Microbiology Laboratory Diagnosis & Data interpretation Prof T Rogers Dept of Clinical Microbiology Trinity College Dublin.

Microbiology Laboratory Diagnosis & Data interpretation

Prof T Rogers

Dept of Clinical Microbiology

Trinity College Dublin

Case 1:

• A 70 year old man with a history of chronic obstructive lung disease is seen in A & E

• He complains of breathlessness, is hypoxic, and has a fever

• He gives a history of increasing sputum production which is purulent

• No one else in his family is ill at this time

How will the clinician proceed?

Factors to consider in this patient:

• His medical history: COPD

• His age: an older patient

• Based on his presentation: likely to have a lower respiratory tract infection

• Maybe he has had repeated courses of antibiotics: implication for treatment?

• How will the diagnosis be established or is it sufficient to treat him with antibiotics?

CHEST X RAY: COMMUNITY ACQUIRED PNEUMONIA WHAT’S CAUSING IT?

PAST & PRESENT

• We know from past Epidemiological studies what the causes of CAP are based on microbiological confirmation

• We need to organise microbiological investigations in this patient: what are they?

Some established approaches to Microbiological diagnosis:

• Examination of the specimen: purulent sputum (will saliva do?)

• Microscopy: what test is done?

• Culture: What information does it provide?

• What if the pathogen we are considering is non-cultivable? Are there other investigations to be done?

SPUTUM GRAM STAIN: TYPICAL APPEARANCE OF Strep pneumoniae

GRAM POSITIVE CHAINSDIPLOCOCCI

CAUSES (%) OF COMMUNITY ACQUIRED PNEUMONIA FROM HOSPITAL BASED

STUDIES (N=3,000)CAP

Severe CAP

• No cause found 36 33• Pneumococcus 25 27• Influenza virus 8 2.3• Legionella spp*. 7 17• Haem. Influenzae 5 5• Other viruses 5 8• Psittacosis/Q fever 3 2• Gram neg. bacilli 2.7 2• Staph aureus* 2 5

CAUSES OF ‘ATYPICAL’ PNEUMONIA

• Mycoplasma pneumoniae

• Chlamydophila pneumoniae

• Legionella pneumophila

• Coxiella burnetii

INFLUENZA: culture is difficult to set up so molecular tests used

Legionella pneumophila is “fastidious” so we rely on detection of an antigen of the bug that’s present in urine

Gram –ve, flagellated rod, aerobicFacultative intracellular parasite in both amoeba and human monocytes/macrophages

HANG ON… what is this antigen, and is it present in all strains of L pneumophila???

HANG ON… what is this antigen, and is it present in all strains of L pneumophila???

• No, it’s only found in L pneumophila serogroup 1, which happens to be the most common cause of LD

• But if the cause is another serogroup of Lp what can we do to confirm the diagnosis?

But what about the other causes of atypical pneumonia- they are difficult to culture too!

• Mycoplasma pneumoniae: serology for complement fixing antibodies

• Coxiella burnetii (Q fever): PCR for early diagnosis, acute and convalescent sera for phase 1 and 2 antibodies

• Chlamydophila pneumoniae: acute and convalescent sera for detection of specific IgG and/or IgM antibodies in serological tests

How reliable is a laboratory diagnostic test?

• Sensitivity; the proportion of individuals with the infection (disease) who are correctly identified by the laboratory diagnostic test.

• No with a positive test, divided by the number with the disease

• If 9 out of 10 with the infection have a positive test in a population of 100 then the sensitivity is 90%

What’s the test’s specificity?

• Specificity: proportion of population studied who do not have the infection who are correctly identified by the test;

• So if among the 90 without the infection 2 have a positive result the specificity is

97% (88/90)

When do we look for high sensitivity and specificity?

• When looking for common diseases that are easily treatable and not so serious high sensitivity

• Or if we are screening for an uncommon infection where we can take the positive and do a second test that is more specific eg, screening blood for infectious agents

• For a serious disease eg, HIV/AIDS we need excellent specificity for confirmation otherwise MISDIAGNOSIS!

What are the implications of a positive or negative result?

• Positive predictive value of a test:

proportion of individuals with a positive test result who have the disease

Negative predictive value:

Proportion with a negative result who don’t have the disease

Case 2:Clinical Symptoms/SignsCase 2:Clinical Symptoms/Signs

• Cough x 2-3 weeks• Purulent Sputum• Haemoptysis• Fevers• Weight Loss• Chest Pain

