Peran Uji Mikrobiologi & sensitivitas test MMDEAHHapsari UKK –IPT- IDAI

Kuntaman ,Loknas PPRA

Mikroba dan manusia Sedikit mikroba yang patogen Banyak mikroba yang potensial untuk patogen Sebagian besar mikroba tidak patogen

FAKTOR BIOLOGIS Flora normal (mayoritas bakteri) pada kulit dan saluran pencernaan mencegah kolonisasi bakteri patogenik dengan mengeluarkan substansi toksik atau dengan bersaing mendapatkan nutrien. Ada 1013 sel dan terdapat 1014 bakteri, yang mayoritas hidup di usus besar. Ada 103-104 mikroba per cm2 di kulit (Staphylococcus aureus, Staphylococcus epidermidis, Diphtheroid, Streptococci, Candida dll.). Berbagai macam bakteri hidup di hidung dan mulut Di lambung dan usus halus terdapat Lactobacilli Di usus halus terdapat 104 bakteri per gram dan di usus besar 1011 per gram, 95-99% di antaranya adalah anaerob. Di saluran kemih terdapat koloni berbagai bakteri dan difteroid. Setelah pubertas, terdapat koloni Lactobacillus aerophilus yang meng-fermentasi glikogen untuk mempertahankan pH asam. Flora normal menciptakan kesesuaian ekologis dalam tubuh, dan menghasilkan baktoriosidin, defensin, protein kationik dan laktoferin yang merusak bakteri lain.

Bagaimana mengetahui patogen tertentu dapat menyebabkan penyakit tertentu? Diagnosis dan terapi infeksi tidak tergantung dari kuman tetapi juga melihat hasil laboratorium yang lain serta gejala klinis pasien Gejala Klinis mis. septicaemia, endocarditis, osteomyelitis meningitis, UTI, pneumonia pharyngitis Kondisi pasien Kuman patogen didapat dari kultur

Alur Pemeriksaan Mikrobiologi Most clinical samples are first grown on general purpose media, media such as blood agar thar suport the growth of most aerobic and facultively anaerobic organism. Enrichment culture, the use if selected culture media and incubation conditions to isolate microorganisms samples, is an important part of clinical microbniology Figure, shows recomended enriched media and selective media for primary isolation of pathogens.

Contents 1 Handling specimen Diagnosis Laboratorium Infeksi 2 3 Peta medan kuman 4 4 Pemilihan AB berdasarkan sensitivitas test Mekmnisme Resistensi 5

Diagnosis of Bacterial Infection Non-microbiological investigations Patient Clinical diagnosis Radiology Haematology Biochemistry Sample Take the correct specimen Take the specimen correctly Label & package the specimen up correctly Appropriate transport & storage of specimen

The specimen must be collected with a minimum of contamination as close to site of infection as possible Mike

Blood Culture Two sets of blood cultures should be drawn. Number of sets positive correlates with true sepsis (except for coagulase negative Staph?) (Clin Microbiol. Rev 19:788-802, 2006) Catheter drawn blood cultures Catheter drawn blood cultures are equally likely to be truly positive (associated with sepsis), but more likely to be colonized (J Clin Microbiol 38:3393, 2001.) One drawn through catheter and other though vein PPV 0f 96% Both drawn from catheter PPV 0f 50% Both drawn through vein PPV of 98% Study of positive coagulase negative Staphylococcus cultures and sepsis (Clin Infect Dis. 39:333, 2004.) Mike

A specimen must be collected at the optimal time(s) in order to recover the pathogen(s) of interest Mary

A specimen must be collected at the optimal time(s) in order to recover the pathogen(s) of interest (cont) Mary

A sufficient quantity of the specimen must be obtained to perform the requested tests Mike/Mary?

Blood Cultures Volume of blood drawn is the single most important factor influencing sensitivity. A single set for an adult blood culture consists of one aerobic and one anaerobic bottle. Optimally 10 mL of blood should be inoculated into each bottle. Volume of blood for a pediatric culture can be related to the infants weight Solitary blood cultures should be less than 5% (Arch Pathol Lab Med. 2001 125:1290-1294) If only enough blood can be drawn for one bottle, inoculate the aerobic bottle. 644 positive blood cultures, 59.8% from both bottles, 29.8% from aerobic bottle only and 10.4% from anaerobic bottle only (J Infect Chemother 9:227, 2003). Mike/Mary?

Pediatric Blood Cultures - Volume Mike/Mary?

