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Module of Infection and Immunology 2013

Interpretation of
Microbiology Results

Yeva Rosana, Anis Karuniawati


Department of Microbiology,
FKUI, Jakarta - 2013

Aims of Diagnostic Microbiology


To provide accurate information about
the presence or absence of
microorganisms in a specimen that may
be involved in patients disease process
Where relevant, to provide information
on the antimicrobial susceptibility of the
microorganisms isolated

Data from Microbiology


Laboratories
Improve
patient
care

Harm
patient

Clinical Microbiology Laboratories

GARBAGE IN,
GARBAGE OUT

QUALITY IN,
QUALITY OUT

Test ordering

Result transcription

Order transcription
Patient preparation

Result delivery
Sample testing

Specimen collection

Result review
Action taken on basis
of result

Specimen identification
Specimen transport

Pre analytical

Analytical

Post analytical

Important requirements
Adequate collection, transport, and
processing of clinical specimens
Cooperation among medical team members :
Clinical practitioners
Nurses
Laboratory practitioners

Ongoing communication and education


among all members of the team

Specimen Collection

Acute phase, before antibiotics


Correct anatomic site
Proper technique, minimal contamination
Appropriate quantity
Container
Labeling
Transport

Important data
Patient identity (name, age, sex, ward)
Clinician identity (name, adress, ph-no.)
Specimen (type, source, time of
collection)
Relevant clinical information
Antibiotics usage
Laboratory test required

Patient-Collected Specimens
Urine, Sputum, Stool, Semen
Never be assumed that the patient
knows how to collect
Printed instruction: will be read?
understand?
Verbal, pictures and written

Urine
Mid stream urine ( Clean catch urine,
urin porsi tengah)
Urine Supra pubic puncture
Urinary Catheter

Swab
Upper respiratory tract, external ear,
eye, genital tract
Tips of swab:
Cotton: excessive fatty acids, toxic to
certain bacteria
Dacron or polyester

Transport media: protect the specimen


from drying

Swabs for Wounds


Aspirate or biopsy sample is much better
Anaerobes on swab die upon exposure to
air but survive in tissue and fluid
Swabs hold only 150 l of fluid
Can become dried out, leading to a loss
of organisms

Upper Respiratory Tract


Throat Swab

Upper respiratory tract, external


ear, eye, genital tract
Tips of swab:
Cotton: excessive fatty acids, toxic to
certain bacteria
Dacron or polyester

Transport media: protect the


specimen from drying

Lower Respiratory Tract

Sputum
Bronchial washing
Bronchial brushing
Bronchoalveolar
lavage
Transtracheal
aspiration
Tracheal aspiration

Juni 2006

GENITAL TRACT SPECIMENS


For Females
Cervical specimens should be
collected after removing
excess mucous from the
cervical os and surrounding
mucosa
Use a second swab to collect
specimen by rotating the
swab for 10 to 30 secs. in
the endocervical canal
Collect vaginal specimens
using a speculum without
any lubricant

GENITAL TRACT SPECIMENS


For males
Urethral specimens are
collected by inserting a
swab 2 to 4 cm. into the
urethra and rotating the
swab for 2 to 3 seconds

GENITAL TRACT SPECIMENS


For HSV lesions
Fluid from lesions should be
aspirated using a syringe
Swab can be used to collect vesicle
fluid or cellular material from the
base of the lesion before crusting
and healing have begun

Genital Specimens
Specimen
source

Potential
Pathogens

Primary plating
media

Cervix

Chlamydia; GC;
herpes

SBA, Choc, TM;


viral transport
media for herpes

Cul-de-sac

Anaerobes, GC, CT,


enterics

SBA, choc, TM,


Mac, ana, thio

Endometrium

Mixed aerobes
/anaerobes

SBA, choc, TM,


Mac, ana, thio

Vagina

Group B strep;
Mixed aerobes
anaerobes BV

SBA; LIM or other


special broth

Storage and Transport of Specimens


Ideally within 30 minutes, preferable
within 2 hours
Refrigerate:
CSF for viruses, outer ear, feces, sputum, urine

