Evaluation of a Loop-mediated Isothermal Amplification Technique for the Detection of Toxigenic Clostridium difficile

strains in Diarrheal Stools J. Van Broeck1, M. Delmée1

1 Belgian Reference Center for Clostridium difficileMicrobiology Unit, Université Catholique de Louvain, Brussels, Belgium

Universitécatholique de Louvain

Ev aluation of a Loop-me diated Isothermal Ampli fication Technique for the De tection ofToxigenic Clostridium difficile Strains in Diarrheal Stools

Van Broeck J.1 and Delmée M.11Microbiology Uni t, Univ ersity Hos pital St-Luc, Catholic Univ ersity of Louv ain, Brussels, Belgium

BackgroundThe illumigene TM C. di fficile (Meridia n Bioscience) is a molecular assay, based on loop-mediated isothermal ampli fication(LAMP) targeting a conserved sequence of the toxin A gene. We ev aluated the performances of illumige ne TM for dia gnosing C. difficile infection on diarrheal stools as a sta ndalone test and also in algori thms where three immunoassays, the C. diff Quik Chek Complete TM (Tec hlab®) toxins A&B and GDH, the Premier C. difficile glutamate dehydrogenase (GDH) and the Immunocard toxin A&B (Meridian Bioscience), were compared as a screening method. Toxigenic culture was used as Gold S tandard. Algorithms were as follows: in case of negativ e results for both GDH and toxin, stool was considered negativ e. In case of positiv e GDH and or positiv e toxin, the stool was considered positiv e. In case of discordant results, the illumigene TM

result was used .Materials and methodsStools were from inpatie nts of the Univ ersity Hospi tal St-Luc -UCL, older than 2 years and suffering from diarrhoea. Cultures were performed on CCFA and on CCFA with added bile salts. In case of positiv e culture, strains were tested for toxin production using cell cytotoxicityassay. The illumigene TM and all immunoassays were performed according to the manufacturer’s instructions. ResultsBetween October 2010 and January 2011, 296 stools were tested. 22 samples were shown to contain toxigenic C. difficile by toxigenic culture (prev alence : 7,4%). The sensitiv ity (SE), speci ficity (SP), PPV and NPV of illumigene TM was : 85,7%, 99,6%, 94,7% and 98,9%. Unresolv ed result was recorded in one insta nce and the sample was excluded from our calculation. Screening with C. diff Quik Chek Comple te TM or with Premier C. difficileGDH c ombined with Immunocard and followed by illumigene TM on GDH positiv e, toxins A and B nega tiv e samples gave SE, SP, PPV and NPV of respectiv ely : 81%, 99,6%, 99,4%, 98,6% and 81%, 98,9%, 85%, 98,5%.ConclusionThe illumigene TM assay is a highly performant molecular test compared to existing immunoassays. The combination of a GDH assay with a v ery high NPV and the illumigene TM assay re duces cost while maintaining a v ery good sensitiv ity a nd specifici ty and allowing results in less than two hours.

Abstract

Introduction

Clostridium difficile associated diarrhea is a global health problem and new virulent strains are emerging. Culture together with the identification of toxin production (toxigenic culture) is still the most sensitive but very slow diagnosticmethod. Quicker and more sensitive tests could help early diagnoseof the disease. In this study, the illumigeneTM C. difficile (Meridian Bioscience Inc. Cincinnati,OH, USA) was evaluated in a one step and in a two steps algorithm towards Toxigenic culture as reference method. illumigeneTM is a molecular assay based on loop-mediated isothermal amplification technology. Amplification is accomplished by the use of specially designed primers under isothermic conditions. The illumigeneTM

detects whithin the pathogenicity locus a well conserved part of the tcdAgene on the 54 region. This region is found in all toxin (A+B+ or A-B+) producing strains. Magnesium-pyrophosphate is produced as a result of the amplification which causes the reaction solution to become turbid. This turbidity is measured after 40 minutes using the Meridian illumipro-10TM

Incubator/Reader. The whole process is in a closed system and doesn’t require special PCR precautions. In the two step algorithm we have screened all samples with Glutamatedehydrogenase (GDH) fromC. diff Quik Chek CompleteTM (Techlab® Blacksburg, VA,USA) or using the Premier GDH test (Meridian Bioscience Inc. Cincinnati,OH,USA). On GDH positive samples we performed the illumigeneTM.Since this study a new GDH Immunocard test (Meridian Bioscience Inc. Cincinnati OH, USA) is available with comparable sensitivity.