Collect all microbiology test samples prior to the institution of antibiotics

Blood Cultures - Volume The magnitude of bacteremia may be low (<1cfu/ml) Higher volumes have higher yield

Urine - General Collection method must avoid contamination Clean catch, midstream voided Catheterized urine Suprapubic aspiration Cultures performed quantitatively Less than 10,000 per ml suggest contamination

Pengambilan spesimen yang benar Urin – mid-stream Hindari kontaminasi dengan flora perineal LCS Cegah kontaminasi Cegah perdarahan Kultur darah Cegah kontaminasi dengan kuman di permukaan kulit Pengiriman spesimen ke laboratorium Keterlambatan pengiriman akan menyebabkan keterlambatan diagnosis dan terapi Pathogen mati Pertumbuhan kontaminan Kultur darah harus segera masuk inkubator Bukan almari es ( refrigerator) LCS segera dikirim ke Lab

Faktor –faktor yang berpengaruh atas hasil kultur darah Sampel yang slalah Sputum – didapat saliva Terlambat kirim LCS Pertumbuhan kontaminan Misal kultur darah Pasien sudah mendapatkan antibiotika

Handling specimen Turn Around Time Lab Mikrobiologi Darah Urin Pus Tinja Sputum Turn Around Time Lab Mikrobiologi

Cara pengambilan, penyimpanan dan pengiriman bahan Petunjuk Umum Petunjuk Khusus Pemeriksaan diambil sebelum diberikan antibiotik Bahasn pemeriksaan diambil saat & lokasi yang tepat( untuk dapat kuman) Tindakan aseptik Jumlah cukup Formulir diisi lengkap(riwayat penyakit, pengobatan,diagnosis Pelabelan yang jelas Air seni –penampungan pagi hari-steril-midstream/ kateter-segera kirim.( Urin diambil < 3 hari MRS) Darah : diambil sesuai perjalan penyakit Dengan media “bactec” Ukuran sesuai dengan aturan

Lanjt..... Tinja LCS Pengambilan pada pagi hari atau tinja yang baru Hapusan rektum kurang dianjurkan Jumlah 10 gramn Segera kirim Pengambilan dengan pungsi Pengiriman segera mungkin

Culture diagnostic of typhoid 100 % patients with pos culture 9 0 8 0 7 0 bloods 6 0 stool 5 0 4 0 3 0 urine 2 0 1 0 2 3 4 5 6 7 8 weeks

Contents 1 Handling specimen Diagnosis Laboratorium Infeksi 2 3 Peta medan kuman 4 4 Pemilihan AB berdasarkan sensitivitas test Mekanisme Resistensi 5

Laboratorium Mikrobiologi

Pemeriksaan Kultur Darah

Agar media are inoculated by evenly spreading a defined density of a suspension of the pure culture on the agar surface. Filter paper disks containing a defined quantity of the antimicrobial agents are then placed on the inoculated agar After a specified period of incubation, the diameter of the inhibition zone around each disk is measured. This figure presents zone sizes for several antibiotics.

Antibiograms are periodic reports that indicate the susceptibility of clinically isolated organisms to the antibiotics in current local use.

Contents 1 Handling specimen Diagnosis Laboratorium Infeksi 2 3 Peta medan kuman 4 4 Pemilihan AB berdasarkan sensitivitas test Mekanisme Resistens 5

Hasil Peta Kuman – sensitivitas PICU-NICU - darah (Jan-Jun 2009)RSDK Chl Gen Cip Ctx Caz DKB FOS MEM MFX SXT VAN Enterobacter.aerogenes 92 7 84 26 34 33 80 100 76 64 Eschericia coli 50 25 87 37 Pseudomonas aeroginosa 81 6 68 Staphylococcus epidemidis 83 Ruang Anak

Contents 1 Handling specimen Diagnosis Laboratorium Infeksi 2 3 Peta medan kuman 4 4 Pemilihan AB berdasarkan sensitivitas test Mekanisme Resistensi 5

Pengamatan Hasil Pemeriksaan Mikrobiologi

Pengamatan Hasil Pengamatan Sebelum Terapi Empirik Sesudah Terapi Definitif Spektrum luas Narrow sp Pengamatan De-escalating aman Data epidemiologi oost

Use Antimicrobials Wisely Treat infection, not contamination 12 Steps to Prevent Antimicrobial Resistance: Hospitalized Adults Use Antimicrobials Wisely Treat infection, not contamination Fact: A major cause of antimicrobial overuse is “treatment” of contaminated cultures. Actions: use proper antisepsis for blood & other cultures culture the blood, not the skin or catheter hub use proper methods to obtain & process all cultures Optimizing skin antisepsis is the first critical step in obtaining blood samples for culture. Proper specimen collection and management is key to preventing contaminated cultures. Link to: CAP standards for specimen collection and management

