감염관리를위한진단검사 다제내성균감시검사 (MULTIDRUG RESISTANT ORGANISMS) 조지현원광의대진단검사의학과
다제내성균감시검사 1. MDRO 정의 (multidrug resistant organisms) 2. MDRO 중요성과국내 MDRO 현황 3. MDRO 검사 (screen, confirm) 1) MRSA, VRSA/VISA, CNS 2) VRE 3) ESBL 4) CRE, MRPA, MRAB 4. 보균자검사 ( 전파예방및관리 ) 5. 요약 참고자료 1) CDC. HAIs; Laboratory Resources (http://www.cdc.gov/hai/settings/lab/lab_settings.html) 2) CLSI 2014 M100 S24 Performance Standards for AST 3) 질병관리본부. 다제내성균감염관리지침 2012.10. 4) 질병관리본부. 카바페넴내성장내균속감염관리지침 2012. 5) 질병관리본부. 의료관련감염증표준진단법 ( 송원근 2013). 6) 원광대병원 2013년감수성통계
MDRO 정의 : No consensus But, Microorganisms, predominantly bacteria, that are resistant to one or more classes of antimicrobial agents G(+) G( ) Staphylococcus MRSA, VISA, VRSA Enterococcus VRE Streptococcus S. S. pneumoniae Enterobacteriaceae K. K. pneumoniae, E. E. coli (ESBL) Enterobacter, Serratia, Citrobacter, Morganella, etc Non fermenter A. A. baumannii, P. P. aeruginosa, S. S. maltophilia, B. B. cepacia, R. R. picketti Clostridium difficile Mycobacterium MTB
중요성 : 감염병원인 1) serious, difficult to treat 2) morbidity, mortality 3) length of stay 4) cost 예방가능 1) Judicious use of antimicrobials decrease incidence of microorgnisms developing antibiotic resistance 2) Acquired via transmission by 근무자 환자 환경 환자 환자 환자
전파방지 : 손위생 (Hand hygiene) 가장중요 격리주의 (Isolation precautions) Follow standard precautions for all patients Personal protection equipment (gloves, gown, mask, goggle, face shield) Develop & utilize systems to identify patients with MDROs & notify infection preventionists, physicians & direct caregivers Place patients with a confirmed MDRO or history of an MDRO in singepatient rooms Group patients with the same MDRO in designated areas if single patient room unavailable Implement contact precautions immediately for all patients infected with MDROs, previously identified MDRO or have a history of being colonized with target MDROs. Notify infection prevention and control after placing the patient in isolation 접촉주의 (Contact precautions) 환경관리 (Environmental measures)
KONIS 의료관련감염 report (2013 7 9; 79 Hosp > 300 bed ) 질병관리본부 : 감염병관리 > 감염병감시 > 의료관련감염리포트 (http://www.cdc.go.kr/cdc/health/cdckrhealth0501.jsp?..)
의료관련감염병 병원체 6 종 ( 법정감염병분류 - 지정감염병 : 다제내성균 ) 1. MRSA ( 메티실린내성황색포도알균, methicillin-resistant S. aureus) 2. VRSA ( 반코마이신내성황색포도알균, vancomycin-resistant S. aureus) VISA 3. VRE ( 반코마이신내성장알균, vancomycin-resistant enterococci) 4. MRPA ( 다제내성녹농균, multidrug-resistant P. aeruginosa) : carbapenem, aminoglycoside, fluoroquinolone 에모두내성 5. MRAB ( 다제내성아시네토박터바우마니균, multidrug-resistant A. baumannii) : carbapenem, aminoglycoside, fluoroquinolone 에모두내성 6. CRE ( 카바페넴내성장내세균속균종 (carbapenen-resistant Enterobacteroceae) 감염병의예방및관리에관한법률 2010.12.30 감염병환자, 의사환자 X, 병원체보유자의진단 or 사체검안경우 7일이내신고
의료관련감염병 병원체 6 종신고현황 2012 감염병감시연보. 보건복지부, 질병관리본부
KONIS 의료관련감염 report (2013, 모든검체 ) http://is.cdc.go.kr/nstat/index.jsp 보고시스템 > 감염병웹통계시스템 > 표본감시통계 http://is.cdc.go.kr/nstat/index.jsp
Gram(+) Cocci MRSA VRSA/VISA, CNS VRE
MRSA (ORSA): methicillin, oxacillin 병원분리 S. aureus 특성 SAU (%) 1,085 주 R Penicillin 96.8 Oxacillin 78.2 PBP2a encoded by the mecagene 변형된 penicillin binding protein, lower avidity to beta lactams MRSA 중요성 Pathogenicity Transmissible ( 모든 VRSA 는 MRSA 임 ) Limited treatment options: All b lactam R ( 예외..)
