Hoffbrand, AV

Kibbler, CC

McHugh, TD

McLaughlin, J

Paterson, PJ

Seaton, S

Yeghen, T

English

PubMed

UCL Discovery

Molecular confirmation ofinvasive infection caused byChaetomium globosumIdentification of the causative organism ininvasive fungal infections is difficult.Accurate and reliable diagnostic methodsare required in light of the increasingincidence of emerging fungal infections.1We recently described a method for speciat-ing fungi in formalin fixed, paraffin waxembedded tissue sections.2 DNA is extractedusing a modification of the protocol for theTaKaRa DEXPATTM kit (TaKaRaBiomedicals), with additional steps usinglyticase and ethanol precipitation.2Amplification of the DNA is performed usingpanfungal primers and a method based onthat described by Einsele et al.3 Identificationto species level can be achieved by Southernhybridisation with a probe that bindsAspergillus fumigatus, A flavus, and A versicolor.Polymerase chain reaction products that donot hybridise with this probe are subse-quently identified by sequence analysis.2We have used this method to confirm thediagnosis in the case of a patient with acutemyeloid leukaemia and pneumonia caused byChaetomium globosum. We previously reportedthe details of this case and discussed theassociated diagnostic difficulties.4 A com-puted tomography chest scan revealed cavi-tating lesions characteristic of an invasivefungal infection in the right upper lobe and aright lobectomy was performed. Histologydemonstrated branching hyphae invadingblood vessels that were ‘‘consistent withaspergillus’’. However, many filamentousfungi resemble aspergillus species histologi-cally, and identification relies on culturingthe organism from the tissue. In this case,C globosum was cultured from the tissuespecimen and identified six weeks later, bywhich time the patient had died. Moreover,such microbiological identification cannotdifferentiate between environmental contam-ination and causative organisms.Immunohistochemical staining of the tissuewas subsequently carried out using a mono-clonal antibody directed specifically againstA fumigatus, A flavus, and A niger. Results werenegative, confirming that the infection wasnot caused by these common aspergillusspecies. Chaetomium globosum is a rare patho-gen but a common contaminant, so we wereleft with a presumptive diagnosis.We have now achieved a definitive, mole-cular diagnosis of an invasive fungal infec-tion caused by C globosum in this case usingthe method described above. Sections fromthe original paraffin wax embedded tissuewere cut with a sterile microtome blade andtwo 10 mm thick sections were subjected toDNA extraction, after discarding the outersection. A positive result was obtained afterpolymerase chain reaction amplification withpanfungal primers, but subsequent hybridi-sation with the aspergillus specific probeyielded a negative result. The DNA waspurified using WizardH PCR Preps DNApurification system (Promega, Madison,Wisconsin, USA), according to the manufac-turer’s instructions. The DNA was commer-cially sequenced (Cytomyx, Cambridge, UK),after which sequence analysis was performedwith the EMBOSS software package (HGMP-RC; Medical Research Council, UK). Thesequence was identified as C globosum usinga BLAST search of Genbank and EMBLnucleotide sequence databases and subse-quently submitted to the EMBL database(accession number AJ781794).The C globosum isolate had been added tothe UK National Collection of PathogenicFungi as NCPF 7115. We subcultured thisisolate then extracted, purified, andsequenced the genomic DNA. The sequencewas identical to that of the DNA extractedfrom the tissue sample, confirming thecausative role of this organism in our case.With the growing population of immuno-compromised patients and the broadeningspectrum of antifungal agents available it isimperative that we can accurately identify theorganisms causing invasive fungal infections.This case illustrates the value of moleculartools to enhance the diagnostic process. ;P J Paterson, S Seaton, T Yeghen, T D McHughDepartment of Microbiology, Royal Free Hospital,Pond Street, London NW3 2QG, UKJ McLaughlinDepartment of Pathology, Royal Free HospitalA V HoffbrandDepartment of Haematology, Royal Free HospitalC C KibblerDepartment of Microbiology, Royal Free Hospital;kibbler@rfc.ucl.ac.ukReferences1 Perfect JR, Schell WA. The new fungalopportunists are coming. Clin Infect Dis. 1996;22:S112–18, .2 Paterson PJ, Seaton S, McLaughlin J, et al.Development of molecular methods for theidentification of aspergillus and emerging mouldsin wax tissue sections. Mol Pathol2003;56:368–70.3 Einsele H, Hebart H, Roller G, et al. Detection andidentification of fungal pathogens in blood byusing molecular probes. J Clin Microbiol1997;35:1353–60.4 Yeghen T, Fenelon L, Campbell CK, et al.Chaetomium pneumonia in patient with acutemyeloid leukaemia. J Clin Pathol 1996;49:184–6.Rev 7.51n/W (Jan 20 2003)Journal of Clinical Pathology cp21683 Module 8 30/11/04 17:02:19 Topics: 78; 180J Clin Pathol 2005;000:1–2 1www.jclinpath.comAuthors QueriesJournal: Journal of Clinical PathologyPaper: cp21683Title: Molecular confirmation of invasive infection caused by Chaetomium globosumDear AuthorDuring the preparation of your manuscript for publication, the questions listed below have arisen. Please attend to these matters and return thisform with your proof. Many thanks for your assistanceQueryReferenceQuery Remarks1 Please provide a location forTaKaRa Biomedicals and a townand county in the UK for the MRC.Rev 7.51n/W (Jan 20 2003)Journal of Clinical Pathology cp21683 Module 8 30/11/04 17:02:20 Topics: 78; 1802www.jclinpath.com

Please provide a location for TaKaRa Biomedicals and a town and county in the UK for the MRC. Rev

Molecular confirmation of invasive infection caused by Chaetomium globosum [1]

Molecular confirmation of invasive infection caused by Chaetomium globosum [1]

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