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Medical Microbiology > 13. Diagnosis, management and treatment of fungal diseases > Flashcards

13. Diagnosis, management and treatment of fungal diseases Flashcards

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1
Q

Why is fast correct diagnosis for fungal infections important?

A
  1. Because fungal infections progress quickly and kill quickly.
  2. Treatment between species in the same genra can vary massively
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2
Q

What needs to be used alongside diagnostic tools to give accurate dianosis?

A
  1. Symptoms
  2. Medical or travel history
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3
Q

Why are superficial fungal infections easier to manage?

A
  1. They are more visible so they are easy to identify
  2. Treatment length depends on the place of infections.
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4
Q

What can cause invasive fungal infections?

A
  1. A range of different fungi
  2. Often, pathogens like candida, aspergillus or cryptococcus.
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5
Q

What are the risk factors for fungal infections?

A

For fungi to cause an infection they normally need a predisposing factor.
1. HIV infection
2. neutropenia
3. Diabetes
4. Antibiotic treatment
5. transplant

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6
Q

What is the exception to fungi needing a predisposing risk factor?

A

Primary fungi pathogens

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7
Q

Why is antibiotic treatment a risk factor for fungal infections?

A

It can wipe out the microbiome that competes with the fungi

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8
Q

How is imaging used to help diagnose fungal infection?

A
  1. You can see what is going on
  2. See masses in the tissue but you don’t know what it is just from images
  3. Use the image along with history and symptoms to help confirm a diagnosis.
  4. Based on a lot of assumptions, it is not used for primary diagnosis very often.
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9
Q

Why is diagnosis at a species level important?

A
  1. There are important differences between infections from species in the same genus.
  2. Disease outcomes like mortality depend on the specific organism.
  3. Different fungal species have susceptibility to different drugs and intrinsic resistance mechanisms.
  4. The species cannot be distinguished by eye.
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10
Q

What does type of specimen for diagnosis depend on?

A

The type of infection like superficial vs invasive

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11
Q

What specimens are needed for diagnosis of invasive fungal infections?

A
  1. Invasive infections need invasive sampling.
  2. Aspergillus infections need lung tissue samples through a bronchoalveolar lavage.
  3. Cryptococcus infections require a lumbar puncture to get CSF.
  4. Candidemia requires blood samples
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12
Q

What are the downsides of invasive fungal infections diagnosis?

A
  1. Invasive samples
  2. Take time during which the patient gets worse.
  3. Uncomfortable
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13
Q

What specimens are used for diagnosis of superficial and subcutaneous fungal infections?

A
  1. Easier to access
  2. Scrapings, clippings, external biopsies.
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14
Q

How is light microscopy used to diagnose superficial fungal infections?

A

To look at cell structures like hyphae or yeast cells.

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15
Q

How is Fluorescence microscopy used to diagnose superficial fungal infections?

A
  1. Using potassium hydroxide
  2. Mostly used on dermatophytes
  3. Good for differentiation from bacteria
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16
Q

How is invasive candida diagnosed using microscopy?

A
  1. Blood smears
  2. Normally looks like budding yeast.
  3. C. albicans form germ tubes which is unique and used to differentiate them.
  4. You can also look for hyphae formation.
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17
Q

How is invasive aspergillus diagnosed using microscopy?

A
  1. Using a Diff-Quik stain
  2. Looking for hyphae formation
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18
Q

How is invasive Mucor diagnosed using microscopy?

A
  1. Looking for non-septate hyphae
  2. This is different from other hyphae
  3. from nasal discharge
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19
Q

How is invasive crytococcus diagnosed using microscopy?

A
  1. India ink stain of the CSF
  2. Normal CSF is quite boring so fungal presence is quite obvious
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20
Q

What does microscopy tell us about an infection?

A
  1. If it is fungal or not
  2. If it is a yeast or hyphae
  3. Any distinct characteristics
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21
Q

What is the disadvantage of using fungal cultures for diagnosis?

A

It takes a long time, and during this, the patient is suffering.

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22
Q

What is the advantage of using fungal cultures for diagnosis?

A

You get more information than from microscopy

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23
Q

Why are most fungal cultures done in tubes not on plates?

A
  1. There is less risk of contamination
  2. There is less aeration of spores
  3. Done on a slant to increase surface area and good for long-term storage
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24
Q

Why are there different types of agar used for fungal culture?

