Microbiology 4

1. CSF
Suggested volumes are 1, 2, and 2 ml for routine, fungal, and mycobacterial cultures respectively

a) Lumbar puncture

1. Clean the puncture site with antiseptic solution and alcohol before needle insertion to prevent introduction of infection.
2. Insert a needle with stylet at the L3-L4, L4-L5, or L5-S1 interspace. When the subarachnoid space is reached, remove the stylet and spinal fluid will appear in the needle hub.
3. Slowly drain the CSF into the sterile tubes. Three tubes are generally required for microbiology, hematology, and chemistry testing. The second tube drawn will generally go to Microbiology, and the last tube drawn will generally go to Hematology.

(In traumatic taps, the CSF will often clear as the later tubes are collected. Always send the most turbid tube to microbiology.)

b) Ommaya reservoir fluid

1. Clean the Ommaya reservoir site with antiseptic solution and alcohol prior to removal of Ommaya fluid to prevent introduction of infection.
2. Remove Ommaya fluid via the Ommaya reservoir unit, and place it in a sterile tube.

2. Other CNS specimens

a) Brain abscess

Ninety percent of brain abscesses will grow anaerobic bacteria. If an anaerobic transport system is unavailable or unsuitable for the specimen obtained, transport the specimen without delay to the microbiology laboratory for immediate processing.

A physician aspirates material from a lesion and sends it to the microbiology laboratory in an anaerobic transport system. See Table 1 for transport of specimens in a syringe.

b) CNS biopsy samples

Obtain a biopsy sample from the lesion at surgery, and send it to the microbiology laboratory in an anaerobic transport system. Do not add formalin.

c) CNS specimen collection considerations are outlined in TABLE 4.

1. Gastric aspirates
   The patient should fast prior to each of the following procedures.
   a) Gastric lavage
   
   Submitted primarily for the defection of Mycobacterium tuberculosis in patients (most frequently children) unable to produce quality sputum. Should be performed after the patient wakes in the morning so that sputum swallowed during sleep is still in the stomach.
   
   Pass a well lubricated tube orally or nasally through to the stomach of the patient, and perform the lavage.  Before removing the tube, release the suction and clamp to prevent mucosal trauma and/or aspiration.
   
   b) Duodenal aspiration
   
   Submitted primarily for the detection of Giardia species and larvae of Strongyloides stercoralis and Ascaris lumbricoides
   
   1) Pass a tube orally through to the duodenum of the patient.
   2) To aspirate a sample for giardiasis, the tube should be at least in the third portion of the duodenum.
   
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2. Gastric biopsies and washings
   The patient should fast prior to each of the following procedures.
   
   strong>a) Esophageal, stomach, or duodenum specimen
   
   Esophageal specimens are primarily used to detect Candida species, cytomegalovirus (CMV), and HSV infections. Stomach and duodenal specimens are used for the detection of Helicobacter pylori. Duodenal specimens can also be used for the detection of Giardia species and the larvae of S.stercoralis and A.lumbricoides.
   
   1) Pass an endoscope orally.
   
   2) Obtain specimens through a channel in the endoscope by using one of the following procedures.
   
   a) Using biopsy forceps, obtain samples from the esophagus, stomach, or duodenum.
   
   b) Using a sheathed brush, brush suspicious areas several times to obtain adequate cellular material.
   
   c) Perform a wash by injecting 25-30ml of sterile non-bacteriostatic isotonic 0.85% NaCl through the biopsy channel onto the lesion. Collect the specimen by aspirating the fluid through the scope into a sterile trap, which is connected to the suction tubing. Note: if a gastric ulcer is seen, obtain biopsy samples from the base, the surrounding gastric mucosa, and each of the >four quadrants of the margin.
   
   b) Rectal biopsy
   
   Submitted primarily for the detection of Entamoeba histolytica, Balantidium coli, and HSV. If lesions are not evident, biopsy the posterior rectal mucosa below the MILY: 'Times New Roman'; mso-fareast-font-family: 'Times New Roman'; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: AR-SA" Small bowel biopsy
   
   Submitted for the detection of Giardia, Cryptosporidum, and Microsporidium species.
   
