Scientific Symposium on New Approaches to

Clostridium difficile Testing

Conference report from the Satellite Symposium held during the

2010 United European Gastroenterology Week (UEGW)

23rd–27th OctoberBarcelona, Spain

Chairwoman:Dr Beryl Oppenheim (Birmingham, UK)

Scientific Symposiumon New Approaches toClostridium difficile Testing23−27 October 2010, Barcelona, Spain

Conference Report

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Account managerAudrey van Oorta.oort@elsevier.com

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Conference report from an Alere International sponsored symposium

Scientific symposium on new approachesto Clostridium difficile testing

A satellite symposium held during the 18th United EuropeanGastroenterology Week (UEGW) of the United European Gas-troenterology Federation (UEGF) in Barcelona, Spain, addressednew approaches to Clostridium difficile testing. The symposiumwas chaired by Dr Beryl Oppenheim of the Sandwell and WestBirmingham Hospitals NHS Trust, UK.

Dr Oppenheim welcomed attendees and provided an introductionto the dilemmas facing clinicians treating patients with possibleC. difficile infection, despite tests for the infection being availablefor many years. There continues to be multiple clinical challenges toquestion with this patient population, such as: Is this a single case?What is the best diagnostic test to use? Is there a possibility of false-positive or false-negative diagnostic tests? Is this a first infection ora recurrence? How severe is the infection likely to be? What is thebest treatment plan for this patient and individuals close to them?

These questions were discussed in the context of the currentand future management of C. difficile infection with presentationsby experts from Europe and North America. Prof. TracyWilkins, Professor Emeritus at Virginia Tech and Co-founderand Chairman of the Board of TechLab®, Blacksburg, USA,discussed the history of C. difficile research and the developmentof commercial assays to detect pathogenic toxins. Prof. MichelDelmee, head of microbiology at the Brussels Saint-Luc UniversityHospital in Belgium, compared the relative benefits of commercialdetection methods and provided a two-step algorithm for diagnosingC. difficile infection. Finally, Prof. Jost Langhorst, of theDepartment of Internal and Integrative Medicine at the Universityof Duisburg-Essen, Germany, reviewed the role of lactoferrinas a biomarker for inflammation in both C. difficile infectionand inflammatory bowel disease (IBD). A summary of thesepresentations follows below.

C. difficile testing: past, present, and futureIdentifying the cause of pseudomembranous enterocolitis

Prof. Tracy Wilkins provided an overview of the discovery ofC. difficile disease and the evolution of diagnostics for detectingthe pathogen and its toxin. New ideas for future diagnostic aidswere discussed, including fecal lactoferrin as a biomarker forinflammation and a new evidence-based protocol utilizing glutamatedehydrogenase (GDH) as a method for eliminating specimensthat are negative for C. difficile. Interest in the management andassessment of C. difficile infection has risen in the past few years,most recently with the increased incidence of C. difficile disease andthe introduction of new detection methods.1 Today, the incidence ofC. difficile disease continues to rise and involves both nosocomialand community acquired infections.

The history of C. difficile disease began with the discovery in theearly 1970s of severe pseudomembranous enterocolitis associatedwith clindamycin antibiotic therapy.2,3 More research into antibiotic-related colitis followed, and C. difficile was eventually identifiedas the cause.4−6 A pivotal report in 1976 of 16 cases of severecolitis, in which four patients died, highlighted the risk of severecomplications, and the need for a specific treatment for thedisease.3

Although pseudomembranous enterocolitis was recognized as adeadly condition, its cause was not determined until years later.Clinicians ruled out allergic reaction or direct toxicity of theantibiotic received. Researchers identified a cytopathic effect ontissue culture cells that was not due to a virus and that could not bepropagated.4 While no pathogenic bacteria, Mycoplasma or viruseswere found, it was suspected that pseudomembranous colitis wascaused by a bacterial toxin and the hunt accelerated to unravel themystery.

Progress for identifying the pathogen was made when it wasdiscovered that C. sordellii antitoxin neutralized the effect ofthe cytotoxin.5 Although C. sordellii was not present in thesesamples, C. difficile, which produces a similar cytotoxin, wasisolated from samples of patients with pseudomembranous colitisand subsequently identified as a potential pathogen (Table 1).