A

Different fungi need different media to differentiate between them

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25
Q

Different fungal culture media: Sabouraud’s dextrose agar

A
  1. Recommended for most fungi as it is generic.
  2. Sugar rich
  3. Lots of fungi grow quite easily on it.
  4. It doesn’t distinguish between species
  5. All yeast grow as white, waxy, colonies on this agar
  6. Contains chloramphenicol to inhibit bacterial growth
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26
Q

Different fungal culture media: Mycosel agar

A
  1. A generic rich media
  2. Gives lots of growth
  3. Contains chloramphenicol to inhibit bacterial growth
  4. Contains cycloheximide to inhibit the growth of Aspergillus and C. neoformans
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27
Q

Different fungal culture media: Brain-heart infusion

A

Similar to Sabouraud’s but more enriched

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28
Q

Different fungal culture media: potato dextrose agar

A

Used to induce spore formation to aid identification

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29
Q

Different fungal culture media: Birdseed agar

A
  1. Used to see more distinct morphology
  2. Used to isolate cryptococcus neoformans from contaminated samples.
  3. It Also contains drugs to inhibit the growth of other fungi you have ruled out
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30
Q

Why are growth conditions for fungal culturing important?

A
  1. This is critical as fungi grow and present differently at different temperatures.
  2. Some fungi won’t grow at certain temperatures like dermatophytes not growing above 28oC.
  3. Some fungi can take weeks to grow as some fungi are very slow growing which is not good for diagnosis.
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31
Q

What helps determine the growth conditions for fungal cultures?

A
  1. The type of sample
  2. Like a nail sample being done at room temp
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32
Q

Why is it hard to distinguish between yeasts in cutlure?

A

They all look the same:
1. White
2. Waxy
3. Domed
4. Raised

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33
Q

What is chromogenic agar?

A
  1. Contains different metabolites that produce different pigments when metabolised by different fungi.
  2. This produces different colours depending on species.
  3. It is very very expensive so not commonly use.
  4. Can be used in research or in complex cases
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34
Q

What is a liquid culture test?

A
  1. Used to diagnose different candida species.
  2. Used bovine or horse serum
  3. C. albicans will produce germ tubes and others don’t so you look for these.
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35
Q

What can you do with all these different culture tests?

A

Run them in parallel so you have lots of information to be able to make a diagnosis

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36
Q

How can mould be diagnosed through culturing?

A
  1. They all have distinct morphologies when grown on different agar.
  2. They are also cultured on different media
  3. They can then be microscopically identified by the spore morphology
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37
Q

How can yeast be characterised using biochemical tests?

A
  1. Using API strips
  2. These contain lots of different tests like what sugar they can use
  3. Standardised version of chemically defined techniques
  4. Tricky to do in the lab as they are not hugely consistent and you need good technique.
  5. There is a specific window you need to read the results in to be valid.
  6. Not used as a primary diagnostic tool
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38
Q

How can fungal infections be diagnosed using PCR?

A
  1. Cost is reducing, so it is becoming more common.
  2. Amplify a specific region of a gene
  3. These are hyper-conversed regions that contain some hypervariable regions that distinguish the species.
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39
Q

What gene region is normally used for fungal diagnosis?

A

The ITS region of the 18s or 28s of rRNA

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40
Q

Why are rRNA genes used for PCR diagnosis?

A
  1. There are 100s of copies of genes in the cell
  2. This means you don’t need a high fungal load to get a good sample.
  3. Better than virulence factors that will have only 1 or 2 copies per cell.
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41
Q

What are the different types of PCR used to diagnose fungal infections?

A
  1. Nested PCR
  2. PCR-ELISA
  3. PCR-blot
  4. Real-time PCR
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42
Q

Why is real time PCR used for fungal infection diagnosis?

A
  1. It is qualitative
  2. You need to start with the same concentration in the samples and have a control.
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43
Q

What are the disadvantages of PCR for fungal diagnosis?

A
  1. There are lots of different kits with different steps and reagents. This introduces variation.
  2. Human error in following testing kits
  3. There is no standardisation of testing.
  4. Equipment and expertise varies.
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44
Q

What needs to be considered when diagnosing a fungal infection?

A

If the patient is on antifungal prophylaxis which can effect results.

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45
Q

What is the process of molecules identification of yeast and moulds?

A
  1. Start with a pure fungal culture
  2. Extract the DNA and purify to remove proteins
  3. PCR of the region of interest using primers
  4. Use of good primers
  5. Validate results on a gel.
  6. Sequence using sanger sequencing
  7. Then use BLAST and select the most likely species for diagnosis.
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46
Q

Why does molecular diagnosis of fungal infections need to start with a pure culture?

A

Because it can’t distinguish between infection and commensal fungi

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47
Q

What do you need to know to molecularly identify fungi?