Room temperature:
Abscess, lesion, wound, body fluids, CSF for
bacteria, inner ear, genital, nasal, throat,
tissue

Factors for successful recovery of


microbes from blood

Possible types of bacteremia


Specimen collection methods
Blood volumes
Number of specimens: 2-3
Timing of blood cultures
Interpretation of results

Bacteremia

When ?
Draw blood cultures as close as possible to the episode of
chills or fever. Do NOT delay, as recovery of
microorganisms diminishes with time after the fever spike.

Chills

Blood Cultures

Bacteremia
Level

Temp
30
0

Time (min)

60

Effect of Volume
100
90
80
70
60
% Relative 50
Yield
40
30
20
10
0
5

10 15 20 25 30 35 40 45 50 55 60
ml

*Reprinted from Infectious Disease Clinics of North America, Vol 16, M.L. Towns and L. B.
Reller, Diagnostics methods: Current best practices and guidelines for isolation of bacteria
and fungi in infective endocarditis, p. 363-376(2002) with permission from Elsevier.

Number of blood specimens

Blood volume
Adults: bacteria <30 CFU/ml, more blood that is
cultured, the greater the chance of isolating the
organism
Infants: bacteria >1000 CFU/ml

Blood Volume in children

26

Anticoagulation
Entrapped bacteria within a clot may go
undetected
Inoculated into sterile tube containing an
anticoagulant for transport to the laboratory
Heparin, EDTA, Citrate : not recommended
SPS (Sodium polyanethol sulfonate)
0,025-0,03%
SPS : anticoagulant, anti-complementary, antiphagocytic, interferes activity of some antibiotics
Inhibit Neisseria spp., Gardnerella vaginalis,
Streptobacillus moniliformis, Peptostreptococcus
anaerobius

Specimen Rejection
Suboptimal specimens
Require a phone call to the person in charge of
collecting specimen
Lab never discharge specimen before contacting
the health care team
In certain situation it may be necessary to process
suboptimal specimen
notation in final report!

A rejected specimen is not


a repudiation of the
health care provider
who submitted it.
It is simply a request for a
new specimen that will
provide the clinically
information necessary
for good patient care.

Microbiology Investigation:
Microscopic
Culture and susceptibility tests
Serology:
Antigen detection
Antibody detection

Molecular:
Detection of nucleic acid

Gram staining : report


The presence of host cells and debris
The Gram reactions, morphologies, and
arrangement of bacterial cells present.
Reporting the absence of bacteria and host
cells can be equally as important
Optionally, the relative amounts of bacterial
cells (rare, few, moderate, many) may be
provided

Post- Analytical Phase of Testing


Result transcription
Result delivery

Result review
Action taken on basis of result

report it all and let the


doctor decide..

does not take into account the


reality of
If the microbiologist names it, the
physician feels obliged to treat it

Factors to consider When Determining


Whether Testing is Warranted

The body site from which the organism


was isolated
The presence of other bacteria and the
quality of the specimen from which the
organism was grown
The hosts status

Pharyngitis
Normal pharyngeal flora:

Staphylococcus aureus and MRSA


Streptococcus pneumoniae
Haemophilus influenzae
Neisseria meningitidis

Should not be reported unless there


is a special communication to
microbiologist

Nose cultures
Not predictive of the etiologic agents of
sinus, middle ear, or lower respiratory tract
infections

Pathogen directed:
Screening of MRSA
Detection of Bordetella pertusis

Urine culture
105 CFU/ml urine in asymptomatic patient
102 CFU/ml urine in patient with clinical
sign and symptom of UTI
group B streptococci:
Pregnant women: any amount have
implications for the fetus
Female 12-55: >50 CFU/ml urine

Relevant clinical information ??