B Lactam Families SIEMENS, CLSI AST Update for 2100, by Lisa Walker Cefoxitin
MRSA, Oxacillin/Cefoxitin 판독기준 Oxacillin disk (unreliable) > Cefoxitin disk Oxacillin MIC overcall resistance, 특히 non S. epidermidis 중증감염 MIC 0.5 2.0 ug/ml > meca/pbp 2a or cefoxitin disk.
Screening Tests for MRSA in Staphylococcus species Cefoxitin > Oxacillin > Methicillin Oxacillin > Methicillin: storage, heteroresistant strains. Cefoxitin > Oxacillin : meca inducer, easy reading (clear endpoints) 2 4 4 8 33 to 35, ambient air. (Testing at temperature above 35 may not detect MRSA.) Transmitted light. > 1 colony. light film of growth => MRSA 21mm = meca(+) 21mm = meca( ) 24mm = meca(+) 25mm = meca( ) 4ug/mL = meca(+) 4ug/mL = meca( )
Tests for MRSA Detection Cefoxitin/Oxacillin test Broth & Agar based Tests Heteroresistance: 내성유전자 (+), 일부만 in vitro 내성표현 Grow more slowly Missed at Tm above 35C. at 33 35 C (maximum of 35 C), ambient air for a full 24 hours NaCl (4% w/v; 0.68 mol/l) Latex agglutination test for PBP2a Nucleic acid amplification tests (PCR) for meca gene meca mediated oxacillin resistance 의 surrogate meca( ) & oxacillin R > oxacillin R, not cefoxitin R : all beta lactam R or No report except anti MRSA activity
VISA/VRSA VRSA/VISA 중요성 Limited treatment options Resistant to oxacillin (MRSA) & other antimicrobial agents. Transmissible (?) SAU (%) 1,085 I Glycopeptides Teicoplanin 0.1 Vancomycin 0.1 Vancomycin Disk test : not differentiate VS VI S. aureus, nor differentiate VS VI VR CoNS ( 유사한억제대 ). For S. aureus, VSSA may become VI during the course of prolonged therapy. MIC 8ug/mL S. aureus, 32ug/mL CoNS : reference Lab 으로보내기. Teicoplanin Disk test : TR & TI from TS is not known because of no reevaluation currently. Interpretive standards for Vancomycin/Teicoplanin For S. aureus For CoNS
VRSA (& VRE) screening tests ( 8ug/mL) 6 ug/ml vancomycin 2 4-8 16 4 8-16 32 Direct colony suspension to obtain 0.5 McFarland turbidity. Preferably, using a micropipette, spot a 10-uL drop onto agar. 35±2, ambient air. Transmitted light for > 1 colony or light film of growth => Presumptive reduced susceptibility 24 hours Validated MIC method 로 MIC confirm. MIC 4ug/mL fail to grow 가능성부담.
일반검사실방법으로 VISA/VRSA 가검출되는가? : 모든검사방법이 VISA 와 VRSA 를검출하지는못한다. VRSA are detected by reference broth microdilution, agar dilution, Etest, MicroScan overnight and Synergies plus ; BD Phoenix system, Vitek2 system, Vancomycin screen agar plate (BHI agar containing 6 µg/ml of vancomycin) Disk diffusion test Disk diffusion, not detect VISA isolates. [VRSA(vanA) no inhibition zone] VISA are detected by non automated MIC methods including reference broth microdilution, agar dilution, and Etest using a 0.5 McFarland standard inoculum. Vancomycin screen agar plates: detect VISA (MICs 8 µg/ml), but (MICs 4 µg/ml). Automated methods: detect VISA (MICs 8 µg/ml), but (MICs 4 µg/ml).
가능하면, Vancomycin agar screen plate 를적용 all MRSA useful for detecting VISA (MIC = 8 µg/ml) After VRSA/VISA (+) screening Verified by repeating a validated MIC method, & the organism identification. Additional antimicrobial agents: : Clindamycin, daptomycin, linezolid, quinupristin/dalfopristin, rifampin, trimetho sulfa. To stock : Minimum repeated subcultures All VRSA = MRSA Most VISA = MRSA
CNS with Decreased Susceptibility to the Glycopeptides CoNS 중요성 difficult to determine the significance, increasing numbers of healthcare associated infections invasive devices(catheters ) impaired host defenses CoNS 의 Decreased Susceptibility to Glycopeptides 중요성 Limited treatment options. Detection can be difficult. heteroresistance. Identifying CoNS to species level about 20 CoNS species (S. haemolyticus and S. epidermidis) some species are more resistant outbreak recognition & tracking resistance trends Can routine susceptibility tests detect these isolates? Not all methods. Should be reported to infection control? Yes!
