Infection Therapies Flashcards
What is selective toxicity?
Process within bacteria which are more susceptible to antibiotics than the equivalent process in the host
Antibiotics should be designed to interrupt biological processes which are not found in humans but are essential for survival in bacteria
Why does bacteria have a cell wall?
A single cell and exposed, membrane is insufficient so needs wall
Protects from mechanical damage and osmotic pressure
Bacteria is high salt concentration cell wall prevents bacterial lysis
What is the wall structure like of a gram-positive cell?
Dense layer composed of numerous rows of peptidoglycan, thick cell wall
What is the wall structure like of a gram-negative cell?
Thin inner layer of peptidoglycan, then an outer membrane on top of that (double membrane structure)
Describe and explain the bacterial cell wall:
Mixture of polysaccharides and lipids:
NAM|NAG|NAM
| a.a chains |
V - — ——- V
peptide chains bound to NAM sugars due to carboxylic acid side chain
—–= cross linking so rigidity increases
Have some D a.a to provide resistance (unnatural)
Why has penicillin got a B lactam structure?
B lactam much more reactive than a typical amide because of the strained ring system defavours resoance, makes it very reactive and susceptible to nucleophilic attack
How does penicillin work?
Inhibit the enzyme transpeptidase in a cell wall that cross links the cell wall as it has a similar structure of peptidoglycan D-Ala-D-ala so can bind in the active site
B lactam sits in the same position in amide bond of natural substrate which binds to seriene in A/S
Covalent inhibitor
What normally occurs in the cell wall formation of a bacteria?
Sereine hydroxyl performs nucleophilic attack at terminal amide bond of D-alanine an displaces it, forms and ester linkage
Ester is reactive and the peptide chain comes along and forms cross link
Why is Penicillin G acid sensitive?
- Carbonyl in B lactam ring is highly susceptible to nucleophilic attack
- Acid catalysed ring opening of the highly strained 4 membered lactam ring releases strain
- The neighbouring acyl group can actively participate in an intramolecular mechanism to open the lactam ring, so can’t be a covalent inhibitor beta-lactam could’ve destroyed
How can penicillin be modified to make it less acid sensitive?
Limiting neighbouring (R side chain) group activity by adding an electron withdrawing group
Pulls electron density away so carbonyl oxygen less likely to donate electron to B lactam
What are penicillin resistant bacteria and how do they work?
Resistance because enzymes catalyse the hydrolysis of penicillin, B- lactamase enzymes
B- lactamase has the same sereine- OH group in the active site so penicillin can bind, but leaves enough space for water to come in and hydrolyse penicillin and inactivate it
What are two ways that B lactamase sensitivity can be reduced?
- Penicillin analogues that are not recognised by B lactamase
- Co-administrate B- lactamase inhibitors
Describe an example of a penicillin derivative which isn’t identified by B lactamase?
Bulky R side chains such as an aromatic group with methyl side chains
Acts as a steric shield
Can bind to transpeptidsae but not B lactamase
What is an example of a B lactamase inhibitor?
Clavulanic acid
Binds in B lactamase A/S and undergoes nucleophilic attack and produces intermediate
Covalent inhibitor
Needs to be administered with penicillin
Why is it difficult to treat Gram -ve bacteria?
Has to get through cell membrane to get to cell wall
Also has to pass through periplasmic space which contain enzymes which drug can be hydrolysed
Passes through using porins which can pump back out the antibiotic as foreign material
What are broad spectrum antibiotics?
Can target both Gram-ve and Gram+ve bacteria
What is semisynthesis of penicillin?
Fermentation of penicillin fungi cultures
Penicillin amylase which can hydrolyse it and remove side chain, got to hydrolyse it acymatically as B lactams are very reactive, can be easily modified after
Why are cephalosporins used and how are they different to penicillin?
Activity against some penicillin resistant bacteria because of resistance against B lactamase
Lower activity than penicillins so need higher doses, but more broader spectrum
Similar to penicillin is but feature a six membered ring fuse to the beta-lactam so has less ring strain, so more stable to acid hydrolysis than the penicillin
Why are cephalosporins more stable in acidic conditions but still reactive?
The six membered ring causes the structure to be less strained so the amide bond is less reactive, so less readily hydrolysed in acidic conditions
Still reactive as curly arrow goes further down to remove acid to make it more reactive
How are cephalosporins inactivated?
Metabolic hydrolysis of the acetate reduces compound activity
Hydrolysis changes acetate to a OH group which isn’t as good of a leaving group as the acetate so reduces activity
How would you decrease the inactivation of cephalosporins?
Remove acetate group
Well absorbed as less polar but reduced activity as methyl group isn’t as good leaving group
OR
change it to a cephaloridine
How does vancomycin work?
A glycopeptide antibiotic inhibits peptidoglycan cross linking by binding to the D-ala, D-ala terminus of the cross linking peptide, similar to penicillins and cephalosporins but does this in a different way
It binds to the substrate rather than the enzyme by binding to the substrate it means the substrate can’t fit in the active site of the transpeptidase enzyme
What is the bonding in vancomycin?
Amide carbonyl on vancomycin structure
Amide NH of D alanine unit, hydrogen bonding
What occurs in vancomycin resistance?
In vancomycin resistant bacteria, instead of the final D-alanine, they have D-lactic acid
This means there is an ester linkage rather than an amide linkage, so no NH for critical binding, instead there are two oxygens so repel each other
The ester linkage is tolerated by transpeptidase but not by vancomycin
How is drug resistance tackled?
Combination therapies
Less chance of bacteria mutation and resisting against both types of antibiotics
How do sulphonamides work as an antibacterial?
Antimetabolite
Inhibit folic acid biosynthesis
Inhibit dihydropteroate synthase so can’t produce tetrahydriofolate which folic acid is essential for bacterial survival
Produces something other than folic acid
Competitive inhibitor
What is prontosil and what does it work for?
Active in vivo but not in vitro
Due to it being a prodrug metabolised in gut to give sulfonamide
Limited applications due to toxicity:
UTIs, mucous membrane infections, Gut infections
What does bacteriostatic mean and how does sulphonamide do this?
Stop bacteria cell growth,
Blocking folic acid synthesis blocks nucleic acid synthesis, hence cell growth and division stops
How can sulphonamides not stop folic acid production in humans?
