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JC WCS > Microbiology JC092: Diagnosis Of Infections > Flashcards

Microbiology JC092: Diagnosis Of Infections Flashcards

1
Q

Case 1:

  • Elderly male
  • X smoke, social drinker
  • HT 30 years
  • DM 15 years on insulin
  • mild CAD (of LAD)
  • Hyperlipidaemia
  • Gout
  • Chronic renal failure on CAPD (Continuous Ambulatory Peritoneal Dialysis)

HPI:

  • Fever, SOB (1 day)
  • 2 doses of Ciprofloxacin by family physician
  • No bowel motion for 1 day
  • worsening of symptoms

Drug history:

  • Anti-HT
  • Anti-HC
  • Aspirin
  • PO4 binder
  • Diuretics
  • Fe + Erythropoietin receptor activator

Social history
- History of travel to hotel and zoo

A

Progress:

  • IV Augmentin
  • Upper endoscopy (∵ Hb drop from 11 to 7) —> aborted ∵ O2 desaturation to 70%, RR 30 —> ICU
  • 7x watery diarrhoea in 24 hours —> Microbiologist

P/E:

  • 39oC
  • HR 120, Irregular
  • BP 160/90
  • RR 25
  • O2 saturation: 70% on room air, 95% on CPAP
  • Slow mentation
  • Pallor
  • Facial puffiness, Bilateral ankle edema (∵ fluid retention)
  • Scratch marks
  • No exit site erythema / tunnel tract / abdominal tenderness
  • PD fluid clear
  • Decreased air entry to left posterior chest, coarse inspiratory crepitus

CBC:

  • Anaemia
  • Neutrophilia
  • Leukopenia
  • High urea, creatinine
  • High LDH

Diagnosis:
- Rapidly progressing acute CAP in elderly with multiple comorbidities e.g. renal failure, heart disease, DM

Management:

  • Microbiological workup for ACS use of acute CAP (typical + atypical) with history of zoonotic contact in a uraemic patient on CAPD
  • Empirical IV Levofloxacin, Meropenem, Zanamivir (afraid Influenza) till Antigenuria (for Pneumococcus and Legionella) + viral PCR back
  • Acute LH failure with edema: draw fluid out by increased PD

Investigation:

  • Blood culture: negative
  • Cold agglutinin: negative
  • NPA viral antigen by IF: negative
  • RT-PCR for 10 viruses: negative
  • Urine antigen: negative
  • Urinalysis: proteinuria, high glucose, occult blood
  • Stool culture and C. difficile cytotoxin: negative
  • PD fluid: normal cell count and culture negative (no CAPD peritonitis)

Recent travel, Acute CAP, Diarrhoea: suspect Legionella pneumophila
—> although Urine antigen negative
—> ∵ renal failure (patient fail to concentrate urine)
—> may cause false -ve
—> Real time PCR for legionella
—> Positive

Subsequent management:
- Stop Meropenem and Zanamivir
—> continue Levofloxacin
—> Notify epidemiologist of CHP (∵ Notifiable disease)

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2
Q

***Common Bacterial causes of febrile respiratory illness

A

Most important bacteria (Encapsulated bacteria):

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

Others:

  1. Enterobacteriaceae: ***Klebsiella pneumoniae (ill health, >65)
  2. Oral aerobes / ***Anaerobes (aspiration pneumonia)
  3. Acinetobacter baumannii (hospital flora)
  4. ***Pseudomonas aeruginosa (hospital flora)
  5. Burkholderia pseudomallei (soil, sputum)

Atypical bacteria (NOT respond to β-lactam, only respond ***Tetracycline, Macrolide, Fluoroquinolone):

  1. ***Legionella pneumophila (sputum, urinary antigen EIA)
  2. ***Mycoplasma pneumoniae
  3. ***Chlamydophila pneumoniae / psittaci
  4. Coxiella burnetii (Q fever)

Endemic (if patient NOT respond to typical / atypical culprit):
13. ***Mycobacterium TB (sputum)

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3
Q

Common Viral causes of febrile respiratory illness

A

Most important in normal adults:

  1. ***Influenza A-C
  2. ***Adenovirus

Children / Elderly:

  1. RSV
  2. Parainfluenza 1-4
  3. Rhinovirus
  4. Metapneumovirus
  5. Coronavirus MERS, SARS, OC43, HKU1, 229E
  6. Enterovirus
  7. Bocavirus
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4
Q

