Microbiology JC092: Diagnosis Of Infections Flashcards
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
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)
***Common Bacterial causes of febrile respiratory illness
Most important bacteria (Encapsulated bacteria):
- ***Streptococcus pneumoniae
- ***Staphylococcus aureus / Streptococcus pyogenes
- ***Haemophilus influenzae / Neisseria meningitidis
Others:
- Enterobacteriaceae: ***Klebsiella pneumoniae (ill health, >65)
- Oral aerobes / ***Anaerobes (aspiration pneumonia)
- Acinetobacter baumannii (hospital flora)
- ***Pseudomonas aeruginosa (hospital flora)
- Burkholderia pseudomallei (soil, sputum)
Atypical bacteria (NOT respond to β-lactam, only respond ***Tetracycline, Macrolide, Fluoroquinolone):
- ***Legionella pneumophila (sputum, urinary antigen EIA)
- ***Mycoplasma pneumoniae
- ***Chlamydophila pneumoniae / psittaci
- Coxiella burnetii (Q fever)
Endemic (if patient NOT respond to typical / atypical culprit):
13. ***Mycobacterium TB (sputum)
Common Viral causes of febrile respiratory illness
Most important in normal adults:
- ***Influenza A-C
- ***Adenovirus
Children / Elderly:
- RSV
- Parainfluenza 1-4
- Rhinovirus
- Metapneumovirus
- Coronavirus MERS, SARS, OC43, HKU1, 229E
- Enterovirus
- Bocavirus
Common Fungal causes of febrile respiratory illness
Usually in immunosuppressed hosts
- ***Cryptococcus
- ***Aspergillus
- ***Dimorphic fungi: Penicillium, Histoplasma, Coccidioides
- Zygomycetes
- Pneumocystis (atypical yeast)
Common Parasite causes of febrile respiratory illness
Usually ***Eosinophilia in blood
- Paragonimus westermanii
- Ascaris lumbricoides
- ***Strongyloides stercoralis
- Others
***Laboratory diagnosis of infection
Microbial factors:
- ***Visualisation in clinical specimens
- Gram stain, ZN smear, Light microscopy for bacteria
- Electron microscopy for viruses - ***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 - ***Biochemical tests (detection of growth)
- 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
- Ab response of host towards microbial components
- IgM
- ***Paired sera showing 4x increase - ***Cell-mediated immune response
- Mantoux test
- IGRA
- Lymphocyte proliferation
- CTL response towards specific Ag
Flowchart: Decision making in management of infectious disease
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)
GIGO syndrome
Garbage in —> Garbage out
Improperly collected specimens —> Misleading test results
Quality of clinical specimens
Depends on:
- Degree of representation of pathophysiological process
- Manner of collection
- Manner of transportation
General principles for specimen collection
- ***Relevant specimens —> according to Clinical indication
- Correct containers —> avoid Leakage / contamination
- Expedient transportation (if not, proper transport / refrigeration should be considered) —> Preserve characteristics
- Request forms —> allow Proper entry (what + why)
- ***Biohazard —> Universal precaution (should consider all patients’ specimens to be biologically hazardous)
- Known hazards (e.g. HIV) —> Inform, clearly state on request form, biohazard labels
- Unusual infections —> Consultation with microbiologists
Blood
Most important test for diagnosis of ***Sepsis (Bacteraemia, Fungaemia, Endocarditis)
- 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) - 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 - 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 - Site
- Amount
- each set should contain 10-15 ml in each of aerobic + anaerobic blood culture broth (blood volume determines sensitivity of the test) - Broth
- ***antibiotics absorbing resins - 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 - ***PCR, Ab test for culture negative endocarditis
- Coxiella burneti, Bartonella henselae, Tropheyrema, Brucella, Leptospira, Mycoplasma, Chlamydia
CSF
- Representative specimen for ***Meningitis
- Lumbar puncture undertaken whenever meningitis suspected + excluded intracranial space occupying lesion
- Indications
- Fever + **Meningism
- **Encephalopathic signs
- Unexplained febrile illness in an irritable infant who is feeding poorly - Aseptic technique
- Non-reused bottles (prevent falsely positive gram smear results) - Amount
