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MBBS IV - Junior Clerkship WCS > JC92 (Microbiology) - Diagnosis of infections > Flashcards

JC92 (Microbiology) - Diagnosis of infections Flashcards

1
Q

Clinical approach to suspected infection

A

1) History, physical examination, preliminary investigation
» clinical diagnosis (suspicion of an infectious disease process)

2) Localize the focus of infection on clinical/ radiological findings
3) Collect, transport relevant clinical specimen(s) for microbiological examination
4) Empirical antimicrobial therapy (if indicated)
5) Modify diagnosis/ therapy when results of rapid tests (e.g. Gram stain)/ final reports (identity, sensitivity test) are available
6) Monitor therapy – collect further clinical specimens for microbiological examination/ antibiotic assays

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2
Q

3 determinants of the quality of clinical specimens

A

1) Its degree of representation of the infectious disease/ pathophysiological process (e.g. sputum vs. saliva)
2) The manner of collection (no contamination)
3) The manner of transportation (storage) before it is analysed

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3
Q

Methods to ensure the quality of collected clinical specimen

  • How to process, collect, transport biological samples…etc
A

1) Only collect relevant clinical specimens under correct clinical indications

2) Put all specimens into the correct container; properly secure (e.g. bagged)
to prevent leakage (biohazard)/ exogenous contamination

3) Transport specimens expediently to the laboratory; if not, consider proper
transport medium/ refrigeration

4) Fill all request forms with legible writings (computer entry), including the clinical features, diagnosis, antibiotics used
5) Consider all patients’ specimens biologically hazardous (universal precaution)
6) State known biological hazards (e.g. HIV positive, Ebola) on the request form; put biohazard labels on the specimen bag
7) Consult microbiologists for unusual infections/ clinical situations

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4
Q

Methods of storing specimen for PCR

A

previously unused (new)/ gamma-irradiated bottle to ensure that there is no carryover of dead microbes

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5
Q

Which types of clinical specimen are NOT useful for microbiological tests?

A

o Bedsore, superficial wound swabs

o Routine culture of superficial swabs, e.g. penile swab, perineal swab, nasal swab

o Some drain fluids, e.g. long-term PTBD

o Urine from patients requiring chronic urinary catheterization

o Colonic biopsy for routine bacterial culture (without providing any clinical information)

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6
Q

List all assays for microbial characteristics

A

Assay for specific microbial characteristics:

o Visualize typical morphology in clinical specimens (e.g. pleural fluid):
 Gram smear, ZN smear and light microscopy for bacteria
 Electron microscopy for viruses

o Detect growth by culture (e.g. blood, pleural fluid), biochemical tests

o Detect specific microbial components:

1) Proteins - EIA (ELISA/LA): Ag/Ab reaction
2) Polysaccharides - EIA (ELISA/LA)
3) Lipids, fatty acids - chromatography (GLC-MS)
4) Specific sequences of genome:
 DNA: PCR &/or probe hybridization
 RNA: RT/PCR &/or probe hybridization
5) Random shotgun sequencing by high throughput next generation sequencing

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7
Q

List all assays for measuring host immune response to infection

A

Antibody response towards microbial components (does not tell latent or active):
 IgM (>3 days), IgG (7 days)
 Paired sera

Cell-mediated immune response:
 Mantoux test
 Lymphocyte proliferation
 Cytokine/chemokine activation/ release from CD4 lymphocytes/ cytotoxic CD8 lymphocytes (CTL)

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8
Q

Blood culture

  • Indications (types of infections and clinical presentation)
A

Types of infections:
 Sepsis (bacteraemia, fungaemia)
 Endocarditis

Clinical presentation:

1) Sudden relative increase in pulse rate, temperature (fever/ hypothermia)
2) Change in sensorium (mental obtundation); onset of chills, prostration (extreme physical weakness), acute hypotension
3) Prolonged, mild, intermittent fever +/- heart murmur

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9
Q

Blood culture

  • Collection technique and volume
  • Storage form
A

Aseptic technique
- Skin disinfection by 70% alcohol, then 30s with Chlorhexidine gluconate 0.5% in alcohol

Volume Take adequate volume of blood to improve sensitivity:

