Historical Perspective

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1 The Diagnosis of C. difficile Infections: Does Molecular Make it More or Less difficile? Stephen M. Brecher Ph.D. VA Boston Health Care System BU School of Medicine The opinions expressed in this presentation are those of the presenter and do not necessarily represent the views of the Veterans Affairs HealthCare System I have no financial disclosures relevant to this presentation 1 Overview C. difficile historical perspective Case Histories The Patients The Tests The Patients and the Tests Treatment and Infection Control Summary and Conclusions Historical Perspective Bacillus difficilis (now C. difficile) was cultured from healthy neonates in In the 1960 s it was noted that patients on antibiotics developed diarrhea 2 Staphylococcal Colitis Originally thought to be caused by S. aureus and treated with oral bacitracin Stool cultures routinely ordered for S. aureus Early 1970 s, a new explanation Clindamycin Colitis Severe diarrhea, pseudomembrane colitis, and occasional deaths documented in patients on clindamycin 1. Hall, J.C. and O Toole E Am J Dis Child. 49: Gorbach S.L NEJM.341: Antibiotic Associated Pseudomembranous Colitis Due to Toxin-Producing Bacteria 1 In 1978, C. difficile was shown to be the cause of many cases of hospital/antibiotic-associated diarrhea Bartlett and co-workers demonstrated cytotoxicity in tissue culture and enterocolitis in Syrian Hamsters with stool isolates of C. difficile isolated originally from patients with pseudomembranous colitis 1. Bartlett, J.G. et al NEJM. 298: Factors That Complicated the Discovery of CDI C. difficile is found in healthy infants who appear to be refractile to CDI 1 Infant intestinal cells do not appear to have receptors for toxins A and B Antibiotics often cause diarrhea unrelated to C. difficile by disrupting the intestinal microbiome You have bacterial cells and human cells The bacterial cells in your intestine are trying to eat and reproduce (by digesting your food) Antibiotics are trying to kill them What would you do if someone was trying to kill you while you were trying to eat and reproduce? 1. Rousseau, C. et al J Clin Microbiol. 49: 858

2 Case 1 # 3 Case Presentations 60 YO male admitted to a hospital for community acquired pneumonia, treated with levofloxacin and discharged 7 days later, seen at another hospital because of pound weight gain over the last few days ( my abdomen has never been so big ) and hypertension (213/106) Afebrile, WBC of 8.5, albumin 3.1, creatinine 0.9, no diarrhea noted Admitted, treated for hypertension and ciprofloxacin given to complete treatment for CAP. Discharged 3 days later 7 Case Study Con t Presents to ER 3 days after discharge Fever (101), diarrhea, generally feeling ill, no abdominal pain WBC 27.8K, albumin 2.9, creatinine 1.2 Admitted with C. difficile colitis listed as a possible dx, but not treated (except for levofloxacin) Day 2, 10 stools/day, altered mental status C. difficile EIA for toxins A/B positive; put on metronidazole 500 mg TID Case 1 Continued Day 3: transferred to SICU, anuric, abdominal pain, distension, developed cardiac complications, ventilated, renal failure. Poor prognosis and colectomy ruled out following surgical consult Oral and rectal vancomycin added WBC > 30K, albumin 2.3, creatinine 3.1 Day 4, WBC 59.6K, toxic megacolon Day 5, WBC 88K, made DNI/DNR, died Case 2 31 yo pregnant female (14 weeks, twins) seen at a local ER with history of 3 weeks intermittent diarrhea 3 days cramping and watery diarrhea Stool + for C. difficile toxin Received T/S for UTI 3 months prior to ER visit Admitted, treated with metronidazole and discharged Readmitted next day with severe colitis Treated in hospital for 18 days with metronidazole, oral vancomycin and cholestyramine, discharged Case 2 Readmitted 4 days later Diarrhea and hypotension Spontaneously aborted her fetuses Subtotal colectomy, aggressive therapy Died on 3rd day Post-mortem showed toxic megacolon with evidence of pseudomembranous colitis MMWR 54:(47);

