comparative diagnosis of pcr and inmunocromatography

8/10/2019 Comparative Diagnosis of PCR and Inmunocromatography

1/8

NEW MICROBIOLOGICA, 36, 57-63, 2013

Comparative analysis of different methodsto detect Clostridium difficile infection

Adriana Calderaro, Mirko Buttrini, Monica Martinelli, Chiara Gorrini, Sara Montecchini,Maria Cristina Medici, Maria Cristina Arcangeletti, Flora De Conto, Silvia Covan, Carlo Chezzi

Department of Clinical and Experimental Medicine, Unit of Microbiology and Virology, University of Parma, Italy

INTRODUCTION

Clostridium difficile is the major causative agent

of healthcare-associated diarrhoea andpseudomembranous colitis (Freemanet al., 2010).Two large toxins, toxin A (TcdA, enterotoxin) andtoxin B (TcdB, cytotoxin), are recognised as themain virulence factors of C. difficile. Both toxinsare localised within a 19.6 kb region of the bac-terial chromosome termed locus of pathogenici-ty (PaLoc) (Braunet al., 1996).For a long time, the distinction between toxigenicC. difficile strains, like those producing both tox-ins (A+ B+) and causing disease, and non toxi-genic strains (A-B-), producing neither toxins nor

causing disease (Rupnik, 2008), was commonly

Corresponding authorProf. Adriana Calderaro MD, PhD

Associate Professor of Microbiology

and Clinical Microbiology

Director of the Unit of Clinical Microbiology

Department of Pathology and Laboratory Medicine

Section of Microbiology

University Hospital of Parma

Viale A. Gramsci, 14 - 43126 Parma, Italy

E-mail address: [email protected]

accepted. However, later studies showed that C.difficile is a very heterogeneous species and manytypes of variant isolates can be differentiated both

among toxigenic and non toxigenic strains. Theterm variant strain describes any strain that dif-fers from the reference C. difficile strain VPI10463 (A+B+) either in the DNA sequence of thePaLoc and/or in the pattern of toxin production(Rupnik, 2008).The incidence and the severity of C. difficile in-fections (CDI) have been increasing for the lastdecade, particularly in North America andEuropean nations. The most virulent variantstrains, such as 027/NAP1/BI strain and ribotype078/toxinotype V strain, were isolated in these

countries. The reported changes in CDI epidemi-ology have brought renewed focus on the mostappropriate method to detect C. difficile and/orits toxins in faecal samples (Freemanet al., 2010).The diagnosis of CDI is usually based on a clini-cal history of recent antimicrobial usage and di-arrhoea in combination with the result of labo-ratory tests (Cohenet al., 2010; Crobachet al.,2009; Delmeeet al., 2005). Currently, the goldstandard methods for the diagnosis of CDI arethe stool cytotoxicity assay and the toxigenic

The increased incidence and severity of Clostridium difficile infection, particularly in North America and Europe, havebrought renewed focus on the most appropriate method to detect C. difficile and/or its toxins in stools. This prospec-tive study evaluated the usefulness of the IllumigeneTM C. difficile assay in diagnostic practice for the detection of tox-igenic C. difficile DNA in clinical samples. A total of 88 out of 306 stool samples analysed were positive both byIllumigene and the combination of toxigenic C. difficile culture (TC) and immunochromatographic assay (IC) with aconcordance of 100%. Of the 218 samples negative by the combination of TC and IC, 204 were negative also byIllumigene with a concordance of 93.57%. In our experience, compared to conventional assays Illumigene assay provedto be easy to perform, accurate and prompt giving results within 1 hour at a cost of 28 euro per sample.

KEY WORDS: Clostridium difficile, Diagnosis, LAMP, Toxins, Diarrhoea.

