1.3 Infection Diagnostics Flashcards
[Bacterial Meningitis]
- Top causes: Streptococcus pneumoniae*, _______________, ____________ (less common; increased immunisation)
- Other causes: Streptococcus agalactiae (GBS), ______________ (in young (neonates/infants), old (>60) and immunocompromised)
- *S. pneumoniae is also found in other organ systems (e.g. causing _____________).
Neisseria meningitidis;
Haemophilus influenzae;
Listeria monocytogenes;
otitis media
[Bacterial Pneumoniae]
- Community-acquired: ________________ (most commonly encountered), Haemophilus influenzae, Staphylococcus aureus
- Atypical causes (important to consider): ________________, _______________, ____________________
- Tuberculosis: Mycobacterium tuberculosis (subacute to chronic form)
Streptococcus pneumoniae;
Mycoplasma pneumoniae; Chlamydia pneumoniae; Legionella pneumophila
[Bacterial Skin infections]
- Staphylococcus aureus, Streptococcus pyogenes (GAS)
- Pseudomonas aeruginosa (tends to cause ecthyma – ________________ beneath which ulcers form – typically seen in __________________)
crusted sores;
neutropenic patients
[Bacterial Eye infections]
- Staphylococcus aureus, ______________ (contact with genital secretions), _______________ (neonatal eye infection – passing through infected vaginal canal)
Neisseria gonorrhoeae;
Chlamydia trachomatis
[Bacterial Sinusitis]
- ____________, ____________
Streptococcus pneumoniae, Haemophilus influenzae
[Bacterial URTI]
- ____________ (GAS), _____________ (epiglottitis)
Streptococcus pyogenes;
Haemophilus influenzae
[Bacterial Gastritis]
- ____________ (may predispose to peptic ulcers and gastric cancer)
Helicobacter pylori
[Bacterial Food poisoning]
- Enteric bacteria: _____________, _______________, ______________
Campylobacter jejuni, Salmonella, Shigella
[Bacterial UTIs]
-Predominantly Gram-negative: E. coli, ___________ (hospital-acquired UTIs), _______________ (common in college-age females)
Pseudomonas aeruginosa;
Staphylococcus saprophyticus
[Bacterial STDs]
- Chlamydia trachomatis (____________ urethritis), Neisseria gonorrhoeae (___________ urethritis), _________________ (syphilis → chancre (painless genital ulcer)), Ureaplasma urealyticum (non-gonococcal urethritis), ________________ (common in the Caribbean → chancroid (painful genital ulcer))
non-gonococcal;
purulent;
Treponema pallidum;
Haemophilus ducreyi
[Bacterial Anaerobic]
- Anaerobes predominate in the GI tract: intra-abdominal infections from _______________ (Bacteroides, Clostridium)
- Lemierre’s syndrome (__________________ often starting out as pharyngitis → Fusobacterium then translocates and seeds into the bloodstream where it enters the jugular vein adjacent to the pharynx)
- Brain abscesses: often due to oral anaerobes (facultative anaerobes: may survive in oxygenated/deoxygenated environments) like _____________
- Diabetic foot infection: usually as part of a polymicrobial process
perforated viscus;
septic jugular thrombophlebitis;
Strep viridans
[Viral infections: Encephalitis/ meningitis]
- Major causes: ___________
- Other causes: ____________ (polyomavirus seen with HIV infections), ______________(subacute sclerosing panencephalitis (SSPE)), __________ (e.g. dengue virus → cause meningoencephalitis occasionally), ___________ (acquired as a zoonosis from infected canines)
HSV-1/2 ;
JC virus;
measles virus;
arboviruses;
rabies virus
[Viral infections: Hepatitis]
- Chronic: Hepatitis _________ viruses; acute (self-limiting): Hepatitis _______ viruses
- HAV and HEV may sometimes cause fulminant hepatitis; Hepatitis E may rarely become a chronic hepatitis in immunocompromised patients
B, C, D ;
A, E
[Viral exanthema]
Some viruses may cause characteristic viral exanthema (widespread rash):
• ______ causes chickenpox (fluid-filled vesicles) and remains latent in the nerve roots before reactivation as shingles (herpes zoster)
VZV
[Viral STDs]
- ______ (genital ulcers), _______ (genital warts), HIV (systemic infection)
HSV-2;
HPV
[Viral Myelitis]
- Neurotropic viruses (infecting nerve cells) causing myelitis (spinal cord inflammation): ____________, _________