They target a metabolic pathway that aren’t found in humans as we don’t biosynthesise folic acid
Bacteria lack transport proteins required for folic acid uptake
What does bactericidal mean and give an example:
Kills bacteria, e.g penicillin as inhibits cell wall synthesis
What does stronger binding of sulfonamides to blood and plasma mean for release of drug and vice versa?
Slower release= long acting
Fast release= short acting
Describe a way a sulfonamide is changed to reduce toxicity:
The 1º NH2 is prone to enzymatic acylation in blood
Can be fatal if the acylated product precipitates and blocks kidney tubule
Replacing the thiazole unit with pyrimidine solves the problem even if molecule doesn’t solubilise, it won’t precipitate
What are two ways of increasing sulfonamide drug resistance?
- Increasing intracellular conc of PABA as sulfonamide reversible competitive inhibitor of dihydropteroate synthase
- Enzyme mutations, mutations which prevent sulfonamide binding but still natural substrate (PABA turnover)
How does trimethoprim work?
Anitmetabolite
Antibiotic and antimalarial
Inhibits dihydrofolate reductase so inhibits conversion of folic acid into tetrahydrofolate
Often used in conjunction with sufamethoxazole to prevent resistance- sequential blocking
Inhibits dihydrofolate reductase which is found in humans, however there are structural differences between human and bacterial enzymes so only bind to bacterial
How do aminoglycosides work?
Protein synthesis inhibitors
Bactericidal, broad spectrum
Made of amino sugar structures with the ring being the drug
e.g streptomycin, kanamycin
How does chloramphenicol work and give details about it:
Protein synthesis inhibitors
Bacteriostatic, broad spectrum
Quite toxic but okay for topical use
Binds to large ribosomal subunit and blocks peptide bond formation
When is chloramphenicol not okay to use?
In newborn babies, inadequately metabolised so causes Grey baby syndrome
How do macrolides work and give details about it:
Inhibit protein synthesis in bacteria
Large cyclic structures with sugars coming off it
Not for use in combo with chloramphenicol as binds to same region of ribosome so would compete
Unstable in stomach acids, can be taken orally and coated tablet formulations
e.g ethromycin, clarithromycin
How do tetracyclines work and give details about it:
Inhibit protein synthesis in bacteria
Most widely prescribed broad-spectrum antibiotic after penicillin
Bind to small ribosome subunit, preventing tRNA binding
Protein release is also inhibited
Have stronger affinity for Mg2+ and Ca2+
Transported into cells by active transport and diffusion
Name 4 ways in which bacteria cause drug resistance:
- Target enzyme mutation, prevent Abs from binding to them
- Enzymatic modification to inactivate the drug
- Modified cell membrane structure preventing getting in or make them get out
- Resistance by genetic transfer
Describe two ways in which drug resistance occurs by genetic transfer:
Transduction- bacteriophages transfer resistance in genes in DNA plasmids by infecting other cells
Conjugation- genetic material exchanged directly via connecting bridges (sex pili) between 2 cells
Describe how HIV infects a cell:
HIV reverse transcriptase changes RNA into ssDNA into DNA
HIV intergrase catalyses viruses DNA into nucleus of a cell, so viral DNA is being produced to make viral RNA into polyproteins
HIV protease cuts polypeptide chain to produce proteins
These gather and bud at the surface of the membrane and virion released
This causes the host cell to rupture
HIV targets immune cells and that’s why HIV patients are immunocompromised
How is the sugar phosphate backbone of DNA elongated?
DNA polymerase catalyses reaction so that the phosphate group nucleotide is phosphorylated eliminate to leave the alpha phosphate and join the hydroxyl group
How do viral DNA polymerase inhibitors work?
Not having the hydroxyl group so no longer elongation of DNA chains
How do nucleoside reverse transcriptase inhibitors work for antiviral chemotherapies?
Competitive inhibitor
Don’t have triphosphate group as too polar to enter, once entered they can be biologically elaborated
Lacks a 3’ hydroxyl group, DNA chain terminators
What are the negative effects of nucleoside reverse transcriptase inhibitors?
Non-selective inhibition of mammalian DNA polymerases
Competing with natural nucleosides
How do non-nucleoside reverse transcriptase inhibitors work for anti-viral chemotherapies?
Reversible Non- competitive inhibitor
Hydrophobic molecule
Bind to an allosteric binding site adjacent to the substrate binding
Show selectivity for HIV reverse transcriptase over host DNA polymerase
What is the resistance in a non-nucleoside reverse transcriptase inhibitor and how is this over come?
Pan-class resistance mutation
Replacement of Lys-103 with asparagine
Combine NNRT with a nucleoside reverse transcriptase inhibitor at the start of treatment as binding sites are distinct
Describe the HIV protease and how it works?
Aspartyl Protease
Subunit A= acidic
Subunit B = basic
How does Saquinavir work?
HIV- protease inhibitor
Peptidomimetic
Replacement of an amide bond with a non hydrolysable bond
Froms a hydroxyethylamine
How does HIV- integrase work?
HIV integrase enzymatically splices the DNA form of the HIV genome into the human DNA
This way the virus utilises the host cell to copy its DNA and also translated into copies of its own proteins in order to replicate
How do HIV- fusion inhibitors work?
Structurally similar to HIV proteins responsible for fusion of the virus to cell membranes and subsequent intracellular uptake
Binds to surface of HIV cells (GP-41 protein)
HIV virus can’t recognise cells it wants to effect so no fusion to CD4 cells
Needs to be administered by injection as large protein structure
e.g enfurvitide
What happens when mutation occurs towards HIV fusion inhibitors?
Mutation in HIV GP-41 protein to prevent enfurvitide to bind but can still fuse to CD4 receptors
How does aciclovir work?
Selective viral DNA polymerase inhibitor
Prodrug
Converted to triphosphate structure and then acts as DNA chain terminator has no hydroxyl group
How is acyclovir a selective antiviral?
The first activation step is only catalysed by thymidine kinase produced by the virus, so only activated in cells which are infected
What are the features of famciclovir?
Pro drug of penciclovir
Longer duration of action, increased stability due to removal of oxygen ring
What is the function of neuraminidase on the influenza virus?
Catalyses the cleavlege of terminal sugar molecule from glycoproteins and glycolipids
Important for releasing the virus from host cells after bidding
Name a neuraminidase inhibitor and how does it work?
Zanamivir
Hydroxyl group replaced to guanine unit
Competitive inhibitor
Poor bioavailability so needs to be inhaled
Name a different neuraminidase inhibitor and how does it work?