  • 10-30 ml (adult) per set in each of the aerobic and anaerobic blood culture broth
  • 5ml per bottle

Broth: antibiotic-absorbing resins

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10
Q

Blood culture

  • Timing of blood collection?
  • Sets required for different types of infections?
A

Timing:
 Before antibiotics
 1h before/ at the onset of chills/ fever for intermittent bacteraemia
 Anytime for continuous bacteraemia (e.g. endocarditis, early stage of typhoid fever)

Sets:
>2 sets for bacterial meningitis
>2 sets for sepsis from different venipuncture sites:
3-4 sets for suspect endocarditis
2 sets for central venous catheter infection (1 through catheter, 1 through peripheral venous puncture)

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11
Q

Blood culture

  • Testing requests (which pathogens for testing)
A

Testing for typical organisms causing sepsis and endocarditis

Special requests for culture-negative endocarditis:
serology (Ab test) and nucleic acid amplification assay (PCR) of:
- Coxiella burneti (Q fever)
- Bartonella henselae (cat scratch disease),
- Chlamydia,
- Brucella,
- Tropheryma,
- Leptospira,
- Mycoplasma

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12
Q

CSF collection

  • Indicated in which infections and clinical presentations
A

Meningitis (after exclusion of intracranial space occupying lesion by CT**)

Clinical presentation:

1) Fever and meningeal irritation
2) Unexplained febrile illness in an irritable infant who is feeding poorly
3) Mental obtundation
4) Focal neurological deficits

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13
Q

CSF collection

  • Site of LP
  • Storage methods
  • Volume of collection
A

Site: L4-5

Storage:
- Use new (non-reused) glass bottles to prevent falsely positive Gram
smear/ ZN stain
- Expedient transport to the laboratory, No storage

Volume:
 >1-2ml of CSF for bacterial meningitis
 5-10ml for mycobacterial/ fungal meningitis

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14
Q

Synovial and serous fluid collection

  • Storage method
  • Collection technique
  • Volume collected
A

Use sterile bottles:
o Tissue sample: put into normal saline with lgoss
o Bone marrow sample (prone to clotting): put into citrate/ heparin (anticoagulated)

Collection: Sterile aspirate: inject saline and re-aspirate

Volume: >5ml for mycobacterial/ fungal infection
Selected cases: inoculate a portion of fluid (2-5 ml each) into aerobic and anaerobic blood culture broth with resin to maximize yield

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15
Q

List CSF assays for bacterial, viral and parasitic infections

A

Bacterial:

  • Gram stain and culture
  • Mycobacteria: ZN smear, PCR, AFB culture
  • Long antibiotic course: Nucleic acid amplification PCR
  • Syphilis: VDRL, EIA test
  • Mycoplasma pneumoniae - IgM

Virus:

  • HSV1, HSV2, VZV - Nucleic acid amplification (PCR)
  • Enterovirus (acute meningoencephalitis) - RT-PCR
  • JEV, other meningo-encephalitic viruses - Paired sera and IgM

Ameba:
- Wet mount

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16
Q

Infections that indicate serum antigen detection test

A

Viral infections (e.g. NS1 protein of Dengue virus)

Fungal:
 Cryptococcus neoformans
 Aspergillus galactomannan
 Fungal D-glucan

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17
Q

Serological tests for Malaria, babesiosis, filariasis

A

Thick and thin smear from EDTA blood

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18
Q

Serological test for CMV infection in immunosuppressed host

A

Immunostain the buffy coat fraction for semi-quantitative assays

  • CMV pp65 antigenaemia (in WBC)
  • EDTA blood

Viral load in whole EDTA blood

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19
Q

Serological tests for disseminated adenovirus and EBV (post-implant LPD)

Indication for pre-emptive testing for these pathogens?

