Interpretation of Microbiology Reports

Dr. Cathal Collins

Objectives

• Have some idea of laboratory processes• Have some idea of the relative importance of

laboratory reports and how they should be interpreted

Workshop Format

• First bit:– Example to demonstrate laboratory processes

• Middle bit– Examples to demonstrate how reports should be interpreted

• Final bit– Lessons learned

First Bit

Example

• Jane Doe, nursing home• Presented to A&E• Fever, frequency, dysuria and right flank pain• Clinical review• Blood cultures and MSU• Co-amoxiclav and gentamicin (both IV) started

Urine microscopy counting chamber

White cell

Epithelial cell

Urine microscopy

Day 1 Laboratory

• Urine microscopy is the only report that will be available soon after specimen arrival– White cells (note significant pyuria >10white cells/mm3)

– Red cells

– Epithelial cells

– Casts/crystals

– Bacteria (present or not)

• Appropriate agar plates are inoculated in attempt to culture pathogens for identification and susceptibilities

Blood cultures

Blood culture bottle Blood culture machine

Day 2 Laboratory

• Blood culture flags up as positive in the blood culture machine

Gram stain

Gram-positive cocci ?staph

Gram-positive cocci ?strep

Gram-negative bacilli

Yeast

Day 2: Blood culture flags +ve

Gram stain and Organisms

• Gram-positive cocci– Staphylococcus spp– Streptococcus spp– Enterococcus spp

• Gram-positive bacilli– Listeria monocytogenes– Clostridium spp– Bacillus spp

• Gram-negative cocci– Neisseria spp– Moraxella catarrhalis

• Gram-negative coccobacilli– Haemophilus spp– Acinetobacter spp– Bordetella pertussis

• Gram-negative bacilli– Escherichia coli– Klebsiella spp– Proteus spp– Enterobacter spp– Serratia spp– Pseudomonas spp

Day 2 Laboratory

• Gram stain of blood: interim report issued and communicated with advice

• Appropriate agar plates are inoculated– Direct susceptibility testing using 5 or 6 key antibiotics:

e.g. co-amoxiclav, pip-tazobactam, gentamicin, ciprofloxacin, cefpodoxime

– Not standardised- a drop of blood is lawned onto an agar plate- don’t know how much bug is in the drop

Day 2 Laboratory

• Good idea of what is growing on the urine agar plates

MacConkey NLF and LF

Chromogenic agar

Day 2 Laboratory

• Urinary isolate:– Set up biochemical

identification test• API• Automated (Vitek,

Phoenix etc)

API 20e

Vitek 2Phoenix

Day 2 Laboratory

• Urinary isolate:– Set up susceptibility tests

(standardised inoculum)• Disc diffusion

• Automated (Vitek,

Phoenix etc)Disc diffusion

Vitek E-test for MIC

Day 3 Laboratory

• Final report on urine specimen• However, additional tests may be indicated to

establish the resistance mechanism• Good idea of what’s in the blood cultures with

unreliable susceptibility results for the 5 key anti-GNB antibiotics

• Identification of and standardised susceptibility testing on the blood culture isolate is performed

Day 3 Laboratory

• The direct non-standardised susceptibility tests suggests that the blood culture organism may have reduced susceptibility to co-amoxiclav, pip-tazobactam, gentamicin, cefpodoxime and ciprofloxacin

• The urinary isolate is an Escherichia coli

• However, the standardised susceptibility pattern on the urinary E. coli is concerning!

Susceptibility pattern of urinary E. coliAntibiotic Susceptibility

Ampicillin R

Co-amoxiclav I

Cephradine R

Cefuroxime R

Cefotaxime I

Ceftazidime S

Cefepime I

Cefoxitin S

Pip-tazobactam I

Meropenem S

Ciprofloxacin R

Nitrofurantoin S

Co-trimoxazole R

Amikacin S

Gentamicin R

http://www.bsac.org.uk/_db/_documents/Chapter_11.pdf

Day 3 Laboratory

• The susceptibility pattern is highly suggestive that the organism is an extended-spectrum beta-lactamase (ESBL) producer

• Confirmatory ESBL tests set up on both urinary and blood culture isolate:– Looking for differences in susceptibility between a 3rd/4th gen

cephalosporin with and without a beta-lactamase inhibitor

Meanwhile…

• The patient has not improved clinically, remaining ill with a persistent fever and rising inflammatory markers

• The clinical team are advised to stop the co-amoxiclav and gentamicin and to start meropenem

• The infection control team are contacted and informed re a probable ESBL-producing isolate

Day 4: Final Urine Report

• Microscopy:– WCC 450/mm3

– RCC 0– No casts/crystals– ++ bacteria

• Culture:– > 105 cfu/mL Pure growth of E. coli– S: Meropenem– R: Ampicillin, Ciprofloxacin, Gentamicin

• Comment:– Similar isolate to that in blood. This isolate is an ESBL producer.