Oseltamivir (tamiflu)
Add NH2 instead of hydroxyl unit
Activated by esterase’s in GIT
What is the cell membrane of a fungi made up of?
Ergosterol
What are the three targets for anti fungal therapy?
Cell membrane
DNA synthesis
Cell wall
How is the cell membrane in fungi made and how is this similar to human cell membrane, what needs to be targeted?
In early stages of ergosterol its the same as making cholesterol up to lanosterol
14a-demthylase is a fungal enzyme which removes methyl group which converts to ergosterol
Need to make a drug which inhibits membrane formation after lanosterol
How do azoles work as an anti fungal?
This is a multistep process, the azole nitrogen binding to the cytochrome p450 co factor, an iron containing macrocycle
Inhibit C-14 demethylation as its a cytochrome p450 and adds hydroxyl group so leaves no space for oxygen binding as the nitrogen azole binding
Describe polyenes and give an example:
Interfere with cell membrane structure
Natural macrocyclic lactones, which are amphipathic in nature
e.g nystatin, natamycin
Long cyclic structure with hydroxyl group on one side (polar) and alkene on other side (hydrophobic)
Has sugar on the end
How do polyenes work?
They inbed within the membrane
Polyenes can interact with ergosterol
The hydrophobic side interact with ergosterol
The polar side interacts with each other
This means creating a hole in the cell membrane so things e.g. salts leak out of the cell
The sugar can interact with hydroxyl group on ergosterol
How do DNA/RNA antifungals work and give an example:
e.g Flucytosine
Looks similar to naturally occurring nucleotide
Taken up to fungal cells as they have enzyme called cytosine permeate, humans lack this
Once inside, enzyme called cytosine deaminase converts it to fluorouracil which can be taken into RNA and changes the structure and affects protein biosynthesis
Flurouracil can be converted to thymidine monophosphate through uridine
If thymidine synthase is inhibited then DNA synthesis is also inhibited
What are the disadvantages of DNA/RNA antifungals?
Restricted spectrum activity
Acquired resistance due to:
- monotherpy
- rapid onset
What are clinical uses of DNA/RNA antifungals?
No mono therapy (resistance)
Used for candidiasis/ cryptococcosis
In combo with amphotericin or fluconazole
What is the viscosity of vaginal discharge influenced by?
Stage of menstruation
Oral contraceptive
Pregnancy
Where does vaginal discharge come from?
From glands
Contains shed epithelial cells and glycogen
Glycogen broken down into lactic acid (pH 4-5)
What can be causes of non infective pathological change in vaginal discharge?
Pain and bleeding away from normal menstries
Foreign body (TSS), contraception
Cervical polyps, cervical erosion
What can be causes of infective pathological change in vaginal discharge?
-Fungal
-Protozoal (TV) yellow/green discharge, foul smell (STI), GP for ab
-Bacterial (BV) grey discharge, fishy smell, GP for ab
-Viral (HIV, warts) Fluid filled vesicles, bumps
What type of infection is vaginal thrush?
Vulvovaginal Candidiasis
Yeast- Candida albicans
What can be the causes of vaginal thrush and why?
Pregnancy (decrease immune system, gestational diabetes)
Diabetes mellitus (increase levels of sugars, food for yeast, change in receptors of albicans so phagocytes can’t remove it)
Contraceptives, immunosuppressants, antibiotics
Other non medical factors (tight fitting clothes, local cleaning products)
What are the clinical symptoms of vaginal thrush in women?
Vulval pruritus (itchy) and white/ cream or curd like discharge, white plaques on vaginal wall or cervix
Dysuria (pain urination) and dyspareunia (pain on intercourse)
What are the clinical symptoms of thrush in men?
Vary from none to itching, burning and redness of glands on foreskin
Thick yellowish discharge
Penile shaft, scrotum, and groin occasionally involved
What are the referral red flags in vaginal thrush?
Under 16 or over 60
Reoccurance within 2 months or treatment failure
Factors that predispose to thrush e.g diabetes
What are OTC topical treatments to vaginal thrush and describe them:
Topical imidazoles:
Clotrimazole, econazole., miconazole
External cream (2%)
Internal cream (10%)
Pessary (500mg)
Use internal preparations at night as absorbs better
Can be used in combination
May affect latex condoms
What are OTC oral treatments to vaginal thrush and describe them:
Fluconazole
150mg single dose
Can be combined with external cream
12-24 hour improvement, 3 days max
Interactions:
-Anticoagulants -Ciclosporins -Phenytoin
Describe the mechanism of action of azoles:
Inhibits 14 a-demethylase
Disrupts ergosterol biosynthesis
Accumulation of methyl sterols- not effective cell wall
Membrane bound enzyme systems disrupted
What are POM treatments for vaginal thrush and describe them:
Oral itraconazole- 200mg BD for 1 day
Oral ketoconazole- 400mg OD cc for 5 days
^ POM as less specific for fungal and used for resistant/chronic cases
Topical nystatin- 14 day treatment, stains clothes yellow, for resistant cases
Topical provide- iodine (rare)
What is the advice for vaginal thrush medication to give to pregnant women?
Not a danger to foetus
Topical azoles preffered
What can oral thrush be caused by?
Breast feeding, dentures (incorrect use), antibiotics, dry mouth, steroids, and immunosuppresents
Is oral thrush common in babies and describe why?
1in 7 babies develop oral thrush in week 4
Clears up in 3-8 weeks
What are symptoms of oral thrush?
Milk curds in mouth, when wiped away leave red sore lesions, similar to vaginal thrush but in mouth
What are the referral red flags of oral thrush?
If there for more than 3 weeks
Lesions are more than 1cm, could be ulcers
Risk factors present but past history of diabetes etc
What is the OTC treatment for oral thrush and describe it:
Miconazole gel (QDS PC) for over 4 months old
Use after food and drink, to a clean mouth
Use for around a week
What are the POM treatment for oral thrush and describe it:
Nystatin and amphoteracin
- lozenge or oral suspension
Fluconazole and itraconazole (reserved)
- 7-14 days treatment
What are examples of fungal skin infections?
Tinea pedis (fungal foot), tinea capitis, tinea corporis
What is the general structure of a fungal skin infection ?
Raised edge, angular defined shape, usually itches, red and angry
What is Tinea Pedis?