A

Viral load in whole EDTA blood

Preemptive treatment of bone marrow transplant recipient with low WBC count

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20
Q

Serological tests performed on clotted blood

A

Monitoring treatment:
Antimicrobial levels; Aminoglycosides, vancomycin
Serum bactericidal titre

Detecting antibodies/ antigens

  • acute and convalescent serum for the definitive diagnosis of infection using 4 fold rise in antibody titre
  • Seroconversion from negative to positive specific IgG by EIA or/& positive IgM by capture EIA
21
Q

Pathogens that are detected by antigen/antibody assays in clotted blood

A

 Widal test (serum agglutinins H and O) for typhoidal and non-typhoidal salmonella

 Brucella IgM ELISA

 antistreptolysin O for GAS

 VDRL for treponema pallidum

 Protozoa: Toxoplasma gondii

 Fungal: Aspergillus

 IgM for viruses

 4X rise in IgG titre for many viruses & rickettsiae

22
Q

Culture of intravascular catheter

  • Indications (type of infection and clinical presentation)
  • Sampling technique
  • Positive result
A

catheter-related sepsis

1) Local signs of phlebitis, inflammation/discharge of subcutaneous tunnel/exit site
2) Persistent fever without localising signs of infection elsewhere

Sampling:

  • Disinfect the exit site of the catheter
  • remove the catheter, aseptically cut off the distal 5cm segment and directly drop into a dry screw-cap container/ culture bottle
  • Send the specimen directly to the laboratory to prevent excessive drying
  • Roll the catheter segment over the blood agar plate 4 times

Positive:
≧15 colony forming units of a single organism after 48 hours of incubation

23
Q

Nasopharyngeal swab

Indicated for which infections

A

 Viral respiratory illnesses (e.g. rhinovirus, adenovirus,
influenza virus A and B, parainfluenza virus 1/2/3/4,
respiratory syncytial virus, coronaviruses,
metapneumovirus)

 Bordetella pertussis (whooping cough), Mycoplasma
pneumoniae, Chlamydia pneumoniae

24
Q

Throat swabs

Indicated for which infections

A

 Viral respiratory illnesses (same as nasopharyngeal swab/ aspirate)
– generally not as sensitive except with pneumonia

 Group A Streptococcus pyogenes (causes 10% pharyngitis) and other
beta-hemolytic streptococcus

 special request: Corynebacterium diphtheriae, Neisseria gonorrhea

25
Q

Nasopharyngeal swab

  • Sampling technique
  • Storage method
A

small dacron swab on a flexible wire handle – pernasal Transwab

  1. Pass through the nose into the nasopharynx
  2. Rotate till the posterior pharyngeal wall is reached and the
    patient is stimulated to sneeze/ cough
  3. Remove and put into (viral/ bacterial) transport medium
    o Viral transport medium contains antibiotics, buffer, pH indicator
    o Charcoal transport medium for B. pertussis

Storage: Refrigerate if anticipate delay (except Bordetella pertussis), but not freeze

26
Q

Nasopharyngeal swab

  • Assays
A

 Multiplex RT-PCR (most sensitive)

 Immunofluorescent antigen detection on the nasopharyngeal cells (stain
fluorescein-conjugated antibody against viral antigen)

 Cell culture

27
Q

Throat swabs

  • Sampling technique
  • Storage method
  • Assays
A
  1. Directly visualize the throat area
  2. Depress the tongue to minimise contamination by oral secretions
  3. Swab both tonsillar areas, posterior pharynx, any areas of inflammation, ulceration, exudation or membrane formation
  4. Transport medium

Storage: Refrigerate if anticipate delay (except Neisseria gonorrhea), but not freeze

Assay: Multiplex RT-PCR

28
Q

Sampling methods for upper and lower respiratory tract

A

Upper:

  • Nasopharyngeal swab/ aspirate
  • Throat swab

Lower: Decreasing amount of oropharyngeal contamination

  • Saliva
  • Expectorated sputum
  • Endotracheal aspirate, tracheostomy aspirate, bronchoscopic aspirate, transtracheal aspirate
  • Bronchoalveolar lavage (BAL)
  • Protected catheter brush
  • Transbronchial lung biopsy
  • Open lung biopsy
29
Q

Saliva sampling

  • Indications
A

Detection of respiratory virus by highly sensitive point-of-care nucleic acid
amplification assays

30
Q

Expectorated sputum

  • Indications
  • Sampling technique
  • Sign of contamination
  • Methods to maximize yield
A