poliovirus, human T-lymphotropic virus 1 (HTLV-1)
[Viral Gastroenteritis]
- ________, ___________, ___________ (most common overall cause)
Adenovirus, Rotavirus, Norovirus
Fungal infections may be classified by their invasiveness or source of fungi:
• Fungi may exist as yeasts (unicellular), moulds (more complex; form ___________) or dimorphic (____________ at lower temperature, ____________ at higher temperature)
hyphae;
mould;
yeast
[Fungi: Endogenous (part of normal human flora)]
- Candida: inhabits the normal GI tract → presents as ____________ in immunocompromised patients (e.g. HIV) and impaired T cell immunity
- _______________: common coloniser of the respiratory tract → presents as pneumonia in immunocompromise (e.g. high-dose steroids/HIV infection)
oral thrush;
Pneumocystis jiroveci
[Fungi: Environment (ubiquitous)]
- Aspergillus, Zygomycetes (Mucorales): cause invasive fungal infections in _________________ patients (e.g. SCTs, chemotherapy)
sick neutropenic
[Fungi: Regional endemic (only in specific regions)]
- Histoplasma (in ______________): mainly in the Midwest, Central and East USA; but present worldwide
- _______________: endemic to the Southwest USA (e.g. California, Arizona)
soil and bat guano;
Coccidioides
The following fungal pathogens are medically important:
Dimorphic fungi
- Various: Mould at environmental temperature (25°C); Yeast at body temperature (37°C)
Opportunistic fungi
- Candida albicans: Form ______________
- Cryptococcus neoformans: Yeast with ___________ (virulence factor)
- Aspergillus fumigatus: True hypha (mould)
pseudohypha, germ tubes and true hypha;
thick capsule
[Anaerobic culture]
Anaerobes are _______________- which often fail to grow due to suboptimal collection and transport (die upon higher oxygen tension exposure):
• Proper specimens: blood (in anaerobic bottle), normally sterile body fluids (e.g. _____, ______), abscess/cyst aspirates, surgically obtained tissues
• Improper specimens (sites exposed to ___________): swabs, sputum, bronchoalveolar lavage, open wounds, voided urine, cervical/vaginal samples
oxygen-sensitive bacteria;
pleural, synovial;
oxygen
[Special cultures]
________ (for Campylobacter cultures), special media (e.g. ___________________ for Legionella)
Microaerophilic;
buffered charcoal yeast extract/BCYE
[Mycobacterial culture]
Acid-fast bacilli (AFB) culture used when suspecting TB or other NTMs:
• Sputum or gastric aspirate (________________ give optimal yield)
2 – 3 early morning specimens
[Streptococus pneumoniae]
- test: _____________
- Sensitivity: 77 – 87% (bacteraemic); 44 – 60% (non-bacteraemic)
- Specificity: 80 – 100%
Pneumococcal urine antigen
[Legionella pneumophila]
- test: _______________
- Only tests for serogroup 1
Legionella urine antigen;
[Mycoplasma pneumoniae, Chlamydophila pneumoniae]
- test: ____________
- Positive serology may only indicate __________ (definitive diagnosis based on paired serology – 4 fold rise in titres or seroconversion) → retrospective diagnosis
Mycoplasma/Chlamydia serology;
past exposure
[Burkholderia pseudomallei]
- test: __________
- Asymptomatic people in endemic areas may be seropositive
B. pseudomallei serology
[Helicobacter pylori]
- Tests
1. ____________ (gastric antrum specimen)
2. Urea breath test
3. Stool antigen test - False negatives possible in patients on _______________
Rapid urease test;
PPIs/antibiotics
DIAGNOSIS OF FUNGAL INFECTIONS
Fungal infections are typically diagnosed using fungal cultures (performed on special media):
• ______________ can grow in regular bacterial blood cultures, while other fungi grow better in special fungal blood culture bottles (e.g. Histoplasma if suspecting disseminated histoplasmosis)
- Fungal antigen tests: Galactomannan (___________ cell wall) , ___________ (fungal cell wall – generic marker), Cryptococcal antigen: (Cryptococcus infections)
- Fungal serology tests (detect antibodies): Histoplasma
- Histopathology (tissue biopsies): Look at morphology under special stains
Candida spp.;
Aspergillus;
β-glucan
DIAGNOSIS OF PARASITIC INFECTIONS
The diagnostics for parasitic infections include:
