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1. Specimen collection and processing

2. Assist. Prof. Dr. Ali Al- Yafai Assistant Professor of Diagnostic & Molecular Microbiology

3. Basic principles of specimen collection, preservation, storage and transportation

4. Basic principles of specimen collection, preservation, storage and transportation The collection, transport, and processing of clinical specimens are preanalytical steps that are key to organism recovery. Microbiological laboratories aid the clinicians for the diagnosis and treatment of disease.

5. Basic principles of specimen collection, preservation, storage and transportation Specimen collection and transportation are critical considerations, because results generated by the laboratory are limited by the quality and condition of the specimen upon arrival in the laboratory. Failure to properly collect specimens for culture may result in the failure to isolate the organism that is actually causing the infection.

6. Specimen collection

7. Specimen collection Specific guidelines on specimen collection are necessary for optimal use of microbiology services. There are general principles that each microbiology laboratory should follow for specimen collection and test ordering. First and foremost, universal precautions are to be used while specimens are aseptically collected and processed.

8. Specimen collection Universal precautions must be used for all specimens collected. Specimens should be collected during the acute (early) phase of an illness (or within 2 to 3 days for viral infections) to increase chances of isolation and identification of the causative organisms. For optimal detection of the pathogens responsible for an infectious disease, specimens should be collected at a time when the likelihood of recovering the suspected agent is greatest

9. Specimen collection Specimens should be collected at optimal times (for example, early morning sputum for AFB culture). Specimens for bacterial culture, wherever possible, should be collected prior to the start of antibiotic treatment. Antibiotics may inhibit the growth of pathogens, which may result in a negative culture even if the patient has a bacterial infection.

10. Specimen collection The history and physiology of the disease must also be considered to determine when to collect a specimen. Collection of a specimen is dependent on the anatomic site of infection or suspected disease, so specimens should be collected from appropriate anatomic site where microorganisms are most likely to be present. The Salmonella typhi , the agent of typhoid fever first attacks the blood and therefore can be isolated from the blood during the first week of illness.

11. Specimen collection During weeks 2 and 3, the highest yield is found in the urine or feces. If a patient has symptoms of a urinary tract infection, the urine should be collected. Endocarditis may be diagnosed through positive blood cultures and other clinical procedures and not necessarily from a specimen collected from the heart. Strict aseptic technique is to be used for specimen collection.

12. Specimen collection Careful skin preparation before procedures such as blood cultures and spinal taps decreases the chance that organisms normally present on the skin will contaminate the specimen. Sterile organs blood, bone, and tissue samples in which organism contamination may be interpreted as infection. Careful handling of specimens, because specimens as cerebrospinal fluid, biopsies, and catheterized urine are more difficult to obtain. There may be no guarantee that a repeat specimen can be collected.

13. Specimen collection Containers Specimens should be collected using appropriate collection devices and should be tightly sealed to prevent leaks. Specimens for microbiology cultures should be collected in sterile, screw-cap containers and they should be transported within sealable and leak-proof container.

14. Specimen collection Containers Specimens should be collected under aseptic conditions to minimizes contamination with indigenous flora and other contaminating microorganisms that are not involved in the infectious process. Contamination from adjacent tissue and any bacteria that normally colonize the site should be avoided during specimen collection.

15. Specimen collection Containers Use of special techniques that bypass areas containing normal flora when feasible (e.g., covered brush bronchoscopy in critically ill patients with pneumonia) prevents many problems associated with false-positive results. The specimen container should not have any visible exterior contamination due to contaminated containers may lead to inaccurate results.

16. Specimen collection Containers Avoid the specimen that was received in a fixative (formalin), because it kills any microorganism in that specimen. Swabs Swabs are appropriate for specimens from the upper respiratory tract, external ear, eye, and genital tract. Swabs should not be used as collection devices unless the specimen source is the throat, cervix, or other difficult-to-reach area.

17. Specimen collection Swabs The tips of swabs that used to collect the specimen may contain cotton, ryon , polyester, Dacron, or calcium alginate. Dacron or polyester swabs have a wide range of uses. Swab on a plastic or wire shaft is acceptable for most organisms. Cotton-tipped swabs tend to have excessive fatty acids, which may be toxic to certain bacteria such as Neisseria gonorrhoeae .