Use Antimicrobials Wisely Treat infection, not colonization 12 Steps to Prevent Antimicrobial Resistance: Hospitalized Adults Use Antimicrobials Wisely Treat infection, not colonization Fact: A major cause of antimicrobial overuse is treatment of colonization. Actions: treat bacteremia, not the catheter tip or hub treat pneumonia, not the tracheal aspirate treat urinary tract infection, not the indwelling catheter Clinical criteria and additional laboratory data can help distinguish infection from colonization. Improving the specificity of diagnostic criteria for infection can help reduce unnecessary antimicrobial use. Link to: IDSA guideline for evaluating fever in critically ill adults

Follow Established Guidelines Consult Specialist Follow Guidelines

Stop Antimicrobial Treatment Use Local Data Know your antibiogram Know your formulary Know your patient population When infection is not diagnosed When infection is unlikely

Hasil Kultur Darah Ruang Anak RSDK Kuman Jumlah Prosentase Candida albicans 1 0.45 % Enterobacter aerogenes 86 38.7 % Escherichia coli 14 6.3 % Pseudomonas aeruginosa 26 11.7 % Salmonella typhi 4 1.8 % Staphylococcus aureus 28 12.6 % Staphylococcus epidermidis 61 27.4 % Streptococcus pneumoniae 2 0.9 %

Pasien sepsis dengan demam selama 10 hari. Pseudo.aero ( darah ) Kleb.pnem ( darah ) L :21.000 L : 8.300 Ampi-sulbactam Amikasin

Kultur Darah : Klebsiella pneumonia Hasil Kuman: Klebsiella Pneumonia Sensitif: Amikacin, cefepim. Imipenem, meropenem, sulbactam cefoperazon Resisten: Ampicilin, ceftazidim, kotrimoksasol, gentamycin, moxifloxacin Kultur Urin : Escherichia Coli Kultur Darah 4/9/2010 Hasil Kuman: Escherichia Coli, > 100.000 Sensitif: Cefepim. Gentamycin, Imipenem, meropenem, fosfomycin Resisten: Amikacin, Ampicilin, Ampicilin sulbactam, ceftazidim, kotrimoksasol, moxifloxacin

Kultur Darah : Pseudomonas aeroginosa Hasil Kuman: Pseudomonas aeruginosa Sensitif: Kotrimoksasol, meropenem Resisten: Amikacin, Ampicilin, Cefepim, gentamicin, moxifloxacin, fosfomycin

Perawatan selama 2 bulan Pasien DSS mengalami : Sepsis VAP + Gagal Nafas Perdarahan Sembuh Perawatan selama 2 bulan Invitro : Chloramphenicol = S Invivio : Pseudomonas tidak bisa dengan Chloramphenicol

Pasien dengan diare kronis Hasil Kultur feses : Escherichia coli EPEC (+), berarti memang didapatkan infeksi di saluran cerna

Contents 1 Handling specimen Diagnosis Laboratorium Infeksi 2 3 Peta medan kuman 4 4 Pemilihan AB berdasarkan sensitivitas test Mekanisme Resistensi 5

Mechanisms of antimicrobial resistance Antimicrobial agents are catagorized according to their principle mechanism of action Interference with cell wall synthesis ( lactams, Glycopeptide agents) Inhibition of protein synthesis (macrolide, tetracycline) Interference with nucleic acid synthesis (fluoroquinolones, rifampin) Inhibition of a metabolic pathway (trimetopim sulfamethoxazole) Disruption of bacterial membrane structure (polymixin) Tenover FC. Am J Med 2006;119(6):S3-S10

1 3 4 2 5

…mechanisms of antimicrobial resistance Table . Pediatric bacterial pathogens, mechanisms of resist …mechanisms of antimicrobial resistance Organism Mech of resist clinical implications ________________________________________________________ Str pneumoniae alteration of PBP relative resistant to -lactam agents (pen cillin, cephalosp) alteration in the resistance to macrolide ribosomal binding site of antibiotics efflux pump to expel relative resist to macro an antibiotics from the lide cyoplasm Pong AL. Pediatr Clin N Am 2005;52:869-94

…mechanisms of antimicrobial resistance Pediatric bacterial pathogens, mechanisms of resist Organism Mech of resist clinical implications ________________________________________________________ S. Aureus alteration in the resistant to all -lactams binding site of a specific transpeptidase (mecA) alteration at resistance to macrolides ribosomal binding and clindamycin site efflux pump to expel relative resist to macro an antib from the cyopl lide Pong AL. Pediatr Clin N Am 2005;52:869-94

