Ocular Diagnostics Flashcards

Question

How should samples for fungal culture be submitted?

Answer 1

Collection of sufficient material = limiting factor Material should be collected from periphery of lesions where more likely to recover viable fungi. Submitted in standard Amies transport medium or whole tissue samples in plain pot.

Answer 2

Immunofluorescent assay (IFA) and enzyme-linked immunosorbent assays (ELISA) Used to detect pathogen-specific antibodies or detect antigen

Answer 3

IFA - antibody detection Test serum incubated with plates or slides containing immobilised antigen. If antibodies to antigen present within the serum they will bind and then are detected by incubation with a secondary antibody that targets the antigen-bound antibody. This secondary antibody is labelled with a fluorescent marker that can then be visualised - results are given as an antibody titre and represent the greatest dilution of the sample that still shows fluorescence.

Answer 4

Exponential amplification of very small amounts of DNA to detectable levels. Very sensitive for infectious and inherited disease In case of infectious disease sensitivity can also be downfall as may amplify small amounts of non clinically significant DNA - risk positive results of uncertain significance (can be inferred by CT value provided by lab) Herpesvirus = prime example where significance on PCR can be difficult to ascertain.

Answer 5

Conventional - heated and cooled cycles, temperature dependent DNA polymerase with code specific primers is used to amplify and therefore detect tiny quantities of DNA. Con = DNA does not need to be intact/organism does not need to be vital, unexpected DNA may be amplified if primer sticks to another part of the target DNA than expected) Nested (2 sets of primers to reduce amplification of inappropriate DNA segment) Real time PCR (qRT-PCR) - after every PCR cycle the amount of product formed is measured and displayed in fluorescent units, greater the target DNA the more quickly product and therefore fluorescence is formed. Once certain threshold reached of fluorescence in a sample it is deemed to be positive and the number of cycles taken to achieve this allows quantitative measurement of DNA in the original sample. Pro = allows estimate of DNA present and thus interpretation of clinical significance Reverse transcriptase PCR (RT-PCR) - reverse transcriptase added to produce complimentary DNA from an RNA sequence. Used for the detection of RNA viruses such as FIV

Answer 6

Impression smear, scrapes, aspirates Scrapes and smears = more superficial lesions than aspiration.

Answer 7

Can either apply asymmetric strips of millipore filter onto the ocular surface or with aid of cytology brush/swab Cytological samples using swab improved with being moistened with local anaesthetic, transport medium or saline. Roll onto conjunctival/corneal surface then roll opposite direction on slide to maintain cell preservation.

Answer 8

Used when larger sample required for submission Local anaesthesia required Handle end of no 11 or 15 Bard Parker scalpel blade Scalpel handle held at an acute angle to tissue surface and gently dragged backwards to collect sample within acute angle Gently place onto submission slide and if necessary smeared with another slide using digital pressure and drawing slides apart.

Answer 9

Thin smear of any material collected Appropriately labelled submission + relevant history Material rapidly air dried to prevent deterioration of cells Slides stored appropriately (room temp, out of light, not sent with formalin pots)

Answer 10

Romanowsky, Wright-Giemsa, Diff Quik (in house)

Answer 11

Eputhelial/round cell = excellent aspiration (adenomas, SCC, MCT) Mesenchymal (connective) tissues = poor aspiration Cystic masses - very acellular unless marked inflammation

Answer 12

Technique in which AH is aspirated from the anterior chamber allowing for cytological or serological evaluation of the fluid or to aspirate cells from masses in the anterior segment or iridial surface. Conducted under GA or deep sedation with topical anaesthesia as incorrect placement of the needle of movement could cause significant intraocular damage. Fluid for culture = plain (paediatric tube if possible) Cytological evaluation = direct smear of fluid or submission in EDTA (paediatric tube if possible), EDTA helps preserve cellular morphology but in high concentration if use just normal EDTA tube it will cause cellular lysis. Most common complications - acute hypotony, haemorrhage Important aqueous is not removed too fast or subsequent severe hypotony can lead to choroidal effusion and retinal detachment. Haemorrhage most likely with aspiration of masses or if iris stroma punctured with needle Normal findings for AH analysis = no cells, total protein 21-37mg/dl, no microbes

Answer 13

Under GA Generally globes where prognosis for vision poor for diagnosis of potential infectious disease e.g cryptococssis, blastomycosis

Answer 14

True positive = positive result in which the animal has the disease

Answer 15

True negative = negative result in which the animal does not have the disease

Answer 16

True positives/number of animals with disease (true positives + false negatives) x 100 Highly sensitive test = good screening test Will almost always detect animals with the disease but there may be a few false positives

Answer 17

True negatives/number of animals without disease (true negatives + false positives) x 100 Highly specific test = unlikely to give a false positive result, if positive = diagnostic A highly specific test may lose some sensitivity so there may be false negatives. If positive = has disease, if negative can't fully rule out

Answer 18

Diagnostic accuracy = frequency with which correctly classifies an animal as having or not having the disease Number correctly classified/number of animals in study x 100

Answer 19

Predictive value of a positive test = True positives/all positives x 100 Predictive value of a negative test = true negatives/all negatives x 100 PV affected by disease prevalence, where cut offs are drawn for positive or negative result, what is the gold standard and what if the new test is actually better than the gold standard?

Answer 20

ROC curves plot diagnostic specificity (false negative rate) on the x axis against sensitivity (true positive rate). ROC curve with 45 degree angle = test no better than tossing a coin Best type of ROC curve = head directly to the top of graph then horizontal - suggests ability of the test to differentiate diseased from non diseased animals is good.

Answer 21

Detection of antibodies = exposure and seroconversion - interpret in light of vaccination status and prevalence within the population. Toxoplasma titre in dogs/cats (care with interpretation as high prevalence in general population - significance of titre levels?) Feline coronavirus - again high normally in population so not synonymous with active infection. Negative titre does not fully exclude FIP - 10% of cats with FIP may have negative titre. Pathology arises due to 2 main reasons - some forms of FCoV mutate and able to enter macrophages rather than being confined to enteroytes. 2 = cats own immune response to this virus is exaggerated- marked production of antibodies, humoural response and vasculitis (wet FIP) or inappropriate cell mediated response leading to granuloma formation (dry FIP)