18. Specimen collection Swabs Because cotton swabs may release toxic fatty acids, calcium alginate or Dacron swabs usually are preferred for collection. Calcium alginate should be avoided for collection of samples for viral culture because it could inactivate herpes simplex virus (HSV). Calcium alginate also inhibits PCR techniques due to calcium has been found to be a Taq polymerase inhibitor, competitively binding to the polymerase in place of magnesium during PCR and as a result reducing the efficiency of amplification.

19. Specimen collection Swabs Wooden shafts should be avoided because the wood may be toxic to Chlamydia trachomatis . Swabs are never appropriate for anaerobic culture because the atmospheric oxygen is toxic to anaerobes, so aspirates are preferred rather than swab for anaerobic cultures. Swabs are also not optimal for detection of mycobacteria, or fungi and they should not be used when these organisms are suspected.

20. Specimen collection Quantity of specimens Properly collected and adequate amounts (quantity) of specimen should be collected to carry out the investigations. Sufficient specimen quantity should be sent for testing ordered. Sufficient blood culture volume is critical to the accurate and timely detection of organisms causing sepsis.

21. Specimen collection Quantity of specimens Very little specimen is obtained with a swab, and much of the specimen is retained within the swab tip. Inadequate amounts of specimen may yield false-negative results. When multiple tests are ordered, sufficient samples or volume should be submitted in order to provide for the optimal recovery of organisms.

22. Specimen collection Labeling of specimens All samples must be labeled with the patient identifiers such as patient name and medical records number and date of birth are to be used to verify the patient, the specimen, and the specimen orders. A mislabeled specimen, in which the name on the specimen and the name on the test requisition do not match, is a common preanalytic error that occurs before the specimen gets to the microbiology laboratory.

23. Specimen collection Labeling of specimens The mislabeled specimen becomes a laboratory error when the laboratory fails to recognize the label mismatch and proceeds to process the specimen. A complete requisition should include the following: The patient’s name Hospital number Age or date of birth Sex

24. Specimen collection Labeling of specimens Collection date and time Ordering physician Exact nature and source of the specimen Diagnosis (may be ICD-9-CM code) Current antimicrobial therapy

25. Specimen preservation, storage and transportation

26. Specimen preservation, storage and transportation Anticoagulants are used to prevent clotting of specimens such as blood, bone marrow, and synovial fluid, because microorganisms will otherwise be bound up in the clot. The type and concentration of anticoagulant is very important because many organisms are inhibited by some of these chemicals. Sodium polyanethol sulfonate (SPS) at a concentration of 0.025% (w/v) is usually used, because Neisseria spp. and some anaerobic bacteria are particularly sensitive to higher concentrations.

27. Specimen preservation, storage and transportation Heparin is also a commonly used anticoagulant, especially for viral cultures, although it may inhibit growth of gram-positive bacteria and yeast. Citrate, ethylenediaminetetraacetic acid (EDTA), or other anticoagulants should not be used for microbiology, because their efficacy has not been demonstrated for a majority of organisms. It is the microbiologist’s job to make sure the appropriate anticoagulant is used for each procedure.

28. Specimen preservation, storage and transportation All specimens must be promptly transported to the laboratory as soon as possible, preferably within 2 h. Any delay may lead to the decline of the pathogen and multiplication of the normal flora. It is important to maintain the specimen as close to its original state as possible, so exposure to heat, cold, and drying should be avoided. The specimen must be promptly transported to the laboratory to preserve the viability of fastidious organisms and to prevent overgrowth of fastidious organisms by more rapidly growing bacteria which may be insignificant.

29. Specimen preservation, storage and transportation A culture tubes with semisolid Stuart’s or Amies transport medium provide excellent systems for collection and transport of specimens. Cerebrospinal fluids should be transported immediately to the laboratory and examined. It is crucial to avoid any delay in the workup of cerebrospinal fluid when one considers the morbidity and mortality of the infection and the fastidious nature of those bacteria associated with meningitis.