SUMMARY

Received June 6, 2012 Accepted September 9, 2012


2/8

C. difficile culture (www.cdc.gov/HAI/organ-isms/cdiff/Cdiff_clinicians.html, accessed on 11th

June 2012). The stool cytotoxicity assay is notstandardized, requires skill and facilities for cell

culture, implies a long time to report and may beaffected by the lability of toxin B (Cohenet al.,2010; Crobachet al., 2009; Delmeeet al., 2005;Doinget al., 2011), so that its usefulness in theclinical setting is limited (www.cdc.gov/HAI/or-ganisms/cdiff/Cdiff_clinicians.html, accessed on11th June 2012). The toxigenic C. difficile cultureis slow and laborious, often requires 72 to 96 h tocomplete, and therefore is unlikely to be adoptedroutinely in the current laboratory diagnosis ofCDI (Cohen et al., 2010; Crobach et al., 2009;Delmeeet al., 2005). Nowadays, faster detection

methods have been developed for the detection ofC. difficile toxins and most laboratories haveadopted enzyme immunoassays for toxins A andB as the routine method of testing. These tech-niques are easier to perform, faster than gold stan-dard assays and do not require specific technicalskills (Barbutet al., 2009; Plancheet al., 2008;Wilkinset al., 2003). However, they are not sensi-tive enough to be used as a stand-alone assay forCDI laboratory diagnosis (Lalandeet al., 2011).Immunoenzymatic assays for the diagnosis ofCDI other than those for toxins A and B have al-

so been commercially available for more than 10years, such as those for the detection of C. diffi-cile glutamate dehydrogenase (GDH).The commercial GDH tests offer a turnaroundtime of 15 to 45 min, and this is one of the rea-sons these tests are used in many laboratories.This antigen has proven to be a good screeningmarker for C. difficile because this essential en-zyme is produced constitutively in large amountsby all C. difficile strains and can be readily de-tected in faecal specimens but it does not distin-guish toxigenic from non toxigenic strains

(Wilkinset al., 2003).The changes in CDI epidemiology have recentlyled to the development of commercial assaysbased on real-time PCR for the detection of tox-ins A and B. In December 2008, the Food andDrug Administration (FDA) approved the firstcommercial real-time PCR assay for the detec-tion of C. difficile toxins (Kvachet al., 2010). Themajority of variant strains show mutations intcdA and tcdB genes, encoding toxins A and B,respectively. The commercially available real-time

PCR assays correctly detect the majority of vari-ant strains of C. difficile on a genetic basis, and aredesigned to detect a conservative region of thetoxin B gene (tcdB). However, it has been report-

ed that in C. difficilevariant strains tcdA is moreconserved than tcdB (Rupnik, 2008). Recently, anew commercial assay based on loop-mediatedisothermal amplification (LAMP) technology andtargeting tcdA gene, the IllumigeneTM C. difficileassay (Meridian Bioscience, Cincinnati, OH), wasdeveloped. The tcdA target region was selected asa conservative region in all known A+B+ and A-B+ toxinotypes of variant strains of C. difficile,which is intact in all toxigenic strains, includingthose with a large deletion in the tcdA gene(Lalandeet al., 2011).

The aim of this study was to assess the useful-ness of the IllumigeneTM C. difficile assay in di-agnostic practice for the detection of toxigenic C.

difficile DNA in clinical samples, compared to thecombination of the toxigenic C. difficile cultureand the detection of the toxin A/B and glutamatedehydrogenase (GDH) by an immunochromato-graphic assay. Its possible application as a pri-mary test in the diagnosis of CDI was also evalu-ated after a subsequent 5-month period in thefield.

MATERIALS AND METHODS

Samples and patientsThree-hundred and six faecal samples collectedfrom 306 patients (291 Italians and 15 foreignersfrom developing countries) attending theUniversity Hospital of Parma (Northern Italy)with a suspicion of CDI over a three-month peri-od in the year 2011 were examined in a prospec-tive study.All faecal samples were subjected to an im-

munochromatographic assay for GDH and tox-ins A and B, toxigenic C. difficile culture andIllumigeneTM C. difficile assay as described below.

Immunochromatographic assay (IC)The simultaneous detection of C. difficile GDHand toxins A/B in faecal samples was performedby the immunochromatographic assay C. DIFFQUIK CHEK COMPLETE (TechLab, USA) ac-cording to the manufacturers instructions(Plancheet al., 2009).