Athletes foot
Thrive in warm moist conditions
More common in adult males
Usually starts near the little toe
Flakes, becomes white, macerated and begin peeling
Itchy and sore
What are lifestyle managements for athletes foot?
Clean, dry feet
Appropriate footwear and socks
Put socks on before underwear
Wear footwear in the gym/ changing room
Antifungal powder for shoe
What are OTC treatments for athletes foot?
Azoles- BD, TDS for 14 days from resolved
Terbinafine- OD-BD 7-14 days
What are referral signs in athletes foot?
Severe infections (large parts of foot)
Secondary bacterial infections -from itching, yellow crust
Diabetic (impaired circulation)
Treatment failure after 2 weeks
What are POM treatments for athletes foot and describe them:
Only necessary for severe/ extensive infections
Oral terbinafine (can affect liver so liver function tests)
-250mg OD for 4-6 weeks for tinea pedis
-250mg OD for 3-6 months for nail infections
Intraconazole, griseofulvin, fluconazole
What should be used to treat ring worm and why?
Canesten HC- not found in a moist area so may be more resistant
What is onychomycosis?
Fungal nail infection by dermatophytes, yeasts and moulds
What are the symptoms of a fungal nail infection?
Starts in the nail bed and spreads causing the nail to become thickened, discoloured, flaky and may separate from nail bed
What is OTC treatment for a fungal nail infection?
Amorolfine nail lacquer (curanail)
Used to treat proximal and distal onychomycosis - proximal (nail bed) may be more resistant
Weekly application for up to 12 months, because it’s a slow-growing nail, often little difference in the first three months
What are the referral red flags in a fungal nail infection?
Nail plates destroyed
More than 2 nails affected
Proximal onychomycosis
Pregnancy/ breastfeeding
Under 18 years old
What is antimicrobial stewardship?
Measures design to ensure the optimal selection of therapy for patients for the best clinical outcome while minimising toxicity, also to minimise resistance
What is the TARGET toolkit in primary care?
Treat Antibiotics Responsibly Guidance, Education, Tools
Why are primary care pharmacists advised to report drug allergies to penicillin?
Some may not be allergies, may be tolerances, so having to use second line treatment rather than penicillin when not needed to
Why does Start Smart then Focus mean in terms of prescribing antibiotics?
Start smart- Allergies, sepsis- prompt effect
Focus- send microspecimen to lab, stop using broad spectrum ab, switch to oral from iv, continue to review in 24 hours
Why does clinical data need to be monitored when a patient is on antibiotics?
To see if the patient has an infection
If the patient is having the right Ab
Can the patient be switched from IV to oral
Can the Ab be stopped
What are near patient investigations?
Anything that can be observed that indicates an infection e.g pus from wound, green discoloured sputum etc
Why are microbiology microscopy and staining useful to look for infections?
Simple and cheap
Staining used to identify potential pathogen presence
Identifies gram -ve and gram +ve
Why are microbiology cultures useful to look for infections?
Enables clear identification of organism
Significance in bacterial count used to confirm diagnosis
e.g UTI= >10^5 organisms per mil
Why are sensitivity investigations useful for using antibiotics and describe:
Used to determine antibiotic sensitivity after identification of the causative organism
Antibiotic discs are selected based on results of the Gram stain looks for zone of inhibition, if zones same size as control means it’s sensitive
If zone of inhibition larger than 26mm, bacteria is suceptible
What does R and S mean on a resistance microbiology report chart?
R= resistant, can’t be used
S= sensitive, can be used
What are advantages and disadvantages of microbiology for infections?
+ gives you specific info about organism and sensitivities
- may be impossible to obtain a sample is the infection seated
- organism may not grow if antibiotics have been started
- may culture colonising bugs (not cause of infection)
- take two days, sometimes longer to grow in the lab
What are four investigations used to demonstrate the presence of an infection and their recovery?
- Haematology results
- Biochemistry results
- Body temp
^MAIN ONES - Scanning techniques (X-rays)
How is haematology used to demonstrate infections?
White cell count (WCC)
Reference range= 4-11 x10^9/L = no infection
A raised WCC indicates the presence of infection
In less acute infections WCC may remain in reference range
How is biochemistry used to demonstrate infections?
-Used in combo with WCC
-C Reactive Protein (CRP)
-Reference range= 0-10
-Non specific indicator of infection but is also affected by other conditions e.g can rise in post surgery, inflammation
-Creatinine can be raised by UTIs/ sepsis
-Liver function tests may be raised in certain infections e.g viral hep
How is body temperature used to demonstrate infections?
Used in combo
Normally raised in an infection
Can also be a sign of other diseases such as neoplasms, acute MI and pulmonary embolism
How are scanning techniques used to demonstrate infections?
E.g shadowing on a chest x ray for a chest infection
Ultrasound and X ray
In extreme cases when unsure:
- White cell scan, white cells are extracted, radio-labeled and re-injected if source of infection unclear, under X ray shows the cells accumulating at the site of infection
When would you switch an antibiotic from IV to oral administration?
Review after 48 hours
If patient:
-haemodynamically stable with no signs of fever
-clinically improving
-able to take oral meds
-functional GI tract with no malabsorption
-no other interactions
Why is oral route of administration better than IV?
Less time consuming
Less chance of infections no canulla
What should be the duration for the course of antibiotics and give examples:
Duration not well defined
Usually less than 14 days
3 days for female uncomplicated UTI, 7 days for male
6 weeks or more for endocarditis or osteomyelitis
5 days adequate for most infections
Which common hospital antibiotics require levels and monitoring and why?
Genamicin IV and Vancomycin IV
Monitor drug levels according to hospital policy
Monitor creatinine as nephrotoxic
What are empirical antibiotics?
Best guest antibiotics , because microbiological results don’t become available for 24-72 hours, initial therapy before then is often empirical
What is targeted treatment?
Antibiotic treatment once microbiology results are available
What are the four main groups of bacteria and which part of the body do these bacteria normally affect?
- Gram-positive- Gut and mouth
- Gram-negative- Skin and mucous membranes
- Anaerobes- GI tract,
- Atypical- Chest, GI and genitourinary
What are the main antibiotics for the four main groups of bacteria?
- Gram-positive- Flucoxacillin, vancomycin
- Gram-negative- ceftazidime
- Anaerobes- Metronidazole
- Atypical-
Broad spectrum- co-amoxiclav
What are patient factors to consider when choosing an antibiotic?
