1. Interpretation of Microbiology Reports
Dr. Cathal Collins

2. Objectives Have some idea of laboratory processes
Have some idea of the relative importance of laboratory reports and how they should be interpreted

3. Workshop Format First bit: Middle bit Final bit
Example to demonstrate laboratory processes
Middle bit
Examples to demonstrate how reports should be interpreted
Final bit
Lessons learned

4. First Bit

5. 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

6. Urine microscopy Urine microscopy counting chamber White cell
Epithelial cell
Urine microscopy counting chamber

7. 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

8. Blood cultures
Blood culture bottle
Blood culture machine

9. Day 2 Laboratory
Blood culture flags up as positive in the blood culture machine
Gram stain

10. Day 2: Blood culture flags +ve
Gram-positive cocci ?staph
Gram-negative bacilli
Gram-positive cocci ?strep
Yeast 10

11. 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

12. 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

13. Day 2 Laboratory Good idea of what is growing on the urine agar plates
MacConkey NLF and LF
Chromogenic agar

14. Day 2 Laboratory Urinary isolate:
Set up biochemical identification test
API
Automated (Vitek, Phoenix etc)
API 20e
Phoenix
Vitek 2

15. Day 2 Laboratory Urinary isolate:
Set up susceptibility tests (standardised inoculum)
Disc diffusion
Automated (Vitek, Phoenix etc)
Disc diffusion
Vitek
E-test for MIC

16. 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

17. 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!

18. Susceptibility pattern of urinary E. coli
Antibiotic
Susceptibility
Ampicillin R
Co-amoxiclav I
Cephradine
Cefuroxime
Cefotaxime
Ceftazidime S
Cefepime
Cefoxitin
Pip-tazobactam
Meropenem
Ciprofloxacin
Nitrofurantoin
Co-trimoxazole
Amikacin
Gentamicin

21. 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

22. 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

23. 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.

24. 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.

25. 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”

26. 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

27. Lesson Slide Microscopy result early, culture result late
More information available in the laboratory than is released in the reports

28. Sterile v Non-sterile Site
These 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-sterile
These 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

29. Sterile v Non-sterile Site
These 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-sterile
These 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
Identify all organisms growing

30. Sterile v Non-sterile Site
These 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-sterile
These 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
Identify all organisms growing
Look for specific pathogens

31. Sputums Upper respiratory tract not sterile
What are the significant organisms?
Depends on patient’s history

32. Sputums Upper respiratory tract not sterile
What are the significant organisms?
Depends on patient’s history

33. 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

34. 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

35. 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

36. Middle Bit

37. 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

38. Urine report Case 1 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
Is the isolate sensitive to ciprofloxacin?
Is the patient still symptomatic?
Does she still need antimicrobial therapy or a change to an alternative agent?
Why was a fluoroquinolone chosen for an uncomplicated lower urinary tract infection?
Was there a pregnancy test at any point?

39. 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

40. Swab of blister report Case 2 Culture report: Staphylococcus aureus
S: Flucloxacillin, Erythromycin
Pseudomonas aeruginosa
S: Ciprofloxacin, Pip-tazobactam
Coagulase-negative staphylococci
Is the cellulitis improving?
Relevancy of each organism- Does he need Pseudomonas cover?
Suboptimal doses
Is the benzylpenicillin required?
Change to orals?

41. 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
On vancomycin but trough level is below recommended therapeutic range of 10-15mg/L
When was level taken?
Any missed doses?
Etc…
Is vancomycin necessary?
Need clinical information!

42. Case 3
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

43. Stop vancomycin! Asymptomatic bacteriuria
Case 3
Scenario 1
Stop vancomycin! Asymptomatic bacteriuria

44. Scenario 2 Case 3 Same patient
However, new onset dysuria and frequency for 2 days
No fever, no flank pain

46. Case 3
Scenario 2
Stop vancomycin! Nitrofurantoin or doxycycline to complete 7-10 days of antimicrobial treatment

47. Scenario 3 Case 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

48. Case 3
Scenario 3
Investigate and treat! 3 sets of blood cultures Trans-oesophageal ECHO Increase vancomycin dose Aim for trough levels of 15-20mg/L Add gentamicin and rifampicin

49. Lesson Slide Treat the patient, not the report!
A laboratory report should always be correlated with the clinical picture

50. 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

52. 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?

53. Sensitivity and Specificity of a Test
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

54. Case 4
Both the sensitivity and specificity of HSV PCR are >95% but they are not 100%
False negative results are possible

55. 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

56. Calculating PPV and NPV

57. 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

58. 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

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

60. Final Bit

61. Objectives Have some idea of laboratory processes
Have some idea of the relative importance of laboratory reports and how they should be interpreted

62. 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

63. 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

64. Thank you
Any Questions?