Indication: Chest infections

Sampling: Instruct/assist patients properly (e.g. by physiotherapist) to avoid
misleading culture result by contamination

Signs of contamination:
Gram stain shows high buccal epithelial cell content + low white cell count suggests oropharyngeal contamination

Maximize yield:

  • Early morning freshly expectorated sputum
  • expectorated sputum induced by heated aerosol of 10% glycerol and 15% sodium chloride, followed by gastric washing
  • Multiple specimens
31
Q

Endotracheal aspirate, tracheostomy aspirate, bronchoscopic aspirate, transtracheal aspirate

  • Indication
  • differentiate contamination with genuine infection?
A

Indication: Intubated patients

Oropharyngeal contamination: Gram stain shows high buccal epithelial cell content + low white cell count

polymicrobial aspiration pneumonia after tracheostomy&raquo_space; do semiquantitative culture

32
Q

Bronchoalveolar lavage (BAL)

  • Indications
  • Sampling technique
  • Positive sign of infection
A

Indication: Severe, persistent, undetermined pneumonia
immunocompromised: diagnose cytomegalovirus, mycobacterial, fungal, Pneumocystitis carinii infection

Sampling:

  1. Wedge the bronchoscope tip into the lingular/ middle lobe bronchus
  2. Lavage the bronchopulmonary segment with normal saline in 20-40 ml samples
  3. Aspirate the fluid for microbiological analysis
  4. Reserve a portion for viral transport medium for viral pneumonia

Positive:
10^5 bacteria/ml = highly suggestive of the usual pyogenic bacterial infection

33
Q

Protected catheter brush (PCB)

  • Indication
A

diagnosis of lower respiratory tract infection for:

Patients who are at risk for unusual infections (alcohol abuser,
diabetics, intubated ICU patients)

Chest radiographic evidence of necrotizing infection

Recurrent/ unresponsive pneumonia

34
Q

Urine collection

  • 4 methods
  • Indication for each method
A

A. Clean-voided (catch), mid-stream urine: for UTI

B. Catheterized urine: for incontinent males, patients with chronic indwelling urethral catheters and clinical infection

C. Suprapubic aspiration

  • clinical evidence of urinary tract infection but the counts in clean-voided specimen are low or indeterminate
  • neonates and infants when catheterization may be contraindicated

D. Early morning urine: collect 3 consecutive samples in patients with
suspected renal tuberculosis/ sterile pyuria

35
Q

Clean catch urine, MSU

  • Collection technique
A

 Wash with cotton wool from front to back (clean introitus to prevent contamination by flora)

 Collect urine “midstream” in the container provided

36
Q

Catheterized urine collection

- Collection technique

A

For incontinent males:
 Clean the glans penis with an antiseptic solution
 apply a new (non-sterile) external catheter and drainage system
 collect the first specimen from the drainage bag

For patients with chronic indwelling urethral catheters:
 Disinfect the wall of the catheter at its junction with the drainage tube
 puncture with a 21-gauge needle attached to a syringe for aspiration

37
Q

Urine collection

  • Storage method
  • Assays
A

Storage: Transport all urine specimens to the laboratory for processing within 1
hour of collection unless refrigerated at 4oC

Detection methods:
o Urinalysis (dipstick test + microscopy) - For midstream and sterile urine

o Bacterial DNA (gonococcus, Chlamydia)

o Urinary bacterial antigen:
 Legionella pneumophila serogroup 1 infection
 Invasive pneumococcal diseases (Streptococcus pneumoniae)

38
Q

Fecal sampling

  • Type of feces for sampling
  • Number of specimen for diagnosis
  • Storage and transport
  • Methods to increase yield
A

Type: Send diarrheal stool only

Number: 3 stool specimens to diagnose infectious diarrhea

Storage: Immediately transport to laboratory for organisms to survive (e.g. Shigella, Campylobacter)

Increase yield:

  • Choose the portion of stool containing mucus/ blood/ pus
  • Increase stool specimen number
  • Fast transport to laboratory
39
Q

Fecal sampling

  • Assays for infectious dx/ requests
A

Virus-induced diarrhea:
 Rotavirus: EIA for viral antigen
 Norovirus, sapovirus, enteric adenovirus 40/41, astrovirus: Viral transport medium for RTPCR

