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M2 Infection > 1.3 Infection Diagnostics > Flashcards

1.3 Infection Diagnostics Flashcards

1
Q

[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 _____________).
A

Neisseria meningitidis;

Haemophilus influenzae;

Listeria monocytogenes;

otitis media

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2
Q

[Bacterial Pneumoniae]

  • Community-acquired: ________________ (most commonly encountered), Haemophilus influenzae, Staphylococcus aureus
  • Atypical causes (important to consider): ________________, _______________, ____________________
  • Tuberculosis: Mycobacterium tuberculosis (subacute to chronic form)
A

Streptococcus pneumoniae;

Mycoplasma pneumoniae; Chlamydia pneumoniae; Legionella pneumophila

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3
Q

[Bacterial Skin infections]

  • Staphylococcus aureus, Streptococcus pyogenes (GAS)
  • Pseudomonas aeruginosa (tends to cause ecthyma – ________________ beneath which ulcers form – typically seen in __________________)
A

crusted sores;

neutropenic patients

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4
Q

[Bacterial Eye infections]
- Staphylococcus aureus, ______________ (contact with genital secretions), _______________ (neonatal eye infection – passing through infected vaginal canal)

A

Neisseria gonorrhoeae;

Chlamydia trachomatis

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5
Q

[Bacterial Sinusitis]

- ____________, ____________

A

Streptococcus pneumoniae, Haemophilus influenzae

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6
Q

[Bacterial URTI]

- ____________ (GAS), _____________ (epiglottitis)

A

Streptococcus pyogenes;

Haemophilus influenzae

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7
Q

[Bacterial Gastritis]

- ____________ (may predispose to peptic ulcers and gastric cancer)

A

Helicobacter pylori

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8
Q

[Bacterial Food poisoning]

- Enteric bacteria: _____________, _______________, ______________

A

Campylobacter jejuni, Salmonella, Shigella

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9
Q

[Bacterial UTIs]
-Predominantly Gram-negative: E. coli, ___________ (hospital-acquired UTIs), _______________ (common in college-age females)

A

Pseudomonas aeruginosa;

Staphylococcus saprophyticus

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10
Q

[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))

A

non-gonococcal;

purulent;

Treponema pallidum;

Haemophilus ducreyi

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11
Q

[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
A

perforated viscus;

septic jugular thrombophlebitis;

Strep viridans

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12
Q

[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)
A

HSV-1/2 ;

JC virus;

measles virus;

arboviruses;

rabies virus

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13
Q

[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
A

B, C, D ;

A, E

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14
Q

[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)

A

VZV

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15
Q

[Viral STDs]

- ______ (genital ulcers), _______ (genital warts), HIV (systemic infection)

A

HSV-2;

HPV

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16
Q

[Viral Myelitis]

- Neurotropic viruses (infecting nerve cells) causing myelitis (spinal cord inflammation): ____________, _________

A

poliovirus, human T-lymphotropic virus 1 (HTLV-1)

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17
Q

[Viral Gastroenteritis]

- ________, ___________, ___________ (most common overall cause)

A

Adenovirus, Rotavirus, Norovirus

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18
Q

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)

A

hyphae;

mould;

yeast

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19
Q

[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)
A

oral thrush;

Pneumocystis jiroveci

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20
Q

[Fungi: Environment (ubiquitous)]
- Aspergillus, Zygomycetes (Mucorales): cause invasive fungal infections in _________________ patients (e.g. SCTs, chemotherapy)

A

sick neutropenic

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21
Q

[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)
A

soil and bat guano;

Coccidioides

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22
Q

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)
A

pseudohypha, germ tubes and true hypha;

thick capsule

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23
Q

[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

A

oxygen-sensitive bacteria;

pleural, synovial;

oxygen

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24
Q

[Special cultures]

________ (for Campylobacter cultures), special media (e.g. ___________________ for Legionella)

A

Microaerophilic;

buffered charcoal yeast extract/BCYE

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25
Q

[Mycobacterial culture]
Acid-fast bacilli (AFB) culture used when suspecting TB or other NTMs:
• Sputum or gastric aspirate (________________ give optimal yield)

A

2 – 3 early morning specimens

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26
Q

[Streptococus pneumoniae]

  • test: _____________
  • Sensitivity: 77 – 87% (bacteraemic); 44 – 60% (non-bacteraemic)
  • Specificity: 80 – 100%
A

Pneumococcal urine antigen

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27
Q

[Legionella pneumophila]

  • test: _______________
  • Only tests for serogroup 1
A

Legionella urine antigen;

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28
Q

[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
A

Mycoplasma/Chlamydia serology;

past exposure

29
Q

[Burkholderia pseudomallei]

  • test: __________
  • Asymptomatic people in endemic areas may be seropositive
A

B. pseudomallei serology

30
Q

[Helicobacter pylori]

  • Tests
    1. ____________ (gastric antrum specimen)
    2. Urea breath test
    3. Stool antigen test
  • False negatives possible in patients on _______________
A

Rapid urease test;

PPIs/antibiotics

31
Q

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

A

Candida spp.;