3 Case # 3 74 YO male with ESRD, DM, seizure disorders, vascular dementia, meningioma, esophageal adenocarcinoma, PVD, and HTN Fecal incontinence Case 3: C. difficile Assays Date WBC Test/comment K Toxin A/B EIA K Toxin B gene PCR K Toxin B gene PCR K Toxin B gene PCR K Toxin B gene PCR K Toxin B gene PCR +, EIA -, GDH K Toxin B gene PCR K Toxin B gene PCR Not done Note written by ID, GI and Microbiologist Not done Toxin B gene PCR - 14 Case 3: Team Meeting Arranged a 3-way meeting with GI and ID Many reasons for this patient to have diarrhea Team wrote a note that patient is most likely colonized with low levels of C. difficile (GDH -, Toxin EIA -, PCR +) Do not order or treat for CDI unless significant leukocytosis Why Did I Present These 3 Cases? Case 1: Rapid recognition and appropriate treatment of severe disease is essential if we are to prevent mortality Case 2: Community based CDI exists and we do not understand the risk factors Pregnancy, lack of protective flora, medications other than antibiotics Community based virulent strains 1 Case 3: False positives with PCR, need to explore alternative explanations for diarrhea 1. Goorhuis, A. et al. CID 2010; 47: Who Should I Test? Acute Infectious Diarrhea million cases/year in USA Why does my patient have diarrhea? Infectious causes vs non-infectious causes Gastroenteritis usually associated with nausea and vomiting Clinically Significant Diarrhea >3 loose unformed stools/24 hours or >250 g of unformed stool/day 1. DuPont, HL. N Engl J Med :

4 Burden of CDI in the US surveillance study of 10 geographical area in US used to predict annual number cases, types, and outcomes 453,000 cases, 83,000 first recurrences 29,300 deaths 65.8% HCA (24.2% while in a hospital) 34.2% CA Risk : female, white, > 65 years old 1. Lessa et. al. N Eng J Med : Goals of Testing Identify cases of CDI and rule out CDI in patients with diarrhea 1 Initiate specific treatment plans for patients with CDI Maximize infection control interventions and environmental cleaning in rooms of CDI patients and carriers to prevent transmission 1. Polage, CR et al. Nosocomial Diarrhea: Evaluation and treatment of causes other than C. difficile. Clin Infect Dis : Changing Difficiliology It used to be easy Hospitalized patients on antibiotics with diarrhea Bad tests but we didn t know better and repeated them until they were positive (CD x 3 or more) No longer easy because Community, healthcare associated and nosocomial CDI Risk factors beyond antibiotics Many reasons for diarrhea, particularly, in hospitalized patients 21 Colonization or Infection Any microbiological result from an assay done on a patient sample that has a microbiome must be interpreted with respect to the findings and the patient s clinical symptoms This is not a new concept Respiratory carriage of S. pneumoniae, H. influenzae and N. meningitidis Multi-site carriage of S. aureus Remember ordering a diagnostic lab test is like picking your nose in public: you must first consider what you will do if you find something Catherine D. DeAngelis. Arch Pediatr Adolesc Med. 148: Who to Test Persons with 3 unformed BM within 24 hours with risk factors for CDI (Clinically Significant Diarrhea) WBC, creatinine, albumin, antibiotics, IBD, surgery, and older age (older than me) Patients who completed therapy who still have CSD Do not perform tests on everyone with diarrhea Laxatives, tube-feeding, diabetes, etc. Do not perform tests on asymptomatic patients Do not get coerced by Test of Cure requests Cured patients can carry toxigenic C. difficile 23 What to Test The Brecher Guidelines 1 Only test loose or liquid stool If it ain t loose, it s of no use Stick test for stool consistency If the stick stands, the test is banned If the stick falls, test them all Bristol Chart 5-7? 1. Brecher mindfart (an idea that slips out on it s own) but for a reference: Brecher, SM et al CID.57:

