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Theme 2.1 Microbiology > Laboratory diagnosis of Infections > Flashcards

Laboratory diagnosis of Infections Flashcards

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Biology 101 flashcards
1
Q

What are the steps to follow when diagnosing infection?

A
  1. History taking
  2. Clinical examination
  3. Radiological testing
  4. Appropriate specimen collection and laboratory testing
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2
Q

What are the infectious agents?

A

Bacteria(including Mycobacteria)
Viruses
Fungi
Parasites

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3
Q

What do we consider when taking or collecting specimens?

A
  1. Site of collection
  2. Collection technique
  3. Volume of specimen
  4. This must be done prior to administration of antimicrobial agents
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4
Q

Site from which we take the specimen

A
  • Directed by from history taking and clinical examination

- Take cognizance of whether the site is usually sterile/contains normal flora

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5
Q

Collection technique

A

Aseptic technique
Sites containing normal flora: minimise contamination by irrigation with normal saline, or disinfect skin eg. For blood culture

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6
Q

Volume

A

Sufficient sample allows for complete analysis of sample. Eg for blood culture in adults require 4-10mls of blood
Pus swabs hold minimum material

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7
Q

Discuss the transportation of specimens

A

Specimens should be transported as soon as possible to the laboratory
To ensure survival of organisms – some organisms do not survive for long periods unless transport media is used eg. Viral transport medium
To prevent overgrowth of organisms that grow easily/survive well eg. Urine samples/sputum. If overgrowth occurs-may impact on the isolation of the true pathogen/affect the interpretation of the culture
Temperature : viruses- 2-8°C
bacteria- 15-25°C for most specimens. Urine 2-8°C

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8
Q

WHAT ARE THE TYPES OF SPECIMENS?

A
  1. Blood culture
  2. Cerebrospinal fluid
  3. Pus
  4. Sputum
  5. Sterile fluids
  6. Stool
  7. Urine
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9
Q

Laboratory testing for the diagnosis of infections

A

Laboratory techniques:

  • Macroscopic examination
  • Direct microscopic examination
  • Culture and antimicrobial susceptibility testing
  • Antigen detection
  • Serology/Antibody detection
  • Genotypic methods (Polymerase chain reaction=PCR)
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10
Q

MACROSCOPIC EXAMINATION

A 
Macroscopic examination
CSF : Turbid, spiderweb clot
Stool : parasites(adult worm/segments)
                 blood/mucus
                 rice water consistency
Sputum: purulent
Urine: blood stained
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11
Q

When performing a Microscopic examination.

they use a light microscope.

Discuss the wet preparation.

A
  1. Wet preparation
    - Specimens = urine, stool
    - leucocytes, erythrocytes, parasites, yeast cells are the cells its used to identify.
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12
Q

Cell count under microscopy

A
  • Polymorphs, Lymphocytes, Erythrocytes
  • Usually done on Cerebrospinal fluid(CSF), Sterile fluids(pleural, pericardial)
  • Assists in diagnosing likely cause of infection: preponderance of polymorphs suggests acute bacterial meningitis, preponderance of lymphocytes suggests Tuberculosis/Cryptococcal infection
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13
Q

Staining techniques

A
  1. Gram stain
  2. Acid fast stains
  3. Auramine - O Stain
  4. Modified Ziehl-Neelsen stain
  5. Giemsa stain
  6. India ink stain
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14
Q

Gram stain

A

One of the most useful stains in Microbiology
Differentiates between Gram positive and Gram negative bacteria
Also demonstrates shape and arrangement of bacteria and yeast cells
Guides management of infection before a culture is available

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15
Q

Acid fast stains

A

Acid fastness is a property of Mycobacteria eg. Mycobacterium tuberculosis. These organisms usually stain poorly with the Gram stain due to the high lipid content in their cell wall structure
Examples = Ziehl-Neelsen; Auramine stains

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16
Q

Auramine - O Stain

A

Auramine stain, acid alcohol, potassium permanganate

View with fluorescence microscope

17
Q

Modified Ziehl-Neelsen stain

A

Used for detection of coccidian parasites eg. Cryptosporidium species, Isospora belli, Cyclospora

18
Q

Giemsa stain

A

Used for detection on parasites on a peripheral blood smear eg. Plasmodium spp(Malaria), Trypanosoma spp

19
Q

India ink stain

A

for encapsulated yeasts eg. Cryptococcus neoformans on cerebrospinal fluid

20
Q

Haemotoxylin and eosin stain

A

Used in histology

21
Q

Discuss culture in microbiology

A

Most bacteria and fungi are able to grow on artificial media(viruses can also be cultured on cell culture media, but the culture method is not widely used)

Artificial media: liquid(broth) or solid(agar) which contain nutrients to allow the growth of the organism

Fastidious vs non-fastidious bacteria/fungi

22
Q

Bacterial colonies on blood agar and chocolate agar. What are the required temperatures?