Table 1 – Major events leading to the discovery ofC. difficile disease

Year Event Research team

1977 Undescribed toxin in pseudomembranous colitis Larson et al.4

1977 Antibiotic-induced colitis implications of a toxin

neutralized by C. sordellii antitoxin

Rifkin et al.5

1977 Clindamycin-associated colitis due to a toxin-

producing species of Clostridium in hamsters

Bartlett et al.6

1978 Identification of C. difficile as a cause of

pseudomembranous colitis

Multiple research teams

Initially, C. difficile was not thought to be responsible for the colitis;it was listed as non-pathogenic because it was a component of thenormal fecal flora in about half of all newborn infants. 7 The reasonswhy the presence of C. difficile is not harmful to infants remainsunknown, but in adults, the organism can cause pseudomembranouscolitis. Wilkins and colleagues successfully produced an antitoxinto C. difficile. 8

Developing a C. difficile toxin assay

It was later determined that C. difficile produced a secondtoxin that could be separated from the cytotoxin by ion-exchangechromatography.9 These findings were subsequently confirmed byWilkins and colleagues,10 and both groups of researchers namedthe Toxins ‘A’ and ‘B’, representing the enterotoxic and cytotoxictoxins, respectively.

The enzyme immunoassay (EIA) for detection of Toxin A wasdeveloped by Wilkins and colleagues early after the discovery ofthe connection between Toxin A and colitis. 11,12 Shortly afterwards,another research group claimed that a latex test, which was alreadyavailable at that time, also detected Toxin A. In 1986, Wilkinsand Lyerly determined that the commercial latex test for Toxin Adid not, in fact, detect the toxin, but instead, detected GDH, anessential metabolic enzyme that is produced by both toxigenicand non-toxigenic strains. 13,14 The detection of fecal GDH turnedout to be a sensitive indicator for the presence of C. difficile.The C. DIFF QUIK CHEK COMPLETE® (TechLab®) is a newlydeveloped diagnostic aid that utilizes the detection of both GDHand Toxins A and B. Because GDH is produced by other bacteria, a

Conference report from an Alere International sponsored symposium 1

monoclonal antibody is necessary to ensure specificity. The resultingassay has a sensitivity of 85−90% compared with toxigenic culture.

Non-toxigenic C. difficile strains will react in the GDH testand can occur in 5−20% of antibiotic associated diarrhea (AAD),depending on the institution conducting the testing. At Prof. Wilkins’institution in Virginia, approximately 11% of strains identified by thetest are non-toxigenic (Wilkins, unpublished data). By comparison,very few strains identified in nursing homes are non-toxigenic.However, a much higher percentage of strains identified in youngpeople are non-toxigenic C. difficile.

The greatest utility of the C. diff Quik Chek Complete® is that itcan be used prior to real-time polymerase chain reaction (PCR) as arapid and inexpensive screening test. Results can be obtained within30 minutes. Real-time PCR emerged in 2009 as a method to detectthe toxin B gene (tcdB) of toxigenic C. difficile, implying that thestrain is a toxin producer. The test can be completed within 2 hoursand detects the presence of the gene, rather than the toxin itself.Real-time PCR testing is very expensive compared to other methodsof GDH/toxin testing.15 Clinicians can save time and expense byusing an algorithm approach, such as screening with the C. diffQuik Chek Complete® and then confirming the diagnosis with real-time PCR only in cases that are neither clearly positive nor negativefor GDH and toxin.

The greatest utility of the C. diff Quik ChekComplete® is that it can be used prior toreal-time PCR as a rapid and inexpensive

screening test

Interpreting results

Does a positive C. difficile test indicate disease? C. difficile isoften found in younger patients without causing disease. In fact,Prof. Wilkins noted that we should remember that babies oftenharbor C. difficile in the absence of disease. There have been reportsof C. difficile-associated colitis in younger patients, but youngerpatients are generally much more resistant to the disease.1,16 It isunclear why younger people tolerate the presence of C. difficilewithout developing colitis, but an intact colonic mucosa and a betterimmune system may play a role.