5 What Clinical Symptoms Help Determine if the Patient has CDI? C. difficile Clinical Picture Clinical symptoms Increased number of unformed bowel movements 3, 6, 9 progression Leukocytosis Increased creatinine (1.5 x baseline) Decreased albumin Increased serum lactate Varying definitions of mild, moderate, and severe disease based on above parameters Do not monitor by fecal leukocytes or related enzymes C. difficile Virulence Factors CDI Pathogenesis 1 Production of Toxins A and B Increased production of toxins in certain ribotypes due to deletions in regulatory genes Why does C. difficile make these toxins? Resistance to non-treatment antibiotics Fluoroquinolones, macrolides, etc. Ability to form spores Some ribotypes do this better than others Antibiotics do not kill spores recurrent disease Environmental spore survival transmission Surface molecules (proteins, lipids) that promote colonization and infection 27 Toxins A and B inactivate Rho guanosine triphosphates Colonocyte death Intestinal barrier function Neutrophilic colitis Extra-GI invasion is rare Age, immune response, antibiotics associated with severity/recurrence 1. Lessa et. al. N Eng J Med : CDI Testing Issues Cost vs Value Test Menus/Test Costs 2-3 step algorithms (difficile dancing) Molecular (PCR/LAMP) alone or in combination Colonization or infection Do we need a positive toxin assay in a PCR/LAMP positive patient? Does time to result improve time to treatment and improve outcome? Cost is of little value if the results are inaccurate Low sensitivity Low specificity Repeat testing Value is measured by impact of the test result on the patient and the facility Increased sensitivity Increased specificity Increased productivity Improved patient care The most expensive test is one that does not work 29 30

6 Ye Old Laboratory Diagnosis of CDI Glutamate Dehydrogenase (GDH) Toxigenic Culture (Culture and CCNA) Cdiff sniff by Cliff Enzyme Immunoassay (EIA) ToxinsA/B Laboratory Diagnosis Molecular Based (PCR Or LAMP) Cell Culture Neutralization Assay (CCNA) Stool Culture The Current Laboratory Diagnosis of CDI Glutamate Dehydrogenase (GDH) Laboratory Diagnosis Enzyme Immunoassay (EIA) for Toxins A/B Molecular Based (PCR Or LAMP) Glutamate Dehydrogenase (GDH) GDH is a metabolic enzyme that is found in strains of C. difficile GDH EIA has High sensitivity (NPV is very high) Low specificity (PPV is low) a + test needs another test (toxin +/- NAAT) Geographical differences in the distribution of certain ribotypes may effect test performance 1 1, 2 or 3 Step Testing Difficile Dancing? A 2-test/1 card EIA for GDH and Toxins A/B with discrepant results resolved by a molecular technique has become a popular alternative to EIA for toxins A/B alone A GDH screen with GDH positives tested by a molecular assay 1. Tenover, F.C. et al J. Clin. Microbiol. 48: C. Diff Quik Chek Complete Lateral flow EIA for GDH and Toxins A/B on one test card Quinn et al 1 reported that if Both + = + Both - = % discrepant, re-test. Use PCR Sharp et al 2 reported that 88% of specimens were both positive or both negative Used random access PCR to resolve remaining 12% 1. Quinn, C. D J Clin Microbiol. 48: Sharp, SE et al J Clin Microbiol. 48: Molecular Testing There are now many molecular assays for detection of toxin A or toxin B genes They do not detect toxins A and B They all work well, cost more and are significantly more sensitive than EIA Is sensitivity more important than specificity? How are they best utilized? 36

