Peran Uji Mikrobiologi

Peran Uji Mikrobiologi
& sensitivitas test
MMDEAHHapsari
UKK –IPT- IDAI
LOGO
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 mengfermentasi 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
Contents
1
Handling specimen
2
Diagnosis Laboratorium
Infeksi
3
Peta medan kuman
44
Pemilihan AB berdasarkan
sensitivitas test
5
Mekmnisme Resistensi
Diagnosis of Bacterial Infection
Patient
Clinical
diagnosis
Non-microbiological
investigations
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
Specimen
Urine Culture
Blood Culture,
bacterial,
mycobacterial,
fungal
Source of
Contamination
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).
Improper cleaning of
skin or catheter prior to
drawing specimen.
Transfer from SPS
tube to blood culture
vial.
Collection from
catheter.
Storage and
Transport
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.
Ambient. Must be
incubated in
automated
system within 12
hours.
Solution/Monitor
Patients must be
instructed to properly
cleanse the peri-urethral
genital skin area prior to
collection of the midstream portion of the
urine stream in order to
get an accurate urine
culture result. Use of
urine preservative tubes.
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.
Education
Prompt
feedback to
individuals or
sites who
collected urine
for culture.
Urine
preservative
tubes should
be used when
appropriate.
Timely
feedback to
individuals
who collected
specimen.
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 19:788802, 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
Specimen
Urine
Optimal Time
First morning specimen preferred.
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
AFB Culture
GC/Chlamydia,
urine
Three consecutive specimens collected 8-24
hours apart, with at least one being an early
AM specimen
First voided urine of day. First stream of urine
optimal. Less sensitive: Patient should not
have urinated for at least 1 hour.
Comments
Or not have urinated in several
hours
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
Sputum not saliva
Not midstream urine.
Place sample in transport tube per
manufacturer’s instructions
Do not use NAT methods as “proof of cure”.
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)
Specimen
Ova and
Parasites
Stool Cultures
Optimal Time
Wait 10 days if barium or oil present.
For multiple samples, collect every other day.
Recommend 2 samples on consecutive days.
Prior to 3 days post admission.
Blood Parasites
Collect during a febrile episode or every 6 hours
for a 24 hour period.
Viral Culture
Collect as soon after onset of symptoms as
possible.
Comments
Place stool in preservative (10%
formalin, PVA, SAF, Ecofix) within
one hour of collection.
Instruct patient.
Place in enteric preservative (CaryBlair) immediately.
Stool specimens that are obtained 3
days after admission are not usually
helpful for the diagnosis of hospital
acquired diarrhea
Submit finger stick Thick & Thin
slides or peripheral blood in an
EDTA tube within 24 hours. Store at
ambient temperature.
The first 3 days is best.
A sufficient quantity of the specimen must be
obtained to perform the requested tests
Culture
Blood Culture
Minimum
Requirements
10 ml of aerobic; 10
ml for anaerobic
bottle
Comment
One swab for
multiple
cultures
CSF Culture
A separate swab(s)
for each culture
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).
Enough material must be submitted for gram stain, if
required.
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
Anaerobic
Cultures
See chart
Cooperation with other departments (laboratory and non
laboratory) is key.
See Table
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).
Pediatric Blood Cultures - Volume
Recommended Pediatric Blood Culture Volumes By Patient Weight
Weight
Weight
Minimum
One
Two Adult
(KG) of
(LB) of
Volume
Pediatric
Bottles
Patient
Patient
(mL)
Bottle
(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
30.7-54.9
10.0 mL
No
Yes (5 mL in
Kg
Lb
each)
>25.0 Kg
>55 Lb.
20.0 Ml
No
Yes (10 mL
in each)
Collect all microbiology test samples prior to
the institution of antibiotics
Specimen
Blood Culture
Hair, skin and nails
Fungal Culture
Urine Culture
Comments
Collect two sets at same time from different sets. DO NOT collect both sets from
the same site (assessment for contamination)
Collect before antifungal therapy or discontinue treatment for at least 5 days.
Antibiotics may cause a transient decrease in bacterial concentration resulting in a
false negative report
Blood Cultures - Volume
The magnitude of bacteremia may be low (<1cfu/ml)
Higher volumes have higher yield
Increase Volume
Increase Yield
10 ml  20 ml
30%  40%
20 ml  30 ml
10%  15%
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
Lab
Mikrobiologi
Darah
Urin
Turn Around
Time
Pus
Tinja
Sputum
Cara 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-sterilmidstream/ katetersegera kirim.( Urin
diambil < 3 hari MRS)
 Darah : diambil sesuai
perjalan penyakit
 Dengan media “bactec”
 Ukuran sesuai dengan
aturan
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 typhoid
100
90
% patients with
pos culture
80
70
bloods
60
stool
50
40
30
urine
20
10
0
1 2
3
4
5
weeks
6
7
8
Contents
1
Handling specimen
2
Diagnosis Laboratorium
Infeksi
3
Peta medan kuman
44
Pemilihan AB berdasarkan
sensitivitas test
5
Mekanisme Resistensi
Laboratorium Mikrobiologi
Pemeriksaan Kultur Darah
Contents
1
Handling specimen
2
Diagnosis Laboratorium
Infeksi
3
Peta medan kuman
44
Pemilihan AB berdasarkan
sensitivitas test
5
Mekanisme Resistens
Hasil Peta Kuman – sensitivitas
PICU-NICU - darah (Jan-Jun 2009)RSDK
Chl
Gen
Cip
Ctx
Caz
DKB
FOS
MEM
MFX
SXT
Enterobacter.aerog
enes
92
7
84
26
34
33
80
100
76
64
Eschericia coli
50
25
87
37
50
87
100
87
50
Pseudomonas
aeroginosa
81
6
68
0
37
0
87
25
50
Staphylococcus
epidemidis
83
33
33
33
50
100
50
33
100 80
Ruang Anak
VAN
100
Contents
1
Handling specimen
2
Diagnosis Laboratorium
Infeksi
3
Peta medan kuman
44
Pemilihan AB berdasarkan
sensitivitas test
5
Mekanisme Resistensi
Pengamatan Hasil Pemeriksaan
Mikrobiologi
Pengamatan Hasil
Sebelum
Terapi Empirik
Sesudah
Terapi Definitif
Spektrum luas
De-escalating
Data
epidemiologi
Narrow sp
Pengamatan
aman
oost
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
 Link to: CAP standards for specimen collection and management
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
 Link to: IDSA guideline for evaluating fever in critically ill adults
Follow Established Guidelines
Consult Specialist
Follow Guidelines
Use Local Data
Stop Antimicrobial
Treatment
 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 %
4
1.8 %
Staphylococcus aureus
28
12.6 %
Staphylococcus epidermidis
61
27.4 %
Streptococcus pneumoniae
2
0.9 %
Salmonella typhi
Perjalanan anak dengan sepsis
39.5
Pasien sepsis
dengan demam
selama 10 hari.