Common Fungal causes of febrile respiratory illness

A

Usually in immunosuppressed hosts

  1. ***Cryptococcus
  2. ***Aspergillus
  3. ***Dimorphic fungi: Penicillium, Histoplasma, Coccidioides
  4. Zygomycetes
  5. Pneumocystis (atypical yeast)
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5
Q

Common Parasite causes of febrile respiratory illness

A

Usually ***Eosinophilia in blood

  1. Paragonimus westermanii
  2. Ascaris lumbricoides
  3. ***Strongyloides stercoralis
  4. Others
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6
Q

***Laboratory diagnosis of infection

A

Microbial factors:

  1. ***Visualisation in clinical specimens
    - Gram stain, ZN smear, Light microscopy for bacteria
    - Electron microscopy for viruses
  2. ***Culture of microbe (detection of growth)
    - Agar plate for bacteria (Sputum: may not be accurate since many oral commensals, importance of Blood, Pleural fluid, BAL cultures)
    - Cell line for viruses
  3. ***Biochemical tests (detection of growth)
  4. Detection of specific microbial components (Rapid test)
    - Proteins ELISA/LA (EIA: Ag/Ab reaction)
    - Lipids GLC-MS (chromatography)
    - Polysaccharide ELISA/LA (EIA)
    - **    Specific DNA and RNA sequences (    *PCR / probe hybridisation)
    - Random shotgun sequencing by high throughput next generation sequencing (expensive but catch all approach)

Host factors

  1. Ab response of host towards microbial components
    - IgM
    - ***Paired sera showing 4x increase
  2. ***Cell-mediated immune response
    - Mantoux test
    - IGRA
    - Lymphocyte proliferation
    - CTL response towards specific Ag
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7
Q

Flowchart: Decision making in management of infectious disease

A

Hx + P/E + Knowledge in microbiology / infectious disease
—> Diagnosis (best guess)
—> Clinical specimen + Organ imaging (locate site of infection) + Tissue biopsy (BAL, invasive procedures etc.)
—> Microbiological examination to confirm / refute —> Modification of diagnosis
—> Treatment (Empirical antimicrobial therapy if indicated)

Microbiological exam:

  • Rapid test (e.g. Gram stain)
  • Final reports (e.g. Identity + Sensitivity test)
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8
Q

GIGO syndrome

A

Garbage in —> Garbage out

Improperly collected specimens —> Misleading test results

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9
Q

Quality of clinical specimens

A

Depends on:

  1. Degree of representation of pathophysiological process
  2. Manner of collection
  3. Manner of transportation
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10
Q

General principles for specimen collection

A
  1. ***Relevant specimens —> according to Clinical indication
  2. Correct containers —> avoid Leakage / contamination
  3. Expedient transportation (if not, proper transport / refrigeration should be considered) —> Preserve characteristics
  4. Request forms —> allow Proper entry (what + why)
  5. ***Biohazard —> Universal precaution (should consider all patients’ specimens to be biologically hazardous)
  6. Known hazards (e.g. HIV) —> Inform, clearly state on request form, biohazard labels
  7. Unusual infections —> Consultation with microbiologists
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11
Q

Blood

A

Most important test for diagnosis of ***Sepsis (Bacteraemia, Fungaemia, Endocarditis)

  1. Indications
    - sudden relative ↑ in HR, Temp
    - change in sensorium + onset of chills, prostration, hypotension
    - prolonged, mild, intermittent fever without / with a heart murmur
    - ***signs of sepsis (fever, tachycardia, tachypnoea, leukocytosis, leukopenia)
  2. Timing
    - taken 1 hour before / at onset of chills / fever in patients with intermittent bacteraemia
    - ***before antibiotic administration
    - not important in continuous bacteraemia e.g. endocarditis, early stage of typhoid fever
  3. Number
    - usually 2 at **different sites
    - patient with central venous catheter: 1 through catheter + 1 by peripheral venous puncture
    - **    infective endocarditis: >=3 cultures repeated within 1 hour
  4. Site
  5. Amount
    - each set should contain 10-15 ml in each of aerobic + anaerobic blood culture broth (blood volume determines sensitivity of the test)
  6. Broth
    - ***antibiotics absorbing resins
  7. Aseptic technique
    - proper skin disinfection by **70% alcohol + 30 seconds with **chlorhexidine gluconate 0.5% in alcohol
    - adequate volume of blood with 5ml per bottle (10ml per set of blood culture)
    - avoid contamination by normal flora
  8. ***PCR, Ab test for culture negative endocarditis
    - Coxiella burneti, Bartonella henselae, Tropheyrema, Brucella, Leptospira, Mycoplasma, Chlamydia
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12
Q