Severity of infection
Immune status
Pharmacokinetic factors (e.g elderly, neonates)
Pharmacogenetic factors (e.g G6PD deficiency)
Epidemiology
Past medical history
Allergy status
What are symptoms of community acquired pneumonia?
Cough, chest pain, dyspnoea, increased in sputum
What are the treatments for community acquired pneumonia?
Non severe (5-7 days) Amoxicillin (doxycycline if allergic) +/- clarithromycin
Severe (7-10 days) Benzylpenicillin (IV) + clarithromycin
When shouldn’t you dipstick to test for a UTI and why?
Over 65 due to them already containing the bacteria naturally
What are the treatments for UTI?
Simple UTI: women (3 days) men (7 days), trimethoprim, niturfuratoin, co-amoxiclav
Penicillin allergy, fosfomycin instead of co-amoxiclav
When should you not prescribe nitufuratoin for a UTI?
If there is poor renal function, under 30ml/min
What is cellulitis?
Tenderness, pain erythema, local abscesses
How can you tell if an antibiotic treatment is working in cellulitis?
Draw around the redness and the redness should shrink when taking an Ab
What is the first line treatment for cellulitis?
7-14 days: flucoxacillin
If allergy, clarithromycin +/- metronidazole
Name 3 transmission types from animals and give examples:
Vector- bite from insect, e.g malaria
Vertebrate reservoir e.g rabies
Vector- vertebrate reservoir e.g plague
Name 3 transmission types from humans:
Venereal spread- genitals
Faecal- oral spread
Resp or salivary spread
What is horizontal transmission and give an example:
From an infected person, infection is spread by air, water, food, vector and then to many people
E.g influenza
What is vertical transmission and give an example:
A transfer of disease to next generation by mother’s milk, contact or placenta
E.g HIV
What are pseudomonas?
Opportunistic bacteria
Term for bacteria which morphologically and physiologically resemble members of the genus pseudomonas
Very diverse group of Gram -ve rods with a strictly respiratory mode of metabolism
Name two pseudomonas and what do they do?
Pseudomonas aeruginoasa- quintessential opportunistic pathogen, leading cause of hospital acquired infection, often develops antibiotic resistance
Borrelia Burgdorferi- spread by bites of ticks, can be treated with antibiotics in first stage, causes Lyme disease
What are the three stages of Lyme disease?
- Distinctive expanding rash at bite site
- Dissemination stage with headache and fever
- Persistent infection: arthritis, nervous system affected
What is the bacteria that causes acute community acquired pneumonia?
Streptococcus pneumonia
What is the main cause of pneumonia in children?
Mainly a virus, a bacterial infection can be secondary
What is the main cause of pneumonia in adults?
Bacterial more common than viruses
How can neonates develop interstitial pneumonitis?
Caused by chlamydia acquired from the mother at birth
What is the most common bacteria causing a UTI in a community setting?
80% due to E. coli
What are endospore forming bacteria and give two examples:
Endospores can survive for many years
Gram +ve and usually rod shaped
Two important ones are Bacillus (aerobe) and Clostridium
Bacillus anthracis causes anthrax (disease from cattle to humans)
Bacillus cerus causes food poisoning
What is Vibrio Cholerae and what are its symptoms?
Disease in intestinal tract
Ingested by unclean water, shellfish (uncooked)
Symptoms are diarrhoea, abdominal cramps, N&V, dehydration
Death is generally due to the dehydration caused by the illness
How does Vibrio Cholerae cause diarrhoea?
VC produces A-B cholera toxin subunits
These bind to GM1 (GM1 ganglioside receptor) on the GI tract
A will enter the epithelial cell and bind and activate the GaS G protein which then activates adenylate cyclase
This produces cAMP which activates Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), which causes the efflux of Cl- ions out of the cell in the GI tract, which causes an efflux of Na+ and H2O out into the GI tract so more water causes diarrhoea
What are pyogenic cocci and give an example of one?
Spherical bacteria which cause various suppurative (pus-producing) infections in animals
Gram +ve bacteria are the leading cause
e.g Staphylococcus aureus- leaves on skin and nasal membranes
Leading cause of hospital acquired infections as can be resistant towards penicillin
What is MRSA?
Methicillin-resistant Staphylococcus aureus
Usually confined in hospital, can be treated by vancomycin (drug is toxic, expensive, has to be given by IV)
Name different types of staphylococcal toxins and describe one of them:
Alpha toxin- produced by most staph aureus strains that cause human disease, toxin disrupts the smooth muscle in blood vessels and is toxic to many cells types
Beta, delta, gamma, exfoliate and TS-1 toxins
What are the symptoms of tetanus and why?
First sign is jaw lock (trismus), followed by a stiffness of the neck and back. The stiffness and spamming of the muscles expands throughout the body- 30% die
Tetanus affects on the nerve muscle transmission, it produces tetanospasm, blocks inhibitory transmitter GABA release therefore continuous stimulation by excitatory transmitter so muscle can’t relax and spasm all the time
What bacteria causes tetanus?
Clostridium Tetani
What are enteric bacteria and give an example:
Gram-negative rods with facultative anaerobic metabolism that live in the intestinal tracts of animals
e.g Escherichia coli- indicator of faeces in water
1 L of drinking water must contain less than five E. coli bacteria
What is salmonella and explain how it causes diarrhoea:
Most common cause of food associated diarrhoea
Salmonella is absorbed to epithelial cells in terminal portion of small intestine
Bacteria penetrate cells and migrate to lamina propria layer of ileocecal region
They multiply in lymphoid follicles causing hyperplasia and hypertrophy
Polymorphonuclear leukocytes confine infection to GIT, inflammatory response mediates release of prostaglandins, which stimulates cAMP and active fluid release which causes diarrhoea
What is an acute infection and give an example of it:
Get ill, get better e.g common cold
Give an example of an acute infection with a rare late complication:
Measles
What is a latent infection and give an example of a latent infection:
Get one disease episode gets better, then another episode and get better
VZV (chickenpox)
What is a chronic infection and give an example of this:
Disease episode and virus is active that never goes away
e.g Hep B
What is a chronic infection with a disease and give an example:
Virus active then different onset again
E.g HIV
What are four types of polio virus? Describe them:
Asymptomatic – 90%
Abortive poliomyelitis- non specific febrile illness (5%)
Non paralytic poliomyelitis or aseptic meningitis (1-2%)
-progression of virus into CNS causes backpain plus muscle spasms plus abortive symptoms
Paralytic poliomyelitis (0.1-2%)- appears 3 to 4 days after first symptoms have subsided, virus spreads from blood to anterior horn cells of the spinal cord and motor cortex of brain, cause paralysis
How can poliovirus be transmitted?