- Microscopy (very important): Blood smears (for blood parasites like _______________)
- Stool examination (for ova and GI parasites)
Serology (detect antibodies)
- For selected parasites (e.g. ______________ → suspected amoebic liver abscess; ____________ → disseminated in immunocompromised patients)
- Histopathology (tissue biopsies): Look at morphology under special stains
malaria and filariasis;
Entamoeba histolytica;
Strongyloides;
DIAGNOSIS OF VIRAL INFECTIONS
Most viral infections are diagnosed via PCR or serology:
- Culture: Slow, labour-intensive, needs special expertise + not all viruses can be cultured
- Direct fluorescence antibody (DFA) tests
- For ____________ (but not as sensitive as PCR)
- Nucleic acid amplification tests (e.g. PCR). Commonly used for viral detection of:
• Respiratory viruses (e.g. nasopharyngeal swabs and other respiratory specimens)
• HIV and Hepatitis viruses (e.g. HIV, HAV, HBV, HCV)
• Herpes viruses (e.g. CMV, EBV, HSV)
- Serology (antibody/antigen): Influenza (_____________; not very sensitive), HIV (__________________) and hepatitis viruses, dengue (__________ antigen → NS1 antigen almost as sensitive as PCR test)
- Histopathology: Viral inclusion bodies may be distinctive (e.g. ________ owl eye inclusions are highly specific)
Respiratory viruses;
rapid antigen tests;
p24 antigen/HIV antibody;
IgM/IgG/NS1;
CMV
The submission of the best specimen type for a particular test or to recover a specific organism is of paramount importance for a successful outcome:
- Specimen type (aspirate/blood/serum/urine), quantity (volume), number needed
- Type of container (+ correct label) or need for transport medium (e.g. viral transport medium/VTM)
- Time taken for specimen to reach lab (may be crucial in some cases → anaerobes become less viable when exposed to oxygen; _________________ require a complex nutritional requirement)
Specimens should be fresh and ideally taken before antimicrobial therapy is initiated:
• All samples should be appropriately labelled (any sample likely containing hazard group 3 organisms (e.g. Mycobacterium tuberculosis) must be processed in a ______________________
fastidious bacteria like N. gonorrhoeae;
Containment Level 3 (CL3) laboratory)
When obtaining a peripheral blood culture, proper aseptic techniques must be used:
• __________ must be worn, and standard precautions must be observed
• ___________________ is used in a circular rub for at least 30 seconds over a 5cm area (solution must be completely dry before skin puncture)
• Do not touch the venepuncture site after skin preparation (except with sterile gloves)
• The needle is inserted into the vein and at least 20cm3 of blood is drawn (10cm3 in aerobic culture, 10cm3 in anaerobic culture) → air cannot be injected into the anaerobic bottle (to prevent false negative results)
• A second set of blood cultures is drawn from a separate site (total 40cm3 drawn) → best diagnostic tool for _____________
Sterile gloves;
Chlorhexidine 2% in 70% isopropyl alcohol;
bacteraemia
BRONCHITIS & BRONCHIOLITIS
The table below shows the laboratory diagnosis of bronchitis and bronchiolitis:
• Atypical bacterial pathogens: ______________
• Bacteria causing bronchitis: _____________
• Viral pathogens: _____________
nucleic acid amplification test (NAAT) or serology;
expectorated sputum specimen;
NAAT
COMMUNITY-ACQUIRED PNEUMONIAS
The aetiologic agents listed include the common bacterial pathogens (e.g. S. pneumonia) and atypical pathogens (e.g. ________________________)
• Must also consider ________________ (in SEA and Northwest Australia) → causes melioidosis (cause of CAP) – GNR associated with soil exposure
Legionella, Mycoplasma, Chlamydophila;
Burkholderia pseudomallei
A 70-year-old male is admitted for cough, fevers and shortness of breath (acute onset over 3 days). He has a past history of diabetes, is a smoker and has a history of a right lobectomy for lung cancer. His chest X-ray shows extensive airspace changes in the right middle and lower zones and left lower zone. The right sided loss of volume is consistent with previous surgery.