C. difficile disease is an inflammatory condition with anappearance similar to that of ulcerative colitis (UC), with respect tothe influx of activated neutrophils in the colonic mucosa (Figure 1).

Figure 1. Colitis: influx of activated neutrophils.

“This is an inflammatory disease,” noted Prof. Wilkins. “Theappearance of small areas blossoming into little volcanoes oferuption of neutrophils coming into the colon is characteristic ofmoderate to severe C. difficile disease”. Tests to detect this increasein neutrophils allow clinicians to determine if patients are presentingwith an inflammatory illness. This inflammatory response, withassociated tissue necrosis, is the primary pathogenic element ofpseudomembranous enterocolitis in severe cases, rather than simplythe presence of the C. difficile organism or toxins.

“This is an inflammatory disease. Theappearance of small areas blossoming intolittle volcanoes of eruption of neutrophilscoming into the colon is characteristic ofmoderate to severe C. difficile disease”

Lactoferrin assays for intestinal inflammation

Lactoferrin is a protein found in high concentrations in activatedneutrophils that infiltrate the intestinal lumen and serve as amonitor for intestinal inflammation. Several lactoferrin assays areavailable for detecting increased levels in feces. The TechLab®

lateral flow tests LEUKO EZ VUE™ and IBD EZ VUE® are rapidand inexpensive, and easy to use. The Leuko EZ Vue™ detects fecalwhite blood cells in acute cases of diarrhea (infectious) and theIBD EZ Vue® is more focused on the differentiation of active IBDfrom irritable bowel syndrome (IBS). The MicroWell® ELISAtests, IBD-CHEK® and IBD-SCAN®, are qualitative and quantitativetests, respectively. These tests are useful when a large number ofpatient specimens must be examined. The IBD-Chek® differentiatesactive IBD from IBS and healthy persons, and IBD-Scan® offers aquantitative result as an indicator of the amount of inflammation inactive IBD.

IBD-Scan® may also be useful for gauging C. difficile diseaseseverity. Recent data show a direct correlation between the severityof disease in C. difficile infection as assessed by clinicians and thelevel of lactoferrin (Table 2). These results show an approximately10-fold differential between patient groups. Such a clear differencein lactoferrin levels can aid clinicians who currently determineseverity based on symptoms, such as abdominal pain and numberof stools per day.

Table 2 – Lactoferrin level in patients with clinicallydefined C. difficile infection

C. difficile disease activity N Mean lactoferrin level (mg/mL)

Severe 13 1,350

Moderate 15 160

Mild 2 12

Healthy 14 2

Recently, a simple laboratory algorithm for diagnosing C. difficileinfection was proposed.17,18 A screening algorithm with C. diff QuikChek Complete®, which allows for simultaneous detection of bothGDH and Toxins A and B, followed by additional testing withthe lactoferrin assay, PCR, or toxigenic culture as needed, wasdeveloped and tested (Figure 2). Approximately 80% of samplesare determined within 30 minutes to be negative by C. diff QuikChek Complete®, as indicated by the absence of GDH or toxin.Another 10% are positive for both GDH and toxin, and are therefore

2 Conference report from an Alere International sponsored symposium

Lactoferrin

+

–

80%

GDH(–) Tox(–)

Report Negative

10%

GDH(+) Tox(–)

Additional testing

10%

GDH(+) Tox(+)

Report Positive

PCR or

Inflammation

Positive = Consider treatment

Negative = Monitor patient

Toxigenic culture

Figure 2. Proposed algorithm for diagnosis of C. difficile infection. GDH, glutamate dehydrogenase; PCR, polymerase chain reaction; Tox, Toxins A and B.

conclusively positive for C. difficile infection. The remaining 10% ofsamples are positive for GDH but negative for toxin, and thereforerequire further testing. This result can indicate the presence of anon-toxigenic strain of C. difficile or a toxigenic strain that has notyet produced enough toxin to test positive.

Using the proposed algorithm, samples requiring further testingfor C. difficile diagnosis can be evaluated for inflammation using thelevel of lactoferrin or can be tested with PCR or toxigenic culture.Testing for elevated lactoferrin offers an inexpensive, rapid option todetect inflammation. Patients negative for lactoferrin (inflammation)can avoid unnecessary treatment with antibiotics, which itself cancause C. difficile infection.