A

The sequence of the gene of interest

48
Q

What do you need to consider when using primers for identifying fungi?

A
  1. Optimum temperature
  2. Correct Tm
  3. Need 90-100% for clinical efficiency
49
Q

How could MALDI-TOF be used to diagnose fungal infections?

A
  1. Uses proteins or spore suspensions.
  2. Needs well curated data bases for identification
  3. Needs minimal sample processing
  4. It cannot tell between commensal and invasive fungi.
  5. Need to use along with symptoms and patient history to give diagnosis
50
Q

What is serology?

A
  1. Looking at the patient’s response to the fungal antigens
  2. This takes time to measure
  3. It cannot tell the difference between active infection and previous commensal activity.
51
Q

What techniques can be used to test serology for diagnosing fungal infection?

A
  1. Double diffusion assay
  2. ELISAs, which take a long time and are technically challenging
52
Q

What is the problem with serological testing?

A
  1. False positives from superficial colonisation
  2. False negatives in deeply immunocompromised that produce no antibodies to the fungal infection.
53
Q

Why does the sensitivity of serological tests matter in fungal diagnosis?

A
  1. Sensitivity massively varies in the tests.
  2. The fungal load needs to be considered as it reduces as the sample it processed
  3. High sensitivity is needed for these tests
    .
54
Q

What are some targets for fungal serological tests?

A
  1. capsular polysaccharides
  2. Mannans
  3. Galacromannan
  4. b-1,3-glucan
  5. JF5 antigen
55
Q

What are some examples of fungal serological tests?

A
  1. Aspergillus lateral flow device against the growing tip antigen
  2. Cryptococcus antigen latex test (not very sensitive)
  3. Candida ELISA kit against b-1,5-oligomannosides
56
Q

Fungal specimen collection and processing: Cerebrospinal fluid

A
  1. 3-5ml is best for fungal investigation
  2. Has to be used quickly and cannot be stored.
  3. Centrifuge and resuspend in sabouraud’s dextrose agar
57
Q

Fungal specimen collection and processing: Sputum, bronchial wash or throat swabs

A
  1. Easy collection
  2. Can be difficult for respiratory infections
  3. need to process immediately
  4. Can be stored for a bit at 4oC
  5. Inoculate on sabouraud’s agar
58
Q

Fungal specimen collection and processing: Blood culture for invasive candidemia

A
  1. Biphasic culture bottle
  2. Requires a lot of blood from very sick people
  3. slant BHI infusion and broth
  4. Only accurate 50% positive results
  5. Takes a few days and must be check and vented daily
59
Q

What is better then treating fungal infections?

A
  1. Preventing them
  2. Prophylaxis for the right patients is important
  3. Education at risk patients is also critical
  4. For examples neutropenia or bone marrow transplant recipients
60
Q

What is empirical treatment?

A
  1. Treatment before a diagnosis
  2. Unresponsive to antibiotic treatment
61
Q

What is definite treatment?

A
  1. The standard treatment to produce the recovery of the patient
62
Q

What is maintenance therapy?

A
  1. Ongoing preventative treatment
  2. Happens after the primary treatment
  3. Important for immunocompromised people or people that had specific infections
63
Q

Why does fungal treatment fail?

A
  1. Poor compliance with treatment both from patients or healthcare provider
  2. Inadequate drug absorption or distribution
  3. Availability of antifungals. Might have to go for a less effective drug, as that is all that’s available
  4. Drug inactivation or interactions especially for pro-drugs
  5. Drug resistance
  6. Pharmacokinetics and pharmacodynamics with other medication
64
Q

Should you focus more on diagnosis or empirical treatment?

A
  1. It is about balance
  2. But ultimately you need a diagnosis for effective treatment
  3. Use all the information you have to start empirical treatment to buy time to get an accurate diagnosis.
65
Q

Why are fungal infections hard to treat?

A
  1. They are also eukaryotic so they share a lot more processes with our cells.
  2. This limits the targets that can be used for drugs
  3. There is lots of acquired and intrinsic resistance.
66
Q

When was the most recent antifungal discovered?

A

2006

67
Q

What are the most common antifungals?

A
  1. Amphotericin B
  2. Flucytosine
  3. Lipid AMB
  4. Fluconazole
  5. Caspofungin
  6. Voriconazole
68
Q

Why is the lack of new antifungal such a problem?

A
  1. Fungi are associated with climate change
  2. This means infections are massively increasing, and we cannot treat them.
69
Q

When was the 1st anti fungal discovered?

A

AmpB in 1957

70
Q

What is the gold standard for antifungals?