• Radiological tests (e.g. CXR, CT) are useful adjuncts for diagnosing infective processes
• Bacterial blood cultures would be appropriate for patients admitted with severe pneumonia (yield for causative organism from blood cultures would be about 20%)
o Proper techniques for obtaining a blood culture should be used (obtain 3 sets of blood if _____________ is suspected)
• Aerobic bacterial cultures and Gram stain from a respiratory specimen
• If the patient is intubated, an _________________ should be done, or a ___________________ involving bronchoscopy with protected brush specimens
o Pure bacterial count > 103 cfu/mL in a brush specimen from BAL correlates with a histological diagnosis of pneumonia
• Other diagnostic tests to consider include:
o Nasopharyngeal swab (for respiratory virus) followed by PCR (e.g. influenza causing severe pneumonia)
o Legionella and pneumococcal urine antigen testing
o Burkholderia pseudomallei: both blood and respiratory cultures/serology
Diagnosis: The patient had a severe Streptococcus pneumoniae pneumonia and bacteraemia with blood cultures. The endotracheal aspirate grew S. pneumoniae and his urinary pneumococcal antigen was also positive.
endocarditis;
endotracheal tube aspirate;
bronchoalveolar lavage;
[STAINING TECHNIQUES- GRAM STAIN]
The Gram stain is an important first step in diagnostic bacteriology, and can be performed directly on specimens (e.g. sputum, blood cultures) or on cultured isolates:
• Gram-positive bacteria are stained purple, while Gram-negative bacteria are stained red
• Gram-positive bacteria have ______________ cell walls (which retain the crystal violet stain)
• Gram-negative bacteria have thinner cell walls, so the crystal violet stain is lost during decolourisation, and __________ is picked up during counterstaining
thick peptidoglycan;
safranin
The bacterial shape and organisation are also observed after Gram staining:
• Cocci (round-shaped) and bacilli (rods) are the two main groups, with an intermediate form (coccobacilli) → arranged in different patterns
• Vibrio species are ____________ bacteria with a _____________ -shape
• Spirochetes are spiral-shaped bacteria which are ________________ (better visualised under dark field microscopy, fluorescence microscopy or special immunohistochemical stains) (e.g. _________________ causing syphilis)
Gram-negative; curved/comma
non-Gram staining;
Treponema pallidum
[Aerobic Gram-positive cocci]
- Staphylococcus spp. (in _________): Common skin or mucosal commensals which may cause cellulitis, vasculitis etc.
- Streptococcus spp. (in ___________): Common respiratory or mucosal commensals which may cause pneumonia, URTIs etc. gram positive dipolococcus (also described as lancet shaped).
- Enterococcus spp. (short ___________): Common GI commensals which may cause UTIs, endocarditis, meningitis, bacteraemia
clusters;
chains;
chains
[Aerobic Gram-positive bacilli (rods)]
- Corynebacterium spp.: Skin commensals which may cause ____________ (most notable), lymphadenitis, endocarditis etc.