Relapse is common in patients and occurs in about 25% ofcases, 19 particularly in those treated with the antibioticsmetronidazole or vancomycin. A subsequent infection may involvea more virulent strain or more severe infection. Recently, anew strain of C. difficile, ribotype 027, was identified that isassociated with an increased infection-related mortality rate. 20 Thisvirulent strain has been reported across Europe and North Americaand is a predominant ribotype.21 The appearance of this newstrain may be largely responsible for the increase in C. difficileinfection observed over the last decade in Europe as well as inNorth America.22

Clinicians now have multiple diagnostic tests for C. difficileand must determine which strategy is most appropriate for theirpatient. Effective management also depends on identifying whichpatients who test positive for C. difficile actually require treatment.Examining a fecal sample for C. difficile toxin alone may not bea reliable diagnostic parameter. With its simultaneous detection ofC. difficile antigen (GDH) and Toxins A and B, the C. diff QuikChek Complete® is a rapid and reliable test that, when coupledwith a lactoferrin test, offers a more complete clinical picture toenable effective patient management. Additionally, assessing thelactoferrin level can help physicians identify rapidly and accuratelythose patients who are exhibiting an inflammatory response and

may therefore require intervention, and those who are tolerating theinfection well and can therefore avoid unnecessary treatment.

With its simultaneous detection of C. difficileantigen (GDH) and Toxins A and B, theC. diff Quik Chek Complete® is a rapid andreliable test that, when coupled with alactoferrin test, offers a more completeclinical picture to enable effective patientmanagement

Comparing C. difficile antigen and toxin testingwith bacterial culture

Prof. Michel Delmee discussed the relative benefits of differentC. difficile testing methods and reviewed a current two-stepalgorithm for C. difficile diagnosis in the clinical setting. Over thelast several years there have been important improvements in thelaboratory diagnosis of C. difficile infections. Clinicians now havemuch better diagnostic tools and are able to streamline the diagnosisand management of this disease. “We are detecting C. difficileantigen and toxin in patients much quicker and earlier,” notedProf. Delmee.

A major cause of hospital-acquired diarrhea is C. difficile indeveloped countries. 23,24 Further, once there is a case of diarrheain the hospital, the environment is rapidly contaminated by sporesthat may persist for weeks or longer, increasing the patient’s riskof exposure or re-exposure to C. difficile. Thus, management ofC. difficile focuses on prompt and effective management of notonly the patient but also the patient’s environment, both to improveindividual patient outcomes and to prevent the spread of infection. Tomeet this need, an accurate and rapid diagnosis of every C. difficileinfection is essential. According to Prof. Delmee, “Every case of

Conference report from an Alere International sponsored symposium 3

hospital-acquired diarrhea should be tested for C. difficile − it’s themain known cause.”

“Every case of hospital-acquired diarrheashould be tested for C. difficile − it’s the

main known cause”

Screening of stool samples for C. difficile infection isrecommended in all patients with at least one of the following:• History of antibiotic therapy• Hospital-acquired diarrhea• Age >65 years• History of C. difficile-associated diarrhea.

Careful consideration should be given to patients of advancedage, which is a risk factor for developing C. difficile-associateddiarrhea.25 Residence in a nursing home represents the mostimportant risk factor for developing diarrhea in older people. 26 Arecent survey of residents of a long-term care facility demonstratedunder-diagnosis of C. difficile infection.27 By contrast, screeningshould be avoided in children aged less than 2 years and in patientswith formed stools, as these patients are very unlikely to haveC. difficile disease.

Available tests for C. difficileFor the past 30 years, the cytotoxicity assay and culture have been

the reference tests for C. difficile infection. More recently, rapid tests,including toxin EIAs, GDH EIA, and PCR, have become available(Table 3).

Table 3 – Summary of available diagnostic tests forC. difficile infection

Test Time to detection

Reference test Cytotoxicity assay 6−48 h

Culture 24−48 h

Rapid test Toxin EIA (A/A+B) 15−60 min

GDH EIA 15−60 min

PCR 2−3 h

EIA, enzyme immunoassay; GDH, glutamate dehydrogenase; PCR, polymerase

chain reaction.

