal

samples, and 475 each ATM swabs from rectal and nasal sites, for an overall 1425 specimens (Table 1).

Methicillin-Susceptible Staphylococcus aureus (MSSA)

Due to the low MRSA prevalence among this predominantly out-patient population, comparison of swab types was

performed using MSSA detection as a proxy for MRSA detection.

Overall MSSA were identified from a total of 118 patients (Table 2). Of these, 102 (21.5%) were detected from the

nasal ATM swab, 37 (7.9%) from the rectal ATM swab, with overall 112 (23.6%) being identified from either nasal or

rectal ATM swabs, while MSSA were detected from 107 (22.5%) of the pooled eSwab specimens.

The difference in overall number of MSSA patients detected by the paired mattress ATM swabs compared to the

pooled eSwabs was not statistically significant: 107 pooled eSwabs were MSSA-positive compared to 112 instances

where at least one of two paired mattress ATM swabs were MSSA-positive (Fisher’s exact test/two tailed P

value=0.7079).

Vancomycin-Resistant Enterococci (VRE)No VRE were identified from any swab processed in the study. This outcome is not surprising since VRE

are typically nosocomial and the population screened was predominantly outpatient in nature.

3---+-

Table 5: Species and resistance profiles associated with R-GNB isolated from the 2 swab types

•Class C (ampC) ββββ-lactamase resulting in resistance to oxyimino-cephalosporins; **Inducible class C ββββ-lactamase inherent in C. freundii, but DHA-1-mediated in K. pneumoniae

***CIP-R, ciprofloxacin-resistant; **** GM-R, gentamicin-resistant

1++-+P. mirabilis

1--+-

1-+-+-

1++--+

1-+--+K. pneumoniae

1-+---

2+--+-

14-+-+-

111---+-

2++-++

4---++

3++--+

1+---+

117-+--+

5----+E. coli

1-++--C. freundii

Pooled

eSwab +

only

Mattress

ATM Swab +

only

Both ATM

and Pooled

eSwabs +GM-R****CIP-R***

Inducible

Class C**

Class C

(ampC)*

Class A

ESBL

Species of R-

GNB isolated

Acknowledgements: The StarSwab-II ATM swabs for this and the pilot study were kindly donated by Susan Finn, StarPlex Scientific; the media used for this and the pilot study other than for the eSwab/WASP arm were generously donated by Brian Kemp, Oxoid; Material for the WASP/eSwabs were supplied by Copan. And definitely not forgotten: our gratitude to all the MSH technicians who eagerly set up eSwabs on

the WASP, and to the MSH ER staff who took the time to obtain informed consent and take extra specimens even while rushed off their feet!