- Bacillus spp.: Bacillus cereus is a skin commensal which may cause ____________, Bacillus anthracis is the agent which causes ________
- Listeria spp.: Intracellular bacteria which causes ________________ in immunocompromised/very old/young
diphtheria;
food-borne illnesses (diarrhoeal);
anthrax;
gastroenteritis & meningitis
[Aerobic Gram-negative cocci]
- Neisseria gonorrhoea & meningitidis: Causes gonorrhoea and meningitis respectively
- ________________: Superficially resembles Neisseria; colonises the respiratory tract and causes bronchitis and respiratory infections in patients with COPD
Moraxella catarrhalis
Aerobic Gram-negative bacilli (rods)
- Enterobacteriaceae: Glucose fermenters (GI commensals)–> ____________________
- Non-fermenters: Non-glucose fermenting (using oxidative pathways in metabolism; healthcare-associated pathogens – in ICU patients with invasive procedures)–> ___________________
- Fastidious: Special growth requirements: _________________
E. coli, Klebsiella pneumoniae, Enterobacter, Serratia, Citrobacter, Proteus, Morganella;
Pseudomonas, Stenotrophomonas, Acinetobacter;
Haemophilus spp., Brucella spp.
FAST STAIN
Acid-fast organisms possess a different cell wall structure, and thus cannot be Gram stained:
• Acid-fast organisms retain the ____________ stain; non-acid-fast organisms lose the stain and pick up the ____________ counterstain
Organism
- Mycobacteria: Weakly Gram-positive rods
- Nocardia and other aerobic actinomycetes:: Nocardia are weakly Gram positive rods; other actinomycetes are also acid-fast (except ________________ – Gram-positive non-acid-fast rod)
- Various parasitic oocysts: Oocysts of Cryptosporidium parvum, Cyclospora, Isospora and hooklets of cysticerci (eggs of tapeworms – T. solium/saginata)
- Some Legionella species: E.g. L. micdadei
carbol fuchsin;
methylene blue;
Actinomyces israelii;
Mycobacterium tuberculosis is a clinically important pathogen (causes TB), which can be stained by the acid-fast stain and the _____________:
Acid-fast stains
- M. tuberculosis tends to cord together on broad cultures upon acid-fast staining due to _____________ in the cell wall (important virulence factor)
Auramine stain
- ___________ stain binding to mycolic acid in the acid-fast cell wall
- Other mycobacteria other than M. tuberculosis are called MOTT/NTM (non-tuberculosis Mycobacteria).
Auramine stain;
mycolic acid;
Fluorescent
FUNGAL STAINS
Fungi may be stained with KOH/calcofluor white, India ink stain, or mucicarmine stain:
KOH/Calcofluor white: Stains _______________ (fluoresces with polarised light)
India ink stain: Stains for ____________ (only 50% sensitivity)
Mucicarmine stain: Stains cryptococcal capsule ___________ (fungi like Candida and Cryptococcus may also stain Gram-positive)
chitin;
Cryptococcus ;
pink
PARASITE STAINS
Parasites are typically stained depending on the suspected organism causing disease:
- Malaria/Filaria Stool: _____________* (intracellular organisms)
ova/cysts/parasites : ____________ (no stain), iodine, iron/haematoxylin, trichrome stains
Special GI pathogens
- Acid-fast/safranin stain (e.g. Cryptosporidium, Cyclospora, Cystisospora, Microsporidium)
*The Giemsa stain of Plasmodium falciparum shows ______________________ (characteristic of P. falciparum).