Interpretation: Blood Culture


Probable contaminant:

Bacillus spp, Corynebacterium spp.,


Propionibacterium acnes, or coagulase(-)
staphylococcis in only one of several cultures
multiple organisms from only one of several
cultures
The clinical presentation and/or course is not
consistent with sepsis
The organism causing the infection at a primary site
of infection is not the same as that isolated from
the blood culture

Interpretation: Blood Cultures


Probable pathogen

the same organism in repeated cultures obtained


either at different times or from different anatomic
sites
certain organisms in culture obtained from patients
suspected of endocarditis
certain organisms such as members of
Enterobacteriaceae, S. pneumoniae, gram-neg
anaerobes, and S. pyogenes
commensal microbial flora from e.g.
immunosuppressed patients or those having
prosthetic devices

How susceptibility is reported:


zone diameter (mm) vs. RIS+3 vs. RIS

There is no very-, super- or hypersensitive organism


Killing of a sensitive bacteria in vivo is
a multi-factorial event
It is NOT recommended to report
S+1~3 or sensitive, very sensitive, or
most sensitive. It is EXTREMELY
misleading!

Interpretative Reading of
Antibiotics Susceptibility Test

Recognizing unusual results


Recognizing drugs best avoided owing
to their risk of selecting resistance in
the particular pathogen

Using indicator drugs


DM.Livermore, TG Winstanley, KP Shannon. J.of
Antimicrobial Chemotherapy (2001) 48, Suppl.S1

Recognizing Unusual Results:


Resistance requiring confirmation
Examples
S.aureus
Any of: vancomycin, teicoplanin. linezolid
Streptococcus pneumoniae
Any of: Meropenem, vancomycin, teicoplanin, linezolid
Enterobacteriaceae
Meropenem, imipenem
Neisseria gonorrhoeae
Any third-generation cephalosporin
Anaerobes in general
metronidazole

Natural Resistance Typical of Common Pathogens


Examples
Acinetobacter baumannii
Ampicillin, amoxycillin, 1st gen. cephalosporin
Pseudomonas aeruginosa
Ampicillin, amoxycillin, 1st and 2nd gen. cephalosporin,
cefotaxime, ceftriaxone, nalidic acid, trimethoprim
Salmonella spp.
Cefuroxime (active in vitro, not active in vivo)
Proteus vulgaris
Ampicillin, amoxycillin, cefuroxime, colistin, nitrofurantoin
Strepococcus pneumoniae
Trimethoprim, amynoglycoside

Using indicator drugs


Examples
MRSA: resistant to all -lactams
ESBL (ceftazidime, cefpodoxime,
cefotaxime, ceftriaxone): avoid all
cephalosporin
N.gonorrhoeae (nalidixic acid): indicate
reduced susceptibility to
fluoroquinolones

Multiple Drugs Resistant Organisms


(MDROs)
Microorganisms, predominantly bacteria,
that are resistant to one or more
classes of antimicrobial agents
Although the names of certain MDROs
describe resistance to only one agent,
these pathogens are frequently resistant
to most available antimicrobial agents

MDROs
Resistant Staphylococcus aureus : MRSA, VISA, VRSA
Vancomycin Resistant Enterococcus (VRE)
Gram Negatif Bacteria (GNB):

Extended Spectrum -Lactamase


Pseudomonas aeruginosa
Acinetobacter baumanii
Stenotrophomonas maltophilia, Bulkhoderia cepacia

Multi-Drugs Resistant Streptococcus pneumoniae


(MDRSP)

Extended Spectrum -lactamases


(ESBLs)
Bacteria have the ability to hydrolyze and cause resistance
to the 3rd-generation Cephalosporins and monobactams but
not the cephamycins and carbapenems
Beta-lactamase inhibitors (clavulanic acid, sulbactam, and
tazobactam) generally inhibit ESBL producing strains
Important reason for therapy failure with cephalosporins and
have serious consequences for infection control

Other MDROs
Plasmid mediated AmpC -lactamases

K.pneumoniae, E.coli, Salmonella spp.


