Interpretation of Microbiology Results

Yeva Rosana, Anis Karuniawati

Department of Microbiology,

FKUI, Jakarta - 2013

Module of Infection and Immunology 2013

Aims of Diagnostic Microbiology

To provide accurate information about the presence or absence of microorganisms in a specimen that may be involved in patient’s disease process

Where relevant, to provide information on the antimicrobial susceptibility of the microorganisms isolated

Data from Microbiology Laboratories

Improve patient

care

Harm patient

GARBAGE IN,

GARBAGE OUT

Clinical Microbiology Laboratories

QUALITY IN,

QUALITY OUT

Test ordering

Order transcription

Patient preparation

Specimen collection

Specimen identification

Specimen transport

Sample testing

Result transcription

Result delivery

Result review

Action taken on basis

of result

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

Mid stream urine ( Clean catch urine,

urin porsi tengah)

Urine Supra pubic puncture

Urinary Catheter

Urine

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 µl 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

Juni 2006

Lower Respiratory Tract

Sputum Bronchial washing Bronchial brushing Bronchoalveolar

lavage Transtracheal

aspiration Tracheal aspiration

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

0

30

60 Time (min)

Temp

Chills Blood Cultures

Bacteremia

Level

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.

Effect of Volume

0

10

20

30

40

50

60

70

80

90

100

5 10 15 20 25 30 35 40 45 50 55 60

ml

% Relative

Yield

*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, anti-phagocytic, 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 host’s 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 hyper-sensitive 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 β-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 β-Lactamase

Pseudomonas aeruginosa

Acinetobacter baumanii

Stenotrophomonas maltophilia, Bulkhoderia cepacia

Multi-Drugs Resistant Streptococcus pneumoniae (MDRSP)

Extended Spectrum β-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 β-lactamases K.pneumoniae, E.coli, Salmonella spp. Spectrum of resistant: Penicillin, Cephalosporine,

Cephamycin, Monobactam

Carbapenem hydrolizing enzymes (CHE) class B: metallo-β-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

Cultivation-dependent

methods entail long delays for many pathogens (fastidious)

Legionella pneumophila

Chlamydia pneumoniae

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 acute-phase 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 non-

viable organisms RNA detection is more accurate indicator of

viable microorganisms