A

35±2˚C =temperature for incubation
16-18 Hours=period of incubation
Aerobic, anaerobic, 5% CO2 conditions for incubation

23
Q

Fungal colonies on saborauds dextrose agar(SDA).

A

Incubated for up to 6 weeks, Aerobically

  1. Candida albicans
  2. Trichophyton terrestre
24
Q

Mycobacteria(M. Tuberculosis/Non tuberculous mycobacteria)

A

Mycobacteria – incubate up to 6 weeks

25
Q

Identification of micro-organisms

A

Gram stain appearance:
Gram-positive/ Gram-negative
Cocci/bacilli/ pairs/chains/curved

Cultural characteristics:
Pigment production
Colony morphology eg. Mucoid, 
Nutritional requirements
Hemolysis
Smell
Ferment sugars eg. Lactose fermenter

Biochemical characteristics:
Range of reactions that help identify the organism

26
Q

Why is antimicrobial susceptibility testing done?

A

Helps guide the selection of appropriate antimicrobials for the infecting microorganism

Kirby-Bauer disc diffusion method

Broth dilution method/Etest/agar dilution = Minimum inhibitory concentration(MIC)

Results are reported as Sensitive, Resistant, intermediate

27
Q

What is serology?

A

An indirect method of diagnosing the cause of an infectious disease
Evidence of antibody response to an infecting microorganism
Useful for microorganisms that cannot be isolated in culture/difficult to culture. Also for patients who have received antibiotic therapy
Examples: Treponema pallidum(cause of Syphilis), Hepatitis B, HIV

28
Q

What is the disadvantage of serology?

A

Disadvantage: antibody response is not immediate. Lag between infection and antibody response
Usually necessary to collect acute- and convalescent-phase sera and compare titres(10-14 days apart). look for rising IgG titre to suspected pathogen( fourfold rise in titre or greater).
Single high titre may also be diagnostic

29
Q

In serology what is the meaning of Positive IgM and IgG respectively?

A

Positive IgM antibody response suggests acute infection

Positive IgG antibody may reflect past infection

30
Q

What are the methods of serology?

A
  1. Latex agglutination
  2. Haemagglutination
  3. ELISA(Enzyme-linked immunosorbent assay)
  4. Immunofluorescent methods(direct/indirect)
31
Q

VIRAL INFECTIONS SUMMARY

A

Specimen collection: use of Viral transport medium especially for specimens collected from non-sterile sites eg. Sputum/urine
Microscopy: immunofluorescence.
Culture: cell culture: not commonly performed. Cytopathic effect(CPE)
Genotypic methods commonly employed eg. Qualitative or quantitative PCR
Serologic methods : important for HIV/Hepatitis B
Cytology/Histology : cytoplasmic inclusions/intranuclear inclusions

32
Q

FUNGAL INFECTIONS (YEASTS/MOULD) SUMMARY

A

Specimens: CSF, Biopsies, nail clippings, hair
Microscopy: wet preparation=KOH preparation, India ink for encapsulated yeasts(Cryptococcus)
Microscopy: stained smears= PAS stain/Gomori-methenamine silver stain on tissue sections
Cultures: prolonged incubation for certain slow growing fungi.
Identification : Germ tube test, urea test
moulds: lactophenol cotton blue preparation
Serology: Cryptococcal antigen test

33
Q

PARASITES SUMMARY

A

Microscopy: wet preparation(stool/urine) – ova, cysts, trophozoites, larvae
Microscopy: stained smears: Modified Ziehl-Neelsen stain for coccidian parasites, Giemsa stain for malarial parasites, trypanosomes on peripheral blood
Serology: Entamoeba histolytica, Cysticercosis, Hydatid disease

34
Q

How do we go about antigen detection?

A

Look for antigen directly on clinical specimens
Immunofluorescent method commonly used, lateral flow assay(LFA) or agglutination
Respiratory viruses(SARS-CoV-2), Chlamydia, bacteria, Pneumocystis jirovecii, Cryptococcal antigen(CRAG)

  • Immunofluorescence
  • CRAG LFA
35
Q

GENOTYPIC METHODS

A

Genetic material of micro-organisms ie. DNA, RNA analysed for diagnostic purposes
Qualitative(positive/negative) or quantitative(bacterial or viral load)
Polymerase chain reaction(PCR) most common method used
Allows for rapid diagnosis of infections
Commonly used for diagnosis of Tuberculosis(TB GeneXpert), viral infections(SARS-CoV-2)

Theme 2.1 Microbiology (10 decks)

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