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Peran Uji Mikrobiologi & sensitivitas test Peran Uji Mikrobiologi & sensitivitas test

MMDEAHHapsariUKK –IPT- IDAI

Kuntaman ,Loknas PPRA

Mikroba dan manusiaMikroba dan manusia

Sedikit mikroba yang patogen

Banyak mikroba yangpotensial untuk patogen

Sebagian besar mikrobatidak patogen

FAKTOR BIOLOGISFAKTOR 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.

Kuman patogen didapat dari kultur

Gejala Klinis

Kondisi pasien

mis. septicaemia, endocarditis,osteomyelitis meningitis,UTI, pneumoniapharyngitis

Bagaimana mengetahui patogen tertentu dapat menyebabkan penyakit tertentu?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

Alur Pemeriksaan Mikrobiologi

Alur Pemeriksaan Mikrobiologi

ContentsContents

Handling specimen 1

Diagnosis Laboratorium Infeksi2

Peta medan kuman3

Pemilihan AB berdasarkan sensitivitas test

44

5 Mekmnisme Resistensi

Diagnosis of Bacterial InfectionDiagnosis of Bacterial Infection

Patient Clinical diagnosis

HaematologyBiochemistry

Non-microbiological investigations

Radiology

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 The specimen must be collected with a minimum of contamination as close to contamination as close to

site of infection as possiblesite of infection as possible

The specimen must be collected with a minimum of The specimen must be collected with a minimum of contamination as close to contamination as close to

site of infection as possiblesite of infection as possible Specimen Source of

Contamination Storage and Transport

Solution/Monitor Education

Urine Culture All non surgical samples become contaminated with urogenital flora during collection. Contaminating bacteria will replicate if specimen is not quickly transferred to a preservative tube or stored (4°C).

Transfer urine to a Urine Preservative tube within 10 minutes of collection (good for 48 hrs. at ambient temp. Less optimal: store/transport urines at 4° C for up to 24 hrs.

Patients must be instructed to properly cleanse the peri-urethral genital skin area prior to collection of the mid-stream portion of the urine stream in order to get an accurate urine culture result. Use of urine preservative tubes.

Prompt feedback to individuals or sites who collected urine for culture. Urine preservative tubes should be used when appropriate.

Blood Culture, bacterial, mycobacterial, fungal

Improper cleaning of skin or catheter prior to drawing specimen. Transfer from SPS tube to blood culture vial. Collection from catheter.

Ambient. Must be incubated in automated system within 12 hours.

Ongoing education program. Monitoring contamination rates. Limit use SPS tubes. Do not draw from catheter unless specifically requested (protocol; discard 5X cath. volume); then one culture set from catheter and one from peripheral.

Timely feedback to individuals who collected specimen.

Blood Culture 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.)

A specimen must be collected at the optimal time(s) in order to

recover the pathogen(s) of interest

A specimen must be collected at the optimal time(s) in order to

recover the pathogen(s) of interest Specimen Optimal Time Comments

Urine First morning specimen preferred. Or not have urinated in several hours

Blood Culture Collect prior to administration of antibiotics. Collect 2-3 sets of blood cultures from different sites. If suspect bacterial endocarditis and initial cultures are negative at 48 hours then collect 2-3 additional cultures from different sites. Suspected bacteremia or fungemia with persistently negative blood cultures

Interpretation of one positive culture problematic, especially if isolate is coagulase negative Staphylococcus. Consider laternative blood culture methods dsigned to enhance recovery of mycobacteria, fungi, and other rare and fastidious microorganisms

AFB Culture Three consecutive specimens collected 8-24 hours apart, with at least one being an early AM specimen

Sputum not saliva

GC/Chlamydia, urine

First voided urine of day. First stream of urine optimal. Less sensitive: Patient should not have urinated for at least 1 hour. Do not use NAT methods as “proof of cure”.

Not midstream urine. Place sample in transport tube per manufacturer’s instructions Lingering DNA may still be present.

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

the pathogen(s) of interest (cont)

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

the pathogen(s) of interest (cont)

Specimen Optimal Time Comments Ova and Parasites

Wait 10 days if barium or oil present. For multiple samples, collect every other day.

Place stool in preservative (10% formalin, PVA, SAF, Ecofix) within one hour of collection. Instruct patient.

Stool Cultures Recommend 2 samples on consecutive days. Prior to 3 days post admission.

Place in enteric preservative (Cary-Blair) immediately. Stool specimens that are obtained 3 days after admission are not usually helpful for the diagnosis of hospital acquired diarrhea

Blood Parasites Collect during a febrile episode or every 6 hours for a 24 hour period.

Submit finger stick Thick & Thin slides or peripheral blood in an EDTA tube within 24 hours. Store at ambient temperature.

Viral Culture Collect as soon after onset of symptoms as possible.

The first 3 days is best.