7 Do I Need to Perform a Toxin EIA With or Without a PCR? Large UK study correlated toxin levels with mortality 1 Single hospital US Study also correlated toxin positivity with poor outcomes 2 US study reported no correlation between EIA assay positivity and severity of disease 3 Issue is that we still do not have a rapid and reliable toxin assay 1. Planche TD et al. Lancet Infect Dis : Polage, CR et al. JAMA Intern Med. Doi 10:1001/jamainternmed Romney, RM et al. J. Clin Microbiol : Positive Toxin Assay With Poor Outcome in CDI 1 Large national study in England Outcome in 6522 inpatient episodes Cytotoxin positive (16.5% Mortality) Cytotoxigenic culture positive (9.7%) Above tests both negative (8.6%) Toxin production correlated with mortality Cytotoxin -, cytotoxigenic culture + = C. difficile excretor 1. Planche,TD et al. Lancet ID : Economic Evaluation of Different CDI Testing Strategies 1 Meta-analysis, 5 studies, 4,582 patients Patient management based on laboratory results CDI testing strategies odpcr, bpcr, LF GDH EIA, plate GDH EIA, EIA Toxin, TC cytotoxicity Cost measured by complex formula odpcr most cost effective, although most expensive 1. Schroeder, LF at al J. Clin Microbiol. 52: Molecular Testing Increases CDI With molecular tests, the number of CDI positive tests increases Important to only test patients with CSD Must differentiate between infection and colonization Sometimes this is very difficult to do Is increased detection of carriers important? Isolation? IC says Yes Treatment? Most say No 40 Molecular Detection of Carriers of CDI at time of Hospital admission 1 Screened rectal swabs from new admissions for tcdb gene by PCR Carriers placed on contact precautions during hospitalization CDI rates were compared for pre and post intervention During intervention CDI rate was 3.0/10000 patients days (down from 6.9) Concluded that detecting and isolating CD carriers was associated with a significant decrease in HA-CDI 1. Longtin, Y. et al. JAMA Intern Med. Doi: /jamainternmed Increased Detection Infection or Colonization Compared PCR to a 3 step algorithm BD GeneOhm PCR for toxin B gene GDH (Diff-Chek-60, then EIA for Toxins A/B (Quik- Chek) then Cell Culture Neutralization (Vero cells) Cases defined by diarrhea or histopathology/direct visualization Results PCR 85 positives 3-Steps 56 positives Positives increased by >50% 29 cases of CDI were there but not be detected by a 3-step assay More severe cases associated with positive toxin detection Longtin, Y. et al CID.56: 67-73

8 Impact of Clinical Symptoms on Interpretation of Diagnostic Assays for CDI 1 Compared 8 methods and 2 gold-standards with and without clinical symptoms in 150 patients TC & CSD 35 Positives TC & No CSD 44 Positives 4 + assays & CSD 40 Positives 4+ assays & No CSD 50 Positives 20 % of patients had laxatives 36% did not have clinically significant diarrhea 1. Dubberke, E. et al J Clin Microbiol. 49: A Sniff by Cliff Will Detect Cdiff Diagnostic accuracy of dog (Cliff) for detecting Clostridium difficile infection (CDI) Next Generation Toxin Testing Cliff detected 50+ stool samples correctly Cliff detected 50- stool samples correctly Nurses noses did not smell as well (3 studies) Bomers M K et al. BMJ 2012;345:bmj.e7396 (great video link) Ultrasensitive single molecule array (Simoa) technology LOD significantly better than current toxin assays Toxin levels 1.5 to 1.7 times higher in patients with CDI attributable severe outcomes 1. Song, L. et al doi: /JCM by British Medical Journal Publishing Group 46 If the First PCR is Negative Should I Order Another PCR? Of 406 tests from 293 patients with a prior negative PCR negative 10 positive Only 3+ in <7 days Exceptions Severe clinical changes 1. Luo RF, Banaei N. J Clin Microbiol. 2010;48: Do We Need to Test for Hypervirulent Ribotypes? NAP1/B1/027 is associated with more severe disease and can have higher treatment failures and relapse rates 1-4 In a recent study of 310 cases of CDI (43 classified as severe), ribotype was not a predictor of severe disease. WBC and albumin were more clinically relevant 5 Should we treat patients based on symptoms and severity of disease or treat based on the strain type? Most FDA approved assays cannot rapidly detect the gene deletion associated with this strain (tcdc) Other strains may also be more virulent Molecular characterization is a valuable tool for big picture epidemiological investgations 6 Ribotype 244 (Australia) shown to be hypervirulent and tests positive as 027 on the Cepheid PCR (both have a tcdc deletion mutation at position 117) 7 1. Louie TJ, et al. N Eng J Med. 2011;364: Cornely, OA et al. Lancet Infect Dis.2012; 12: Figueroa, I. et al CID; 55: S104-S109, 4. See, I CID. 58: Walk, S.T. et al CID. 55: Wilcox, M.H. et al CID. 55: Lim, SK, et al. CID :

