STDs/HVSs Flashcards
What sample types do you usually see for STD screens
Charcoal transwabs are most common -> necessary for N. Gonorrhea
Gene Xpert swabs or first void urine are required for Chlamydia trachromatis investigation
IUDs might be sent down as part of a PID investigation
How does the charcoal transwabs work
They contain inorganic buffer which ensures the maintenance of bacteria without their overgrowth
This allows for the excellent recovery of both aerobic and anaerobic bacteria
Charcoal is necessary for the recovery of aerobes, anaerobes and fastidious organisms e.g. N. Gonorrhea
What plates do STD screens get?
NYC agar for gonorrhea
PDA for query thrush
Blood agar -> Gonorrhae will take days to grow
PCR for Chlamydia
How is MNYC agar incubated
5-10% CO2
What plates do HVSs get
Blood agar
PDA
NEO/FAA if necessary
-> if post operative or post partum a NEO is put up + Met disc
-> a FAA is put up if query anaerobes
What plates are put up for PID investigation
Blood
NYC (if query STD caused PID)
FAA
MacConkey
Extended blood agar
PDA if query thrush
What plates are put up if given an IUD?
Same plates as PID screen
Blood, FAA, MacConkey, Extended blood, NYC (? STD)
What samples get a gram stain?
All of our swabs get a gram stain other than rectal or pharyngeal swabs -> these tend to be too dirty
=> HVSs, STDs, PIDs all get grams
Talk about the use of extended blood agar
○ PID investigation, IUD infection
Extended incubation time to enhance growth of more fastidious organisms
Talk about Blood agar
○ Agar used for the cultivation of most pathogenic organism
○ Contains 5% defibrinated horse blood which is added to autoclaved basal/nutrient media
○ The blood enriches the media to support the growth of fastidious bacteria while inhibiting certain bacteria such as Haemophilus (require blood agar)
Useful for organisms which demonstrate haemolysis such as Strep species
How do you make a gram from the swabs?
Smush the tip of the swab onto the glass slides directly
Talk about MacConkey agar
○ Used in PID/IUD investigation mostly for GNBs such as the Enterobacteriaceae and Pseudomonas
○ A selective and differential medium, selective for gram negative bacteria and enteric/GIT bacteria, differential based on lactose fermentation
○ Made selective by bile salts and crystal violet which inhibit gram positives
○ Neutral red is the pH indicator
▪ Lactose fermenters cause a change in pH which turns the medium pink such as E. Coli
▪ Non lactose fermenter GNBs such as Pseudomonas aeruginosa grow a yellow colour
Sodium chloride maintains the osmotic balance of the medium
Talk about Potato Dextrose agar
○ Potato dextrose agar is a versatile growing medium for bacteria and fungi (yeasts and molds).
Often uses for candida species e.g. C. albicans in thrush
Talk about FAA
○ Anaerobes such as actinomyces during PID or IUD infection
○ Fastidious anaerobe agar capable of supporting most clinically significant fastidious anaerobes
○ Contains a wide range of additional substrates such as Pyruvate, Vitamin K, Cysteine, arginine etc
Grows organisms such as Bacteroides and Fusobacterium
Talk about NEO agar
○ + Met disc for post operative or post partum HVSs
○ Anerobic neo blood agar is used to grow anaerobic GNBS while preventing the growth of other organisms such as E. Coli, and aerobic bacteria such as staph etc
○ Neomycin can also be used aerobically for the isolation of Group A strep such as S. pyogenes and Group B strep such as S. agalactiae (S, aureus is susceptible to neomycin)
Basically neomycin blood agar works by suppressing the most common commensals such as S. aureus and E. Coli, by adding a metronidazol disc and incubating anaerobically you are ensuring only the growth of anaerobic bacteria
Talk about MNYC agar
○ A selective media for the isolation of N. gonorrhoeae and N. meningitidis
○ GC agar base supplemented with lysed or chocolatised horse blood with the addition of Oxoid Vitox
○ Made selective through the use of an antibiotic cocktail containing vancomycin, colistin, trimethroprim and yeast autolysate
▪ Vancomycin inhibits gram-positive bacteria
▪ Colistin inhibits gram negative bacteria including Pseudomonas species
▪ Trimethoprim inhibit Proteus
▪ Trimethoprin + colistin act synergistically to inhibit GNBs
▪ Yeast autolysate fulfils the CO2 requirement needed to enhance Neisseria growth which enhances both the size and number of colonies
○ The horse plasma, haemoglobin and proteose peptone provide nutrients for the growth of N. gonorrhoeae and N. meningitidis
○ Phosphate buffers the medium
○ Starch neutralises Neisseria toxin
What are the common organisms found in HVSs?
Beta Haemolytic Strep (S. pyogenes and S. agalactiae)
Fungi
S. aureus
Listeria
GNBs
Proteus
Talk about Beta Haemolytic Streps in HVS
▪ Such as S. pyogenes (Group A Strep) and S. agalactiae (Group B Strep)
○ S.pyogenes
® Group A Streps such as S. pyogenes can cause toxic shock syndrome
○ S.agalactiae
® Can only release a positive S. agalactiae after the sens has been done, if penicillin susceptible you can release early but if resistant you will have to wait for the full sens to be done
® Group B BHS like S. agalactiae cause infections during the 3rd trimester of pregnancy
* Usually screen for BHS during 3rd trimester so as to not pass infection on to baby through birth cannal -> treated with antibiotics
▪ Always check blood agar against the light for any signs of Beta haemolysis
All BHSs will go for sens
Why is listeria so important in HVSs
Can cause stillbirths if patient is pregnant
Talk about GNBs in HVSs
Often seen where there is disruption in the microbiome I.e. decrease in lactobacillus
Talk about proteus in HVSs
▪ Will swarm the plate
▪ There will be a noticeable bad smell
Whole plate will be coated in a cloudy film or cloudy rings seen around the colonies
How do you report the scanty growth of HVS plates and what should you do
If very scanty then incubate for an additional day
▪ CA1: scanty growth in primary inoculum ▪ CA2: scanty growth in secondary inoculum CA3: scanty growth in tertiary inoculum
Talk about fungi in HVSs
Candida species most common cause of thrush
○ Very large white colonies across the plate ○ Always keep any PDA positive plates in case the patient has recurrent thrush -> might want to carry out sens on it -> why recurrent -> resistant etc Can always do a wet prep if not 100% confidenf a colony is yeast etc
What organism would expect to see on the gram stain of a HVS?