30. Specimen preservation, storage and transportation If specimens cannot be processed as soon as they are received, they must be stored Several storage methods are used: Refrigerator temperature [4°C] Room temperature [22°C], Body temperature [37° C], Freezer temperature [either –20° or –70° C])

31. Specimen transport and storage Cerebrospinal fluid (CSF) and other body fluids, blood, and specimens collected for recovery of Neisseria meningitidis should be held at room temperature because refrigeration adversely affects recovery of potential pathogens from these sources. Never refrigerate spinal fluid, genital, eye, or internal ear specimens unless for viral cultures. If a delay is unavoidable, urine sputum and other respiratory specimens, stool, and specimens for detection of C. trachomatis or viruses should be refrigerated to prevent overgrowth of normal flora.

32. Specimen transport and storage Urine, stool, viral specimens, sputa, swabs, and foreign devices such as catheters should be stored at 4° C. Serum for serologic studies may be frozen for up to 1 week at –20° C, and tissues or specimens for long-term storage should be frozen at –70ºC. Stool specimens for bacterial culture that are not transported immediately to the laboratory can be refrigerated, but if the delay is longer than 2 hours, the specimen can be added to Cary-Blair transport media.

33. Specimen transport and storage Stools for Clostridium difficile toxin assay should be collected without a preservative and can be refrigerated; if the delay will be longer than 48 hours, the specimen should be frozen at −70° C. Many microorganisms are susceptible to environmental conditions such as the presence of oxygen (anaerobic bacteria), changes in temperature ( Neisseria meningitidis ), or changes in pH ( V.cholerae ).

34. Specimen receipt and processing

35. Specimen receipt and processing Rejection of unacceptable specimens Criteria for specimen rejection should be set up and distributed to all clinical practitioners. In general, specimens are unacceptable if any of the following conditions apply: The information on the label does not match the information on the requisition or the specimen is not labeled at all (patient’s name or source of specimen is different).

36. Specimen receipt and processing Rejection of unacceptable specimens The specimen has been transported at the improper temperature. The specimen has not been transported in the proper medium (e.g., specimens for anaerobic bacteria submitted in aerobic transports). The quantity of specimen is insufficient for testing (the specimen is considered quantity not sufficient [QNS]). The specimen is leaking

37. Specimen receipt and processing Rejection of unacceptable specimens The specimen transport time exceeds 2 hours post collection or the specimen is not preserved. The specimen was received in a fixative (formalin), which, in essence, kills any microorganism present. The specimen has been received for anaerobic culture from a site known to have anaerobes as part of the normal flora (vagina, mouth). The specimen or swab is dried.

38. Specimen receipt and processing Rejection of unacceptable specimens More than one specimen of urine, stool, sputum, wound, or routine throat specimens submitted on the same day from the same source. Only one swab submitted with multiple requests for various organisms (bacteria, AFB, fungi, virus, ureoplasmas , etc.).

39. Specimen receipt and processing Specimen processing When multiple specimens arrive at the same time, priority should be given to those that are most critical, such as cerebrospinal fluid (CSF), tissue, blood, and sterile fluids. Urine, throat, sputa, stool, or wound drainage specimens can be saved for later. Acid-fast, viral, and fungal specimens are usually batched for processing at one time.

40. Specimen receipt and processing Specimen processing When a specimen is received with multiple requests but the amount of specimen is insufficient to do all of them, the microbiologist should call the clinician to prioritize the testing. Anytime a laboratory staff member contacts the physician or nurse, the conversation and agreed-upon information should be documented to ensure proper follow-up.

41. Specimen receipt and processing Specimen processing On arrival in the laboratory, the time and date received should be recorded. Depending on the types of specimens, the microbiological investigations include: macroscopic and direct microscope exanimation of and culture and antimicrobial sensitivity testing of isolated bacteria or fungi. Other techniques such as molecular and serological for raid diagnosis of microbial infections as well as tissue culture for viruses and demonstration of some toxins.

42. Communication of lab findings This is assignment for students!

43. Thank you