CSF

A
  • Representative specimen for ***Meningitis
  • Lumbar puncture undertaken whenever meningitis suspected + excluded intracranial space occupying lesion
  1. Indications
    - Fever + **Meningism
    - **    Encephalopathic signs
    - Unexplained febrile illness in an irritable infant who is feeding poorly
  2. Aseptic technique
    - Non-reused bottles (prevent falsely positive gram smear results)
  3. Amount
    - >=2 ml for bacterial meningitis
    - >5 ml for mycobacterial / fungal meningitis
  4. Transport
    - ***expedient

Investigations:

  1. **Gram / ZN smear / **Wet mount for amoeba
  2. ***Bacterial culture
  3. ***PCR / Ag detection assay if suspect difficult-to-grow organism / patient already on antibiotics

Special request:

  • Mycobacteria, Treponema pallidum, Cryptococcus neoformans, Borrelia burgdorferi, Dimorphic fungi, Brucella, Amoeba should be considered if onset is insidious
  • ***Paired sera: Meningoencephalitis virus + Mycoplasma pneumoniae

Atypical pathogens

  1. ***Mycobacteria: PCR, AFB culture (for TB)
  2. ***Fungi: Cryptococcal antigen + Fungal culture (Latex agglutination)

Acute encephalitis

  1. ***PCR (for HSV1, HSV2, VZV)
  2. RT-PCR (for Enterovirus)
  3. CSF ***IgM (for Japanese encephalitis + Mycoplasma pneumoniae)
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13
Q

Other normally sterile body fluids

A
  • Pleural
  • Peritoneal
  • Pericardial
  • Synovial
  1. Aseptic technique
    - ***Percutaneous needle aspiration
  2. Volume:
    - more the better (>5ml for mycobacterial / fungal infection)
  3. Use of bottles containing **citrate / **heparin necessary if specimen prone to clotting (e.g. bone marrow)
  4. Inoculations of portion of fluid (2-5ml each) into aerobic + anaerobic blood culture broth with resin should be done if have sufficient volume
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14
Q

Indication for Semiquantitative culturing of IV catheter

A

Indications:

  1. Local signs of ***phlebitis
  2. Persistent fever without other localising signs of infection
  • Exit site disinfected —> when area dry —> ***remove catheter + cut off distal 5cm segment aseptically —> direct drop into dry screw cap container —> send directly to lab to prevent excessive drying
  • Catheter segment is rolled over blood agar plate 4 times (Roll-plate culture)
  • Presence of ***>=15 CFU of single organism after 48 hours of incubation —> ↑ risk of catheter-related infections
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15
Q

Diagnosis of Skin / Soft tissue infections

A

Degree of representation (Pathophysiological process of infection) —> Degree of contamination (Superficial colonising flora)

  1. **Tissue biopsy (Gold standard)
    - put into sterile, wide mouth, screw cap container (e.g. universal bottles)
    - sterile **    isotonic saline added to prevent drying (sterile cotton gauze for small samples)
    - expedient transportation important if no transport medium used
  2. Sterile aspirate
  3. ***Abscess fluid
  4. Drain fluid
  5. ***Wound swabs (disinfect wound with alcohol first)
  6. Other normally sterile body fluids: joint, pleural, peritoneal, pericardial (inoculate also into blood culture broth to maximise yield)

Smear + Culture for **mycobacteria / **fungi must be considered if lesion not responding to broad spectrum antibiotics

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16
Q

Eye, Ear

A

Eye swabs:

  • wetted with sterile normal saline
  • taken ***before topical anaesthetic (avoid antimicrobial activity of topical anaesthetic)

Corneal scraping:

  • testing of fungi, acanthoamoeba, mycobacteria, microsporidia
  • taken ***after topical anaesthetic

Middle ear aspirate (***tympanocentesis):

  • recurrent / persistent otitis media
  • elderly / neonates
  • nasopharynx / pharynx culture NOT useful in establishing etiology of middle ear infection

Ear discharge / External auditory meatal swabs:
- only for management of otitis externa

17
Q

Faeces

A

Indications: ***Infectious diarrhoea

  • Portion of stool containing **mucus / **blood / ***pus should be chosen
  • most can be diagnosed after evaluation of 3 stool specimens

False negative due to:

  1. ***Overgrowth of commensal flora (e.g. E. coli, Bacteroides, Enterococcus)
  2. ***Death of some pathogenic bacteria (e.g. Shigella, Campylobacter cannot survive if not immediately transferred)
  3. ***Loss of architecture of Parasites (protozoa, ova etc.)