58 A. Calderaro, M. Buttrini, M. Martinelli, C. Gorrini, S. Montecchini, M.C. Medici, M.C. Arcangeletti, F. De Conto, S. Covan, C. Chezzi


3/8

Toxigenic C. difficile culture (TC)Specific medium plates, containing cycloserine-cefoxitin-fructose agar (CCFA) (Kima, Italy) andSchaedler agar (Kima, Italy), respectively, were

streaked with stool samples and incubated anaer-obically at 37C for 48-72 h. To enhance the sen-sitivity of the TC, an aliquot from all the faecalsamples tested was also inoculated in cooked-meat broth (CMB) (Kima, Italy) for 48 h in ananaerobic atmosphere and pre-treated by heat-shock at 100C for 3 minutes before inoculationonto CCFA agar plates according to standard pro-cedures (Jousimies-Somer et al., 2002). Thespecies identification of putative C. difficilecolonies was based on the biochemical test RapidID 32A (bioMrieux, France) and/or performed

by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry(MS) (Bruker Daltonics Microflex LT system,Becton Dickinson, Italia S.p.A.) according to themanufacturers instructions. In order to investi-gate the in vitro production of toxins A and B, theidentified C. difficile isolates were also culturedanaerobically at 37C for 72 h in CMB, and 0.5 mlof the broth culture was tested by C.DIFF QUIKCHEK COMPLETE, according to the manufac-turers instructions.

IllumigeneTM C. difficile (Illumigene) assayThe Illumigene (Meridian Bioscience, Cincinnati,OH) assay uses LAMP technology to amplify anddetect a conserved 204-bp sequence in the 5 por-tion of the tcdA gene (Lalande et al., 2011).Genomic DNA was extracted from faecal samplesby heat at 95C for 10 minutes. The assay wasrun according to the manufacturers instructions.In each vial provided, the Illumigene assay con-tains an internal control of extraction and inhi-bition of amplification constituted by formalin-in-activated Staphylococcus aureus and its specific

primers set. External quality controls supplied bythe manufacturer, including a positive control (asolution containing plasmidic DNA with S. au-

reus and C. difficile DNA insert) and a negativecontrol (a solution containing plasmidic DNAwith S. aureus DNA insert), were run for eachnew reagent batch. The Illumigene assay reportsa positive, negative, or invalid result. In case ofan invalid result (that may be caused by inhibi-tion of amplification, incorrect sample prepara-tion, or failure in internal control preparation)

the Illumigene assay was repeated according tothe manufacturers instructions.

Analytic sensitivity and specificity

of the Illumigene assayThe analytic sensitivity of the Illumigene assay wastested using faecal samples experimentally seededwith a toxigenic C. difficile reference strain (ATCCVPI10463/ToxA+ Tox B+/wild type/toxinotype 0)cultured in CCFA medium. Serial dilutions of aknown concentration of C. difficile were mixedwith an equal quantity of human faeces obtainingexperimentally seeded samples from 1.5107

c.f.u./g (colony forming units per gram) of faecesto 1 c.f.u./g of faeces. The same samples were alsoused for a comparative evaluation of the detection

limit of the IC, both for GDH and toxins A/B.The analytic specificity of the Illumigene assaywas tested using faecal samples containing en-teropathogenic bacteria other than C. difficile suchas Salmonella spp., Helicobacter pylori andStaphylococcus aureus, or intestinal protozoa suchas Giardia intestinalis,Entamoeba coli,Entamoeba

dispar, andBlastocystis hominis. Moreover, ex-perimentally seeded samples containing bacte-ria/parasites together with C. difficileVPI10463were tested by the Illumigene assay to verify theabsence of their influence on the detection of C.

difficile DNA. Analytic specificity was also deter-mined by testing a non toxigenic C. difficile strain(ATCC 70057/Tox A- Tox B-). Moreover, the toxi-genic C. difficile strain ribotype 078/toxinotype Vwas tested by the Illumigene assay.

Comparative evaluation of the resultsof Illumigene assay, TC, and ICThe results of the Illumigene assay were com-pared with those obtained by the combination ofTC and the detection of toxins A/B by IC. A sam-ple was defined as positive by the combination

of IC and TC when TC was positive and/or whenIC assay for toxins A/B was positive.Discordant results between the Illumigene assayand the combination of IC and TC were investi-gated by an additional test, a PCR assay for thedetection of tcdA/tcdB genes (Spigaglia et al.,2002), as described below.On the basis of the results of the comparativeevaluation, a primary test for the detection of C.

difficile was selected among those evaluated anda new algorithm was developed for the diagnosis

LAMP for toxigenic C. difficile detection 59


4/8

of CDI. This algorithm was applied to a total of718 samples belonging to 718 patients (681Italians and 37 foreigners from developing coun-tries) over a five-month period.

