1 / 52

Screening methods for Carbapenemase-producing Enterobacteriaceae

Screening methods for Carbapenemase-producing Enterobacteriaceae. Adrian Brink Clinical Microbiologist, Ampath National Laboratory Services, Milpark Hospital, Johannesburg and Department of Clinical Microbiology & Infectious Diseases, University of Witwatersrand, Johannesburg, South Africa.

buckminster-hays
Télécharger la présentation

Screening methods for Carbapenemase-producing Enterobacteriaceae

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Screening methods for Carbapenemase-producing Enterobacteriaceae Adrian Brink Clinical Microbiologist, Ampath National Laboratory Services, Milpark Hospital, Johannesburg and Department of Clinical Microbiology & Infectious Diseases, University of Witwatersrand, Johannesburg, South Africa

  2. Scope of presentation • Introduction: Pro-active vs Reactive strategies to combat spread of CPE • Screening methods for GIT colonization of CPE- Culture vs PCR • The clinical relevance of a positive screening PCR and/or culture • Screening methods for carbapenem-resistant clinical isolates of Enterobacteriaceae • Conclusions

  3. Introduction

  4. Introduction CurrOpin in Inf Dis 2010;23:327–33

  5. Proactive vs Reactive strategies • Successful infection control measures are based on early detection and containment through isolation and cohorting. • A reactive approach to infection control will advocate directed action after the problem is detected, that is a cluster of CPE infections has been detected. This approach has been applied traditionally in most parts of the world to confront various MDR organisms. • The reactive approach does not advocate allocating resources upfront before the problem has become evident, and resource allocation to confront the outbreak is proportional to the local epidemiology. • This approach is aimed at control of an outbreak (and is often appealing to healthcare administrators who are under competing demands for resource allocation).

  6. Proactive vs Reactive strategies • On the contrary, the proactive approach assumes that allocating resources upfront will allow earlier detection and containment. • The proactive approach assumes that due to logarithmic escalation of such an outbreak, it is more cost-effective to combat the problem before it has been established. • The proactive approach will aim to achieve eradication even when difficult and resource-consuming, and when not feasible will aim at containment at the lowest achievable levels of spread

  7. Proactive vs Reactive strategies • Active screening included ICU patients on admission & weekly there-after which is currently not included in our SASCM guideline • Netcare (n=54 hospitals): 1st Oct 2013-1st Sept 2014 • 1004 new colonized pts identified with PCR/culture • 20.3% (204/1004) were weekly ICU screens after 2 weeks in ICU • 42% (86/204) were VIM and of those cultured 55% were CPE & 45% were P.aeruginosa • Courtesy D vd Bergh, L Devenish, K Swart Saidel-Odes et al. Infect Drug Resistance 2014;7:9-14

  8. ESCMID guideline for the management of infection control measures

  9. Proactive vs Reactive strategies Schwaber et al. Clin Infect Dis 2014;58:697–703

  10. Proportion of newly detected carbapenem-resistant Enterobacteriaceae carriers identified via active (PCR& culture) surveillance vs clinical cultures by year, 2007–2012 Schwaber et al. Clin Infect Dis 2014;58:697–703

  11. Impact of active surveillance on detection of carriers and subsequent isolation of CPE in clinical specimens • “Although the preferred microbiological method for screening was not initially specified, there is a requirement that the test chosen provides at least a preliminary result within 24 hours, which allows a decision regarding isolation”. Schwaber et al. Clin Infect Dis 2014;58:697–703

  12. “ If you know the enemy and know yourself, you need not fear the result of a hundred battles. If you know yourself but not the enemy, for every victory gained you will also suffer a defeat. If you know neither the enemy nor yourself, you will succumb in every battle”

  13. Genetics as the source of high transfer frequency of the OXA-48 gene amongst Enterobacteriaceae Potron et al. Antimicrob Agents Chemother 2014;58:467-71

  14. Genetics as the source of high transfer frequency of the OXA-48 gene amongst Enterobacteriaceae • The current emergence of the carbapenemase OXA-48 among Enterobacteriaceae is related to the spread of a single IncL/M-type • plasmid, pOXA-48a. • This plasmid harbors the blaOXA-48 gene within a composite transposon, Tn1999, which is inserted into the tirgene, encoding a transfer inhibition protein. • Patron et al showed that the insertion of Tn1999 into thetirgene negatively impacts on transfer inhibition and hence is involved in a higher transfer frequency of plasmid pOXA-48a. • This may likely be the key factor for the successful global dissemination of this plasmid. Potron et al. Antimicrob Agents Chemother 2014;58:467-71