Infection control precautions in a healthcare setting are indicated. Please contact the clinical microbiology team if any concerns.

Day 4: Final Blood Culture Report

• Gram:– Gram-negative bacillus both bottles at 12 hours

• Culture:– Escherichia coli– S: Meropenem– R: Ampicillin, Ciprofloxacin, Gentamicin

• Comment– Significance as discussed. Similar isolate to that in urine.

This isolate is an ESBL producer. Infection control precautions in a healthcare setting are indicated. Please contact the clinical microbiology team if any concerns.

Antimicrobial stewardship

“Prioritization of tested antimicrobials and selective reporting of susceptibility profiles (e.g., not routinely reporting susceptibility of S. aureus to rifampin to prevent inadvertent monotherapy with rifampin) can aid in the prudent use of antimicrobials and direct appropriate therapy based on local guidelines”

Antimicrobial stewardship

“…there is an association between antibiotic susceptibility reporting from microbiology laboratories and antibiotic prescribing for the treatment of urinary tract infections.”

Ciprofloxacin and risk of resistant organisms e.g. C. difficile

Lesson Slide

• Microscopy result early, culture result late• More information available in the laboratory than is

released in the reports

Sterile v Non-sterile Site

• SterileThese sites normally do not contain any bacteria so any bacteria found there are significant– Urine– Blood– CSF– Bile– Fluids: Pleural, peritoneal,

synovial, pericardial, amniotic, bursa, CAPD

– Deep tissue samples?

• Non-sterileThese sites are open to the external environment and normally contain bacteria (normal flora, colonisers)– Throat swabs– Skin swabs– Wound swabs– Ear swabs– Nasal swabs– Sputum samples– Nail clippings– Faeces

Sterile v Non-sterile Site

• SterileThese sites normally do not contain any bacteria so any bacteria found there are significant– Urine– Blood– CSF– Bile– Fluids: Pleural, peritoneal,

synovial, pericardial, amniotic, bursa, CAPD

– Deep tissue samples?

• Non-sterileThese sites are open to the external environment and normally contain bacteria (normal flora, colonisers)– Throat swabs– Skin swabs– Wound swabs– Ear swabs– Nasal swabs– Sputum samples– Nail clippings– Faeces

Iden

tify

all o

rgan

isms g

row

ing

Sterile v Non-sterile Site

• SterileThese sites normally do not contain any bacteria so any bacteria found there are significant– Urine– Blood– CSF– Bile– Fluids: Pleural, peritoneal,

synovial, pericardial, amniotic, bursa, CAPD

– Deep tissue samples?

• Non-sterileThese sites are open to the external environment and normally contain bacteria (normal flora, colonisers)– Throat swabs– Skin swabs– Wound swabs– Ear swabs– Nasal swabs– Sputum samples– Nail clippings– Faeces

Iden

tify

all o

rgan

isms g

row

ing

Look

for s

peci

fic p

atho

gens

Sputums

• Upper respiratory tract not sterile

• What are the significant organisms?– Depends on patient’s

history

Sputums

• Upper respiratory tract not sterile

• What are the significant organisms?– Depends on patient’s

history

Sputums

• Bronchitis, chest infection, COPD, pneumonia: – H. influenzae, S. pneumoniae, S. aureus, M. catarrhalis,

other organisms in pure growth may be significant

• Bronchiectasis, cystic fibrosis, immunocompromised, ICU:– As above

– Enterobacteriaceae, Pseudomonads, fungi

• Cystic fibrosis– All the above

– B. cepacia complex

Lesson Slide

• Only organisms that are considered potentially pathogenic are worked up from specimens from non-sterile sites– Same applies to wound swabs, faecal samples etc

CSFs

• Initial microscopy including Gram stain performed urgently on sample when it arrives in the lab and the results are communicated immediately

• Culture plates are examined daily but may not get a definitive result for a number of days

• Many reasons for no growth in a patient with bacterial meningitis– Antibiotics before sample was taken– Delicate organism– Fastidious organism

Middle Bit

Case 1

• 28-year old female admitted for management of Crohn’s disease exacerbation

• Day 3 of admission• Dysuria, frequency and suprapubic pain for one day

prior to admission• No fever or flank pain on admission• Commenced on ciprofloxacin 500mg BD PO by

team; now day 3

Urine report

• Microscopy:– WCC 450/mm3

– RCC 0– No casts/crystals– ++ bacteria

• Culture:– > 105 cfu/ml Pure growth of Escherichia coli– R: Ampicillin, Trimethoprim– S: Co-amoxiclav, Nitrofurantoin