Cytotoxicity assay and cultureWith the cytotoxicity assay, a sterile filtrate of a stool sample

is added to a monolayer of tissue cultured cells and observed toidentify the specific cytopathic effect of C. difficile infection. Itappears often, within 6−48 hours. This is the most specific test forC. difficile infection, but it has only moderate sensitivity (60−70%)when compared to toxigenic culture. In addition, it is very timeconsuming, is technically difficult to accomplish, requires humancell cultures, and is not very well standardized.

Culture is still performed using the same cycloserine cefoxitinfructose agar media originally described in the 1970s.28 It is themost sensitive test available for diagnosing C. difficile infection andidentifies virtually every positive case. Many laboratories no longeruse this test owing to its labor-intensive and technical requirements,such as correct atmosphere, cell medium and pH. Once a colony isisolated, it can be tested for toxin production in about 20 minutes.This is termed “toxigenic culture”.

A prospective study evaluated the effectiveness of toxigenicculture in diagnosing C. difficile-associated diarrhea.29 Over 7 years,physicians at the Brussels Saint-Luc University Hospital collected10,552 stool samples from 7,042 patients for analysis using toxigenicculture. A total of 1,058 samples (10%) tested positive in the culture,and 460 (43.4%) of these tested positive for fecal cytotoxin. Theremaining 598 cultures were tested for toxigenicity, and 355 (59.4%)were found to be positive. Thus, toxigenic culture diagnosed overhalf of the cases of C. difficile-associated diarrhea that would havebeen missed using cytotoxicity assay alone. These results support aprotocol using toxigenic culture in addition to routine cytotoxicityassay. They also demonstrate that toxigenic culture remains the “goldstandard” in the evaluation of new diagnostic tests.

Rapid tests in the current clinical environmentIn the 1990s, a series of EIAs for C. difficile were developed

to improve accuracy and decrease turn-around time. Most of theseassays detected Toxin A, but some were also designed to detect bothToxins A and B, as some strains producing only Toxin B have beenreported in both Europe and North America.30,31 In addition, someEIAs were developed to detect GDH. Finally, PCR testing has alsobeen used to detect C. difficile infection.

EIAs are easier to perform and much faster than traditionaltests, yielding results in 20−40 minutes. Sensitivity with ToxinA and B EIAs is only slightly improved over cytotoxicity assays.GDH EIA has a much higher sensitivity (88.9%) compared with bothEIAs and cytotoxicity assays. Specificity is low, however, yieldinga low positive predictive value that makes the test unacceptableas a diagnostic test on its own (Table 4). 32,33 Nevertheless, thenegative predictive value is high, allowing the test to be used as

Table 4 – GDH versus PCR as C. difficile screening32,33

GDH

TC+ TC−

PCR

TC+ TC−

Versus toxigenic culture

number of positive (+) fecal samples GDH+ 16 21 PCR+ 44 7

number of negative (−) fecal samples GDH− 2 161 PCR− 10 204

Sensitivity, % 88.9 81.5

Specificity, % 88.5 96.7

Positive predictive value, % 43.2 86.3

Negative predictive value, % 98.8 95.3

GDH, glutamate dehydrogenase; PCR, polymerase chain reaction; TC, toxigenic

culture.

a helpful screening tool. In 20 minutes, clinicians can excludeC. difficile infection as a possible diagnosis if the test is negative. Bycomparison, PCR testing yields both good sensitivity and specificity,but the test detects the toxin gene, rather than the toxin itself or livingbacteria (Table 4). Further, PCR is expensive, limiting its usefulnessin screening and diagnosis.

