Microbiology - Clinical Testing

Clinical Testing Sampling: taking patient history and clinical details name age NHS number gender foreign travel antibiotic therapy symptoms Take appropriate sample: blood, urine, faeces, sputum, aspirates, swabs, cerebrospinal fluid. Relevant details: when sample was taken how much was taken correct storage container Transport conditions: time taken to get specimen to the lab temperature storage type charcoal swabs or viral transport maintains specimen in similar state until it is processed Culturing considerations: indicators enrichment time temperature gas moisture light Analysis: culture microscopy detection of bi-products molecular techniques Selective enrichment increases the number of pathogen. Eg- selenite broth promotes growth of Salmonella spp. Alkaline peptone water promotes growth of Vibrio cholerae. Selective incubation: eg- Campylobacter spp. grows at 42degrees in reduced oxygen. C. difficile is an anaerobe. Agar Plates: Columbia blood agar Chocolate Fastidious (fussy) anaerobe agar CLED - for urine pathogens SDA for fungi and yeast Hoyle’s Tellurite for Corynebacterium diphtheriae MRSA selective Charcoal blood Chromogenic agar aka UTI clarity agar stains pathogens differently eg. E. coli will be pink etc. Blood agar causes alpha-hemolysis (partial blood lysis), causing it to turn green. Beta-hemolysis causes complete blood lysis, so there is a yellow zone of clearance. In gamma-hemolysis, there is no blood cell lysis. Note the smell of the plate. MIC = minimum inhibitory concentration. This is the minimum concentration of an antibiotic required to inhibit the growth of bacteria. MBC = minimum bactericidal concentration. This is the minimum concentration of antibiotic required to kill the bacteria. Time- and concentration-dependant killing is where there is a threshold to the amount of time or concentration of exposure that ensures killing of the bacteria. Antibiotic susceptibility: bacteriostatic is where the growth of bacteria stops and colony numbers are the same, bactericidal is when the bacteria is being killed and numbers are decreasing. E-test strips have gradually decreasing concentrations of antibiotic. Types of microscopy light detects parasites, fungi and bacteria. Gram staining: crystal violet and iodine stain when specimen is treated with acetone/alcohol. Gram-positive bacteria will be purple. Safranin is the counterstain, and Gram-negative bacteria will be red. Acid-fast staining: for bacteria with a waxy outer layer which resists most stains. Carbol fuchsin stain after treatment with acid alcohol (HCL in ethanol). Counterstain with methylene blue which binds to the DNA. Flagellar staining, India ink stains slide background, and endospore staining which colours spores red and vegetative cells blue. fluorescence Some specimens are auto-fluorescent, or you’d need dyes. Will be seen under UV. Detects fungi and bacteria. phase contrast detects unstained living cells. E lectron Biochemical Tests Catalase test - presence of catalase enzyme. Add hydrogen peroxide in the solution. If bubbles (oxygen) are produced, there is catalase present. Urease test - presence of urease enzyme which breaks down urea into ammonia, co2 and water. Phenol red indicator goes from orange/yellow to pink when it is alkaline (ammonia is present). Bacteria API multi-test strips have for 20 separate tests on one strip. There may be a colour change, and pH change or an end-product is produced. Identify species using an online database. Test strips - detects pathogens and toxins. Sample pad for sample which migrates towards absorbent pad. With antigen present, test strip will show with control strip. Otherwise only control strip shows. ELISA testing - antigen is fixed to test tube. Add sample, add enzyme substrate and a colour product is formed to show a positive or negative result. Serological Tests Haemagglutination inhibition test (HIA) tests for antibodies in blood. Positive result is clumping of the RBCs by the virus (eg- measles, rubeola viruses). With the presence of antibodies, the RBCs will instead bind with the antibodies and cause haemagglutination inhibition. Complement fixation test tests for antibodies in blood. Antibodies will bind to viruses and complements, so when RBCs are added, they cannot bind to the antibodies. In a serum sample without the virus, the antibodies are available to bind to the RBCs so they undergo cell lysis. Colour change is a positive result. Other tests: agglutination, precipitation, flocculation etc. The Legionellaceae family has 52 species which will all give positive tests but we cannot identify which species it is. We can use MALDI-TOF MS and NAATs tests to identify the species. NAATs = Nucleic Acid Amplification Tests Eg- PCR - uses specific primers to bind to DNA, then undergoing cycles of denaturing and annealing, and then polymerising and producing more DNA. NAATs - a very sensitive method, which can detect malaria infections of below 1 parasite/microL. Can detect which species of malaria, and how much is in the sample. Types of PCR: nested (n), quantitative (q), real-time/reverse transcriptase (RT), LAMP (loop mediated isothermal amplification), (QT-NASBA) (quantitative nucleic acid sequence-based amplification). These all require some sort of DNA extraction. Detection is via running sample on agarose gel, using fluorescent labels, or checking the turbidity using a spectrophotometer. MALDI-TOF MS (matrix assisted laser desorption/ionisation - time of flight mass spectroscopy). Burn the sample on the target plate, the molecules will move through the matrix at different speeds, the ion detector will identify the charge of the molecule and the time it takes for the molecule to move from the target to detector plate will allow identification of the molecules present on the sample. Even subspecies of different bacteria will give different profiles to allow identification. Lab Testing for parasites: Stool exam: taken on three separate days to catch the parasites at different stages (eg in their egg or mature parasite stages) To visualise larger parasites, carry out colonscopies , x-rays, MRI and CAT scans. Check blood serology - carry out antigen detection, for malaria or babesiosis etc.