For antibiotic-related diarrhea, consider detection of Clostridium difficile:
 Cytotoxin (tissue culture assay)**
 DNA (PCR)
 Antigen (EIA)
 Microscopy for bacteria

Special requests:
mycobacteria, Cryptosporidium, Cyclospora cayetanensis, Isospora
belli, Microsporidia, Yersinia enterocolitica, Bacillus anthracis

40
Q

Sampling methods for dx of skin and soft tissue infections

A

Decreasing order of representation of infection:

Tissue (most diagnostic)
sterile aspirate, 
abscess (pus) fluid, 
drainage fluid, 
wound swabs
41
Q

Skin and soft tissue infection samples

  • ## Storage method
A

 Add sterile isotonic saline (on a sterile cotton gauze for small specimens) to prevent drying
 Expedient transportation to laboratory (especially if no transport medium is used)

42
Q

Sampling methods for eye and ear infections

A

Eye swabs (wetted with sterile normal saline):
o For culture
o Taken Before topical anaesthetic (has antimicrobial activity)

Corneal scrapings:
o For testing of fungi/ acanthoamoeba/ mycobacteria/ microsporidia
o Taken After the anaesthetic

Middle ear aspirate (tympanocentesis):
o For recurrent/ persistent otitis media (elderly, neonates)

Collect to establish the etiology
 Ear discharge (for otitis externa)
 External auditory meatal swabs (for otitis externa)

43
Q

Sampling methods and assays for STDs

A

Urethral/ endocervical discharge swab

Urethral scrapings: cells (intracellular Chlamydia)

Gram stain of vaginal swabs: vaginitis and cervicitis

Initial stream urine (males)/ first void urine: Bacteria DNA for PCR (of Chlamydia trachomatis, Neisseria gonorrhea)

Triple swabs for suspected gonococcus:
 Urethral/ cervical swab
 Anal swab
 Throat (nasopharyngeal) swab

Special request:
 Giemsa staining for Klebsiella granulomatis (cause of Granuloma inguinale or Donovanosis)
 Dark field examination for Treponema pallidum
 Special culture (if relevant): Haemophilus ducreyi, Herpes simplex, Trichomonas vaginalis (protozoa)

44
Q

Triple swab for gonococcus STD

A

 Urethral/ cervical swab
 Anal swab
 Throat (nasopharyngeal) swab

45
Q

Bacterial causes of febrile respiratory illness/ pneumonia

A

Encapsulated bacteria – well-treated by beta-lactams:

  1. Streptococcus pneumoniae
  2. Staphylococcus aureus/ Streptococcus pyogenes
  3. Haemophilus influenzae/ Neisseria meningitidis

ill health, >65: anaerobic flora replaced by Gram -ve bacilli
o Enterobacteriaceae: Klebsiella pneumoniae

impaired consciousness (aspiration):
o Oral aerobes/ anaerobes

hospitalized patients:
o Pseudomonas aeruginosa
o Acinetobacter baumannii

Soil flora:
o Burkholderia pseudomallei (sputum)

 Legionella pneumophilia (sputum, urinary antigen EIA)
 Mycoplasma pneumoniae
 Chlamydophila pneumoniae/ psittaci
 Coxiella burnetii (Q fever)
 Mycobacterium tuberculosis
46
Q

Fungal causes of pneumonia

A 
 Cryptococcus
 Aspergillus
 Dimorphic fungi: Penicillium, Histoplasma, Coccidioides
 Zygomycetes
 Pneumocystis
47
Q

Viral causes of pneumonia

A 
Most common:
o Influenza A H3N2, H1N1, H5N1, H9N2, H7N9
o Influenza B
o Influenza C
o Adenovirus
 RSV
 Parainfluenza 1, 2, 3, 4
 Rhinovirus Clade A, B, C
 Metapneumovirus
 Coronavirus: e.g.  MERS, SARS
 Enterovirus
 Bocavirus
48
Q

Parasitic causes of pneumonia

A

 Paragonimus westermanii
 Ascaris lumbricoides
 Strongyloides stercoralis

MBBS IV - Junior Clerkship WCS (131 decks)

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