Giemsa stain;
Wet mount;
RBCs with ring trophozoites within them and banana shaped gametocytes
HISTOCYTOPATHOLOGY
- Bladder biopsy with rounded structures with terminal spines (characteristic of ________________ → trematodes with infect the urinary tract)
- Duodenal biopsy with Giardia intestinalis infection (_______________ anteriorly; ____________ appearance laterally)
- H&E stained lung section showing _______________ (typical of CMV) → CMV immunostains can also be performed
- Grocott’s methenamine silver (GMS) stained tissue section of a lung showing dichotomously branched, septate, narrow hyphae of Aspergillus fumigatus
- H&E stain of nasal tissue showing a zygomycosis (e.g. Rhizopus, Mucor invasions) with a broad ribbon-like pauci-septate (few/no septate) hyphae
Schistosoma haematobium;
pear-shaped trophozoite;
fallen leaf;
owl eye inclusions
Aspergillus fumigatus
CULTURES
Cultures can be performed in the clinical microbiology lab for bacteria (aerobic/anaerobic), fungi, and mycobacteria:
• Specimens may be collected from normally sterile body fluids (blood, CSF, ocular fluids, pleural, abdominal (e.g. ascitic)) or other non-sterile sites (sputum, BAL, wounds, urine)
• ________________ are the best choice for enteric fevers (e.g. typhoid fever caused by Salmonella typhi)
Blood cultures
PROCESS OF BLOOD CULTURE
Blood cultures obtained from the patient are transported to the laboratory, where they are placed on an automated blood culture system (incubate at 37°C):
• Blood cultures are flagged positive either through _________________ (pH drops when organism grows) or _______________ (change in bottle pressure)
• When blood cultures are flagged, an aliquot of blood is sampled, and a _____________ is performed (then placed on culture media)
• After the organism grows, it is identified via MALDI-TOF (mass spectrophotometry), biochemical tests, PCR, DNA sequencing or fluorescent probe test
calorimetric pH;
CO2 detection;
Gram stain
[Blood agar]
Different species of Streptococcus (Gram-positive cocci growing in chains) can be distinguished using blood agar (testing for haemolytic enzymes):
- alpha haemolysis: greenish partial lysis of blood e.g. ________________
- beta haemolysis- clear complete lysis of blood e.g. grp a,b,c,f,g streptococci
- gamma haemolysis: non hemolytic e.g. ______________
viridans streptococci, S. peumoniae;
enterococci
After identification of Gram-negative organisms via Gram staining, culture on MacConkey agar helps to differentiate lactose fermenters from non-lactose fermenters:
Lactose fermenters
(Lac operon +)
- ___________, _________, ___________
- Utilise lactose to produce acid (lowers pH) → appears ____________ (precipitation of bile salts in the culture medium)
Non-lactose fermenters (Lac operon -)
- Other Enterobacteriaceae, Pseudomonas
- Unable to utilise lactose → do not turn pink
E. coli, Enterobacter, Klebsiella;
pink and hazy
Chocolate agar is used to culture ____________ (e.g. Haemophilus), which require special nutrients to grow:
• H. influenzae grows on chocolate agar (lysed blood),
but not blood agar
fastidious organisms
Mycobacterial and fungal cultures require specialised media, while viral cultures require _____________ (not commonly used → PCR more widely used today).
Lowenstein-Jensen media
- Greenish agar used to grow ______________ colonies of M. tuberculosis (takes about 4 – 5 weeks to grow; longer than regular bacteria)
Mycobacteria growth in tube (MGIT)
- ________ media used in the MGIT system instead
*Both media allow for automation and faster detection as well as susceptibility testing for mycobacteria.
specialised cell lines;
yellow-buff, crumbly, bread crumb-like;
Liquid;
The Czapek-Dox medium and Sabourad dextrose agar are two media used for culturing fungi:
Sabourad dextrose agar (SDA)
- ______________ and smooth colonies of Candida albicans shown
- Typically used to select for _____________ and some other types of fungi
Czapek-Dox agar
- ____________ surface pigmentation with a suede-like surface (dense felt of conidiophores) of Aspergillus fumigatus
(wet mount with lactophenol blue stain shows conidia/fruiting heads)
- Works well for many ___________ fungi (Aspergillus, Candida, Penicillium, Paecilomyces) and soil bacteria
Large white glabrous;
dermatophytes;
Blue-green;
saprophytic
Viruses must be grown on a normal monolayer of cells as they cannot replicate on their own:
- Positive viral culture: ______________ occurs (with clustering of cells due to cross-linking by viruses)
Cytopathic effect (CPE)
SUSCEPTIBILITY TESTING
Susceptibility testing (to an antimicrobial) can be performed with an organism culture:
• ______________: lowest antibiotic concentration that inhibits the growth of an organism (classified into susceptible, intermediate, resistant)
• May be determined using macro-broth dilution, micro-broth dilution, Kirby-Bauer disc diffusion, agar dilution, epsilometer strip (E-test) or automated systems
• All the tests performed help to generate a culture report
Macro-broth dilution
- _________ appearance with microbial growth (MIC refers to the concentration of the test tube with no macroscopic growth)