Duplex PCR for the detection of tcdA/tcdBgenesA tcdA fragment of 624 bp and a tcdB fragment of412 bp were amplified using the primers set de-scribed by Spigaglia and Mastrantonio (Spigaglia

et al., 2002). The PCR reaction mixture in a vol-ume of 50 l contained: 10x PCR reaction buffer,1.5 mM MgCl2, each deoxynucleotide (SocietItaliana Chimici, Italy) at a concentration of 200M, 125 nM of primers TA1 and TA2 and 62.5 nMof primers TB1 and TB2 (Primm, Italy), 1.5 units

of Taq DNA polymerase (Roche Diagnostics,Mannheim, Germany). PCR conditions were: 2min at 95C, followed by 30 cycles consisting of30 s at 95C, 30 s at 60C and 30 s at 72C and afinal step at 72C for 5 min. Duplex PCR prod-ucts were analysed by gel electrophoresis.The analytic sensitivity of the tcdA/tcdB PCR as-say was evaluated using the same experimental-ly seeded faecal samples used to assess the de-tection limit of the Illumigene assay.

RESULTS

Analytic sensitivity and specificityof the Illumigene assayThe analytic sensitivity of the Illumigene assay incomparison with that of IC (GDH - toxins A/B)

and duplex PCR is reported in table 1. The de-tection limit was 5 cfu/g of faeces for theIllumigene assay, 1000 cfu/g of faeces for IC assayboth for GDH and toxins A/B and 100 cfu/g of

faeces for duplex PCR.The Illumigene assay was negative when the DNAof enteropathogenic bacteria other than C. diffi-cile as well as intestinal protozoa DNA was usedas the template. Moreover, the detection of theDNA of C. difficile was not affected when it waspresent in faecal samples also containing the DNAof other bacteria and/or parasites. Finally,Illumigene gave a negative result when the DNAfrom the non toxigenic C. difficile strain ATCC70057/ToxA- Tox B- was used as the template, andgave a positive result when the DNA from the tox-

igenic C. difficile strain ribotype 078/toxinotypeV was used as the template.

Comparative evaluation of the resultsof Illumigene, TC, and ICThe results of the comparative evaluation ofIllumigene, IC for toxins A/B and TC on the 306analysed faecal samples are reported in table 2.A total of 88 out of the 306 stool samples analysedwere positive both by Illumigene and the combi-nation of TC and IC with a concordance of 100%.Forty-one (46.6%) of the 88 samples were posi-

tive for all the assays whilst 47 (53.4%) were pos-itive only for 2 of the compared assays, alwaysincluding Illumigene and only TC (19) or IC (28),alternatively.The 88 patients with positive samples were 82Italians, and 6 foreigners from developing coun-


TABLE 1 - Comparative analytical sensitivity of the different assays used in this study.

Concentration Illumigene assay IC for GDH IC for toxin A/B Duplex PCRof C. difficile strain for toxin A/BATCC VPI 10463

1.5107

c.f.u./g + + + +1000 c.f.u./g + +/- +/- +

100 c.f.u./g + - - +

10 c.f.u./g + - - -

5 c.f.u./g + - - N.P.

1 c.f.u./g - - - N.P.

ATCC VPI10463: C. difficile positive control ToxA+ Tox B+, wild type, toxinotype 0; +: Positive; -: Negative; +/-: Weakly positive; N.P.: Not performed; c.f.u./g : co-

lony forming units per gram of faeces.