VRE : Enterococci 에서 2 가지 Vancomycin 내성양상 Intrinsic resistance Enterococcus gallinarum, E. casseliflavus/e. flavescens low level resistance vanc (intrinsic) low vancomycin MICs of 2 to 16 µg/ml, S to teicoplanin EFA (%) EFM (%) 587 428 Glycopeptides Teicoplanin 1.4 23.4 Vancomycin 1.7 23.4 Acquired resistance. E. faecium, E. faecalis vana, vanb, vanc, vand, vane 그외 E. raffinosus, E. avium, E. durans, 기타 enterococcal species vana high vancomycin (>128 µg/ml) & teicoplanin ( 16 µg/ml) vanb lower vancomycin (MICs 16 to 64 µg/ml), S to teicoplanin (MICs 1 µg/ml). vand, vane moderate vancomycin (MICs 64 to 128) & teicoplanin (MICs 4 8 µg/ml)
VRE: Enterococcus species 수준까지동정? Aids in confirming intrinsic (vanc) or acquired resistance (vanaor vanb). 감염관리에서중요성 vanaand vanb: transferable, spread from organism to organism. vanc genes : not transferable, less serious infections, no outbreaks. 감별 : Motility, Yellow pigment, Susceptibility profile E. faecium, E. faecalis : non motile, no pigment, E. casseliflavus/e. flavescens : motile, yellow E. Gallinarum : motile, no pigment
Interpretive Standards for Vancomycin for Enterococci Test/Report Group Antimicrobial Agent Disk Content Zone diameter Interpretive Criteria (nearest whole mm) MIC Interpretive Criteria (ug/ml) S I R S I R B Vancomycin 30ug 17 15-16 14 4 8-16 32 Inv. Teicoplanin 30ug 14 11-13 10 8 16 32 For VRE detection, a full 24hr incubation needed. Transmitted light: a haze or any growth in inhibition zone => confirm MIC MIC 8 16ug/mL: species identification
(VRSA &) VRE screening tests ( 8ug/mL) 6 ug/ml vancomycin 2 4-8 16 4 8-16 32 Direct colony suspension to obtain 0.5 McFarland turbidity. Preferably, using a micropipette, spot a 10-uL drop onto agar. 35±2, ambient air. Transmitted light for > 1 colony or light film of growth => Presumptive reduced susceptibility 24 hours Validated MIC method 로 MIC confirm. Motility & Pigment procedure E. casseliflavus, E. gallinarum; vanc, 8 16ug/mL
Gram(-) bacilli ESBL CRE MRPA, MRAB
ESBLs (extended spectrum b lactamase) ESBLs (extended spectrum β lactamases) destroy extended spectrum cephalosporins (G3: ceftazidime, cefotaxime, ceftriaxone), Narrow spectrum/broad spectrum b lactams (Penicillin, Ampicillin, 1 st G cephalosporins) monobactams (aztreonam) not cephamycins (cefoxitin, cefotetan) not carbapenems (meropenem,imipenem) ECO (%) KPN (%) 1,326 657 R R 3rd Cephalosporins Cefotaxime 29 34.4 Ceftazidime 28.3 35.9 Cefepime 26 32.2 Aztreonam 27 34.3 E.C KL.P No. 1,326 657 ESBL(+) 26.3 31.2 Isolates other than K. pneumoniae, K. oxytoca, E. coli or P. mirabilis produce ESBLs? Yes. Other isolates of Enterobacteriaceae (Enterobacter spp, Morganella morganii, Serratia marcescens, Salmonella spp), P. aeruginosa
Changes in Interpretive Standards for Enterobacteriaceae Disk diffusion MICs until 2010 since 2010 Cefotaxime 23, 15-22, 14 26, 23-25, 22 Ceftriaxone 21, 14-20, 13 23, 20-22, 19 Ceftazidime 18, 15-17, 14 21, 18-220, 17 Aztreonam 22, 16-21, 15 21, 18-220, 17 Cefepime 18, 15-17, 14 25, 19-24, 18 [2014] until 2010 since 2010 8, 16-32, 64 1, 2, 4 8, 16-32, 64 1, 2, 4 8, 16, 32 4, 8, 16 8, 16, 32 4, 8, 16 8, 16, 32 2, 4-8, 16 [2014] * SDD (Susceptible Dose Dependent)
ESBLs: Standard Breakpoints Did Not Detect Resistance SIEMENS, CLSI AST Update for 2100, by Lisa Walker
Screening & Confirmatory Tests for ESBLs in E. coli, K. pneumoniae, K. oxytoca, P. mirabilis Using > 1 antimicrobials for screening improves the sensitivity of ESBL detection. Confirmatory testing requires use of both cefotaxime & ceftazidime, alone & in combination with clavulate.