Spectrum of resistant: Penicillin, Cephalosporine,
Cephamycin, Monobactam

Carbapenem hydrolizing enzymes (CHE) class B:


metallo--lactamases

P.aeruginosa, Acinetobacter spp. Enterobacteriaceae (rare)


Spectrum of resistant: Penicillin, Cephalosporine,
Cephamycin, Carbapenems

CHE class A: Klebsiella pneumoniae carbapenemase


K.pneumoniae, E.coli
Spectrum of resistant: Penicillin, Cephalosporine,
Cephamycin, Carbapenems

Clinical Importance of MDROs


Clinical manifestations are similar to infections
caused by susceptibility pathogens
Option for treating patients with these infections
are often extremely limited
Patients with MDROs stay in hospital longer, at
higher cost
Higher mortality

Factors,
affect transmission and spread
environmental factors, both in the
hospital and the community
the use of antibiotics
the antimicrobial fitness of the
pathogen

Phenotypic Identification: Problem


Many significant
pathogens cannot be
grown reliably by current
methodologies
Chlamydia pneumoniae

Cultivation-dependent
methods entail long
delays for many
pathogens (fastidious)

Legionella pneumophila

Phenotypic Identification: Problem


Some phenotypes displayed by a single
organism may vary as a function of the
particular growth condition used in the laboratory
A single phenotype may be generated by any of
a number of highly variable gene products or
combination thereof
Detection of toxin or other virulence factors

Problem Solving?
Alternative tests:
Serology
Nucleic Acid Amplification : PCR

Antigen detection
Microbial-specific structural components (antigens) are
identified in specimens obtained from an infected host
Can be used to identify microorganisms once they have
been recovered in culture
Methods depends on the fact that some microbial
components are chemically unique and form areas on
the molecule known as antigenic determinants
Antigen can be recognized by and can combine
specifically with antibody molecules to form stable
products

Antibody detection
IgM antibodies
detected earlier in the infection (7-10 days)
Usually indicative of active, as opposed to past,
infection

IgG antibodies
Previous infections or immunization
Serodiagnosis of an infectious diseases requires
measurement of IgG concentration on both acutephase and convalescent-phase serum specimens
Chronic infections, epidemiology

False-Negative Serologic test results


Negative result for a patient who really is infected
May not have an intact immune system, and therefore
may not be able to respond to an antigenic stimulus
Congenital or acquired immunodeficiency diseases
Receiving either immunosuppressive therapy after
organ transplantation or cancer chemotherapy
Neonates may not always respond to an infectious agent
because their immune system are not fully mature
For some infections (e.g. legionaires disease) antibody
titers may not rise until months after acute infection

False-Positive Serologic test results


Positive result for a patient who is not infected by the specific
agent for which the test is design
Production of cross-reacting antibody
Some antigen associated with different microorganisms
are closely related and a host may respond by
producing antibody not only to the invading organism
but also to antigenically closely related organism
Reactivation of a latent organism due to infection by a
different organism
Receiving intravenous immunoglobulin

Gene amplification by PCR


PCR can be used to amplify a specific DNA sequence to
produce millions of copies within a few hours
Post-PCR analysis
Confirmation of the PCR product by agarose-gel
electrophoresis
Hybridization with oligonucleotide probes
real time PCR
The use of specifically constructed PCR primers that
fluoresce when incorporated into PCR-generated
amplicons
RT-PCR
RNA may be amplified after it has been converted
to DNA by the enzyme reverse transcriptase

Limitations of PCR:

Clinical significance of positive PCR


PCR assays may detect pathogens at
concentration below those of previously
established gold standard reference
methods
Development of reliable quantitative
measures of pathogen load

PCR assay detect both viable and nonviable organisms

RNA detection is more accurate indicator of


viable microorganisms