LABORATORYLABORATORYSMEAR-STAINSMEAR-STAIN

CULTURECULTURESENSITIVITY PROFILESENSITIVITY PROFILE

MOLECULAR METHODSMOLECULAR METHODS

Laboratory DiagnosisLaboratory Diagnosis

Pulmonary TB-Samples

Sputum-early morning x 3/7

Bronchoalveolar lavage or biopsies

Aspirate of Pleural Fluid

• Non –Pulmonary TB• Early Morning Urines x 3/7 Pus/Tissue for TB of soft

tissue or Bone Liver Biopsy; bone

marrow aspirate for Miliary TB

Blood mycobacterial culture esp. MAC

CSF- TB Meningitis

Laboratory StainingLaboratory Staining• Mycobacteria are

acid-fast so originally used Ziehl-Neelsen stain

• Fluorescence techniques using a fluoresence stain auramine is widely used

ACID ALCOHOL FAST RODS SUGGESTING TUBERCULOSIS

Auramine Stain of mycobacteriaAuramine Stain of mycobacteria

e.g. Middlebrook media-Bactec

Laboratory Diagnosis c’tdLaboratory Diagnosis c’td

• Polymerase Chain Reaction- used to amplify DNA or RNA of Mycobacterium tuberculosis complex

• Very sensitive but problems with dead mycobacteria , environmental contaminants and false positives

• May speed up identification after initial isolation as no re-growth required for i.d.

Principal Antituberculous drugsPrincipal Antituberculous drugs

• (Streptomycin)

• Isoniazid

• Rifampicin

• Ethambutol

• Pyrazinamide

Case 3: You are the intern on the Medical team

• The Microbiologist phones to say the blood culture you took yesterday on a patient with fever and a heart murmur has a staphylococcus like organism growing in one bottle of the blood culture two-bottle set

Case 3: Questions

• What is your interpretation of this preliminary verbal report?

• What further tests do you expect will be performed by the lab?

• How will you manage the patient in the meantime?

Another call from Microbiology

• Next day they call to say the preliminary identification is that of a coagulase positive Staph ( so the organism has grown in culture!)

• They add that the second bottle and 2 further bottles of a second BC set are also growing a staphylococcus (no further information yet)

• What is your interpretation now?

Case 3: continued

• An ECHO cardiogram reveals a vegetation on the aortic valve

• The Microbiology report has been made available: MRSA sensitive to: vancomycin, gentamicin, daptomycin, linezolid, rifampicin;

• Resistant to flucloxacillin/meticillin

What is the treatment plan and what lab tests will it be based on?

• How has the lab determined that the MRSA is susceptible to these antibiotics?

• He is prescribed vancomycin +gentamicin

• Each requires Therapeutic Drug Monitoring (who performs that?)

What is MRSA?

• Staph. aureus that is resistant to meticillin– (flucloxacillin/cloxacillin/nafcillin/oxacillin)

• Altered target site – penicillin-binding proteins– Resistant to all ß-lactams

• mecA gene– encode for production of a low-affinity

penicillin-binding protein (PBP-2´)

Why worry about MRSA?

o Predominantly a hospital-acquired pathogen– Additional burden of infection

o More difficult to treat– Less effective drugs– Often require IV therapy– Evidence of increased mortality with MRSA versus

MSSA

o More expensive to treat– Associated with increased length of stay– Increased drug costs

© July Issue of Epi-Insight, Vol 6, Issue 7, Health Protection Surveillance Centre, Ireland

Community-acquired MRSA

Clinical Characteristics of CA-MRSA

• Skin infection– Furunculosis and skin abscesses (but not

impetigo or folliculitis)

• Necrotising pneumonia and severe sepsis

Weekly August 20, 1999 / 48(32);707-710

Four Pediatric Deaths from Community-Acquired Methicillin-Resistant Staphylococcus aureus -- Minnesota and North Dakota, 1997-1999Methicillin-resistant Staphylococcus aureus (MRSA) is an emerging community-acquired pathogen among patients without established risk factors for MRSA infection(e.g., recent hospitalization, recent surgery, residence in a long-term–care facility[LTCF], or injecting-drug use [IDU]) ( 1 ).

The Spectre of

Glycopeptide Resistance

S. aureus

Penicillin

[1950s]

Penicillin-resistant

S. aureus

Evolution of Drug Resistance in S. aureus

Methicillin

[1970s]

Methicillin-resistant

S. aureus (MRSA)

Vancomycin-resistant

enterococci (VRE)

Vancomycin

[1990s]

[1997]

Vancomycin

intermediate-resistantS. aureus (VISA)

Vancomycin-

resistantS. aureus

Glycopeptide resistance in Staph. aureus

1. Vancomycin resistant S. aureus (VRSA)

2. Vancomycin-intermediate S. aureus (VISA)

– Hetero-glycopeptide resistant S. aureus (hGISA)

Top Five significant BSI isolates as % of Total significant isolates 2006 - 2010(one isolate per patient)

S. aureus

E. coli

0

5

10

15

20

25

30

35%

of

sig

nif

ican

t is

ola

tes

S. aureus 24 24 18 22 18

E. coli 24 22 26 27 29

E. faecium 8 6 7 6 8

E. faecalis 6 4 7 7 5

S. pneumoniae 7 6 6 5 4

2006 2007 2008 2009 2010

St James’s Hospital

Case 4: A two year old child brought in to the Emergency Dept

• Parents say the child has been irritable, vomiting, and feverish

• Examination reveals some neck stiffness and the child is febrile

• She has been vaccinated according to national guidelines (what are they?)

• What is the likely diagnosis?