  15. Plasmid NDM-HK Encoding NDM-1 Plos ONE March 2011; 6(3) e17989

  16. Screening methods for GIT colonization of CPE- Culture

  17. The problem with detecting the carbapenemases

  18. Published evaluations of media / methods for detecting CPE in various patient populations

  19. Published evaluations of media / methods for detecting CPE in various patient populations • In all such studies, the calculation of sensitivity and specificity is based on the supposition that all isolates of CPE will be successfully detected by at least one of the methods under evaluation – although this may not actually be the case. • The performance of a particular method may also be exaggerated if it is assessed alongside a relatively poor comparator. • Finally, most studies are performed in a single location where a single type of carbapenemase may predominate, and different media may show different performances in different geographical locations.

  20. Published evaluations of media / methods for detecting CPE in various patient populations • A Drigalski agar-based culture medium containing ertapenem, cloxacillin, and zinc sulfate • Overall sensitivity 95.6% (NDM, OXA-48, KPC, VIM, IMP) • So called Supercarba medium Nordmann et al. J Clin Micro Dis 2012;50:2761-66.

  21. Published evaluations of media / methods for detecting CPE in various patient populations Girlich et al. Diagn Microbiol Infect Dis 2013;75:214-7.

  22. Published evaluations of media / methods for detecting CPE in various patient populations Girlich et al. Diagn Microbiol Infect Dis 2013;75:214-7.

  23. Published evaluations of media / methods for detecting CPE in various patient populations • There are no published evaluations of SUPERCARBA with samples from colonized patients in routine practice at this time. • Its shelf life is at least 7 to 10 days Girlich et al. Diagn Microbiol Infect Dis 2013;75:214-7.

  24. Screening methods for GIT colonization of CPE- PCR

  25. PCR for detecting CPE carriage

  26. PCR for detecting CPE carriage

  27. PCR for detecting CPE carriage

  28. PCR for detecting CPE carriage

  29. PCR for detecting CPE carriage • PCR-based assays with their rapid turnaround time and high sensitivity are an attractive alternative to culture • However, the role of PCR-based screening methods has yet to be fully elucidated and has mainly been used in outbreaks and/or in high endemicity setting • PCR (multiplex) is an excellent method for excluding CPE carrier status in patients and has high sensitivity, specificity, and negative predictive values • Specificity for CPE “compromised” by non-fermentors “hosting” carbapenemases such as VIM or GES

  30. The clinical relevance of a positive PCR and/or culture screening

  31. The clinical relevance of pos screening PCR +/-culture In ICU patients using culture, 46% of screen-positive patients isolated a carbapenem-resistant K. pneumoniae from a clinical specimen Calfee et al. Infect Ctrl Hosp Epidem 2008;29:966-968

  32. The clinical relevance of pos screening PCR +/-culture 20% of patients with clinical infections due to KPC-KP had a positive screening culture that was preceded by a positive PCR result Giani et al. JHosp Infect 2012:81:119-122

  33. The clinical relevance of pos screening PCR +/-culture • Similarly 47% of PCR + culture screen-positive patients in an Israeli ICU went on to isolate CR-KP from clinical cultures. • The median duration between screening cultures and the recovery of clinical microbiologic cultures was 13.6 days (range 2-66 days)

  34. Proportion of newly detected carbapenem-resistant Enterobacteriaceae carriers identified via active (PCR& culture) surveillance vs clinical cultures by year, 2007–2012 Schwaber et al. Clin Infect Dis 2014;58:697–703

  35. The clinical relevance of pos screening PCR +/-culture • Thus, in addition to potential infection control benefits, does the information gained from surveillance PCR/culture also assist in medical decision making? • Similar to a Candida colonization index to predict ICs? • Hence, additional studies of the impact of detection of asymptomatic colonization on subsequent infection and outcome is needed • Increasing school of thought that broad-spectrum antibiotic use may select for overgrowth and invasion, and hence AMS crucial in this regardsubsequent infection and outcome is needed

  36. Screening methods for carbapenem-resistant clinical isolates of Enterobacteriaceae

  37. Detection of carbapenemase producers in infected pathogens (clinical specimens) • Susceptibility testing: • Imipenem, ertapenem, meropenem. • CLSI vs EUCAST breakpoints • Phenotypic detection: • Modified Hodge-test • EDTA, clavulanic acid, boronic acid • Carba NP test • Carbapenem hydrolysis • UV spectrophotometry & mass spectrometry • Molecular biology • Single or multiplex PCRs +/- sequencing • Real-time PCR • DNA Micro-array