A sufficient quantity of the specimen must be obtained to perform the requested tests A sufficient quantity of the specimen must be obtained to perform the requested tests

Culture Minimum Requirements

Comment

Blood Culture 10 ml of aerobic; 10 ml for anaerobic bottle

Sensitivity of a blood culture is directly related to the volume of blood submitted. Two blood culture sets (10 mL in both aerobic and anerobic bottles) before administration of antibiotics is 98% sensitive (J. Clin.

Microbiol. 1998 36: 657-661). One swab for multiple cultures

A separate swab(s) for each culture

Enough material must be submitted for gram stain, if required.

CSF Culture 2 mL from tube 2,3, or 4

Submit most turbid tube. At least 0.5 mL of CSF is required for cytospin gram stain.

Surgical and Shared Specimens

See chart Cooperation with other departments (laboratory and non laboratory) is key.

Anaerobic Cultures

See Table

Blood CulturesBlood 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).

Recommended Pediatric Blood Culture Volumes By Patient Weight Weight (KG) of Patient

Weight (LB) of Patient

Minimum Volume (mL)

One Pediatric Bottle

Two Adult Bottles (aerobic and anaerobic)

<1.0 Kg 2.2 Lb. 1.0 mL Yes No 1.5-3.9 Kg 2.3-8.6 Lb. 1.5 mL Yes No

4.0-13.9 Kg 8.7-30.6 Lb 3.0 mL Yes No 14.0-24.9

Kg 30.7-54.9

Lb 10.0 mL No Yes (5 mL in

each) >25.0 Kg >55 Lb. 20.0 Ml No Yes (10 mL

in each)

Pediatric Blood Cultures - VolumePediatric Blood Cultures - Volume

Collect all microbiology test samples prior to the institution of antibiotics

Collect all microbiology test samples prior to the institution of antibiotics

Specimen Comments Blood Culture Collect two sets at same time from different sets. DO NOT collect both sets from

the same site (assessment for contamination) Hair, skin and nails Fungal Culture

Collect before antifungal therapy or discontinue treatment for at least 5 days.

Urine Culture Antibiotics may cause a transient decrease in bacterial concentration resulting in a false negative report

Blood Cultures - VolumeBlood Cultures - Volume

Increase Volume Increase Yield

10 ml 20 ml 30% 40%

20 ml 30 ml 10% 15%

The magnitude of bacteremia may be low (<1cfu/ml)

Higher volumes have higher yield

Urine - GeneralUrine - 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 benarPengambilan 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 laboratoriumPengiriman 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 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 Handling specimen

Pus

DarahUrin

Tinja

Sputum

Turn Around Time

Lab Mikrobiologi

Cara pengambilan, penyimpanan dan pengiriman bahanCara pengambilan, penyimpanan dan pengiriman bahan

Petunjuk Umum 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

Petunjuk Khusus 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.....Lanjt.....

Tinja Pengambilan pada pagi

hari atau tinja yang baru Hapusan rektum kurang

dianjurkan Jumlah 10 gramn Segera kirim

LCS Pengambilan dengan

pungsi Pengiriman segera

mungkin

Culture diagnostic of typhoidCulture diagnostic of typhoid

0

1 0

2 0

3 0

9 0

4 0

5 0

6 0

7 0

8 0

100

1 2 3 4 5 6 7 8 weeks

% patients with pos culture

urine

stool

bloods

ContentsContents

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Peta medan kuman3


44

5 Mekanisme Resistensi

Laboratorium MikrobiologiLaboratorium Mikrobiologi

Pemeriksaan Kultur DarahPemeriksaan Kultur Darah

ContentsContents

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Peta medan kuman3


44

5 Mekanisme Resistens

Hasil Peta Kuman – sensitivitas PICU-NICU - darah (Jan-Jun 2009)RSDK 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 50 87 100 87 50Pseudomonas aeroginosa

81 6 68 0 37 33 0 87 25 50

Staphylococcus epidemidis

83 33 33 33 100 80 50 100 50 100

Ruang Anak

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Pengamatan Hasil Pemeriksaan Mikrobiologi

Pengamatan Hasil Pemeriksaan Mikrobiologi

Pengamatan HasilPengamatan Hasil

Pengamatan

SebelumTerapi Empirik

Spektrum luas

De-escalating

Data epidemiologi

Narrow sp

aman

oost

SesudahTerapi Definitif

Use Antimicrobials Wisely Treat infection, not contaminationUse 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 hubuse proper methods to obtain & process all cultures

Link to: CAP standards for specimen collection and management

12 Steps to Prevent Antimicrobial Resistance: Hospitalized Adults

Use Antimicrobials WiselyTreat infection, not colonizationUse Antimicrobials WiselyTreat 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

Link to: IDSA guideline for evaluating fever in critically ill adults

12 Steps to Prevent Antimicrobial Resistance: Hospitalized Adults

Follow Established GuidelinesFollow Established Guidelines

Consult Specialist

Follow Guidelines

Use Local DataUse Local Data

Know your antibiogram Know your formulary Know your patient

population

When infection is not diagnosed

When infection is unlikely

Stop Antimicrobial TreatmentStop Antimicrobial Treatment

Hasil Kultur Darah Ruang Anak RSDKHasil 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 %

Ampi-sulbactam Amikasin

Kleb.pnem( darah )

Pseudo.aero( darah )

L :21.000 L : 8.300

Pasien sepsis dengan demam selama 10 hari.