Dr Oppenheim and colleagues recently evaluated C. difficiledetection by lateral flow assay for antigen and toxin (C. diffQuik Chek Complete®), an EIA (Toxin A&B assay alone), andtwo different commercial PCR methods. All four tests wereperformed on 150 consecutive liquid stool specimens collectedfrom patients aged �65 years who developed diarrhea at least48 hours after hospital admission. Samples were also evaluatedagainst two reference standards (cell cytotoxicity neutralization assayand toxigenic culture). Table 5 summarizes the performance of thefour investigated tests compared with toxigenic culture. 34

4 Conference report from an Alere International sponsored symposium

Table 5 – Performance of C. diff Quik Chek Complete®, Toxin A&B assay alone, and PCRcompared with toxigenic culture34

C. diff Quik Chek Complete®

GDH CDT

Toxin A&B assay alone PCR 1 a PCR 2 a

Sensitivity, % 100 61 44 100 94

Specificity, % 97 100 100 99 99

Positive predictive value, % 82 100 100 95 94

Negative predictive value, % 100 95 93 100 99

a Two different commercial PCR tests (1 and 2) were evaluated.

CDT, C. difficile toxin; GDH, glutamate dehydrogenase; PCR, polymerase chain reaction.

The C. diff Quik Chek Complete® two-stepdiagnostic algorithm

Similar to Prof. Wilkins, Prof. Delmee devised a simple algorithmfor diagnosing C. difficile in the clinical setting. The algorithm wasfirst designed as a three-step process, with GDH screening followedby Toxin A and B screening, and final diagnosis by toxigenicculture. 35 Validation by toxigenic culture at each stage showed that,of the 295 total samples, three positive samples were missed byGDH screening and 10 positive samples were missed by ToxinA and B screening.

In a simplified, two-step protocol, patients are first screened forC. difficile infection with the combined GDH and Toxin A&Bassay C. diff Quik Chek Complete®, followed by toxigenic cultureor PCR testing in patients with contradictory results (Figure 3). 32

Those with positive or negative results for both GDH and ToxinsA and B are reported as positive or negative, respectively. Thisconstitutes the vast majority of samples (80−90%), so that onlya small proportion of samples must be further evaluated withadditional testing. The protocol was validated with clinical testingand showed a sensitivity of 82.6%, a specificity of 98.1%, apositive predictive value of 82.6%, and a negative predictive valueof 98.1%.33 A two-step algorithm for C. difficile detection usingGDH and Toxin A&B assay followed by stool culture had a turn-around time of less than 4 hours for 92% of the specimens.36 A two-step algorithm involving GDH screening followed by PCR testingalso showed a high degree of accuracy.37 Notably, only 20% ofsamples required PCR confirmatory testing.

Prof. Delmee concluded that C. diff Quik Chek Complete® isa rapid, cost-effective screening tool with an excellent negativepredictive value, using combined GDH and Toxin A and B detection.

GDH and Tox A&B

+ +

+

+ – – – –

–

+

Report Pos

Report Pos

Report Neg

Report Neg

Culture or PCR

Figure 3. A two-step algorithm for C. difficile diagnosis in the clinical setting.GDH, glutamate dehydrogenase; NEG, negative; PCR, polymerase chainreaction; POS, positive.

According to the 2010 practice guidelines for C. difficile infectionin adults developed by the Society for Healthcare Epidemiology ofAmerica and the Infectious Diseases Society of America, a two-stepmethod for diagnosis is recommended, in which EIA screening withGDH detection is followed by cytotoxicity assay or toxigenic cultureas confirmation in positive samples. 38

“C. diff Quik Chek Complete® is a rapid,cost-effective screening tool with anexcellent negative predictive value, usingcombined GDH and Toxin A and B detection”

Lactoferrin: biomarker for C. difficile and IBD

Prof. Jost Langhorst discussed new concepts in the role oflactoferrin in C. difficile infection and in IBD and the utility oflactoferrin level in assessing these patients.

C. difficile infection begins with alteration of normal gut floradue to nosocomial or community-acquired C. difficile followed bygrowth of the pathogen and production of toxins. Tissue damageand inflammation caused by Toxins A and B follow, resulting indiarrhea and colitis due to influx of neutrophils and fluids. Patientswith IBD, including Crohn’s disease (CD) and UC, form a groupwho are especially vulnerable to gastrointestinal disease.

