Sterile Sites Lecture PDF

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Summary

Infections of the CNS The blood brain barrier is a host defense that minimizes the entry of infectious agents and large molecules. This also means that large antibiotics like macrolides cannot get through it. Typically, CSF is clean, clear, and sterile. CNS lab findings in CSF infections include low...

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MLSC-3131U, Clinical Microbiology II Infections of the CNS -

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The blood brain barrier is a host defense that minimizes the entry of infectious agents and large molecules. This also means that large antibiotics like macrolides cannot get through it. Typically, CSF is clean, clear, and sterile. CNS lab findings in CSF infections include low glucose, high proteins, and increased WBC count. Very young and old people are at risk of these infections. Meningitis, inflammation of the subarachnoid space. o Newborns, caused by:  Group B Streptococcus  Escherichia coli  Listeria monocytogenes o Infants and Children, caused by:  Streptococcus pneumoniae  Neisseria meningitidis  Haemophilus influenzae Type B o Adolescents and Young Adults, caused by:  Streptococcus pneumoniae  Neisseria meningitidis o Older Adults, caused by:  Streptococcus pneumoniae  Neisseria meningitidis  Listeria monocytogenes o Immunocompromised, caused by:  Cryptococcus neoformans  Listeria monocytogenes Encephalitis, inflammation of the brain itself, usually viral. Meningoencephalitis, combination of meningitis and encephalitis. Brain Abscesses, localized infection of the brain.

Meningitis Causing Bacteria Haemophilus Influenzae -

Tested with VX disks (growth around only the VX disk), ALA-Porphyrin (Negative for ALA is positive for H. influenza), and satellitism is shown around S. aureus. Send to PHL for susceptibility.

Streptococcus pneumoniae -

Most common in adults and 2nd most common in children. Has a mucoid capsule that can be identified as clearing around the bacterial capsule. Bile solubility is also done on this organism for identification.

MLSC-3131U, Clinical Microbiology II -

AST, Cefotaxime ETest, Erythromycin, Penicillin ETest MIC and Vancomycin susceptibilities are done. Do not report macrolides for drug panel because it cannot pass the BBB.

Neisseria meningitidis -

Has 12 serogroups with B being the most prevalent. There is a vaccine available for anyone above the age of 2. Sugar testing is done (Glucose +, Maltose +, Sucrose -, and Lactose -) Oxidase positive fat GNC kidney beans in pairs. AST testing is sent to the PHL for susceptibility.

Aerobic GNBs -

Include E. coli, Serratia, and Salmonella. Neonates and older adults are at increased risk. ASTs, vitek and API20e is done.

Listeria Monocytogenes -

More common in neonates, older adults and immunocompromised. Small GPB, slightly beta hemolytic, grey, translucent. Catalase positive and shows tumbling motility. ASTs are not performed with a comment added in its place (ASTs are not reliable for this organism).

Streptococcus agalactiae -

Grey, translucent, beta-hemolytic GPC. Catalase negative, PathoDX Group B and AST is done.

AST Considerations -

Macrolides, clindamycin, 1st and 2nd gen cephalosporins, tetracyclines, fluoroquinones and aminoglycosides should not be reported for use against CNS infection. Fluoroquinones and Tetracycline are not reported for patients under the age of 8 or pregnant women. Carbapenems should be reported on ESBL and Amp C from sterile sites.

MLSC-3131U, Clinical Microbiology II CSF Shunt Infections -

Coagulase negative staphylococcus often cause problems with any indwelling plastic devices like shunts and catheters. Some gram negative can cause infection through shunts. Propionibacterium acnes is seen to have an increasing incidence and immunocompromised patients usually have infections caused by candida.

Specimen Collection and Transportation -

CSF is collected in sterile, clean, leak proof tubes. Do not refrigerate CSF specimens. Keep at RT but always set it up STAT. If viral, refrigerate or freeze at -70 degrees. Comment on volume and appearance. Ideally, all specimens are processed in a BSC to avoid aerosol spread and the BSC should be decontaminated afterwards. Spill kits should be readily available for the decontamination and clean-up of biohazardous CSF with bleach (10% for spills and 1% for clean-up).

Specimen Set Up -

If the volume of CSF is greater than 1 mL, it is centrifuged for 10 minutes at 1000 g. The supernatant is collected in a sterile tube and sent to chemistry. 0.5 mL of fluid is left to resuspend for gram staining and culturing. CSF is analyzed for glucose, protein, cell counts, gram staining, cryptococcal antigen detection, culturing and viral testing.

Microscopic Examination -

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Uses a cytospin centrifuge to make smears. Smears are air dried and then methanol fixed. Then a gram stain is done and the absence or presence of bacteria and WBC are reported. The cytospin separates and deposits a very thin layer of cells and microorganisms. It spins very slowly to minimize organism distortion and produces a small button for easy analysis. A negative stain can also be done to detect cryptococcus neoformans capsules. Not clinically used anymore. PCR is done on CSFs especially for viral infections and fastidious organisms.