Faecal- oral transmission
What is smallpox?
Cox viruses are the largest viruses come up to 230–300nm
Viral genome consists of double-stranded linear DNA
What occurs in the measles virus up to the outcome of recovery?
Inoculation of respiratory tract, local replication in respiratory tract, lymphatic spread, viraemia (virus in blood), wide dissemination of virus, leads to many infections (conjunctivae, RT, UT, CNS, lymphatic system, small BV), virus infected endothelial cells plus immune T cells activated which leads to a rash and therefore life long immunity
What are rare outcomes of measles?
Postinfectious encephalitis (swelling of brain)
No resolution of acute infection
Subacute sclerosing panencephalitis (neurological disorder)
All can be fatal
How many hepatitis viruses are there and name then:
At least 6
A, B, C, D, E, G
What are the similarities in the different types of hepatitis viruses?
The target organ for each other viruses is the liver and symptoms are similar
What are the differences in the different types of viruses for hepatitis?
They greatly different in structure, moderate location, transmission and time cause
Which Hepatitis viruses are the classical ones?
Hep A and B
What are the other nonclassical hepatitis viruses called?
non A non B hep viruses (NANBH)
What is Hep A virus and how is it spread through the body?
Spread by faecal- oral route
Results from consumption of contaminated water, shellfish etc.
Picornavirus- small RNA virus that have a naked capsid structure
Target cells are parenchymal cells in the liver and replicate in Hepatocytes and Kupffer’s cells, they are released into bile and stool 10 days before symptoms appear
Can Hep A virus cause a chronic infection?
No, not associated with Hepatic cancer
What are the clinical symptoms of Hep A?
-Fever, fatigue, nausea, loss of appetite, abdominal pain
-Jaundice in 70-80% adults, 10% children
-Fulminant hep occurs in 1-3/1000 people, 80% mortality
What are the treatment/ preventative measures for Hep A?
Prophylaxis with immune serum globulin 80-90% effective
Killed HAV vaccine approved
What is the % of people with Hep A virus compared to the total of Hep viruses?
40%
Describe the Hep B virus:
Small enveloped DNA virus
Genome is a small, circular partly double stranded DNA
Encodes for reverse transcriptase and replicates through an RNA intermediate
How does the Hep B virus infect the host?
The virus enters the cell, DNA is released into the nucleus, transcribed and protein MRNA is transcribed back to DNA, put with viral proteins, viral core forms and leaves cell and takes with it bits of plasma membrane, destroying it
What are the ways of transmission for Hep B virus?
Blood
By injection into the blood stream
Also found in saliva, semen and milk
So by sexual contact and birth
What is the timescale for Hep B Virus infection?
Virus starts to replicate in liver 3 days after infection
Symptoms may not be observed for 45 days or longer
Infection proceeds for a long time without any liver damage, during this time HBV genome integrates into hepatocyte chromatin and remains latent
What are the symptoms and outcome of acute HBV?
Leads to resolution (90%)
Fulminant Hep (1%)
Carries HBsAg+ (HBV antigen) in blood stream for more than 6 months, asymptomatic (9%)
Jaundice
What are the symptoms of chronic HBV?
Fulminant Hepatitis
1º hepatocellular carcinoma
Cirrhosis
What is the treatment and prophylaxis of acute HBV?
No treatment
HBV immune globulin may be administered within a week of exposure or to newborn babies to prevent disease
What is the treatment for chronic HBV and give examples:
May be treated with drugs targeted to the polymerase or nucleoside analogues
e.g adefovir dipivoxil, famciclovir
Who is the HBV vaccine recommend for?
Children and people in high risk groups
Describe the Hep C Virus:
Contains a positive sense RNA genome
How is HCV transmitted?
Similar way to HBV, blood and sexually
What are the outcomes of HCV?
15% Recovery
15% Cirrhosis
Greater potential for chronic hep
Leads to cirrhosis (scarring of liver) 20%
Can lead hepatocellular carcinoma (1º liver cancer) 4%
What are the three different types of influenza virus and what family do they belong to?
A, B and C- mainly A and B in humans
Orthomyxoviridae family
Describe the influenza virus and how new strain occur:
Virus envelope and has segment a negative sense RNA genome
Segmentation of genome facilitates development of new strain through rotation and reassortment of the gene segments among different humans and animals strains
Genetic instability is responsible for the annual epidemics (mutation) and periodic pandemics (re-assortment)
What is genetic drift and shift?
Drift- where a mutation occurs (more minor)
Shift- where re- assortment occurs (pandemics)
How does the influenza virus infect the host cell?
Virus binds and enters the cell, virus is uncoated and eight segments of RNA is released, enters the nucleus and transcribed into positive RNA which is replicated, RNA leaves the nucleus and segments assemble to make a viral core, budding at cell surface causing it to damage cell
Positive RNA also transcribed into mRNA then transported to cytoplasm and protein synthesised
Describe the structure of the influenza virus:
Lipid membrane envelope which has Hemagglutinin (HA) spikes and Neuraminidase (NA) spikes
Matrix protein
What is the function of Hemagglutinin on influenza:
Each particle has around 500 HA spikes which bind to host cell and fuse the virus envelope to the cells plasma membrane and initiate infection
Mutation in HA are responsible for minor and major changes in antigenicity, shifts only occur with A viruses
What is the function of Neuraminidase on influenza:
Each particle has around 100 NA spikes, they release virus from cell surface by cleaving silica acid on glycoproteins which allows it to leave (enter)
Can also undergo antigenic changes
Name two antiviral drugs that target Neuraminidase:
Zanamivir
Oseltamivir
Effective in first 24-48 hours
How was the labatory diagnosis of influenza obtained?
Respiratory secretions isolated in primary monkey kidney cell cultures overtime
Rapid antigen assay, PCR
What are the main differences between influenza and the common cold?
Fever is common in influenza, uncommon in cold
Muscle and joint pain (myalgia and arthralgia) severe and common in flu, uncommon in cold
Fatigue and weakness is more common and lasts longer than cold
Stuffy nose, sneezing and sore throat is common in cold but only occasionally in flu
What is a rhinovirus?