A

AmpB

71
Q

Why are there no new antifungals?

A
  1. Hard to find suitable targets
  2. Resistance
  3. Lack of funding and development for new antifungals
72
Q

What are the 4 classes of antifungals?

A
  1. Azoles
  2. Polyenes
  3. 5-flucytosine
  4. Echinocandins
73
Q

What do Azoles and polyenes target?

A

Fungal cell membrane integrity

74
Q

What do echinocandins target?

A

Fungal cell wall synthesis

75
Q

What do 5-flucytosines target?

A

fungal nucleic acid synthesis

76
Q

How do polyenes work?

A
  1. It disrupts the stability of the cell membrane.
  2. Contains 7 conjugated double binds that can intercalate with ergosterol.
  3. This creates K+ channels that lyse the cell
  4. This means polyenes are fungicidal
  5. Primary therapy for cryptococcosis
77
Q

What is an example of a polyene?

A

Amphotericin B

78
Q

How are polyenes delivered to patients?

A
  1. In Micelles
  2. This makes delivery easier
  3. Must be given by IV
  4. Easy to give if you have good healthcare infrastructure.
  5. Lots of fungal infections happen in places without healthcare systems so treatment is not accessible
79
Q

What are the strengths of polyenes like AmpB?

A
  1. Broad spectrum of activity
  2. Fungicidal
  3. Low drug resistance
  4. Cost effective
  5. Long half life
  6. Higher doses and concentration in tissues.
80
Q

What are the disadvantages of polyenes like AmpB?

A
  1. Hard to administer
  2. Infusion toxicity
  3. Kidney toxicity (another reason it must be given in hospital)
  4. can cause anaemia
81
Q

How do azoles like fluconazole work?

A
  1. Targets ergosterol biosynthesis by inhibiting cytochrome P450 enzyme lanosterol-14-alpha-demethylase
  2. Fungistatic so it requires a functioning immune system to clear the infection.
82
Q

How is fluconazole mostly used?

A
  1. As a single dose usually 1 pill
  2. Normally used for yeast infections
  3. Can be given as pills, pastes, gels or mouthwash
  4. Often used to treat candida and C. neoformans
83
Q

What are the strengths of fluconazole?

A
  1. IV or oral administration
  2. Easy to administer
  3. Few drug interactions
  4. Side effects are uncommon
  5. Good tissue penetration which is good for superficial infections that are starting to penetrate into the tissues
84
Q

What are the disadvantages of Fluconazole?

A
  1. Limited spectrum of action so you need an accurate diagnosis.
  2. There is emerging and innate resistance.
85
Q

How do azoles like voriconazole work?

A
  1. Targets ergosterol biosynthesis by binding to lanosterol demethylase and a 2nd enzyme in the pathway.
  2. Fungicidal
  3. Expanded spectrum of activity
86
Q

How is voriconazole used?

A
  1. Fungicidal activity similar to AmpB
  2. Given by IV or orally
  3. Active against opportunistic moulds
  4. More downsides
87
Q

What are the strengths of Voriconazole?

A
  1. IV or Oral administration
  2. Easy to administer
  3. Broad spectrum
  4. Fungicidal
  5. Low toxicity
  6. Good brain penetration
88
Q

What are the disadvantages of voriconazole?

A
  1. Variable metabolism
  2. Normally given in a healthcare setting to monitor drug levels and side effects
  3. Side effects include retinal, cardiac, and cerebral
  4. Lack of activity against mucormycotina
  5. Potential for drug interactions normally with heart medication
89
Q

How do echinocandins like caspofungin work?

A
  1. Blocks cell wall synthesis by inhibiting ß-(1,3)-d-glucan synthase
  2. fungal cels lose shape and become fragile
  3. Similar mechanism to penicillins in bacteria
  4. Good target as the target is not in human cells
90
Q

How is caspofungin used?

A
  1. Fungicidal against C. albicans
  2. C. neoformans not susceptible to echinocandins (shows importance of accurate diagnosis)
  3. Fungistatic in moulds
  4. Fungicidal for yeasts
90
Q

What are the strengths of caspofungins?

A
  1. Unique mode of action
  2. Fungicidal against candida
  3. Reasonably broad spectum
  4. Low toxicity
  5. Few drug interactions
  6. potential for combination therapy
91
Q

What are the disadvantages of caspofungins?

A
  1. IV only
  2. No activity against cryptococcus and mucormycotina
  3. Safety not established for children or pregnant people.
  4. Limitations in hepatic insufficiency
92
Q

How does 5-Flucytosine work?