Screening & Confirmatory Tests for ESBLs in E. coli, K. pneumoniae, K. oxytoca, P. mirabilis ESBL(+) in E. coli, K. oxytoca, K. pneumoniae 8ug/mL Cefpodoxime, 2ug/mL Ceftazidime, Aztreonam, Cefotaxime, Ceftriaxone ESBL(+) in P. mirabilis 2ug/mL Cefpodoxime, Ceftazidime, Cefotaxime ESBL(+) : 5mm increase (eg.) Ceftazidime 16mm Ceftazidime clavulate 21mm ESBL(+) : 3 twofold conc. Decrease (eg.) Ceftazidime 8ug/mL Ceftazidime clavulate 1ug/mL 다제내성균표준검사법
Carbapenem: imipenem, meropenem, ertapenem 중요성 Treat a variety of serious infections by highly resistant organisms Treat nosocomial and mixed bacterial infections. R Limits therapeutic options, Transmissible Drug Enterobacteriaeae Nonfermentors Anaerobes MSSA & Strep spp. Imipenem + + + + Meropenem + + + + Ertapenem + Limited + + Doripenem + + + + Activity : species dependent. Ertapenem Enterobacteriaceae Meropenem slightly more active than imipenem against G( ) 내성 (+) 세균 Intrinsic R : Stenotrophomonas maltophilia, Morganella morgagnii, P. mirabilis, Providentia, Serratia marcescens Proteus species, Serratia marcescens, Enterobacter species, Klebsiella pneumoniae. Pseudomonas aeruginosa, Burkholderia cepacia, Acinetobacter species,
Carbapenem 내성기전 Enterobacteriaceae Cephalosporinase + porin loss Carbapenemase P. aeruginosa Porin loss Up regulated efflux Carbapenemase Acinetobacter Cephalosporinase + porin loss Carbapenemase > 1 enzyme & > 1 porin modification : high MIC (>16 µg/ml). porin changes alone : lower MICs (2 8 µg/ml). AmpC type enzyme or an ESBL, combined with porin loss. Carbapenem resistant vs Carbpenemase producing Carbapenemase Enzyme Most Common Bacteria Class A Class B (metallo b lactamse) KPC, SME, IMI, NMC, GES IMP, VIM, GIM, SPM, NDM Enterobacteriaceae (rare reports in P. aeruginosa) P. aeruginosa Enterobacteriacea Acinetobacter spp. Class D OXA Acinetobacter spp. Carbapenemase (outbreak, transmissible) vs non carbapenemase produsing
Carbapenem: Changes in Interpretive standards Disk diffusion until 2010 Since 2010 Enterobacteriaceae Ertapenem 19,16 18, 15 22,19 21, 18 ECO (%) KPN (%) R R 1,326 657 Carbapenems Meropenem 0 1.4 Ertapenem 0.7 4.9 Meropenem 23,20 22, 19 Imipenem 16,14 15, 13 Doripenem MICs until 2010 Since 2010 Ertapenem Imipenem Meropenem 2, 4, 8 4, 8, 16 Enterobacteriaceae 0.25, 0.5, 1 0.5, 1, 2 [2012] 1, 2, 4 Doripenem Imipenem degrades easily, meropenem more stable than imipenem. store carbapenems at the coldest temperature range.
Carbapenem : Standard Breakpoints Did Not Detect Resistance SIEMENS, CLSI AST Update for 2100, by Lisa Walker
Screening & Confirmatory test for CRE for Old Criteria User
Screening & Confirmatory test for CRE continued Not all carbapenemase producing Enterobacteriaceae are MHT(+) other resistance mechanisms. Old criteria user MHT(+), all carbapenem R; MHT( ), according to criteria. New criteria user Rt confirmatory test 만 infection control & epidemiological investigation 목적으로사용. 역학조사대상 Carbapenemase producing isolates: 1 or more carbapenems 에 I or R, 1 or more cephalosporin subclass III 에 I or R.