Associated with at least 30% of common colds
Self limiting, it doesn’t cause serious disease
More than 100 serotypes
Incubation period 8-10 hours
What is a coronavirus?
Similar to that caused by rhinovirus but has longer incubation period (three days)
Spread by aerosols
Antibodies formed by adult hood, reinfections common
What are complications of coronaviruses?
SARS (severe acute respiratory syndrome)
Atypical pneumonia out with a high fever, chills, rigours
Up to 20% patients have diarrhoea
Mortality at least 10%
What is zoonosis?
Transferred through animals e.g. bites, scratches, vectors, contact, faeces
Describe the structure of a Marburg and Ebola virus:
Filoviruses
Filamentous, enveloped, -ve stranded RNA virus
What are the two species of ebola viruses and their fatality?
Zaire (ZEBOV) - 80% fatal
Sudan (SEBOV) - 54% fatal
How is Ebola virus transmitted?
Direct contact with infected body fluids E.G blood, vomit, semen
Can survive for several days outside the body so also on clothing and equipment
What are the initial symptoms of a Ebola?
High fever, severe headache, muscle joint pain, severe weakness, sore throat, nauseous, dizziness
Are secondary symptoms of Ebola?
Involve bleeding both internally and externally from any opening in the body
e.g dark or bloody stools and diarrhoea, vomiting blood, red eyes from swollen BV, red spots on skin from subcut bleeding, from nose, mouth rectum and genitals
Low BP, fast but weak pulse, organ damage, proteinuria (protein in blood)
Hyperglaemic shock
How fast does death occur with Ebola?
From onset of symptoms 7-14 days
What are retroviruses?
All RNA viruses
Envelope positive stranded RNA genome
Contain RNA dependent DNA polymerase (reverse transcriptase)
They then are integrated into host chromosome and won’t leave the body
What is the genome structure for retroviruses and describe them:
LTR- GAG- POL- ENV- LTR
3 major genes:
GAG- Group specific antigen, capsid, matrix and nucleic acid binding proteins
POL- polymerase, protease, integrase
ENV- envelope, glycoproteins
LTR- long-terminal repeats, Controls how viruses replicated inside the cell, contain promoter sequences, enhances and oncogenes
Describe how retroviruses infect a cell:
GP120 glycoprotein binds to CD4 receptor and CCR5, CXCR4 binds to cytokines
Binding of GP120 releases GP41 which allows virus to enter cell
Virus enters cell by penetration and uptake into the vacuole, core of virus is released and RNA genome reversed transcribed into DNA
Double stranded DNA ligated into double stranded plasmid which can integrate into host DNA, so now a provirus
Transcription leads to single-stranded RNA, translation into viral proteins and assembly plus nucleocapsid and budding to infect another cell
What does a retrovirus do to the body to affect immune cells?
Virus sits inside macrophages and transported into brain, dendritic cells and the macrophage don’t work anymore
Overtime affect CD4+ T cells and kills them leading to immunodeficiency
What is the concentration of CD4+ T cells in the blood meaning AIDS?
Less than 200 CD4+ T cells per cubic ml of blood
What are treatments for retroviruses?
Reverse transcriptase inhibitors
What can occur when a person gets AIDS and give examples:
Opportunistic infections due to suppression of immune system:
Viruses, bacteria, fungi, tumours
What are prions:
Short for proteinaceous infectious particles
Infectious agent only made of protein
Abnormally structured forms of a host cell protein
Prions quite resistant to denaturation by protease, heat and radiation but potency and effectiveness can be reduced
What is the main difference that prions can make?
Change a- helices into B sheets
Name the types of herpes viruses and their subtypes:
Alphaherpesvirus- HSV1, HSV2
Gammaherpesvirus- HSV4, HSV8
Betaherpesvirus- HSV5, HSV6, HSV7
What is HSV1 target cell, where is it latent in and how is it spread?
Target cell: mucoepithelial cells
Latent in neuron
Spread by close contact
What is HSV2 target cell, where is it latent in and how is it spread?
Target cell: mucoepithelial cells
Latent in neuron
Spread by close contact (sexually)
What is HSV3 target cell, where is it latent in and how is it spread? Give an example of a HSV3 virus:
Target cell: mucoepithelial cells
Latent in neuron
Spread by close contact and respiratory
Varicella- zoster virus (VZV)- chicken pox
What is HSV4 target cell, where is it latent in and how is it spread? Give an example of a HSV4 virus:
Target cell: B cells and epithelial cells
Latent in B cells
Spread by saliva (kissing)
Epstein-Barr virus (EBV)- glandular fever
Give and example of a HSV8 virus:
Kaposi sarcoma related virus- in people with HIV
What can cause a reactivation of a virus?
Sun, immune system already fighting off something else
Describe the stages in infection of the varicella- zoster virus:
1º infections begin in the mucosa of the upper respiratory tract, virus replication in the lung is a major cause of contagion, virus replication in regional lymph nodes
1º viremia- reproduction
Virus enters the bloodstream and lymphatic system to cells of recticuloendothelial system
A second viremia occurs 11-13 days after the body and skin where is causes a dermal vesiculopusticular rash that develops over time in successive crops, fever occurs too
How soon before is a person infected with VZV contagious?
A couple of days before symptoms
What is the process for the infection of the Epstein-Barr virus?
The virus in saliva initiates infection of oral epithelia and spreads to B cells in lymphatic tissue
There is a productive infection of epithelial and B cells and virus promotes growth of B cells
T cells kill and limit B cell outgrowth, T cells are required for controlling the infection
EBV establishes latency in memory B cells and is re-activated when the B cells are activated
What are the symptoms of EpBV and why?
T cell repose leads to symptoms of infectious mononucleosis
Causative association with lymphoma in immunosuppressed people
Fever, pharyngitis, malaise and fatigue, lymphadenopathy
What is the incubation period for EBV?
Up to 2 months
How is HIV transmitted?
During vaginal and anal intercourse
Anal is easier as less of a barrier as single layer of cells
How does HIV infect a host?
HIV infects and sticks to Langerhans and dendritic cells in the epithelium
The cells can travel to lymph cells as it uses dendritic cells to present itself to CD4 T cells and starts to infect them
On infection into blood, virus likely to infect dendritic and other monocyte E.g macrophage cells
Macrophages are persistently infected and are probably the reservoir for the virus
How does the affinity go from macrophages to CD4 T cells in HIV?