A
  1. It inhibits nucleic acid synthesis by inhibiting purine and pyrimidine uptake.
  2. It is a fluorinated pyrimidine
  3. It is a pro-drug that needs to be metabolised by the fungal cells by cytosine deaminase
  4. Causes unbalanced growth and cell death.
93
Q

What is 5-flucytosine used for?

A
  1. It is not as good as AmpB
  2. Alternative therapy for UTIs caused by fluconazole-resistant candida species
94
Q

What are the strengths of flucytosine?

A
  1. Good absorption and distribution
95
Q

What are the disadvantages of Flucytosine?

A
  1. Limited spectrum of activity
  2. Resistance is common
  3. Toxic side effects
  4. Need to monitor the patient
96
Q

Uses and adverse effects of AmpB

A

Uses: Most fungal infections
Adverse effects: acute infusion reactions, neuropathy, GI upset, renal failure, hearing loss, rash

97
Q

Uses and adverse effects of Fluconazole

A

Uses: Mucosal and systemic candidemia, cryptococcal meningitis
Adverse effects: GI upset, hepatitis, QT prolongation

98
Q

Uses and adverse effects of voriconazole

A

Uses: invasive aspergillosis, fusariosis, scedospoiosis
Adverse effects: GI upset, transient visual disturbances, rash, hepatitis

99
Q

Uses and adverse effects of caspofungin

A

Uses: Aspergillus and candidiasis
Adverse effects: Phlebitis, headache, GI upset, rash

100
Q

Uses and adverse effects of flucytosine

A

Uses: Candidiasis, cryptococcosis
Adverse effects: bone marrow toxicity, neuropathy, nausea, vomiting, hepatic and renal injury, colitis

101
Q

What is the MIC?

A
  1. The minimum concentration of an antifungal agent required to prevent the in vitro growth of yeasts of moulds.
  2. It determines what drugs you use to treat fungal infections
102
Q

What is the MIC50?

A
  1. The lowest concentration of an antifungal that inhibits growth of 50% of the population.
  2. Used in clinical diagnosis
103
Q

What is MIC90?

A
  1. The lowest concentration of antifungal that inhibits growth of 90% of the fungal population.
  2. Used in research
104
Q

What is a disk diffusion assay?

A
  1. A disk of antifungal
  2. Measure the zone of clearance to determine drug susceptibility.
105
Q

What are E-tests?

A
  1. A strip of antifungals that have different concentrations as you go down the strip.
  2. Results vary depending on the test strip which needs to be accounted for.
  3. Not good for clinics as they lack consistency
106
Q

What are the main points regarding antifungal susceptibility testing?

A
  1. It needs to be standardised
  2. Culturing the fungi first takes time
  3. The tests themselves can take up to 2 days which is more time
  4. In the meantime the patient is in critical condition and deteriorates
107
Q

What causes intermediate resistance in fungal populations?

A
  1. Some cells have acquired resistance over a short period of time
  2. Some are small colony variants that express different virulence factors and characteristics that allow them to persist
108
Q

How does azole resistance work?

A
  1. Target site modification of surface targets
  2. Over expression of drug specific efflux pumps
109
Q

How does echinocandin resistance work?

A
  1. Altering of cell wall components to prevent drug binding
  2. Like an antigenic shift
110
Q

Mechanisms of AmpB resistance

A
  1. Change in ergosterol content
  2. Replacement of different sterols
  3. Reorientation of ergosterol to interfere with AmpB binding
111
Q

Mechanisms of azole resistance

A
  1. Over-expression of ERG11
  2. Numerous point mutations in ERG11 to change the binding site
  3. Efflux pumps
  4. Changes in ergosterol biosynthesis
  5. interspecies variability in cytochrome p450
112
Q

Mechanism of echinocandin resistance

A
  1. Remove target
  2. Resistance due to mutation in FKS1, which encodes that cyclic subunit of the synthase drug target
113
Q

Mechanisms of flucytosine resistance

A
  1. 10% of C albicans are intrinsically resistant
  2. Defect in intracellular accumulation
  3. 30% of fungi exposed acquire resistance
  4. Altering of the metabolic pathway
  5. Increase of pyrimidines that compete with drug
114
Q

What are intrinsic fungal resistance mechanisms?

A
  1. Biofilm formation
  2. Target incompatibility
  3. Stress response
  4. Drug transporters
  5. Cell permeability
115
Q

What are acquired fungal resistance mechanisms?

A
  1. Target overexpression
  2. Target incompatibility
  3. Stress response
  4. Drug transporters
  5. Pathway alteration

Medical Microbiology (16 decks)

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