- >=2 ml for bacterial meningitis
- >5 ml for mycobacterial / fungal meningitis - Transport
- ***expedient
Investigations:
- **Gram / ZN smear / **Wet mount for amoeba
- ***Bacterial culture
- ***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
- ***Mycobacteria: PCR, AFB culture (for TB)
- ***Fungi: Cryptococcal antigen + Fungal culture (Latex agglutination)
Acute encephalitis
- ***PCR (for HSV1, HSV2, VZV)
- RT-PCR (for Enterovirus)
- CSF ***IgM (for Japanese encephalitis + Mycoplasma pneumoniae)
Other normally sterile body fluids
- Pleural
- Peritoneal
- Pericardial
- Synovial
- Aseptic technique
- ***Percutaneous needle aspiration - Volume:
- more the better (>5ml for mycobacterial / fungal infection) - Use of bottles containing **citrate / **heparin necessary if specimen prone to clotting (e.g. bone marrow)
- Inoculations of portion of fluid (2-5ml each) into aerobic + anaerobic blood culture broth with resin should be done if have sufficient volume
Indication for Semiquantitative culturing of IV catheter
Indications:
- Local signs of ***phlebitis
- 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
Diagnosis of Skin / Soft tissue infections
Degree of representation (Pathophysiological process of infection) —> Degree of contamination (Superficial colonising flora)
-
**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 - Sterile aspirate
- ***Abscess fluid
- Drain fluid
- ***Wound swabs (disinfect wound with alcohol first)
- 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
Eye, Ear
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
Faeces
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:
- ***Overgrowth of commensal flora (e.g. E. coli, Bacteroides, Enterococcus)
- ***Death of some pathogenic bacteria (e.g. Shigella, Campylobacter cannot survive if not immediately transferred)
- ***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
Urine
Indications:
- Bacteria
- Yeast
- ***UTI —> Bacterial DNA (Gonococcus, Chlamydia)
- 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)
-
**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 - ***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
Genital tract specimens
Indications:
- STD
- PID
N. gonorrhoeae / Chlamydia trachomatis:
- PCR
- Culture (slower)
- Urethral / ***Endocervical discharge
- sent on swab with transport medium
- Gram smear + Gonococcal culture - ***First void urine (rich in urethral discharge)
- PCR for N. gonorrhoeae / Chlamydia trachomatis - Urethral / ***Endocervical scrapings of cells are useful (Chlamydia ∵ intracellular organism)
- if discharge is scanty - 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:
- ***Giemsa stain —> Klebsiella granulomatis (causing Donovanosis / Granuloma inguinale)
- ***Dark field examination —> Treponema pallidum
Upper respiratory tract
e.g. Acute pharyngitis / Nasopharyngitis
- ***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 - ***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
Lower respiratory tract
Yield in ascending order (Oropharyngeal contamination —> Predominant pathogens):
Indications (Usual chest infections —> Intubated patients —> Severe / Persistent undetermined pneumonia (immunocompromised)):
- Saliva
-
Expectorated sputum (instructed / assisted by physiotherapist)
- interpret with caution (high epithelial cell + low white cell count on Gram stain suggest oropharyngeal contamination) - Endotracheal aspirate
- Bronchoscope aspirate
- ***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) - ***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) - ***Transbronchial lung biopsy
- 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
Serum and Blood
- ***Therapeutic drug monitoring
- Aminoglycosides (Gentamicin)
- Vancomycin
- Serum bactericidal titre (in Infective endocarditis) -
**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) - ***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
Specimen for PCR, RT-PCT, Other critical tests (e.g. CSF for smear)
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)
Summary
- 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) - Clinical correlation
- May need histological confirmation of infection