Materials and methodsStools: were from inpatients (>2y) suffering from

antimicrobial- or chemotherapy associated diarrhea.

296 stools collected over a 4 months period (between Oct 2010 and Jan 2011) were tested.

Gender distribution: 144M / 152 F

The prevalence of positive culture samples was 7.4%.

Cultures: on CCFA (twice overnight anaerobic incubation)

CTA: sterile faecal filtrate on MRC-5 cells

CCFAT: CCFA + sodium taurocholate.

Two algorithmes were evaluated :

A The illumigeneTM on all stool samples.

B All samples screened for GDH with C. diff Quik Chek CompleteTM or Premier GDHTM followed by

illumigeneTM on GDH + samples. Culture was performed on all samples and Toxigenic culture (TC)

was considered as the Gold Standard.

CultCultCult CTACTACTAand

+++ ++ +++ --- -- +++ -- ---

CDICDICDIno CDIno CDIno CDI

CCFAT

+++ ---

CDICDICDI no CDIno CDIno CDI

Tox A/Bon colonies

CDICDICDI no CDIno CDIno CDI

+++ ---

Fig. 1 Usual scheme for the diagnosis of Fig. 1 Usual scheme for the diagnosis of CDI, toxigenic cultureCDI, toxigenic culture

ReferencesDiscussionResultsLabora tory Dia gnosis of Clostridium difficile infectionFred tenover et al.The Journal of Molecular Diagnostics Vol 13 nr 6 Nov 2011

Reactiv ity of Clos tridium di fficile Toxinotypes with the illumigene TM C. di fficile Molecular AssayCoyle et al.110th ASM poster 219

A New Molecular Approach for the Detec tion of Clostridium difficileS. Elagin et al.Poster at 26TH Annual Clinical Virology Symposium

A Nov el New Molecular Approach for the Detection of Clostridium difficileJ.L.Wince et al.110th ASM general meeting poster 204

Comparison of Three Clostridium di fficile Molecular Assays to Cul tureD.J.Mayne et al.110th ASM general meeting poster 1090Labora tory diagnosis of Clostridium difficile-associate d diarrhoea : a plea for c ultureDelmée M, Van Broeck J, Simon A, Janssen s M, Avesani J Med Microbiol. 2005 Feb;54(Pt 2):187-91. Review.Ev aluation of a two step algori thm using a rapid antigen and toxin screening test in combina tion with toxigenic culture for the detection of toxigenic Clostridium difficileJ. Van Broeck1, J. Ve rhaegen2, S. Resseler, S. D’hollanders1, C. Hubert1, M. Delmée1

ECCMID Helsinki 2009

A Two Step Algorithm for the Diagnosis of Clostridium di fficile infection: Screening with a rapid Immunoassay for the Detection of Glutama tedehydroge nase and toxins A and B followed by a real-time PCR for Clostridium di fficileJ.Van Broeck, T. Sanli1, V. Philips1 and M. Delmée1 (Brussel s, BE)3rd International Clostridium difficile Symposium, 2010, Bled, Slovenia

A two step algorithm for the diagnosis of Clostridium di fficile infection: screening with a rapid immunoassay for the detection of gluta matedehydrogenase and toxins A and B followed by a real-time PCR for Clostridium di fficileJ. Van Broeck, C. Hubert, M. Va st and M. Delmée (Brussels, BE)ECCMID Vienna 2010Epidemiology of Clostridium difficile toxinotype III, PCR-ribotype 027 associated disease in Belgium, 2006. Delmee M, Ramboer I, Van Broeck J, Suetens C. Euro Surveill. 2006 Sep 14;11(9):E060914.2.