9 Recurrence Relapse or New Infection? Recurrence associated with age>65 and failure of host to make antibodies to toxin B 1 Is recurrence associated with the same strain or a different strain? Of patients with second episodes within 8 weeks, 88% (75/85) had the same strain 2 Of patients with second episodes > 8 weeks, 65% (32/49) had the same strain 1 Similar results from Figueroa et al 3 Diarrhea after an initial episode of CDI may not be CDI 4 1. Gupta, SB et al Clin Infect Dis. 63: Kamboj, M. et al Clin Infect Dis.53: Figueroa, I et al Clin Infect Dis. 55: S104-S10 4. Polage, CR et al Clin Infect Dis. 55: Guidelines for the Diagnosis, Treatment, and the Prevention of Clostridium difficile infections From Table 1: Diagnostic Tests 1. Only stools from patients with diarrhea should be tested 2. NAATs for C. difficile toxin genes such as PCR are superior to toxins A+B testing as a standard diagnostic test for CDI 3. GDH screening tests for C. difficile can be used in 2-3 step screening algorithms with subsequent toxin A+B EIA testing, but the sensitivity of such strategies is lower than NAATs 4. Repeat testing should be discouraged 5. Testing for cure should not be done Am J Gastroenterol advance online publication. 26 February 2013; doi: /ajg Recommendations 2016 Acceptable strategies EIA for GDH and/or toxins A/B with a molecular assay for discrepant results A molecular test with or without a confirmatory toxin assay as long as results are coordinated with clinical data Unacceptable A stand-alone EIA for toxins A/B A stand alone EIA for GDH without a second test for positives Treatment and Management Stop Concomitant Antibiotics (Whenever Possible) Continued use of non-c. difficile antibiotics after diagnosis of CDI associated with an odds ratio of 4.23 (P<0.001) for recurrent disease 1 Phase 3 study of fidaxomicin and vancomycin linked concomitant antibiotics with lower rates of global cure (initial cure without recurrence at 30d): 2 Patients, % P<0.001 FDX VAN Total FDX VAN Total GLOBAL CURE SHEA/IDSA Treatment Recommendations Clinical scenario Supportive clinical data Recommended treatment Mild to moderate Leukocytosis (WBC < 15,000 cells/ul) or SCr level < 1.5 times premorbid level Severe Leukocytosis (WBC 15,000 cells/ul) or SCr level 1.5 times premorbid level Severe, complicated Hypotension or shock, ileus, megacolon Metronidazole 500 mg 3 times per day PO for days Vancomycin 125 mg 4 times per day PO for days Vancomycin 500 mg 4 times per day PO or by nasogastric tube plus metronidazole 500 mg IV q 8 hrs 1. Garey KW, et al. J Hosp Infect. 2008;70: Johnson S, et al. DDW 2010; Abstract 711c. Cohen SH, et al. Infect Control Hosp Epidemiol. 2010;31(5):

10 European Society of Clinical Microbiology and Infectious Diseases: update of the treatment guidance document for Clostridium difficile infection Clinical Microbiology and Infection pages 1-26, 20 JAN 2014 DOI: / Fecal Microbiota Transplantation Superior treatment for recurrent CDI 1 Most cost effective strategy for recurrent CD1 2 NG tube, colonoscopy, other Fresh, frozen, spouse, professional, etc. Intestinal repoopulation (pills) Bottom Line Usually works within 24 hours > 90% long term cure rate 1. Moore, T. et al CID.58: Konijeti, GG. et al CID CDI Treatment Based on Family Relationships First Episode Mother in Law: Metronidazole Step-Mother: Vancomycin Mother: Fidaxomicin Recurrence Mother in Law: Poop Step-Mother: Fecal Transplant Mother: Microbiome Enhancement Prevention of CDI Transmission between patients and healthcare professionals within hospitals represents major source of C. difficile acquisition Prevention associated with Hand hygiene policies Duration of isolation Environmental cleaning practices Antimicrobial stewardship programs Better detection = improved prevention 57 APIC 2010 Clostridium difficile Pace of Progress Survey. Available at: Accessed January 31, Create Team CDIFF Members ID physician, IC guru, GI physician, microbiologist, pharmacist, building management specialist, hospital administrator,?cliff Mission Communication and education for value effective test and treatment strategies, CD transmission control, and antibiotic stewardship 59 Summary and Conclusions No laboratory test can diagnose Clostridium difficile infection (CDI). 1 Correlate test results with the patient Limit testing to at risk patients with clinically significant diarrhea Pick the assay or assays that best fit the needs of your patients and facility Treat patients with severe disease aggressively Consider creating a difficile management team 1.Dubberke, ER and CA Burnham JAMA. Published online