Culturing of CSF for Bacteria -

A few drops of sediments are placed on the agar (CHOC in CO2 and BAP). Always work in a BSC. If N. meningitidis is suspected work up primary culture in the BSC.

MLSC-3131U, Clinical Microbiology II Working and Reporting CSF -

Do not report quantitative growth. Set up any test that requires overnight incubation on day two. Report only the presence or absence of WBC and microorganisms (do not quantitate). The direct test you can do from a CSF is cryptococcal latex agglutination.

Brain Abscesses -

Can be bacterial or fungal. Bacterial abscesses are secondary to ear or sinus infections (strep, Bacteroides, anaerobes) or following trauma, surgery, or infective endocarditis (S. aureus). Fungal abscesses are increasing in incidence (from immunosuppressive drugs) and are usually caused by candida and aspergillus (disseminates from lung/sinuses).

Specimen Collection of Abscesses -

Abscess swabs are transported under anaerobic conditions. If it is a tissue, it will be homogenized with a mortar, pestle and thioglycolate broth. The paste is quickly inoculated onto BAP, Brucella ANO2 and CHOC CO2.

Normally Sterile Body Fluids Pleural Fluid -

Almost looks like serum and comes from the pleural space. Normally there are little to no cells in it. Effusion is seen as excess pleural fluid with high WBC. Suggests infection. Infection usually caused by severe pneumonia that goes uncontrolled.

Peritoneal Fluid -

Comes from the peritoneal cavity and usually does contain WBCs. The lab receives multiple peritoneal fluid samples from dialysis patients. Increased fluid volume, increased WBCs and an elevated protein level is a sign of infection. Primary Peritonitis, rare and spread by the blood or lymph nodes. Secondary Peritonitis, caused by organ perforation from surgery or trauma resulting in a loss of bowel wall integrity. Causes poly-microbial peritonitis. Pelvic Inflammatory Disease, STIs that have migrated through the pelvic floor and into the peritoneal space. Peritoneal Dialysis Fluid, done in end stage renal disease for continual ambulatory peritoneal dialysis. Peritoneal fluid is constantly being replaced and through osmosis, impurities are diffused out of solution when it leaves the body.

MLSC-3131U, Clinical Microbiology II

Pericardial Fluid -

Comes from the space between the pericardium and the outer surface of the heart. Infection will cause pericardial distention which will interfere with cardiac function and circulation. Pericarditis, inflammation of the pericardium usually caused by viral infection. Myocarditis, inflammation of the heart muscle.

Joint Fluid -

Comes from the spaces between the joints. Infection is known as septic infectious arthritis. Most common agent in adults is S. aureus, in younger adults its N. gonorrhoeae, and in children it is Haemophilus influenza. Other possible agents include Streptococci, pneumococci and Viridans. Prosthetic Joint Infections, usually due to skin normal flora like S. epidermidis or other CNST.

Collection, Transport and Storage of Sterile Fluids -

Gram stains are always methanol fixed for sterile fluids and are always STAT. Gram stain is always called to the floor if organisms are seen and is a critical result. Containers used are sterile screw-cap tubes or anaerobic transport swabs. Transported to the lab immediately for plating. Kept at RT. Do not reject samples from sterile sites because they are often coming from traumatic recollections or collections that cannot be repeated. Fluids are often centrifuged, decanted and resuspended for plating. Inoculated onto enrichment broths, BAP and CHOC plates. Cytocentrifugation is done on sterile fluids and all are processed for gram stains. Never throw away sterile fluids. Store at -20 degrees for 1 week after testing in case any other tests need to be run later.

Blood Culture Collection -

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Body weight and age determine how much blood culture we can take from the patient. Each anaerobic/aerobic bottle take 8-10 mL of blood. For people less than 10 years and more than 30 kg, one aerobic and one anaerobic bottle is taken from the left arm (site 1) and one anaerobic bottle (site 2) is taken from the right arm. This makes an entire set of blood cultures (3 bottles). Children less than 10 years old get pediatric bottles (1-5 mL) taken from them (one at site one and 1 at site 2) for a total set number of 2 bottles. Neonates only get one pediatric bottle at site 1 for a total set number of one.

MLSC-3131U, Clinical Microbiology II -

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The most important criteria for blood collection is the correct volume. Do not refrigerate these collections and place them in the blood culture machine within 12 hours. The blood culture analyzers monitors growth through CO2 production detected through colorimetric and fluorescent methods. The bottle can be contaminated with normal skin flora (2-3%). Aseptic collection is very important to avoid this. The skin should be prepared with alcohol and then antiseptic. Never take above the IV line to avoid taking in antibiotics. If only one bottle has normal flora and another doesn’t, it is most likely contamination....