Proposed Diagnosticalgorithms

Fig. 2 OneFig. 2 One--step algorithm step algorithm illumigeneillumigeneTMTM on all sampleson all samples

Fig. 3 TwoFig. 3 Two--step algorithm: GDH on all samples step algorithm: GDH on all samples followed by illumigene on GDH + samples followed by illumigene on GDH + samples

illumigeneTM

on all samples

+

CDI + CDI -

illumigeneTM

on GDH + samples

CDI +

GDHon all samples

-

CDI -

+

+ -CDI -

illumigene on all samplesN=296

1 (sample 2 X unresolved) sens.: 85,70%spec.: 99,60%

N=295 TC+ TC- p.p.v.: 94,70%illumigene + 18 1 n.p.v.: 98,90%illumigene - 3 273

Premier GDHTM on all samplesand illumigene on GDH +

N=295 TC+ TC+ TC- TC- sens.: 81,00%GDH + and illumigene + 17 17 1 1 spec.: 99,60%GDH - and illumigene + 1 0 p.p.v.: 99,40%GDH - and illumigene - 2 4 238 273 n.p.v.: 98,60%GDH + and illumigene - 1 35

Quik Chek Complete GDHTM on all samplesand illumigene on GDH +

N=295 TC+ TC+ TC- TC- sens.: 81,00%GDH + and illumigene + 17 17 1 1 spec.: 99,60%GDH - and illumigene + 1 0 p.p.v.: 99,40%GDH - and illumigene - 2 4 252 273 n.p.v.: 98,60%GDH + and illumigene - 1 21

Toxigenic culture

Results

We used Toxigenic culture as a reference method to evaluate the performances of illumigeneTM for diagnosing C. difficile infection on diarrheal stools as a standalone test and in a two steps algorithm where we screened with GDH and perform illumigeneTM on GDH positive samples. In testing illumigeneTM on all samples, three stayed negative. These three samples stayed also negative using another molecular biology test. We found 12 different ribotypes among the toxigenic strains. They all belonged to the most frequent ribotypes in Belgium. One dark-colored sample was twice “invalid”. After defreezing it was tested again and came out positive with illumigeneTM and another molecular biology test. One illumigeneTM

positive sample was negative for toxigenic culture, even when plated on a CCFAT medium, containing sodiumtaurocholate, known to have a desporulating effect. The sample was also positive using another molecular biology test. The performance of the classic ELISA test, the Premier GDHTM and the GDH of the Quik Chek CompleteTM have comparable sensitivities, but the Premier GDHTM

generates more false positives towards culture and induces a higher amount of PCR’s. Since we did the study, a new ImmunocardGDHTM from Meridian is available with the same sensitivity as theGDH in the Quik Chek CompleteTM. The combination of a GDH assay with a very high negative predictive value and the illumigeneTM

assay, reduces cost while maintaining a very good sensitivity and specificity and allowing results in less than two hours.The presence of GDH in the stool sample could be an extra indication for the presence of the vegetative form of C. difficile and help to differentiate colonised from infected patients.

ECCMID London 2012

P-2259

-

CDI -CDI+

Nbr pos. sensitivity FP FNToxigenic culture: 21 100% 0 0

illumigene on all samples: 18 85,70% 1 3

Premier GDH and illumigene : 17 80,90% 1 4

Quik Chek Complete GDH and illumigene : 17 80,90% 1 4

Immunocard Toxin A&B (ICTAB): 15 71,40% 2 6

Quik Chek Complete GDH + TOX A&B: 13 61,90% 0 8

Cellcytotoxicity (MRC-5): 13 61,90% 0 8

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