Disc diffusion test
- Clear _______________ with no microbial growth (diameter of zones indicates susceptibility to antibiotic)
E-test
- One end of strip has low concentration of antibiotic with gradually increasing concentration
Minimum inhibitory concentration (MIC);
Turbid;
circles (zones of inhibition)
SEROLOGIC METHODS
Serology has an important role in the diagnosis of infectious diseases and may be used to detect antibodies and antigens:
• Antibodies (e.g. IgM detection may indicate early infection; IgG detection indicates later infection/post-vaccination response)
• Antigens: pathogen proteins usually indicating early infection (e.g. __________ in dengue, __________ in acute/early HIV) or chronic infection (e.g. HBsAg in chronic Hep B)
Advantages
1. Non-invasive
2. Useful when:
• Causative agent non-culturable
• Causative agent not detectable by other traditional/molecular methods (e.g. syphilis, Q-fever)
• Patient already treated with ___________ which inhibits growth
3. Provides knowledge of exposure history and immunisation status
Disadvantages
- Not rapid (detectable Ab response ≥ _______ → usually retrospective, confirmatory)
- Unreliable Ab response in ________________
- IgG unreliable for neonates (maternally derived)
- May not be as specific (e.g. IgM), cross-reactions are possible
- Inherent inter-individual variability of Ab response
- Possible lack of standardisation between labs (different methodologies & manufacturers)
NS1 protein;
p24;
antimicrobials;
7 days;
immunocompromised hosts
1) COMPLEMENT FIXATION TEST (CFT)
Complement is a group of proteins which interact with _________________ to lyse cells as part of the innate immune system (used in CFT on a microtitre plate):
• Positive test (no haemolysis) indicates that the serum has antibodies against the antigen
• Antigens and complement are added to the serum, and if antibodies are present, ___________________
o Sheep RBCs and antibodies to the sheep RBCs are added, and if complement proteins were previously fixed, no reaction occurs → positive test
o If complement proteins were not fixed, they cause lysis of sheep RBCs and thus diffuse haemolysis in the well (red colour) → negative test
antigen-antibody complexes;
complement is fixed to the antigen:
2) HAEMAGGLUTINATION & HAEMAGGLUTINATION INHIBITION (HAI)
Haemagglutination is useful in investigating viral titres, while haemagglutination inhibition is useful in investigating viral antibody titres (typically used in influenza virus infections):
• Row A: normal RBCs with no interaction with viruses or antibodies (sediment to bottom of well – _________)
• Row B: viruses adhere to RBC surface, causing RBCs to clump together to form a lattice network (______________ – haemagglutination)
• Row C: if patient has antibodies to the virus, they bind to the viruses and prevent haemagglutination (RBCs form dense plug at bottom of well – red dot)
In a haemagglutination assay, influenza viruses are mixed with chicken RBCs:
• In sample A, haemagglutination occurs up to the 1 : 256 dilution, beyond which a red dot can be seen (negative result) → _________________
• In sample B, haemagglutination does not occur → __________________
• In sample D, haemagglutination occurs up to 1 : 512 dilution → _____________
red dot;
diffuse redness;
HA titre of virus stock is 256;
no detectable virus;
HA titre is 512
3) ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA)
The enzyme-linked immunosorbent assay (ELISA) can be used to detect antigen (direct) or antibody (indirect): • Microplate ELISA: coloured well indicates reactivity → the ___________ the colour of the wells, the higher the reactivity
Direct ELISA
- ________________ is coated to bottom of the well
- Patient’s sample (containing antigen) is added to the well → antigen binds to antibody on bottom of well
- _________________ added → binds to antigen (forms sandwich)
- Enzyme’s substrate is added → reaction’s product causes a visible colour change (positive test)
Indirect ELISA
- Antigen is coated to the bottom of the well (instead of antibody)
- Patient’s serum (containing antibody) is added to the well → antibody binds to antigen on bottom of well
- 2nd specific antibody (enzyme-linked) added → binds to _________
- Enzyme’s substrate is added → reaction’s product causes a visible colour change (positive test)
darker;
Antibody specific for a particular antigen of interest;
2nd specific antibody (enzyme-linked);
antibody
4) IMMUNOCHROMATOGRAPHIC TESTS Immunochromatographic tests may also be performed as a serological diagnostic tool:
• Patient sample is added to a chromatography strip, and if it contains virus, it binds to the virus-specific antibodies
• Antibody-virus complexes move via capillary action to the test line (causing colour change to a darker line)
• Test validity is ensured by a ________________
*This test is used in ________________________
colour change from binding of virus-specific antibodies to the control line;
RSV infections, the dengue dual test (tests for dengue NS1 and dengue IgG/IgM), and for Clostridium difficile.