…mechanisms of antimicrobial resistance Pediatric bacterial pathogens, mechanisms of resist Organism Mech of resist clinical implications ________________________________________________________ Escherichia coli, -lactamases with ceftazidim Klebsiella activity extended resistant to cefotaxim, beyond ampic ceftriaxone, (ESBL) ceftazidim Enterobacter, chromosomal resistant to cefotaxim, Seratia, other -lactamases that ceftriaxone Enterobacteriaceae are deregulated and ceftazidim hyperproduced (ampC) Pong AL. Pediatr Clin N Am 2005;52:869-94

…mechanisms of antimicrobial resistance Pediatric bacterial pathogens, mechanisms of resist Organism Mech of resist clinical implications ________________________________________________________ Pseudomonas multipel -lactamases resistant to cefotaxime aerug each with activity ceftriaxone against different ceftazidim -lactam antibiotics cell wall porin protein carbapenem resist deficient bacteria multiple efflux pumps resistance to -lactam to expel antib from fluoroquinolones the cytoplasm Pong AL. Pediatr Clin N Am 2005;52:869-94

MAJOR ANTIBIOTIC RESISTANCE MECHANISMS Produce antibiotic inactivating enzymes Reduce intracellular antibiotic concentration Alter antibiotic target site Eliminate antibiotic target site

Table Major Antibiotic Resistance Mechanisms Specific examples Antibiotic's effected Produce antibiotic inactivating enzymes β-lactamase, extended spectrum β-lactamases β-lactamase Adenyl, phosphoryl or acetyl transferases Aminoglycoside Reduce intracelluler concentration of the antibiotic Efflux pump Macrolides, tetracyclines, fluoroquinolones Reduce outer membrane permeability Penicillins, macrolides, fluoroquinolones Alter the antibiotic target site Altered penicillins binding proteins β-lactamases Change peptidoglycans termini Glycopeptides Mutations in gyrases or topoisomerase genes tRNA methylases Fluoroquinolone Macrolides Eliminate the antibiotic target site Encode an alternative penicillin binding protein Methicillin Produce less enzyme or an alternative enzyme Trimethoprim, Sulphonamides

Mechanisms of Antibiotic Resistance Enzymatic destruction of drug Prevention of penetration of drug Alteration of drug's target site Rapid ejection of the drug

Antibiotic Resistance Figure 20.20

Proses Resistensi bakteria Mutation Gene exchange Selection Transmission proses biologi alamiah

Emergence of Antimicrobial Resistance Campaign to Prevent Antimicrobial Resistance in Healthcare Settings Emergence of Antimicrobial Resistance Susceptible Bacteria New Resistant Bacteria Mutations XX Resistant Bacteria Resistance Gene Transfer Bacteria have evolved numerous mechanisms to evade antimicrobial drugs. Chromosomal mutations are an important source of resistance to some antimicrobials. Acquisition of resistance genes or gene clusters, via conjugation, transposition, or transformation, accounts for most antimicrobial resistance among bacterial pathogens. These mechanisms also enhance the possibility of multi-drug resistance.

Mutation

R

Gene exchange Konjugasi Transduksi

Gene exchange R

Gene exchange R R R

Selection for antimicrobial-resistant Strains Campaign to Prevent Antimicrobial Resistance in Healthcare Settings Selection for antimicrobial-resistant Strains Resistant Strains Dominant Antimicrobial Exposure x Resistant Strains Rare x Once resistant strains of bacteria are present in a population, exposure to antimicrobial drugs favors their survival. Reducing antimicrobial selection pressure is one key to preventing antimicrobial resistance and preserving the utility of available drugs for as long as possible.

Selection

ANTIBIOTIC

Transmission

Air Droplets Contact Water Food Blood Vectors

Antibiotic Selection for Resistant Bacteria

Rangkuman Pemeriksaan mikrobiologi khususnya biakan dan sensitifitas test sangat berperan dalam menegakkan suatu penyakit infeksi Handling dan koleksi spesimen haruslah mengikuti kaidah yang sudah ditentukan Pelaporan peta medan kuman disetiap RS dengan rutin sangat mendukung dalam pengelolaan pasien infeksi di RS tersebut Penentuan pemberian antibiotik berdasarkan hasil biakan haruslah hati-hati, mengingat kadang ada perbedaan antara invivo dan invitro

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