Mutation in GP120 gene
What does HIV do once in the bloodstream?
Macrophages can act as either:
- reservoir and transporter for HIV
- can be dysfunctional to release cytokines
It can cause clinical latency until CD4 T cells in lymph nodes die due to increase in virus load in blood
This causes the host to lose B cell control and T-cell function which causes immunodeficiency and can get into the brain and cause dementia
What are the clinical symptoms of HIV?
Initial symptoms are those of flu
Then symptoms subside after 2 to 3 weeks and are followed by a period of asymptomatic infection
Deterioration to immune response indicated by increase in susceptibility to opportunistic pathogens
What is HIV wasting syndrome and when does the occur?
Weight loss, diarrhoea and indicator diseases
Occurs in AIDS
In which part of the body do rhinoviruses affect?
UPPER respiratory tract
Otitis media
Sinusitis
Describe how a rhinovirus infects the host:
Virus enters through nose, mouth, eyes
Protein binds tightly to I CAM-1 (surface receptors) on cell or cilia and doct onto it and get inside the cell
Bound tightly so not washed away by secretions
Once inside, more viruses produced so cell bursts causing cell damage and infection to spread
Histamine is released therefore cytokines are (innate)
What are the effects of rhinoviruses and give the symptoms:
Swelling induced by capillary permeability – blocked nose
BV dilation- blocked nose/ sneeze and cough
Fluid secretion- sneeze and cough
Nerve fibre stim- sneeze and cough
What are the signs and symptoms of streptococcal respiratory disease?
Strepthroat- inflammation of pharynx
Back of pharynx appears red with swollen lymph nodes and pus containing abscesses covering tonsils
Fever, Malisse and headache common
Spread via respiratory droplets
Penicillin effective against
What virus causes rabies and describe it:
Rhabdovirus
Single-stranded RNA virus
Where is the rabies virus found and how is it transmitted?
Secreted in saliva of certain animals e.g wolves, foxes
Transmission follows a bite of salivary contamination or other types of skin abrasion
Foxes more infectious as higher conc of virus in saliva
Which countries are free of rabies?
Australia, UK, Japan, Hawaii
What is the incubation period of rabies?
4- 13 weeks
Can be up to 6 months
How does rabies virus infect the host?
Virus is inoculated, replicated in the muscle, enters peripheral nervous system, passive ascent via sensory fibres, replication, infection in spinal cord, brain stem and brain, descending from brain via nervous system to eye, salivary glands, skin and other organs
What is a treatment and prophylaxis for rabies virus?
No treatment, fatal unless treated with postexposure prophylaxis
Prophylaxis should be initiated for anyone exposed by bite or by contamination to an animal suspected to be infected
First protective measure is to wash with soap and water
Then immunisation with the vaccine in combination with one dose of human rabies antibody is recommended (passive)
A series of immunisation is then administered over a month (active)
What is protection against reinfection of influenza associated with?
Development of antibodies to HA, can also be NA
How does influenza hinder immune response?
Depresses macrophage and T cell function
How are strains of influenza named and classified?
- Type of influenza (A,B,C)
- Place of original isolation
- Date of original isolation
- Antigen (HA and NA)
How would you treat, prevent and control influenza?
Antihistamines to relieve symptoms
Anti-viral drug amantadine and rimantadine inhibit uncoating step of influenza A but don’t affect B or C
Immunisation
What bacteria causes syphilis?
Treponema pallidium
Describe the three stages in syphilis:
- 10 to 60 days after infection, chancre, heals after a couple of weeks
- Lasts 3 to 6 months, fever, headache
- After 2 to 40 years, leads to death by variable causes
How can syphilis be treated?
Chloramphenicol and penicillin in the first stages
3rd stage can’t really get rid of the damage
Can syphilis be transmitted to a newborn?
Yes
How is syphilis transmitted?
Humans are the only known host
Virtually always by direct contact with infectious lesions- sexual contact
Only 30% shows symptoms but can still pass it on
What bacteria causes chlamydia and where does it replicate?
Chlamydia trachomatis
Unable to produce ATP in amount required to sustain metabolism outside host cell so needs to infect cells
What are the clinical symptoms of chlamydia in men and which complications can this lead to if untreated?
Urethritis
Epididymitis- tube in testicle becomes swollen
Proctitis- inflammation of anus
Conjunctivitis
Can lead to- systemic spread, Reiters syndrome
Are the clinical symptoms of chlamydia in women and which complications can this lead to if untreated?
Urethritis
Conjunctivitis
Cervicitis
Can lead to- Ectopic pregnancy, infertility, dermatitis, systemic spread, arthritis
What are the clinical symptoms of chlamydia in neonates and which complications can this lead to if untreated?
Conjunctivitis
Can lead to interstitial pneumonia
How does neonatal meningitis occur?
Group B streptococci are normal inhabitants of the female genital tract which may be acquired by the neonate
What are risk factors causing an early onset of meningitis in neonates?
Pre-term delivery, prolonged labour, heavy colonised mother lacking specific Ab
What are outcomes of early onset of meningitis in neonates?
60% Fatal
What is the prevention for early onset of meningitis in neonates?
‘Blind’ treatment of sick baby who has those risk factors
What are risk factors causing a late onset of meningitis in neonates, in the nursery?
Lack of maternal Abs
Poor hygiene in nursery
What are outcomes of late onset of meningitis in neonates, in the nursery?
20% fatal
What is the prevention for late onset of meningitis in neonates, in the nursery?
Good hygiene practice in the nursery
Don’t allow mothers to handle other babies
What is a virus called if it doesn’t have an envelope?
Naked virus
What is the innermost portion of a virus structure made up of?
RNA OR DNA, not both
What are oncogenic viruses?
Have LTRs which can disrupt gene and cause cancer
Which type of hepatitis causes jaundice?
Acute Hep B
Hep A
What is pathogenicity?
The ability of a microorganism to induce a disease
What are the 4 roles of the immune system?
Defence against infections
Recogizes and responds to tissue grafts and newly introduced systems
Defence against tumours
Abs are highly specific reagents, identify foreign tissue
What is the resistance in a non-nucleoside reverse transcriptase inhibitor and how is this over come?
Pan-class resistance mutation
Replacement of Lys-103 with asparagine
Combine NNRT with a nucleoside reverse transcriptase inhibitor as binding sites are distinct
