JC92 (Microbiology) - Diagnosis of infections

Question 1

Clinical approach to suspected infection

Answer 1

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

Question 2

3 determinants of the quality of clinical specimens

Answer 2

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

Question 3

Methods to ensure the quality of collected clinical specimen

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

Answer 3

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

Question 4

Methods of storing specimen for PCR

Answer 4

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

Question 5

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

Answer 5

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)

Question 6

List all assays for microbial characteristics

Answer 6

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

Question 7

List all assays for measuring host immune response to infection

Answer 7

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)

Question 8

Blood culture

• Indications (types of infections and clinical presentation)

Answer 8

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

Question 9

Blood culture

• Collection technique and volume
• Storage form

Answer 9

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

Question 10

Blood culture

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

Answer 10

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)

Question 11

Blood culture

• Testing requests (which pathogens for testing)

Answer 11

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

Question 12

CSF collection

• Indicated in which infections and clinical presentations

Answer 12

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

Question 13

CSF collection

• Site of LP
• Storage methods
• Volume of collection

Answer 13

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

Question 14

Synovial and serous fluid collection

• Storage method
• Collection technique
• Volume collected

Answer 14

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

Question 15

List CSF assays for bacterial, viral and parasitic infections

Answer 15

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

Question 16

Infections that indicate serum antigen detection test

Answer 16

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

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

Question 17

Serological tests for Malaria, babesiosis, filariasis

Answer 17

Thick and thin smear from EDTA blood

Question 18

Serological test for CMV infection in immunosuppressed host

Answer 18

Immunostain the buffy coat fraction for semi-quantitative assays

• CMV pp65 antigenaemia (in WBC)
• EDTA blood

Viral load in whole EDTA blood

Question 19

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

Indication for pre-emptive testing for these pathogens?

Answer 19

Viral load in whole EDTA blood

Preemptive treatment of bone marrow transplant recipient with low WBC count

Question 20

Serological tests performed on clotted blood

Answer 20

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

Question 21

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

Answer 21

 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

Question 22

Culture of intravascular catheter

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

Answer 22

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

Question 23

Nasopharyngeal swab

Indicated for which infections

Answer 23

 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

Question 24

Throat swabs

Indicated for which infections

Answer 24

 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

Question 25

Nasopharyngeal swab - Sampling technique - Storage method

Answer 25

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

Question 26

Nasopharyngeal swab - Assays

Answer 26

 Multiplex RT-PCR (most sensitive)  Immunofluorescent antigen detection on the nasopharyngeal cells (stain fluorescein-conjugated antibody against viral antigen)  Cell culture

Question 27

Throat swabs - Sampling technique - Storage method - Assays

Answer 27

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

Question 28

Sampling methods for upper and lower respiratory tract

Answer 28

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

Question 29

Saliva sampling - Indications

Answer 29

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

Question 30

Expectorated sputum - Indications - Sampling technique - Sign of contamination - Methods to maximize yield

Answer 30

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

Question 31

Endotracheal aspirate, tracheostomy aspirate, bronchoscopic aspirate, transtracheal aspirate - Indication - differentiate contamination with genuine infection?

Answer 31

Indication: Intubated patients Oropharyngeal contamination: Gram stain shows high buccal epithelial cell content + low white cell count polymicrobial aspiration pneumonia after tracheostomy >> do semiquantitative culture

Question 32

Bronchoalveolar lavage (BAL) - Indications - Sampling technique - Positive sign of infection

Answer 32

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

Question 33

Protected catheter brush (PCB) - Indication

Answer 33

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

Question 34

Urine collection - 4 methods - Indication for each method

Answer 34

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

Question 35

Clean catch urine, MSU - Collection technique

Answer 35

 Wash with cotton wool from front to back (clean introitus to prevent contamination by flora)  Collect urine “midstream” in the container provided

Question 36

Catheterized urine collection | - Collection technique

Answer 36

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

Question 37

Urine collection - Storage method - Assays

Answer 37

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)

Question 38

Fecal sampling - Type of feces for sampling - Number of specimen for diagnosis - Storage and transport - Methods to increase yield

Answer 38

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

Question 39

Fecal sampling - Assays for infectious dx/ requests

Answer 39

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

Question 40

Sampling methods for dx of skin and soft tissue infections

Answer 40

Decreasing order of representation of infection: ``` Tissue (most diagnostic) sterile aspirate, abscess (pus) fluid, drainage fluid, wound swabs ```

Question 41

Skin and soft tissue infection samples - Storage method -

Answer 41

 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)

Question 42

Sampling methods for eye and ear infections

Answer 42

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)

Question 43

Sampling methods and assays for STDs

Answer 43

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)

Question 44

Triple swab for gonococcus STD

Answer 44

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

Question 45

Bacterial causes of febrile respiratory illness/ pneumonia

Answer 45

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 ```

Question 46

Fungal causes of pneumonia

Answer 46

```  Cryptococcus  Aspergillus  Dimorphic fungi: Penicillium, Histoplasma, Coccidioides  Zygomycetes  Pneumocystis ```

Question 47

Viral causes of pneumonia

Answer 47

``` 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 ```

Question 48

Parasitic causes of pneumonia

Answer 48

 Paragonimus westermanii  Ascaris lumbricoides  Strongyloides stercoralis