• What investigations should be performed

Case 4; Microbiology calls you, the ED doctor, after a lumbar puncture has been

performed• Spinal fluid looks cloudy, >200 white cells/cu

mm; > 90% polymorphs• High CSF protein and low glucose reported by

Biochemistry• No organisms seen on gram stain• What is your interpretation of these preliminary

results?• What additional investigations should have been

done?• What is the correct management of the case?

Case 4: A 27 year old Bangladeshi student admitted on take presenting with a fever and

vomiting• Initially he is observed on the ward.

• Blood haematology analysis shows a low white cell count (but he is not neutropenic)

• Continues to have fevers and feel unwell, no diarrhoea, no rash no abnormality found on physical examination

• Various microbiological investigations have been requested

Case 5, day 2

• Microbiology contact the team to say a blood culture taken yesterday has flagged positive with gram negative rods in both bottles

• What is your interpretation?

• What treatment/management is indicated?

• Any further investigations?

Case 5: next day

• Microbiology report states:

• Salmonella Typhi isolated

• What antibiotics should be effective for treatment?

Bacteria contaminating chickens that we consume

o Food-poisoning strains of Salmonella

o Campylobacter

o Escherichia coli

© March Issue of Epi-Insight, Vol 6, Issue 4, Health Protection Surveillance Centre, Ireland

Case 6: A 40 year old man with acute myeloid leukaemia undergoing an allogeneic

stem cell transplant• At wednesday case conference this

patient was discussed• He is 22 days after the SCT and has not

yet engrafted. He has become febrile and CT scan of the chest shows two pulmonary infiltrates

• What differential diagnosis are you considering?

• What further investigations?

Case 5 continued…

• A bronchoalveolar lavage was sent yesterday to Microbiology and Histopathology

• Microbiology contact the team to say that microscopy using lactophenol cotton blue shows hyphal structures ? Fungus

• Interpretation?

Invasive Pulmonary Aspergillosis

Case 5 continued

• Next day a report goes out from Microbiology: “Aspergillus species isolated”

• Other investigations are pending, what are they?

• What tests did Histopathology do?

• Are there any other tests you might order to aid interpretation of the above report?

Branching hyphae of Aspergillus

Case 6: A patient on the Intensive Care Unit

• A 65 year old man has been on the ITU for 2 weeks. He had major abdominal surgery, for a colonic cancer, but post-operatively he had an intestinal leak, and went into septic shock. He is being ventilated, is on broad spectrum antibiotics and noradrenaline

On the Microbiology liaison round in ITU

• It’s reported from the lab that a tracheal aspirate taken 3 days ago has produced a heavy growth of a Pseudomonas aeruginosa

• Chest X ray taken earlier shows a consolidation in the right mid and lower zones

• How do you interpret these findings?

HOSPITAL ACQUIRED PNEUMONIA: Pseudomonas aeruginosa

Case 6 continued…

• He is being treated with ciprofloxacin and gentamicin (what are the doses?)

• In the evening of that day he develops profuse watery diarrhoea

• Should any thing be changed at this time?

• Any further investigations warranted?

Case 6 continued…

• Microbiology contacts ITU team next day to advise the Pseudomonas is

• resistant to: ciprofloxacin, tazocin, gentamicin

• Sensitive to meropenem and amikacin

• Stool examination report:

“Clostridium difficile toxin detected”

Interpretation and further action?

Case 6 continued

• His treatment is changed to meropenem and he shows some improvement over the next 24 hours, but still has diarrhoea

• Could he be given a different antibiotic?

Case 6 continued

• 3 days later he is still being ventilated but is more haemodynamically stable

• A subsequent tracheal aspirate was processed in the micro lab

• Report: “Stenotrophomonas maltophilia heavy growth”

• Any change in management

Next day….

• A blood culture taken 2 days ago

• Microbiology reports by phone initially:

“ yeast seen in aerobic bottle”

Interpretation/management/further investigations?

Candidaemia/candidiasis

Case 7: A lady who presents to her GP with a vaginal discharge

• A 25 year old lady who is not pregnant, but is taking a contraceptive pill complains of an “itchy” vaginal discharge

• Speculum examination reveals an inflamed vaginal mucosa

• Differential diagnosis?

• Investigations?

The laboratory report

• Microscopy: gram negative rods and a few yeasts seen

• Culture: Candida species isolated

• Interpretation?

• Management of the case?

Vaginal Candidiasis

Common in women of child bearing years

Symptoms are itchy vaginal discharge

Diagnosed by vaginal examination (white plaques) and microscopy/culture

Treatment is initially topical antifungal (nystatin or azole) pessary or cream

Case 7 continued

• She is given a prescription for an oral antifungal fluconazole which she takes but only with partial resolution of her symptoms

• 2 weeks later she returns to the GP with a recurrence of symptoms

• What further investigations should be done?

Case 7 continued

• A repeat HVS produces the same result but with more information this time:

• Candida glabrata isolated

• No Neisseria gonorrhoeae

• No clue cells (what is this for?)