Carbapenemase 검출시험 Modified Hodge test Carbapenemase inhibition test : 내성기전감별 ROSCO confirmative tablets MBL E test (imipenem or meropenem ± EDTA) Carba NP test: Patrice N et al, 2012 Emerg Inf Ds Rapid detection of. 37 C, 2hr sensitivity, specificity (100%) PCR (real time PCR), Sequencing 2012 JCM Diana D et al JCM Lab detection of Enterobacteriaceae that produce carbapenemase
P. Aeruginosa (MRPA), Acinetobacter baumannii(mrab) Changes in Carbapenem Interpretive standards Disk diffusion until 2010 Since 2010 PAE,ABA PAE,ABA Ertapenem Imipenem 19,16 18, 15 PAE 2012 16,14 15, 13 Im 22,19 21, 18 AB 2014 Meropenem Me 18,15 17, 14 AB 2014 Doripenem Do 18,15 17, 14 AB 2014 Ertapenem Imipenem Meropenem Doripenem MICs until 2010 Since 2010 PAE,ABA 4, 8, 16 PAE,ABA 2, 4, 8 PAE (%) ABA (%) 585 541 R R Carbapenems Imipenem 31.7 56.0 Meropenem 23.8 53.6 Aminoglycosides Gentamicin 20.3 51.1 Tobramycin 51.6 Amikacin 11.8 41.2 Quinolones Ciprofloxacin 34.4 59.0 Levofloxacin 35.6 51.7
실제어떤방법으로내성을검사할것인가?
Algorithm for Testing S. aureus with Vancomycin (VA) 병원에따른자체적인흐름도규정하기
실제 Outbreak 에어떻게대처할것인가?
보균자감시검사 ( 집단발생관리 ) Infection vs Colonization Infection Colonization Microorganism (+) (+) Tissue invasion (+) (-) Symptoms (+) (-) 보균자감시배양 1D 1D 배양순서 : 검체 배양 ( 집락 ) 동정 보균자 증균 / 선별 감수성 screen, confirm 감염관리
질병발생 (Concept of Disease Occurrence)
다제내성균선별검사의시행과격리에대한알고리즘 질병관리본부 2012.10.
Carbapenemase 생성 CRE 감염관리지침
Carbapenemase 생성 CRE 감염관리지침 1. 과거입원 (3개월이내 ) 당시균이분리되었던사실이확인되는경우에는즉시격리조치를취하고선별검사 (1 2일간격 ) 를실시 vs 가능한격리조치를취하고 2. 능동감시대상 ( 선별검사대상 ) 지정 3. 집단감염발생시관리
Carbapenemase 생성 CRE 에대한검사실내감시지침
다제내성균별감염관리 과거입원 (3 개월이내 ) 에서균이분리되어선제격리된환자는감시배양에서 2-3 회음성 (1-2 일간격 ) 이면격리해제 CRE 환자의퇴원여부에대해서는임상판단에따르며, 다제내성균의보균상태로인해퇴원을연기할근거는없음. 다만퇴원시접촉주의지침에대한교육을시행하고, 타의료시설로전원할경우전원대상시설에다제내성균에관한정보제공.
CRE/ 카바페넴분해효소생성 CRE 감시배양 질병관리본부 CDC CRE screening test 2008 Dec.
Vancomycin R Enterococcus Modification 환자 보균자 Any Specimen Rectal swab Stool 배양 동정, 감수성 Bile esculin broth (VAN. 6µg/ml) Multiplex PCR vana, vanb, vanc ChromID VRE E. Faecalis E. faecium
Chromogenic agar 정의 : Culture media for the simple and fast detection of bacteria The chromogenic mixture contains chromogenic substrates Certain enzymes, produced by some bacteria, cleave substrates > coloration of colonies.
Chromogenic agar MRSA, VRE: ChromID MRSA, ChromID VRE CRE: ChromID CARBA, ChromID OXA ESBL: ChromID ESBL MDR Acinetobacter C. difficile
마무리?
세균동정과내성검사방법 각검사실에서사용하는방법 표현형검사 : 생화학적방법, 항원검사 정확한검사방법과한계숙지 내성 (+) 세균검출 순수집락분리 재검 표준방법의준수 검사실에서가능한방법으로내성확인 균주보관및질병관리본부로이송준비 감염관리위원회에통보 병원차원에서의준비 검사방법에대한제안 격리및주의환기 전파방지를위한조치 감시배양과격리해제를위한배양
요약 MDRO 정의및감수성기준설정이해 감수성 screen & confirm 방법이해 목표설정 병원환경과현실적인기대수준설정 실현가능한형태로수정및보완