39
Esch.coli
( urin)
38.5
Suhu
38
Pseudo.aero
( darah )
Kleb.pnem
( darah )
37.5
Series1
37
36.5
L : 19.300
L :21.000
L : 19.000
L : 8.300
36
35.5
1
2
3
4
Ampi-sulbactam
5
6
7
8
9
10
11
Hari perawatan
12
Amikasin
13
14
15
16
17
18
Kultur 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 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
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 = 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
2
Diagnosis Laboratorium
Infeksi
3
Peta medan kuman
44
Pemilihan AB berdasarkan
sensitivitas test
5
Mekanisme Resistensi
Mechanisms 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
48
1
3
4
2
5
Table . Pediatric bacterial pathogens, mechanisms of
resist
…mechanisms of antimicrobial resistance
Organism
Mech of resist
clinical implications
________________________________________________________
Str pneumoniae
alteration of PBP
alteration in the
ribosomal binding
site of antibiotics
efflux pump to expel
an antibiotics from the
cyoplasm
relative resistant to
-lactam agents (pen
cillin, cephalosp)
resistance to macrolide
relative resist to macro
lide
Pong AL. Pediatr Clin N Am 2005;52:869-94
50
…mechanisms of antimicrobial resistance
 Pediatric bacterial pathogens, mechanisms of resist
Organism
Mech of resist
clinical implications
________________________________________________________
S. Aureus
alteration in the
binding site of a
specific
transpeptidase
(mecA)
alteration at
ribosomal binding
site
efflux pump to expel
an antib from the cyopl
resistant to all -lactams
resistance to macrolides
and clindamycin
relative resist to macro
lide
Pong AL. Pediatr Clin N Am 2005;52:869-94
51
…mechanisms of antimicrobial resistance
 Pediatric bacterial pathogens, mechanisms of resist
Organism
Mech of resist
clinical implications
________________________________________________________
Escherichia coli,
Klebsiella
Enterobacter,
Seratia, other
Enterobacteriaceae
-lactamases with
activity extended
beyond ampic
(ESBL)
chromosomal
-lactamases that
are deregulated and
hyperproduced (ampC)
ceftazidim
resistant to cefotaxim,
ceftriaxone,
ceftazidim
resistant to cefotaxim,
ceftriaxone
ceftazidim
Pong AL. Pediatr Clin N Am 2005;52:869-94
52
…mechanisms of antimicrobial resistance
 Pediatric bacterial pathogens, mechanisms of resist
Organism
Mech of resist
clinical implications
________________________________________________________
Pseudomonas
aerug
multipel -lactamases
each with activity
against different
-lactam
antibiotics
cell wall porin protein
deficient bacteria
multiple efflux pumps
to expel antib from
the cytoplasm
resistant to cefotaxime
ceftriaxone
ceftazidim
carbapenem resist
resistance to -lactam
fluoroquinolones
Pong AL. Pediatr Clin N Am 2005;52:869-94
53
MAJOR ANTIBIOTIC RESISTANCE
MECHANISMS
 Produce antibiotic inactivating enzymes
 Reduce intracellular antibiotic concentration
 Alter antibiotic target site
 Eliminate antibiotic target site
54
Table 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
Altered penicillins binding proteins
β-lactamases
Change peptidoglycans termini
Glycopeptides
Mutations in gyrases or topoisomerase genes
tRNA methylases
Fluoroquinolone
Macrolides
Encode an alternative penicillin binding protein
Methicillin
Produce less enzyme or an alternative enzyme
Trimethoprim,
Sulphonamides
Alter the antibiotic
target site
Eliminate the
antibiotic target site
55
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
proses
biologi
alamiah
Mutation
Gene exchange
Selection
Transmission
Campaign to Prevent Antimicrobial Resistance in Healthcare Settings
Emergence of Antimicrobial Resistance
Susceptible Bacteria
Resistant Bacteria
Resistance Gene Transfer
New Resistant Bacteria
Mutation
R
Gene exchange
Konjugasi
Transduksi
Gene exchange
R
Gene exchange
R
R
R
Campaign to Prevent Antimicrobial Resistance in Healthcare Settings
Selection for antimicrobial-resistant
Strains
Resistant Strains
Rare
Antimicrobial
Exposure
Resistant Strains
Dominant
Selection
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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