Identification of biomarkers helps in diagnosing patients withC. difficile-associated diarrhea and determining the severity of theinflammatory reaction. Lactoferrin is a glycoprotein found in manybody fluids and is an important part of the secondary granulesof activated neutrophils. 39 During intestinal inflammation, activatedneutrophils infiltrate the mucosa, increasing the level of lactoferrinin the intestinal lumen.39 Lactoferrin is a very useful biomarker,because it is stable in feces for several days at room temperatureand for months at −20ºC. As previously mentioned, research byWren and colleagues demonstrated that lactoferrin level directlycorrelates with the severity of C. difficile infection. According toProf. Langhorst, “As soon as inflammation caused by Toxins A and Bappears and diarrhea and colitis take place, lactoferrin is measurablyelevated in the feces.” According to Prof. Langhorst, lactoferrinlevel is a biomarker for intestinal inflammation, facilitating earlyintervention to ensure optimal outcomes.

Lactoferrin level is a biomarker for intestinalinflammation, facilitating early interventionto ensure optimal outcomes

Conference report from an Alere International sponsored symposium 5

Table 6 – Lactoferrin level in patients with UC, CD, and IBS46

UC

No inflammation Inflammation

CD

No inflammation Inflammation

IBS

No inflammation

N 15 27 10 33 54

Median lactoferrin, mg/mL 4.34 51.1 6.4 55.1 1.82

Lactoferrin range, mg/mL 0–104 1–1,669 0.01–103 1.3–1,795 0–90

CD, Crohn’s disease; IBS, irritable bowel syndrome; UC, ulcerative colitis.

Lactoferrin and IBDOngoing research is investigating lactoferrin in IBD, particularly

its role in monitoring treatment outcome and disease activity. 39−45

The presence of lactoferrin differentiates active IBD fromfunctional disease such as IBS. Prof. Langhorst and his colleaguesshowed an increased median lactoferrin level with inflammationin both active UC and CD patients compared to those with UCor CD in remission, or IBS and no inflammation (Table 6). Thedifferences in lactoferrin level between UC or CD patients withactive inflammation and patients with IBS were highly significant. 46

“High lactoferrin significantly discriminates active inflammatorybowel disease from functional IBS,” noted Prof. Langhorst.

“High lactoferrin significantly discriminatesactive inflammatory bowel disease from

functional IBS”

Vieira and colleagues showed that lactoferrin is an accuratemarker of intestinal inflammation in both UC and CD, with anoverall accuracy of 91% (sensitivity, 90%; specificity, 92%; positivepredictive value, 96%; negative predictive value, 83%). These resultswere more accurate than those of the clinical symptom index andC-reactive protein (CRP), and similar to those of the CD EndoscopicIndex of Severity for diagnosing intestinal inflammation.43

A separate study conducted by Prof. Langhorst and colleaguesevaluated the combination of biomarker, symptoms, and serummarkers in diagnosing UC and CD.45 Diagnostic accuracy of thecombination of biomarkers such as lactoferrin, symptoms such asdiarrhea or bloody stools, and CRP, was very high, especially forUC (95.3%). Diagnosis is more complicated in CD, which includesseveral subgroups of disease.

Lamb and colleagues monitored lactoferrin level postoperativelyin patients with CD. In uncomplicated postoperative courses,lactoferrin level normalized within 2 months. In patients with activedisease, the marker was elevated, and this proved to be more accuratein predicting clinical activity than were CRP, platelet count, orendoscopic appearance.42

Lactoferrin and C. difficile in IBDThe potential role of C. difficile as a confounding factor in

the clinical course of IBD was first discussed by Trnka andcolleagues three decades ago,46 and it remains a clinical dilemmatoday. Retrospective studies have shown an increase in the rate ofC. difficile infection in patients with IBD, particularly those withUC; infection rates of IBD inpatients have doubled or even tripledin recent years. 47−49 The link between C. difficile infection and IBDis inflammation (Figure 4). While lactoferrin level can indicate thatinflammation is occurring, it cannot determine the cause.

Inflammation

IBDC. difficile

infection

Figure 4. C. difficile infection and inflammatory bowel disease (IBD) bothlead to inflammation, which can be detected by raised lactoferrin levels.