Case 1

Case 2

• 37-year old male• Admitted with cellulitis of left lower limb

surrounding left ankle and extending proximally• Was ice-skating 5 days previously- healing blister on

left ankle • No past medical history of note• On flucloxacillin 500mg QDS IV and

benzylpenicillin 600mg QDS IV

Swab of blister report

• Culture report:– Staphylococcus aureus

• S: Flucloxacillin, Erythromycin– Pseudomonas aeruginosa

• S: Ciprofloxacin, Pip-tazobactam– Coagulase-negative staphylococci

Case 2

Case 3

• 66-year old male• On vancomycin 500mg BD IV day 2 because of the

urine report below; trough level today 7.5mg/L• Mid-stream urine sent to the laboratory 4 days earlier

– Report:• White cell count 20/mm3

• No red cells• No casts• Culture: >105 orgs/mL Pure growth MRSA

– R: Flucloxacillin, Erythromycin– S: Nitrofurantoin, Trimethoprim, Linezolid, Vancomycin

Scenario 1

• Admitted as an emergency 25 days previously with a perforated bowel

• Required a laparotomy and a course of antibiotics (amoxicillin, gentamicin, metronidazole)

• Was admitted to ICU (central line etc), now on the wards

• Finished course of antibiotics over 2 weeks earlier• MRSA screen persistently positive (nose and groin)• Urinary catheter removed 4 days previously• Was always afebrile and well with no urinary or

systemic symptoms

Case 3

Scenario 1

Stop vancomycin!Asymptomatic bacteriuria

Case 3

Scenario 2

• Same patient• However, new onset dysuria and frequency for 2 days• No fever, no flank pain

Case 3

Stop vancomycin!Nitrofurantoin or doxycycline to complete 7-10 days

of antimicrobial treatment

Scenario 2

Case 3

Scenario 3

• Same patient• No urinary symptoms• However, fever for the last 10 days not settling

despite empiric pip-tazobactam (which was stopped that morning)

• Complains of dyspnoea, chest pain• New systolic murmur on auscultation

Case 3

Investigate and treat!3 sets of blood cultures

Trans-oesophageal ECHOIncrease vancomycin dose

Aim for trough levels of 15-20mg/LAdd gentamicin and rifampicin

Scenario 3

Case 3

Lesson Slide

• Treat the patient, not the report!– A laboratory report should always be correlated with the

clinical picture

Case4

• 32 year old female, BIBA to A&E• 2 day hx of malaise, headache, fever, nausea• Became lethargic and confused and had a focal

seizure• LP:

– WCC 67, 98% lymphocytes – RCC 0– Glucose normal– Protein slightly raised– Gram stain: no organisms seen– Culture: no growth

Case 4

• PCR for viral pathogens (HSV 1 and 2, VZV, Enterovirus) negative

• Sensitivity and specificity of PCR assay for HSV >95%

• Patient was started on high dose IV acyclovir on admission for presumed HSV encephalitis

• Would you stop the acyclovir?

Sensitivity and Specificity of a Test

• Sensitivity– The proportion of people with the disease that the test

correctly classifies as having the disease

• Specificity– The proportion of people without the disease that the test

correctly classifies as not having the disease

Case 4

• Both the sensitivity and specificity of HSV PCR are >95% but they are not 100%

• False negative results are possible

PPV and NPV of a Test

• Positive predictive value– The probability of a disease being present assuming a

positive result is obtained (true positives/ test positives)

– The post-test probability of being infected after a positive test result

• Negative predictive value– The probability of not having a disease assuming a

negative result is obtained (true negatives/ test negatives)

– The post-test probability of being uninfected after a negative test result

Calculating PPV and NPV

Case 4

• Pre-test probability of HSV disease approx 60%• Worst case scenario: sensitivity and specificity of test

95%• NPV 93%• Post-test probability of HSV disease with a negative

HSV PCR approx 7%• Acyclovir should be continued

Case 4

• If patient did not have confusion or focal neurological findings, the pre-test probability of HSV disease would be approx 5%

• The post-test probability of HSV disease with a negative HSV PCR result now would be approx 0.3%

• Acyclovir can be stopped

Lesson Slide

• Results don’t always give definitive answers– In many ways relates to second Lesson Slide

Final Bit

Objectives

• Have some idea of laboratory processes• Have some idea of the relative importance of

laboratory reports and how they should be interpreted

Lessons

• Microscopy result early, culture result late• More information available in the lab than is released

in the reports• Only organisms that are considered potentially

pathogenic are worked up from specimens from non-sterile sites

• Treat the patient, not the report• Results don’t always give definitive answers

Lessons

• Microscopy result early, culture result late• More information available in the lab than is released

in the reports• Only organisms that are considered potentially

pathogenic are worked up from specimens from non-sterile sites

• Treat the patient, not the report• Results don’t always give definitive answers

Thank you

Any Questions?