Lactobacillus -> GPBs
Why are we concerned with lactobacillus in HVS gram stains?
○ Any disregulation in the microbiome results in a decreased in lactobacilllus and an increase in GNBs such as Gardnerella or Mobiluncus
○ The lactobacillus bacteria keep the cervix at the correct pH which keeps these GNBs at bay
What organisms are responsible for BV?
GNBs such as Gardnerella or Mobiluncus
How is HVS disregulation graded
▪ Grade 1/H1: normal -> lactobacilllus bacteria predominate
▪ Grade 2/H2: intermediate -> mixed flora of some lactobacillus + some GNBs
▪Grade 3/H3: abnormal -> predmoninantly GNBs with few/absent lactobacillus
What does Grade 3/H3 HVS disregulation mean for the patient
- Indicative of bacterial vaginosis
* Presence of clue cells (epithelial cells covered in GNBs) -> very indicative of BV
* Fishy smelling discharge etc
* Treated with antibiotics + probiotics for the cervix
Gardonella presence indicative of BV
For what cohort of patients do we carry out HVS grading and why?
○ Grading is only carried out for women younger than 60 years old
○ For those 60+ everything seen on the HVS gram stain must be described
Women 60+ tend to have a very dirty gram stain or have very little hence there is no reason to grade the stain
Other than lactobacillus disregulation what do we note on the HVS gram stain?
The presence of white blood cells
What might cause increased white blood cells on a HVS?
Infection
Inflammation
Just the point in the woman’s menstrual cycle
Hormonal changes
When during a woman’s cycle are you more likely to see WBCs on their HVS gram?
WBCs are increased from the menstrual phase to the follicular phase
WBCs reach highest levels around mid-cycle at approx day 14
WBCs decrease again from day 22 i.e. mid-luteal phase
What hormonal changes make it less likely to see WBCs on a HVS gram stain
WBCs decrease after menopause due to a decrease in estrogen
What should you do with a query N. gonorrhea on MNYC agar?
Carry out an oxidase test -> if + then
= Gonorrhea
What are the criteria for a positive N. Gonorrheae
Four + different positives are necessary for diagnosis with Gonorrheae
Oxidase positive
MALDI positive
API positive -> displayed pattern must perfectly match that of Gonorrhea
Gram stain positive
AST positive
Talk about the trends in Gonorrhea
Gonorrhea increasing
Resistant gonorrhea also increasing
Why is it important to test all swabs in a Gonorrhae screen
Some swabs may be negetive from certain sites while other sites are positive
Talk about reading the MNYC agar plates
If N. Gonorrhea positive it will usually be the predominant organism
Plates can be very dirty if a rectal swab has been used
If plates are left in air for too long or in the fridge the Gonorrhea will dry out
How do we process Chlamydia
Chlamydia diagnosed through PCR
Chlamydia won’t grow on NYC agar
If PCR positive then we can try and culture for sens
What might you see on a blood agar for an STD screen?
Beta Haemolytic Strep -> send for sens
GNBs -> coliforms
S. aureus -> send for sens
If plate is 3+ days old you might wee Gonorrhea on blood agar
How would N. gonorrhea look on gram stain
Intracellular diplococci within the white blood cells
What is the API Kit
Biochemical test used to confirm ID of N. Gonorrhea
How does the API Kit work
▪ Biomerieux API KIT used for the identification of N. Gonorrhea and Haemophilus (wounds)
▪ Cards contain substrates for a series of biochemical tests
▪ Different organisms display different characteristic patterns of colour changes for each biochemical test
By comparing the produced colour pattern to that of known organisms we can identify an organism
How do you use the API Kit
▪ You will need to put up 2 chocolate agars with your oxidase positive query vN. Gonorrhea
▪ Using these two plates you will make up a 4.0 MacFarland (heavy) inoculum
* Collect colonies onto a damp swab by swiping across the entirety of both plates
▪ Add the inoculum to each reaction well on the card
* Any test which is underlined is only fillled half way and mineral oil added ontop to create anaerobic conditions
▪ The card is incubated for 2 hours 10 minutes
▪ The colour change is read after incubation and assigned a code
▪ This code must perfectly match to the code of N. Gonorrhea to support a diagnosis
* Must match N. Gonorrhea and not just Neisseria species
What are the most common bacteria in PID
invasive group A streptococcal disease (GAS)1 and toxic shock syndrome (TSS), due to Staphylococcus and Streptococcus spp,2–4 as well as to Actinomyces infections
Gardenella cause BV when there is an imbalance of Lactobacillus
STDs also cause PID
what are the most common anaerobes in HVSs
Bacteroides species (GNB)
Gardnerella vaginalis
What is the gram results of Gardnerella vaginalis
Gram-variable coccobacilli
Most common GNB in HVSs?
Bacteroides species
Why do we put up an extended blood agar plate for HVSs/PID etc?
Actinomyces