As with the general population, risk factors for C. difficile-associated diarrhea in patients with IBD include hospitalizationand antibiotic therapy. In patients with IBD, immunomodulatorytherapy represents an additional risk factor for C. difficile-associateddiarrhea.50 The potential for IBD patients to harbor pathogenicbacteria may also serve as an independent predictor of C. difficileinfection. C. difficile carriers (GDH-positive patients) may developC. difficile infection over time. A prospective study showedC. difficile carrier status was more common in outpatients withIBD (8.2%), including UC (9.4%) and CD (6.9%), comparedwith outpatient controls (1%). Further, the higher carrier statusrate remained, regardless of known risk factors for C. difficileacquisition.51

Prof. Langhorst and colleagues conducted an additional study,which was presented at the 18th UEGW.52 Among 92 patientswith UC in clinical remission not receiving immunosuppressivemedication, 7% were C. difficile carriers (GDH positive). Amongthese, 66% exhibited elevated lactoferrin levels, indicating atleast low-grade inflammation. Of the entire study population of92 patients, 45 (49%) experienced a flare-up within 12 months. Ofthese patients, 11% were C. difficile carriers (GDH positive), andall showed elevated lactoferrin levels, consistent with a flare-up. Ofnote, none of the C. difficile carriers were positive for C. difficileinfection by cytotoxicity assay, and none of the carriers either inremission or during a flare-up required additional treatment withantibiotics. Thus, the presence of C. difficile without stool toxin wasa secondary phenomenon, rather than a risk factor for relapse, inUC outpatients not receiving immunosuppressive medication. Noneof these patients experienced C. difficile infection, demonstrating thepotential for over-treatment of C. difficile with antibiotics.

Summary and conclusionsInterest in the management of C. difficile infection continues

to grow as rates of infection increase in both Europe andNorth America, and as new detection methods become available.Cytotoxicity assay and toxigenic culture remain the reference testsfor diagnosis, but these methods require up to 48 hours forcompletion. Rapid screening tests include toxin EIA and GDH EIA,which can be completed in less than 1 hour, and PCR, whichcan be completed in less than 3 hours. While PCR yields bothgood sensitivity and specificity, the test is expensive to perform.In a simple, two-step algorithm, combined GDH and Toxin A&B

6 Conference report from an Alere International sponsored symposium

screening is followed by toxigenic culture or PCR only in samplesthat are only positive for GDH in the initial screening.

Lactoferrin is an important biomarker in C. difficile infection.Lactoferrin level increases with neutrophil infiltration in theintestines and is proportional to the degree of intestinalinflammation. The protein is a valuable biomarker of inflammationin C. difficile-associated diarrhea, and may be useful in combinationwith C. diff Quik Chek Complete® GDH/Toxin A&B screening todiagnose infection.

Lactoferrin level can differentiate inflammatory from functionalgastrointestinal disorders, and may detect and quantify intestinalinflammation in IBD. Recent results show that C. difficile carrierstatus is more common in inpatients as well as outpatients withIBD, and that this may be a secondary phenomenon, rather thana risk factor for C. difficile infection, in patients not receivingimmunosuppressive medication. Thus, in patients with active IBD,diagnosis of C. difficile requires proof of stool toxin, even withelevated levels of lactoferrin.

References1. Bartlett JG. Detection of Clostridium difficile infection. Infect ControlHosp Epidemiol 2010; 31(Suppl 1): S35−7.

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Conference report from an Alere International sponsored symposium 7

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8 Conference report from an Alere International sponsored symposium

Techlab C. DIFF QUIK CHEK COMPLETE® is the only device available to simultaneously detect

Clostridium difficile glutamate dehydrogenase (GDH) antigen and toxins A & B in a single reaction. It is an

accurate and easy to use method with results within 25 minutes and an NPV of 99%.

When Techlab C. DIFF QUIK CHEK COMPLETE® is used with a Techlab Lactoferrin test such as LEUKO

EZ VUE, it is a cost effective way of giving patients a faster diagnosis, while reducing the need for PCR.

The perfect partnership

Lactoferrin (LF) Test

Negative GDH

Negative Toxin

No further testing required C. difficile Infection

Positive GDH

Positive Toxin

Positive GDH

Negative Toxin

Further testing recommended

Positive LF

C. difficile Infection

"Window" to monitor toxins

Negative LF

Colonised

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