   Biopsies of the small intestine provide the highest diagnostic yield for Microsporidia species. Biopsies from other gastrointestinal sites (stomach, colon, rectum) have a much lower yield m comparison. Obtain biopsy sample of lesion at surgery.
   
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3. Sigmoidoscopy
   
   Useful in the detection of E. histolytica and mycobacterium species and the diagnosis of pseudomembranous colitis associated with C. difficile and possibly Staph. aureus.
   
   a) Perform flexible or rigid sigmoidoscopy.
   
   b) Obtain endoscopic punch biopsy samples of any lesions seen. Aspirate liquid from the inflamed bowel with a pipette passed through the sigmoidoscope. Transport specimens in a sterile screw-cap container. If biopsy samples are small, add a small amount of sterile non-bacteriostatic 0.85% NaCl to prevent the specimen from drying.
   
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4. Fecal specimens
   
   Acute infectious diarrhea is caused by a number of different agents such as viruses, bacteria, and protzoa. In order to achieve optimal isolation of these organisms, we have adopted well established criteria for the tests we perform on stool.
   
   Handling Conditions : All patient specimens received in the lab should be handled as potential biohazards. Stool should be sent fresh within 1 to 2 hours of passage or placed in the appropriate preservative(s) and temperatures for prolonged transport time. Do not use toilet paper to collect stool. Toilet paper may be impregnated with barium salts, which are inhibitory for some fecal pathogens.
   
   4.1 Bacterial Culture for Enteric Pathogens:
   
   4.1.1 Transport - Stool should be sent fresh within 1 to 2 hours of passage or placed in Cary Blair preservative at refrigerator temperatures for prolonged transport time
   
   4.1.2 Collection recommendation: Submit two or three specimens on separate days to increase the probability of isolating a pathogen
   
   4.2 Clostridium Difficile Toxin A/B:
   
   4.2.1 Transport - Stools should be sent fresh within 1 to 2 hours of passage. They should be placed into the Stool-PREP preservative immediately upon arrival at the lab and refrigerated until testing. Stools in formalin are not acceptable, stools in Cary Blair are acceptable as long as they arrive in the correct dilution (1:5), frozen samples are acceptable.
   
   4.2.2 Collection recommendation: Submit two or three specimens on separate days to increase the probability of isolating a pathogen
   
   4.3 OP (Ova and Parasite):
   
   4.3.1 Transport - Stools should be sent fresh within 1 to 2 hours of passage. Routine O&P should be placed into OP preservative immediately upon arrival at the lab and refrigerated until testing. Giardia antigens should be sent in 10% formalin to our reference lab. Microsporidium should be sent in the 10% formalin/PVA set to be sent to our reference lab.
   
   4.3.2 Collection recommendation: Minimum of three specimens collected over a 7 to 10 day period is recommended. Infections with Entamoeba histolytica or Giardia lablia may require the examination of up to six specimens before the organism is detected. Ova and parasites should not be ordered on patients with over a 4-day stay without an ID consultation.
   
   4.4 Miscellaneous: Refer to reference laboratory handbook (Quest / Mayo) for collection requirements.
   
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5. Rectal swabs
   
   Submitted primarily for the detection of Neisseria gonorrhoeae, Shigella species, Herpes simplex virus (HSV), and anal carriage of Streptococcus pyogenes (Gp. A Beta strep.).
   
   Pass the tip of a sterile swab approximately 1 in. beyond the anal sphincter. Carefully rotate the swab to sample the anal crypts, and withdraw the swab. Send the swab in a swab transport (Aerobic culturette).
6. Gastrointestinal specimen collection considerations are summarized in Table 5.