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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
Handling specimen 1
Diagnosis Laboratorium Infeksi2
Peta medan kuman3
Pemilihan AB berdasarkan sensitivitas test
44
5 Mekanisme Resistensi
Laboratorium MikrobiologiLaboratorium Mikrobiologi
Pemeriksaan Kultur DarahPemeriksaan Kultur Darah
ContentsContents
Handling specimen 1
Diagnosis Laboratorium Infeksi2
Peta medan kuman3
Pemilihan AB berdasarkan sensitivitas test
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
ContentsContents
Handling specimen 1
Diagnosis Laboratorium Infeksi2
Peta medan kuman3
Pemilihan AB berdasarkan sensitivitas test
44
5 Mekanisme Resistensi
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
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 Urin : Escherichia ColiKultur Urin : Escherichia Coli
Kultur Darah : Pseudomonas aeroginosa
Kultur Darah : Pseudomonas aeroginosa
Kultur Darah 7/9/2010 Hasil
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
ContentsContents
Handling specimen 1
Diagnosis Laboratorium Infeksi2
Peta medan kuman3
Pemilihan AB berdasarkan sensitivitas test
44
5 Mekanisme Resistensi
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
…mechanisms of antimicrobial resistance…mechanisms of antimicrobial resistance
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
Pong AL. Pediatr Clin N Am 2005;52:869-94
52
…mechanisms of antimicrobial resistance…mechanisms of antimicrobial resistance
Pediatric bacterial pathogens, mechanisms of resistOrganism 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
53
…mechanisms of antimicrobial resistance…mechanisms of antimicrobial resistance
Pediatric bacterial pathogens, mechanisms of resistOrganism Mech of resist clinical implications
________________________________________________________ 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
Pong AL. Pediatr Clin N Am 2005;52:869-94
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
R
Konjugasi Transduksi
Gene exchangeGene exchange
R
Gene exchangeGene exchange
R
Gene exchangeGene exchange
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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