Molecular Epidemiology of Invasive Aspergillosis: Lessons Learned from an OutbreakInvestigation in an Australian Hematology Unit • Author(s): Sarah E. Kidd, PhD; Li Min Ling, MBBS; Wieland Meyer, PhD; C. Orla Morrissey,PhD; Sharon C. A. Chen, PhD; Monica A. Slavin, MDSource: Infection Control and Hospital Epidemiology, Vol. 30, No. 12 (December 2009), pp.1223-1226Published by: The University of Chicago Press on behalf of The Society for Healthcare Epidemiologyof AmericaStable URL: http://www.jstor.org/stable/10.1086/648452 .

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infection control and hospital epidemiology december 2009, vol. 30, no. 12

c o n c i s e c o m m u n i c a t i o n

Molecular Epidemiology of InvasiveAspergillosis: Lessons Learnedfrom an Outbreak Investigationin an Australian Hematology Unit

Sarah E. Kidd, PhD; Li Min Ling, MBBS;Wieland Meyer, PhD; C. Orla Morrissey, PhD;Sharon C. A. Chen, PhD; Monica A. Slavin, MD

Suspected nosocomial Aspergillus fumigatus infections in an Austra-lian hematology unit were investigated by molecular typing of clin-ical and environmental isolates using polymerase chain reaction fin-gerprinting, CSP typing, and multilocus microsatellite typing. Onlymultilocus microsatellite typing revealed that all isolates were ge-netically distinct. The selection of an appropriate typing method isessential for effective outbreak investigations.

Infect Control Hosp Epidemiol 2009; 30:1223-1226

Invasive aspergillosis (IA) in immunocompromised hosts isassociated with significant morbidity and mortality. Clustersof IA are common in hospitals, and molecular typing tech-nologies are needed to rapidly and accurately track infection.Because typing methods for Aspergillus fumigatus differ intheir ability to discriminate between strains,1 it is crucial toselect appropriate method(s). Here, we describe our inves-tigation of a suspected outbreak of IA at the Peter MacCallumCancer Centre (PMCC; Melbourne, Victoria, Australia) withuse of 3 typing techniques. We review the strengths and weak-nesses of frequently used typing techniques and make rec-ommendations about their use in investigations of outbreaksof A. fumigatus infection.

ia cases

From March 19 through April 27, 2006, 3 inpatients receiveda diagnosis of proven pulmonary IA after their admission tothe PMCC up to 3 months earlier. Patients A and B wereadmitted for total body irradiation as part of the stem celltransplantation conditioning regimen, and patient C had re-ceived chemotherapy and mediastinal irradiation for relapsedlymphoma. A. fumigatus was cultured from lung tissue orsputum specimens. All patients died from IA-related respi-ratory failure. Common areas attended by these patients in-cluded the intensive care unit (ICU) and radiotherapy unit.

methods

Air sampling was undertaken in and near the ICU and ra-diotherapy units 2 weeks after diagnosis of the third IA case.

A MAS-100 air sampler (Merck) was used to collect 11 sam-ples of 1000 L per 10 minutes. Thirty swab samples werecollected from surfaces, such as window sills, curtain rails,televisions, computer monitors, wall tiles, and air vents, withuse of sterile transport swabs. Samples were sent to the De-partment of Microbiology, Box Hill Hospital (Melbourne),for further processing and morphological identification of A.fumigatus isolates.

Molecular typing of isolates was initially conducted usingpolymerase chain reaction (PCR) fingerprinting,2 by whichgenotypes were assigned according to observed differences inbanding patterns. CSP typing3,4 and multilocus microsatellitetyping (MLMT) using the STRAf protocol5 were later appliedto these isolates as part of a different study. CSP types wereassigned according to differences in tandem repeat sequencesof the CSP locus and to point mutations in the flankingregions, in line with a recently proposed CSP typing nomen-clature.4 MLMT profiles were assigned according to the num-ber of repeat units detected in each of 9 A. fumigatus–specificmicrosatellite loci.5 For 1 environmental isolate that was un-typeable by both CSP typing and MLMT, the internal tran-scribed spacer ribosomal DNA regions were sequenced usinguniversal fungal primers,6 and these sequences were comparedwith sequences submitted to the National Center for Bio-technology Information database (http://www.ncbi.nlm.nih.gov/) to determine the species.

results

Three A. fumigatus isolates recovered from the patients, 5from air samples, and 3 from surface swab samples wereavailable for study. All environmental isolates, with the ex-ception of one from an air sample, were obtained in the ICU.