Antibiotic-related diarrhoea
- Tissue culture assay of C. difficile ***cytotoxin (only show C. difficile is no use ∵ normal commensal) (EIA antigen + culture + DNA by PCR)

Virus-induced diarrhoea:

  • Rotavirus, Enteric adenovirus 40, 41 —> EIA for Ag detection
  • Norovirus, Sapovirus, Astrovirus —> Faeces in viral transport medium for RT-PCR

Contact microbiologist:
- Mycobacteria, Cryptosporidium, Isospora, Cyclospora cayetanensis, Microsporidia, Culture for Yersinia enterolitica, Bacillus anthracis

18
Q

Urine

A

Indications:

  1. Bacteria
  2. Yeast
  3. ***UTI —> Bacterial DNA (Gonococcus, Chlamydia)
  4. Pneumonia —> Bacterial antigenuria (Pneumococcus, Legionella pneumophila serogroup 1)

3 types:
1. ***Mid-stream urine (need careful instruction to avoid contamination by normal commensal at distal urethra + external introitus)

  1. **Catheterised urine (done only in specific indications)
    - clean glans penis with antiseptic solution —> collect **    first specimen in drainage bag
    - wall of catheter at junction with drainage tube is disinfected —> puncture with 21-gauge needle attached to syringe for aspiration
  2. ***Suprapubic aspirate (gold standard)
    - patients with clinical evidence of UTI but counts in clean-voided specimen are low / indeterminate
    - neonates / infants when catheterisation is CI

Transport:
- within 1 hour unless refrigerated at 4oC

Midstream / Sterile urine:
- Dipstick test + Microscopy should also be performed

Suspected renal TB / Sterile pyuria (pus in urine):
- collect ***3 consecutive early morning urine

19
Q

Genital tract specimens

A

Indications:

  • STD
  • PID

N. gonorrhoeae / Chlamydia trachomatis:

  • PCR
  • Culture (slower)
  1. Urethral / ***Endocervical discharge
    - sent on swab with transport medium
    - Gram smear + Gonococcal culture
  2. ***First void urine (rich in urethral discharge)
    - PCR for N. gonorrhoeae / Chlamydia trachomatis
  3. Urethral / ***Endocervical scrapings of cells are useful (Chlamydia ∵ intracellular organism)
    - if discharge is scanty
  4. Gonococcal infection suspected (+ve risk factors e.g. sex workers, homosexuals)
    - ***Triple swabs: Urethral, Throat (Nasopharyngeal), Anal

Gram stain of vaginal swabs + High WBC:

  • Vaginitis
  • Cervicitis
  • Bacterial vaginosis: paucity of Gram +ve bacilli (Lactobacilli) + abundance of Gram -ve bacilli / Gram variable coccobacilli studded on epithelial cells (Clue cells)

Haemophilus ducreyi, Herpes simplex, Trichomonas vaginalis
- request special culture

Contact microbiologist:

  1. ***Giemsa stain —> Klebsiella granulomatis (causing Donovanosis / Granuloma inguinale)
  2. ***Dark field examination —> Treponema pallidum
20
Q

Upper respiratory tract

A

e.g. Acute pharyngitis / Nasopharyngitis

  1. ***Nasopharyngeal swab / aspirate (good)
    - Bordetella pertussis
    - Atypical pneumonia (Mycoplasma pneumoniae, Chlamydia pneumoniae: RT-PCR, Immunofluorescent Ag detection, cell culture)
    - Respiratory virus (need to put in viral transport medium)
    —> Rhinovirus
    —> RSV, Metapneumovirus
    —> Adenovirus
    —> Influenza A, B
    —> PIV 1, 2, 3, 4
    —> Coronavirus
    —> For RT-PCT / Ag detection
  2. ***Throat swab (less good) —> RT-PCT
    - Streptococcus pyogenes
    - Neisseria gonorrhoeae
    - Corynebacterium diphtheriae
    - Bordetella pertussis
    - All respiratory viruses (generally not as sensitive as NP specimens except with pneumonia)

Throat swab:

  • depress tongue to minimised contamination by oral secretions
  • Both ***Tonsillar areas
  • ***Posterior pharynx
  • ***Areas of inflammation, ulceration, exudation, membrane formation
21
Q