5) LATEX AGGLUTINATION Latex agglutination can detect both antibodies and antigens (e.g. Cryptococcal antigen):
- Antibody detection : Latex beads are coated with antigens (if antibodies present, agglutination occurs)
- Antigen detection: Latex beads coated with _________________
monoclonal antibodies
6) IMMUNOFLUORESCENCE
Immunofluorescence is used to detect antigens and may be direct/indirect (using primary/secondary antibodies):
• Used in testing of ______________ for respiratory viruses (e.g. RSV, influenza A/B, adenoviruses), detection of _____________ in faeces, the pp65 CMV antigenaemia test, and the detection of _________ in skin scrapings
nasopharyngeal aspirates;
rotavirus antigen;
HSV/VZV
7) WESTERN BLOT
The Western blot (protein immunoblot) is composed of _____________ of native proteins (based on 3D conformation or length) then transfer onto a membrane:
• Immunostaining procedure (via antigen-enzyme-linked antibody binding) similar to ELISA procedure is used to visualise certain proteins on the blot membrane
gel electrophoresis
HEPATITIS B SEROLOGY
The antigens and antibodies present on the serology of hepatitis B can be used to determine whether the disease is acute or chronic, and whether the patient has been immunised:
• Measuring _____________(via PCR) is the most useful test to monitor treatment efficacy following treatment
• ________________________ are found acutely after HBV infection → body makes IgM anti- HBcAg antibodies quickly (persist through window period with ______________)
• IgG anti-HBcAg antibodies are eventually made, but is insufficient to eradicate HBV as the infection is now in the chronic state
• _______________ are a sign of immunisation (either by natural infection or Hepatitis B vaccination) → presence of anti- HBc antibodies indicates natural infection
hepatitis B DNA levels ;
HBV surface antigens (HBsAg) and core antigens (HBcAg;
low IgG and HBsAg;
Anti-HBs antibodies
HIV SEROLOGY
After a person acquires HIV sexually or via body fluid exposure, there is an eclipse period of about _________ (no test can pick up the infection):
• ____________ first rises in plasma, and viral detection (nucleic acid test) is most sensitive
• HIV-1 p24 antigen then starts to rise (detectable around day 15 – 17 of infection)
• HIV antibodies (IgM/IgG) only appear shortly after __________
*A short window period is inevitable even in the best of tests (____________ period).
10 days;
HIV RNA;
day 20;
eclipse
MOLECULAR METHODS
There are 3 main molecular methods used in microbiological diagnostics:
1) Nucleic acid amplification tests (NAAT)
- Polymerase chain reaction (PCR) – most common
- Other tests: RT-PCR, strand displacement amplification (SDA), loop-mediated amplification (LAMP), helicase dependent amplification (HDA), nucleic acid sequence based amplification (NASBA)
2) Signal amplification methods: ___________________
3) Probe-based hybridisation tests (fluorescent complementary probe): Culture confirmation for mycobacteria (e.g. TB)
There are many nucleic acid-based assays available for the detection of microbial pathogens:
Advantages
1. Generally high sensitivity (detect down to
one target copy per sample volume)
2. Fast turnabout time (45 minutes – 1 hour)
3. May detect _______________________
4. Can be multiplexed (e.g. respiratory viruses multiplex)
Limitations
- Liable to contamination by amplicons (false positives)
- False negatives possible if PCR reaction is impaired by _____________(e.g. stool)
- May be more costly
- Need skilled operators
- Cannot distinguish live from dead organisms
- Only detects what is included in the assay
Branched DNA, hybrid capture;
non-cultivable/fastidious organisms
inhibitors