PCR fingerprinting revealed identical banding patterns forthe isolates from patients A and B and for 4 environmentalisolates; the isolate from patient C had a unique bandingpattern (Table 1). CSP typing, however, indicated that patientsA and B had genetically distinct strains, and the isolates frompatients B and C shared the same CSP type. The isolate frompatient A shared a CSP type with 3 environmental isolates,and the isolates from patients B and C isolates shared a CSPtype with 1 environmental isolate. MLMT distinguished allclinical and environmental isolates as genetically distinctstrains (Table 1).

CSP typing and MLMT failed to provide a result for en-vironmental isolate 06.587, although PCR fingerprintingyielded a unique banding pattern for this isolate. Internaltranscribed spacer sequencing indicated that 06.587 had beenmisidentified and was actually Aspergillus terreus; therefore,it was irrelevant to this outbreak investigation.

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molecular typing of an outbreak of a. fumigatus infection 1225

table 2. Strengths and Weaknesses of Commonly Used Techniques for Aspergillus fumigatus Strain Typing

TechniqueDiscriminatory

power ReproducibilityEase of

performance Ease of interpretationA. fumigatus–

specific protocolAmenability to

data sharing Reference(s)

Probe-based fingerprintingAfut1-RFLP Moderate Moderate Good Difficult, subjective Yes Poor 1

PCR-based fingerprintingPCR fingerprinting Moderate Poor Good Moderate, subjective No Poor 2

RAPD Moderate Poor Good Difficult, subjective No Poor 1, 2, 7

AFLP High Moderate Moderate Difficult, subjective No Poor 1, 7

Specific locus typingCSP typing Moderate Good Good Easy, objective Yes Good 3, 4

MLST Poora Good Moderate Easy, objective Yes Good 1, 7, 8

MLMT High Good Good Easy, objective Yes Good 1, 5, 9, 10

note. Adapted from de Valk et al.1 AFLP, amplified fragment length polymorphism; MLMT, multilocus microsatellite typing; MLST, multilocussequence typing; PCR, polymerase chain reaction; RAPD, random amplification of polymorphic DNA; RFLP, restriction fragment lengthpolymorphism.a MLST has been demonstrated to be highly discriminatory in many fungal species, but the only scheme developed for A. fumigatus to date hasvery poor discriminatory power.8

discussion

Nosocomial IA is associated with significant morbidity amongpatients, and this has important implications for infectioncontrol practice. In this study, the initial results showed thatisolates from patients A and B and several environmentalsamples shared identical PCR fingerprint profiles, suggestingthat nosocomial infection was likely. In response, the ICUwas closed and thoroughly cleaned, faulty structures (ie, airvents, cracked ceiling tiles, and light fittings) were replaced,and more-stringent infection control measures were imple-mented. There have been no IA cases related to the ICU atPMCC since the outbreak.

In contrast to the initial findings, the subsequent analysisusing MLMT indicated no evidence for common sources ofinfection. However, despite all isolates eventually being foundto be genetically distinct, it is possible that the ICU was thesource of one or more infections and that environmentalsampling was insufficient to detect all strains in the vicinity.Up to 3 months had elapsed between the patients’ admissionto PMCC and the environmental sampling. Therefore, it isalso possible that the collected environmental isolates werenot representative of those that were in the patients’ sur-roundings at the time of acquisition, if the infections werehospital acquired. With the expanding spectrum of moleculartechniques in diagnostics and infection control, it is impor-tant for clinicians to understand their use and clinical rele-vance. This is particularly the case for A. fumigatus, for whichthere is no gold standard typing technique. This study illus-trates the significant variation in discriminatory power be-tween techniques, and although not directly addressed here,techniques also differ in their reproducibility, ease of perfor-mance and interpretation, and amenability to data set com-parison. The strengths and weaknesses of different techniquesmust be considered before their implementation in an infectioncontrol program. Table 2 summarizes some of the moleculartyping techniques commonly applied to A. fumigatus.