Kultur Darah : Klebsiella pneumoniaKultur Darah : Klebsiella pneumonia

Kultur Darah 28/8/2010 Hasil

Kuman: Klebsiella Pneumonia

Sensitif: Amikacin, cefepim. Imipenem, meropenem, sulbactam cefoperazon

Resisten: Ampicilin, ceftazidim, kotrimoksasol, gentamycin, moxifloxacin


Kuman: Escherichia Coli, > 100.000

Sensitif: Cefepim. Gentamycin, Imipenem, meropenem, fosfomycin

Resisten: Amikacin, Ampicilin, Ampicilin sulbactam, ceftazidim, kotrimoksasol, moxifloxacin

Kultur Urin : Escherichia ColiKultur Urin : Escherichia Coli

Kultur Darah : Pseudomonas aeroginosa

Kultur Darah : Pseudomonas aeroginosa


Kuman: Pseudomonas aeruginosa

Sensitif: Kotrimoksasol, meropenem

Resisten: Amikacin, Ampicilin, Cefepim, gentamicin, moxifloxacin, fosfomycin

Pasien DSS mengalami : -Sepsis-VAP + Gagal Nafas -Perdarahan

Sembuh Perawatan

selama 2 bulan

Invitro : Chloramphenicol = SInvivio : Pseudomonas tidak bisa dengan Chloramphenicol

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

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48

Mechanisms of antimicrobial resistanceMechanisms of antimicrobial resistance

Antimicrobial agents are catagorized according to their principle mechanism of action

1. Interference with cell wall synthesis ( lactams, Glycopeptide agents)

2. Inhibition of protein synthesis (macrolide, tetracycline)3. Interference with nucleic acid synthesis

(fluoroquinolones, rifampin)4. Inhibition of a metabolic pathway (trimetopim

sulfamethoxazole)5. Disruption of bacterial membrane structure (polymixin)

Tenover FC. Am J Med 2006;119(6):S3-S10

1

3

2

5

4

50

…mechanisms of antimicrobial resistance…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

macrolideribosomal binding site of antibioticsefflux pump to expel relative resist to

macroan antibiotics from the lide

cyoplasm

Pong AL. Pediatr Clin N Am 2005;52:869-94

Table . Pediatric bacterial pathogens, mechanisms of resist

51


Pediatric bacterial pathogens, mechanisms of resistOrganism 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


52



________________________________________________________

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)


53



________________________________________________________ Pseudomonas multipel -lactamases resistant to

cefotaxime aerug each with activity ceftriaxoneagainst different ceftazidim

-lactamantibiotics

cell wall porin protein carbapenem resist

deficient bacteria

multiple efflux pumps resistance to -lactam

to expel antib from fluoroquinolones

the cytoplasm


54

MAJOR ANTIBIOTIC RESISTANCE MECHANISMSMAJOR ANTIBIOTIC RESISTANCE MECHANISMS

Produce antibiotic inactivating enzymes Reduce intracellular antibiotic concentration Alter antibiotic target site Eliminate antibiotic target site

55

Table Major Antibiotic Resistance MechanismsTable Major Antibiotic Resistance Mechanisms

Resistance Mechanism

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

• Enzymatic destruction of drug

• Prevention of penetration of drug

• Alteration of drug's target site

• Rapid ejection of the drug

Mechanisms of Antibiotic ResistanceMechanisms of Antibiotic Resistance

Figure 20.20

Antibiotic ResistanceAntibiotic Resistance

Proses Resistensi bakteriaProses Resistensi bakteria

MutationGene exchange SelectionTransmission

proses

biologi

alamiah

New Resistant Bacteria

Mutations

XX

Emergence of Antimicrobial ResistanceEmergence of Antimicrobial Resistance

Susceptible Bacteria

Campaign to Prevent Antimicrobial Resistance in Healthcare Settings

Resistant Bacteria

Resistance Gene Transfer

MutationMutation

Konjugasi Transduksi

Gene exchangeGene exchange

R


R


R

R

Resistant StrainsRare

xx

Resistant Strains Dominant

Antimicrobial Exposure

xxxx

xx

xx

xx

Selection for antimicrobial-resistant StrainsSelection for antimicrobial-resistant Strains

Campaign to Prevent Antimicrobial Resistance in Healthcare Settings

SelectionSelection

TransmissionTransmission

• Air

• Droplets

• Contact

• Water

• Food

• Blood

• Vectors

Antibiotic Selection for Resistant Bacteria

Antibiotic Selection for Resistant Bacteria

RangkumanRangkuman

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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