5/8


6/8

both by the Illumigene assay and the combina-tion of TC and IC in this comparative study, 28were negative for TC and 19 for IC. In these cas-es, the negativity of TC was probably due to the

known difficulties encountered in bacteria isola-tion based on the intrinsic complexity when us-ing faeces as the matrix sample (Cohen et al.,2010; Delmeeet al., 2005), while the negativity ofthe IC could be due to the concentration of tox-ins that could have been lower than the detectionlimit of the assay, or due to the degradation ofthese proteins, or an incorrect sample collection.The same aspects could also explain the discrep-ancy found in 14 samples between the results ofthe Illumigene assay (positive) and of IC and TC(both negative). In these 14 samples the positive

result of the Illumigene assay was confirmed in 9cases by the positive result of duplex PCR whilstthe negativity of this assay in the remaining 5 cas-es could be due to the higher analytic sensitivityof the Illumigene assay. Moreover, in all the 14 dis-cordant samples a positive result by GDH IC as-say was obtained (data not shown). Taking into ac-count that the GDH assay does not distinguish be-tween toxigenic and non toxigenic strains, all thesedata taken together likely corroborate the hypoth-esis of false negative results by the conventionalassays TC and IC for toxins A/B and emphasize

that, for these samples, only the Illumigene assayled to an accurate diagnosis of CDI.Interestingly, 2 out of the 204 concordant negativesamples were positive by IC for GDH only (datanot shown): in these cases it can be supposed thatnon toxigenic C. difficile strains were involvedsince the presence of GDH enzyme alone doesnot discriminate between toxigenic and non tox-igenic strains. According to this hypothesis, a C.difficile non toxigenic strain was isolated fromone of these samples.Furthermore, 1 sample out of the 88 concordant

positive samples was positive both by theIllumigene assay and TC but negative by IC, bothfor toxins A/B and GDH: in this case, the nega-tive results of both IC assaysversus the positivi-ty of Illumigene and TC was probably due to thesensitivity of Illumigene that is higher than thatof IC as also demonstrated in this study.According to this hypothesis, the duplex PCR,previously demonstrated to be less sensitive thanIllumigene, was also found to be negative in thiscase (data not shown).

A rapid and accurate diagnosis of CDI is impor-tant since it may result in the prevention of noso-comial transmission and allows a specific antibi-otic therapy to be promptly administered to pa-

tients. The combination of a quick turnaroundtime with a high performance might result in abetter management of CDI and in a timely im-plementation of control measures.From a practical point of view, as also reported byDoing and Hintz (2011), in our hands theIllumigene assay proved easy to perform, not re-quiring particular skills or facilities, and offereda noteworthy rapidity giving results within 1 hour.Based on this rapidity and on the demonstratedexcellent sensitivity of the new diagnosticIllumigene assay, we conclud that it can be ad-

vantageously used in our setting as a primary as-say for CDI diagnosis as a replacement for the ICassay for GDH, previously used in our laborato-ry for the same purpose though not distinguish-ing between toxigenic and non toxigenic strains.Therefore the algorithm we propose for CDI di-agnosis is the following: to apply the Illumigeneassay as a primary assay on all samples sent tothe laboratory with a suspicion of CDI and, onlyon the Illumigene-positive samples (addressingpatients with a negative Illumigene result to oth-er investigations) proceed to IC for toxins A/B and

TC to assess the viability of the strain and the pro-duction of toxins. The current application of thisalgorithm in the diagnostic practice of our labo-ratory on 719 patients allowed prompt and ac-curate diagnosis of CDI cases and administrationof targeted therapy. Moreover, by usingIllumigene, thanks to its excellent sensitivity, anaccurate diagnosis of CDI was established re-quiring no more than one sample per patient. Inour experience, after the introduction of theIllumigene assay, even if there was a small rise inthe cost for the laboratory diagnosis of CDI (28

versus 26 euros), we consider that the better effi-ciency of the Illumigene assay as a primary test isadvantageous, because subsequent investigationsare required only for positive samples leading toless involvement of laboratory staff with a con-sequent general reduction of costs.

ACKNOWLEDGMENTThis study was supported by the Ministry ofUniversity and Scientific Research Grant FIL,Parma, Italy, and with the contribution of the



7/8

Interdepartmental Center CIPACK, Parma, Italy.Part of the content of the manuscript was presen-ted as preliminary results in a scientific contribu-tion (poster) at the 22nd European Congress of

Clinical Microbiology and Infectious Diseases -London 31st March - 4th April 2012.