Molecular typing techniques may be broadly classified as

probe-based fingerprinting, PCR-based fingerprinting, or spe-cific locus typing methods. Probe- or PCR-based fingerprintingtechniques are simple to implement and are relatively cost andlabor efficient.1,2,7 The primers and enzymes used in these meth-ods are often not species specific and can be applied to anyfungal species, without developing and validating new proto-cols. However, the discriminatory power of these techniquesvaries between species. In our experience, PCR fingerprinting,which has successfully been used to distinguish strains withinmany fungal species,2 did not distinguish between all strainsof A. fumigatus in this investigation, leading initially to thelikely erroneous conclusion that this outbreak was nosocomial.Furthermore, because the protocols are not species specific, itis not possible to discern that a culture has been misidentifiedusing fingerprinting methods alone. Subjective interpretationof bands of varying intensity may also lead to problems ofreproducibility.

Multilocus sequence typing, the preferred technique fordistinguishing strains of many fungal species because of itssensitivity, reproducibility, and amenability to data sharing,1,7

was not applied to isolates in the present study, because at-tempts to define a useful multilocus sequence typing schemefor A. fumigatus have thus far failed to achieve sufficientdiscriminatory power.8 The single-locus CSP typing methodshares many of the advantages of multilocus sequence typingand has been recommended for use as a first-line typingmethod for A. fumigatus isolates.3 However, it did not dis-tinguish between all strains in the present study.

The recently developed MLMT STRAf protocol is rapidlybecoming the preferred technique for molecular typing of A.fumigatus because of its extremely high discriminatory powerand reproducibility.1,5,9 With the increasing availability of com-mercial genotyping services, MLMT can be conducted withoutthe need for specialized laboratory equipment and at relativelylow cost. Furthermore, with use of allelic ladders, this methodcan be standardized for interlaboratory or longitudinal datacomparisons (Table 2).10 In light of ours and others’ experience

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1226 infection control and hospital epidemiology december 2009, vol. 30, no. 12

with this method for genotyping A. fumigatus case clusters,9

we recommend its use for investigations of suspected outbreaksof A. fumigatus infection wherever resources permit.

The application of any molecular typing method in theclinical setting requires knowledge of the intrinsic propertiesof the organism and of the resources available. In addition,if additional outbreaks occur, consideration should be givento the capacity of any technique for long-term data collectionand comparison.

acknowledgments

We thank Susan Harper and Steve Burden, for their assistance with envi-ronmental sampling; the Department of Microbiology, Box Hill Hospital,Melbourne, for identification of clinical and environmental isolates; KrystynaMaszewska, for polymerase chain reaction fingerprinting of isolates; andKarin Thursky, for reviewing the manuscript.

Potential conflicts of interest. All authors report no conflicts of interestrelevant to this article.

From the Department of Medicine, Monash University (S.E.K., C.O.M.),Infectious Diseases Unit, Alfred Hospital (S.E.K., L.M.L., C.O.M., M.A.S.),MacFarlane Burnet Institute (S.E.K., C.O.M.), Infectious Diseases Unit, PeterMacCallum Cancer Centre (L.M.L., M.A.S.), and Centre for Clinical ResearchExcellence in Infectious Diseases, Victorian Infectious Diseases Service(M.A.S.), Melbourne, Victoria, and Centre for Infectious Diseases and Mi-crobiology, Westmead Hospital (W.M., S.C.A.C.), and Department of Med-icine, University of Sydney Western Clinical School, Westmead MillenniumInstitute (W.M., S.C.A.C.), Sydney, New South Wales, Australia; and Infec-tious Diseases Department, Tan Tock Seng Hospital, Singapore (L.M.L.)

Address reprint requests to Assoc. Prof. Monica Slavin, Locked Bag 1,A’Beckett Street, Melbourne, VIC, 8006, Australia (monica.slavin@petermac.org).

Received May 20, 2009; accepted July 22, 2009; electronically published Oc-tober 22, 2009.

� 2009 by The Society for Healthcare Epidemiology of America. All rightsreserved. 0899-823X/2009/3012-0014$15.00. DOI: 10.1086/648452

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