Aspergillus;

β-glucan

32
Q

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

A

malaria and filariasis;

Entamoeba histolytica;

Strongyloides;

33
Q

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)

A

Respiratory viruses;

rapid antigen tests;

p24 antigen/HIV antibody;

IgM/IgG/NS1;

CMV

34
Q

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:

  1. Specimen type (aspirate/blood/serum/urine), quantity (volume), number needed
  2. Type of container (+ correct label) or need for transport medium (e.g. viral transport medium/VTM)
  3. 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 ______________________

A

fastidious bacteria like N. gonorrhoeae;

Containment Level 3 (CL3) laboratory)

35
Q

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 _____________

A

Sterile gloves;

Chlorhexidine 2% in 70% isopropyl alcohol;

bacteraemia

36
Q

BRONCHITIS & BRONCHIOLITIS
The table below shows the laboratory diagnosis of bronchitis and bronchiolitis:
• Atypical bacterial pathogens: ______________
• Bacteria causing bronchitis: _____________
• Viral pathogens: _____________

A

nucleic acid amplification test (NAAT) or serology;

expectorated sputum specimen;

NAAT

37
Q

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

A

Legionella, Mycoplasma, Chlamydophila;

Burkholderia pseudomallei

38
Q

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.

A

endocarditis;

endotracheal tube aspirate;

bronchoalveolar lavage;

39
Q

[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

A

thick peptidoglycan;

safranin

40
Q

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)

A

Gram-negative; curved/comma

non-Gram staining;

Treponema pallidum

41
Q

[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
A

clusters;

chains;

chains

42
Q

[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
A

diphtheria;

food-borne illnesses (diarrhoeal);

anthrax;

gastroenteritis & meningitis

43
Q

[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
A

Moraxella catarrhalis

44
Q

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: _________________
A

E. coli, Klebsiella pneumoniae, Enterobacter, Serratia, Citrobacter, Proteus, Morganella;

Pseudomonas, Stenotrophomonas, Acinetobacter;

Haemophilus spp., Brucella spp.

45
Q

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
A

carbol fuchsin;

methylene blue;

Actinomyces israelii;

46
Q

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).
A

Auramine stain;

mycolic acid;

Fluorescent

47
Q

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)

A

chitin;

Cryptococcus ;

pink

48
Q

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).

A

Giemsa stain;

Wet mount;

RBCs with ring trophozoites within them and banana shaped gametocytes

49
Q

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
A

Schistosoma haematobium;

pear-shaped trophozoite;

fallen leaf;

owl eye inclusions

Aspergillus fumigatus

50
Q

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)

A

Blood cultures

51
Q

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

A

calorimetric pH;

CO2 detection;

Gram stain

52
Q

[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. ______________

A

viridans streptococci, S. peumoniae;

enterococci

53
Q

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
A

E. coli, Enterobacter, Klebsiella;

pink and hazy

54
Q

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

A

fastidious organisms

55
Q

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.

A

specialised cell lines;

yellow-buff, crumbly, bread crumb-like;

Liquid;

56
Q

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

A

Large white glabrous;

dermatophytes;

Blue-green;

saprophytic

57
Q

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)

A

Cytopathic effect (CPE)

58
Q

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

A

Minimum inhibitory concentration (MIC);

Turbid;

circles (zones of inhibition)

59
Q

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

  1. Not rapid (detectable Ab response ≥ _______ → usually retrospective, confirmatory)
  2. Unreliable Ab response in ________________
  3. IgG unreliable for neonates (maternally derived)
  4. May not be as specific (e.g. IgM), cross-reactions are possible
  5. Inherent inter-individual variability of Ab response
  6. Possible lack of standardisation between labs (different methodologies & manufacturers)
A

NS1 protein;

p24;

antimicrobials;

7 days;

immunocompromised hosts

60
Q

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

A

antigen-antibody complexes;

complement is fixed to the antigen:

61
Q

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 → _____________

A

red dot;

diffuse redness;

HA titre of virus stock is 256;

no detectable virus;

HA titre is 512

62
Q

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)
A

darker;

Antibody specific for a particular antigen of interest;

2nd specific antibody (enzyme-linked);

antibody

63
Q

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 ________________________

A

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.

64
Q

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 _________________

A

monoclonal antibodies

65
Q

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

A

nasopharyngeal aspirates;

rotavirus antigen;

HSV/VZV

66
Q

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

A

gel electrophoresis

67
Q

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

A

hepatitis B DNA levels ;

HBV surface antigens (HBsAg) and core antigens (HBcAg;

low IgG and HBsAg;

Anti-HBs antibodies

68
Q

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).

A

10 days;

HIV RNA;

day 20;

eclipse

69
Q

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

  1. Liable to contamination by amplicons (false positives)
  2. False negatives possible if PCR reaction is impaired by _____________(e.g. stool)
  3. May be more costly
  4. Need skilled operators
  5. Cannot distinguish live from dead organisms
  6. Only detects what is included in the assay
A

Branched DNA, hybrid capture;

non-cultivable/fastidious organisms

inhibitors

M2 Infection (16 decks)

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