REFERENCES

BARBUT F., BRAUN M., BURGHOFFER B., LALANDE V.,ECKERT C. (2009). Rapid detection of toxigenicstrains of Clostridium difficile in diarrhoeal stools byreal-time PCR.J. Clin. Microbiol. 47, 1276-1277.

BRAUNV., HUNDSBERGER T., LEUKEL P., SAUERBORN M.,EICHEL-STREIBER C. (1996). Definition of the singleintegration site of the pathogenicity locus in

Clostridium difficile. Gene. 181, 29-38.COHEN S.H., GERDING D.N., JOHNSON S., KELLY C.P., LOOV.G., MCDONALD L.C., PEPIN J., WILCOX M.H.,SOCIETY FOR HEALTCARE EPIDEMIOLOGY OF AMERICA,INFECTIOUS DISEASES SOCIETY OF AMERICA. (2010).Clinical practice guidelines for Clostridium difficileinfection in adult: 2010 update by the Society forHealtcare Epidemiology of America (SHEA) andthe Infectious Diseases Society of America (IDSA).Infect. Control. Hosp. Epidemiol. 31, 431-455.

CROBACH M.J., DEKKERS O.M., WILCOX M.H., KUIJPERE.J. (2009). 2009. European Society of ClinicalMicrobiology and Infectious Diseases (ESCMID):data review and reccomendations for diagnosing

Clostridium difficile-infection (CDI). Clin. Microbiol.Infect. 15, 1053-1066.

DELMEE M., VAN BROECKJ., SIMON A., JANSSENS M.,AVESANI V. (2005). Laboratory diagnosis ofClostridium difficile-associated diarrhoea: a plea forculture.J. Med. Microbiol. 54, 187-191.

DOING K.M., HINTZ M.S. (2011). Prospective evaluationof the Meridian Illumigene loop-mediated amplifi-

cation assay and the Gen Probe ProGastro Cd poly-merase chain reaction assay for the direct detec-tion of toxigenic Clostridium difficile from faecalsamples.Diagn. Microbiol. Infect. Dis. 72, 8-13.

FREEMAN J., BAUER M.P., BAINES S.D., CORVER J.,FAWLEY W.N., GOORHUIS B., KUIJPER E.J., WILCOXM.H. (2010). The changing epidemiology ofClostridium difficile infections. Clin. Microbiol. Rev.23, 529-549.

KVACH E.J., FERGUSON D., RISKA P.F., LANDRY M.L.(2010). Comparison of BD GeneOhm Cdiff real-time PCR assay with a two-step algorithm and atoxin A/B enzyme-linked immunosorbent assay fordiagnosis of toxigenic Clostridium difficile infec-tions.J. Clin. Microbiol. 48, 109-114.

JOUSIMIES-SOMER H., SUMMANEN P., CITRON D.M., BARONE.J., WEXLER H.M., FINEGOLD S.M., EDS. (2002).Wadsworth-ktl anaerobic bacteriology manual. 6th

edn. Star Publishing Company.LALANDEV., BARRAULT L., WADEL S., ECKERT C., PETITJ.C., BARBUT F. (2011). Evaluation of a Loop-Mediated Isothermal Amplification Assay forDiagnosis of Clostridium difficile Infections.J. Clin.Microbiol. 49, 2714-2716.

PLANCHE T., AGHAIZU A., HOLLIMAR R., RILEY P,POLONIECKY J., BREATHNACH A., KRISHNAS S. (2008).Diagnosis of Clostridium difficile infection by toxindetection kits: a systematic review.Lancet Infect. 8,777-784.

RUPNIK M. (2008). Heterogeneity of large clostridialtoxins: importance of Clostridium diffcile toxino-types. FEMS Microbiol. Rev. 32, 541-555.

WILKINS T.D., LYERLY D.M. (2003). Clostridium difficiletesting: after 20 years, still challenging. J. Clin.Microbiol. 41, 531-534.

SPIGAGLIA P., MASTRANTONIO P. (2002). Molecular analy-sis of the pathogenicity locus and polymorphismin the putative negative regulator of toxin produc-tion (TcdC) among Clostridium difficile clinical iso-lates.J. Clin. Microbiol. 40, 3470-3475.

LAMP for toxigenic C. difficile detection 63


8/8

comparative diagnosis of pcr and inmunocromatography

Documents