Lower respiratory tract

A

Yield in ascending order (Oropharyngeal contamination —> Predominant pathogens):

Indications (Usual chest infections —> Intubated patients —> Severe / Persistent undetermined pneumonia (immunocompromised)):

  1. Saliva
  2. Expectorated sputum (instructed / assisted by physiotherapist)
    - interpret with caution (    high epithelial cell + low white cell count on Gram stain suggest oropharyngeal contamination)
  3. Endotracheal aspirate
  4. Bronchoscope aspirate
  5. ***BAL
    - considered in patients at risk of unusual infection (e.g. immunosuppressed, alcohol abuser, DM, intubated ICU, radiographic evidence of necrotising infection, unresponsive pneumonia)
    - often used in immunosuppressed hosts, useful for diagnosis of Cytomegalovirus, Mycobacterial, Fungal, PCP infection
    - 10^5 bacteria per ml of BAL: highly suggestive of usual pyogenic bacterial infection
    - tip of bronchoscope wedged into lingular / middle lobe bronchus / involved segment
    - lavage with normal saline 20-40ml samples
    - aspirate fluid for analysis —> part of it should be put in viral transport medium for Viral respiratory diseases (e.g. avian Influenza A H5N1, H7N9, SARS, MERS)
  6. ***Protected catheter brush
    - considered in patients at risk of unusual infection (e.g. immunosuppressed, alcohol abuser, DM, intubated ICU, radiographic evidence of necrotising infection, unresponsive pneumonia)
  7. ***Transbronchial lung biopsy
  8. Open lung biopsy

Mycobacteria / Fungi:

  • ***early morning freshly expectorated sputum / sputum induced by heat aerosol of 10% glycerol + 15% NaCl followed by gastric washing after 1 hour
  • multiple specimens (>=3) + neutralisation of gastric acid —> maximise yield
  • **Deep throat saliva (Posterior oropharyngeal secretion):
  • can be used for detection of respiratory virus by highly sensitive point-of-care nucleic acid amplification assays
22
Q

Serum and Blood

A
  1. ***Therapeutic drug monitoring
    - Aminoglycosides (Gentamicin)
    - Vancomycin
    - Serum bactericidal titre (in Infective endocarditis)
  2. **Serum Ab
    - **    Typhoid fever: Widal’s test
    - Rickettsial infection: Weil-Felix test
    - Brucella detection
    - **S. pyogenes infection: Anti-streptolysin O (acute + convalescent)
    - **    VDRL
    - Toxoplasma gondii
    - Aspergillus
    - **IgM for viruses (by Capture EIA)
    - **    4x rise in IgG for many viruses and rickettsiae (Acute + Convalescent serum) (by EIA)
  3. ***Serum / Blood Ag
    - Cryptococcus neoformans (immunocompromised)
    - Aspergillus galactomannan (immunocompromised)
    - Fungal D-glucan (immunocompromised)
    - Dengue virus NS1 antigenaemia
    - HIV Ag/Ab assay
    - CMV pp65 Ag in WBC (buffy coat fraction of EDTA blood —> immunostaining for semi-quantitative assays esp. in organ transplant recipient)
    - Malaria, Babesiosis, Filariasis (EDTA blood for thick + thin smear)

Bone marrow transplant patients with low WBC count:
- whole EDTA blood to monitor for preemptive treatment:
—> CMV (invasive / disseminated disease)
—> Adenovirus (disseminated disease)
—> EBV (post-transplant lymphoproliferative disorder)

Clotted blood:

  • antimicrobial levels
  • serum bactericidal titre
  • detection of Ag/Ab of various microbes

Sterile containers:
- necessary for assay of peak + trough serum bactericidal titres in patients with endocarditis while on treatment

23
Q

Specimen for PCR, RT-PCT, Other critical tests (e.g. CSF for smear)

A

MUST use:
1. Previously un-used (new)
2. Gamma-irradiated bottle
—> ensure no carryover of dead microbes (corpse / DNA / RNA may contaminate causing false +ve staining / PCR results)

24
Q

Summary

A
  1. Specimens of dubious significance
    - bedsore, superficial wound swabs
    - routine culture of superficial swabs e.g. penile swab, perineal swab, nasal swab
    - some drain fluids e.g. long term PTBD
    - urine from patients requiring chronic urinary catheterisation
    - colonic biopsy for routine bacterial culture (without any clinical information)
  2. Clinical correlation
  3. May need histological confirmation of infection

JC WCS (134 decks)

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