  38. Screening cut-off EUCAST • MIC Susceptibility testing • Imipenem > 1 mg/L • Meropenem > 0.12 mg/L • Ertapenem > 0.12 mg/L

  39. Modified Hodge Test (MHT) • The modified Hodge test has an excellent sensitivity for detecting enterobacterial isolates producing Ambler class A (KPC) and class D (OXA-48) carbapenemases. • Its sensitivity is low for NDM-1 producers (50%), but is increased to 85.7% by adding ZnSO4(100 µg/ml) in the culture medium. • MHT may lack sensitivity for detecting carbapenemase activity in Enterobacterspp • However, this test has a low specificity and is time-consuming. Gerlich et al. J Clin Micro 2012;477-479

  40. Inhibition-based carbapenemase detection • The added value of the inhibition-based carbapenemase detection tests remains variable. • These tests are, for example, based on inhibition by tazobactam, clavulanic acid or boronic acid for detecting the production of Ambler class A carbapenemases (KPC), and inhibition by EDTA or dipicolinic acid for detection of MBL activity. • They are time-consuming and have variable sensitivity and specificity & require trained microbiologists Nordmann et al. J Antimicrob Chemother 2013;68:487-489

  41. Carba-NP test • The Carba NP test is inexpensive, rapid (<2 hours) and easy to perform on colonies and to interpret • It utilises imipenem hydrolysis visualised by a colour change to identify carbapenemase production in Enterobacteriaceae and Pseudomonas species • This test is 100% sensitive and specific, as are molecular techniques. • Needs no equipment • It detects not only all known carbapenemases (belonging to Ambler A, B and D classes) in Enterobacteriaceae but should also identify virtually any new emerging carbapenemase, in contrast to molecular techniques. Nordmann et al. Emerging Infect Dis 2012;18:1503-7

  42. Carba-NP test • 5-hour old cultures • Sensitivity from early cultures was 94% for detection of carbapenemase production in Enterobacteriaceae.

  43. CARBA NP (CNP)-test vs MHT • Sensitivities were comparable (CNP, 100%, versus MHT, 98%;P0.08), but CNP was more specific (100% versus 80%;P<0.0001) and faster Vasoo et al. J Clin Micro 2013;51:3097-3101

  44. UV spectrophotometry • UV spectrophotometry has a 100% sensitivity and 98.5% specificity for detecting any kind of carbapenemase activity. • This cheap technique can accurately differentiate carbapenemase producers from non-carbapenemase producers among carbapenem-non-susceptible isolates [outer membrane permeability defect, overproduction of cephalosporinases or/and extended-spectrum b-lactamases (ESBLs) • Only in reference laboratories Bernabeu et al. DiagnMicrobiol Infect Dis 2012;74: 88– 90.

  45. Mass spectrometry MALDI-TOF • Broth culture of the strain to be tested + carbapenem-3-6 hours • Mass spectrometry • If carbapenemase +, hydrolysis of the carbapenem molecule leads to a degradation product • Advantage: Specific/Sensitive & cheap if you already have one • Disadvantage: Capital expense & expertice

  46. MALDI-TOF vs CARBA NP • Using PCR as the reference method, both tests demonstrated a sensitivity of 87% and a specificity of 100%

  47. Molecular biology • Molecular techniques remain the gold standard for the precise identification of carbapenemase genes & most of these techniques are based on PCR and may be followed by a sequencing step if a precise identification of the carbapenemase gene is needed (e.g. VIM type, KPC type, NDM type or OXA-48 type). • They are either single or multiplex PCR techniques. • A PCR technique performed directly on colonies can give results within 4–6 h (or less when using real-time PCR technology) with excellent sensitivity and specificity. Similarly, other molecular techniques are useful for this purpose. Nordmann et al. J Antimicrob Chemother 2013;68:487-489

  48. Molecular biology • The main disadvantages of the molecular based technologies are besides the cost and the requirement for trained microbiologists: • Lack of detection of carbapenemase activity due to novel unidentified genes or infrequent carbapenemase genes • Detection of carbapenemase-like gene without carbapenemase activity e.g OXA-48-like with narrow spectrum activity such as OXA-163, OXA-204, OXA-232 & OXA-405 Nordmann et al. J Antimicrob Chemother 2013;68:487-489

  49. Screening criteria for Multiplex PCR of carba NS Enterobacteriaceae?

  50. Conclusion

More Related