JC92 (Microbiology) - Diagnosis of infections Flashcards
Clinical approach to suspected infection
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
3 determinants of the quality of clinical specimens
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
Methods to ensure the quality of collected clinical specimen
- How to process, collect, transport biological samples…etc
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
Methods of storing specimen for PCR
previously unused (new)/ gamma-irradiated bottle to ensure that there is no carryover of dead microbes
Which types of clinical specimen are NOT useful for microbiological tests?
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)
List all assays for microbial characteristics
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
List all assays for measuring host immune response to infection
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)
Blood culture
- Indications (types of infections and clinical presentation)
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
Blood culture
- Collection technique and volume
- Storage form
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
Blood culture
- Timing of blood collection?
- Sets required for different types of infections?
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)
Blood culture
- Testing requests (which pathogens for testing)
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
CSF collection
- Indicated in which infections and clinical presentations
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
CSF collection
- Site of LP
- Storage methods
- Volume of collection
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
Synovial and serous fluid collection
- Storage method
- Collection technique
- Volume collected
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
List CSF assays for bacterial, viral and parasitic infections
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
Infections that indicate serum antigen detection test
Viral infections (e.g. NS1 protein of Dengue virus)
Fungal:
Cryptococcus neoformans
Aspergillus galactomannan
Fungal D-glucan
Serological tests for Malaria, babesiosis, filariasis
Thick and thin smear from EDTA blood
Serological test for CMV infection in immunosuppressed host
Immunostain the buffy coat fraction for semi-quantitative assays
- CMV pp65 antigenaemia (in WBC)
- EDTA blood
Viral load in whole EDTA blood
Serological tests for disseminated adenovirus and EBV (post-implant LPD)
Indication for pre-emptive testing for these pathogens?
Viral load in whole EDTA blood
Preemptive treatment of bone marrow transplant recipient with low WBC count
Serological tests performed on clotted blood
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
Pathogens that are detected by antigen/antibody assays in clotted blood
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
Culture of intravascular catheter
- Indications (type of infection and clinical presentation)
- Sampling technique
- Positive result
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
Nasopharyngeal swab
Indicated for which infections
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
Throat swabs
Indicated for which infections
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
Nasopharyngeal swab
- Sampling technique
- Storage method
small dacron swab on a flexible wire handle – pernasal Transwab
- Pass through the nose into the nasopharynx
- Rotate till the posterior pharyngeal wall is reached and the
patient is stimulated to sneeze/ cough - 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
Nasopharyngeal swab
- Assays
Multiplex RT-PCR (most sensitive)
Immunofluorescent antigen detection on the nasopharyngeal cells (stain
fluorescein-conjugated antibody against viral antigen)
Cell culture
Throat swabs
- Sampling technique
- Storage method
- Assays
- Directly visualize the throat area
- Depress the tongue to minimise contamination by oral secretions
- Swab both tonsillar areas, posterior pharynx, any areas of inflammation, ulceration, exudation or membrane formation
- Transport medium
Storage: Refrigerate if anticipate delay (except Neisseria gonorrhea), but not freeze
Assay: Multiplex RT-PCR
Sampling methods for upper and lower respiratory tract
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
Saliva sampling
- Indications
Detection of respiratory virus by highly sensitive point-of-care nucleic acid
amplification assays
Expectorated sputum
- Indications
- Sampling technique
- Sign of contamination
- Methods to maximize yield
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
Endotracheal aspirate, tracheostomy aspirate, bronchoscopic aspirate, transtracheal aspirate
- Indication
- differentiate contamination with genuine infection?
Indication: Intubated patients
Oropharyngeal contamination: Gram stain shows high buccal epithelial cell content + low white cell count
polymicrobial aspiration pneumonia after tracheostomy»_space; do semiquantitative culture
Bronchoalveolar lavage (BAL)
- Indications
- Sampling technique
- Positive sign of infection
Indication: Severe, persistent, undetermined pneumonia
immunocompromised: diagnose cytomegalovirus, mycobacterial, fungal, Pneumocystitis carinii infection
Sampling:
- Wedge the bronchoscope tip into the lingular/ middle lobe bronchus
- Lavage the bronchopulmonary segment with normal saline in 20-40 ml samples
- Aspirate the fluid for microbiological analysis
- Reserve a portion for viral transport medium for viral pneumonia
Positive:
10^5 bacteria/ml = highly suggestive of the usual pyogenic bacterial infection
Protected catheter brush (PCB)
- Indication
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
Urine collection
- 4 methods
- Indication for each method
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
Clean catch urine, MSU
- Collection technique
Wash with cotton wool from front to back (clean introitus to prevent contamination by flora)
Collect urine “midstream” in the container provided
Catheterized urine collection
- Collection technique
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
Urine collection
- Storage method
- Assays
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)
Fecal sampling
- Type of feces for sampling
- Number of specimen for diagnosis
- Storage and transport
- Methods to increase yield
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
Fecal sampling
- Assays for infectious dx/ requests
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
Sampling methods for dx of skin and soft tissue infections
Decreasing order of representation of infection:
Tissue (most diagnostic) sterile aspirate, abscess (pus) fluid, drainage fluid, wound swabs
Skin and soft tissue infection samples
- ## Storage method
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)
Sampling methods for eye and ear infections
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)
Sampling methods and assays for STDs
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)
Triple swab for gonococcus STD
Urethral/ cervical swab
Anal swab
Throat (nasopharyngeal) swab
Bacterial causes of febrile respiratory illness/ pneumonia
Encapsulated bacteria – well-treated by beta-lactams:
- Streptococcus pneumoniae
- Staphylococcus aureus/ Streptococcus pyogenes
- 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
Fungal causes of pneumonia
Cryptococcus Aspergillus Dimorphic fungi: Penicillium, Histoplasma, Coccidioides Zygomycetes Pneumocystis
Viral causes of pneumonia
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
Parasitic causes of pneumonia
Paragonimus westermanii
Ascaris lumbricoides
Strongyloides stercoralis
