lecture 1-2..pptx Ahmed and mouuhmmed ali

طب الأسنان جامعة عدن كلية الطب

1. General Sample Collection Techniques

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

3. General Sample Collection Techniques Microbiological laboratories aid the clinicians for the diagnosis and treatment of disease. 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.

4. General Sample Collection Techniques The recovery of contaminants or normal flora which could lead to inappropriate or unnecessary antibiotic therapy. Each microbiology laboratory establishes criteria for specimen collection and for rejecting specimens that do not meet the requirements.

5. General Sample Collection Techniques Dealing with different types of specimens Specific guidelines on specimen collection are necessary for optimal use of microbiology services. 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

6. General Sample Collection Techniques Dealing with different types of specimens 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.

7. General Sample Collection Techniques Dealing with different types of specimens Specimens should be collected from appropriate anatomic site where microorganisms are most likely to be present. The history and physiology of the disease must also be considered to determine when to collect a specimen. 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. During weeks 2 and 3, the highest yield is found in the urine or feces.

8. General Sample Collection Techniques Dealing with different types of specimens The specimen obtained must be representative of the disease process, for example in the collection of a specimen for the diagnosis of bronchitis or pneumonia as lower respiratory tract infection, sputum should be collected rather than throat swab or saliva. 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.

9. General Sample Collection Techniques Dealing with different types of specimens Properly collected and adequate amounts (quantity) of specimen should be collected to carry out the investigations. 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. Swabs should not be used as collection devices unless the specimen source is the throat, cervix, or other difficult-to-reach area.

10. General Sample Collection Techniques Dealing with different types of specimens Anaerobic cultures - aspirates are preferred rather than swab. Swabs are never appropriate for anaerobic culture because the atmospheric oxygen is toxic to anaerobes. Swabs are also not optimal for detection of mycobacteria, or fungi and they should not be used when these organisms are suspected. Why ?

11. General Sample Collection Techniques Dealing with different types of specimens Swabs are also appropriate for specimens from the upper respiratory tract, external ear, eye, and genital tract. If a swab is used to collect the specimen, the tips of swabs may contain cotton, ryon , polyester, Dacron, or calcium alginate. Dacron or polyester swabs have a wide range of uses.

12. General Sample Collection Techniques Dealing with different types of specimens Cotton-tipped swabs tend to have excessive fatty acids, which may be toxic to certain bacteria such as Neisseria gonorrhoeae . 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).

13. General Sample Collection Techniques Dealing with different types of specimens 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. Swab on a plastic or wire shaft is acceptable for most organisms. Wooden shafts should be avoided because the wood may be toxic to Chlamydia trachomatis .

14. General Sample Collection Techniques Dealing with different types of specimens Specimens for microbiology cultures should be collected in sterile, screw-cap containers and they should be transported within sealable and leak-proof container. 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. General Sample Collection Techniques Dealing with different types of specimens 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. 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.

16. General Sample Collection Techniques Dealing with different types of specimens Careful handling of specimens, because specimens as cerebrospinal fluid, biopsies, and suprapubic aspiration urine are more difficult to obtain. There may be no guarantee that a repeat specimen can be collected. The specimen container should not have any visible exterior contamination due to contaminated containers may lead to inaccurate results. Avoid the specimen that was received in a fixative (formalin), because it kills any microorganism in that specimen.

17. Specimen transport and storage

18. Specimen transport and storage 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.

19. Specimen transport and storage 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.

20. Specimen transport and storage 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])

21. 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. 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.

22. 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.

23. 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 ).

24. What are the criteria for specimen rejection?

25. Nasal Swab

26. Nasal Swab Purposes Nasal swabs are usually collected to diagnose certain pathogens of the upper respiratory tract. Screening for Staphylococcus aureus either because the patient is suffering from recurrent boils/folliculitis or for infection control management when methicillin resistant Staphylococcus aureus (MRSA) It is useful to detect carriers for meningococci

27. Nasal Swab Possible pathogens Bacteria Gram positive bacteria Gram negative bacteria Staphylococcus aureus Hemophilus Influenzae Streptococcus pneumoniae Neisseria meningitides Group A Streptococcus  

28. Nasal Swab Possible pathogens Viruses Coronaviruses   Sever acute respiratory syndrome Coronavirus 1 and 2 (SARS-CoV-1 and2) Middle East respiratory syndrome coronavirus (MERS-CoV) Respiratory syncytial virus(RSV) Rhinoviruses Influenza viruses Echoviruses Parainfluenza viruses Adenoviruses

29. Nasal Swab Norma flora The flora of nose include: Diptheroids Straphylococcus aureus Straphylococcus epidermidis Streptococcus Haemophilus Moraxella lacunata

30. Nasal Swab Collection of specimens Sterile, Dacron, or Rayon swabs with plastic shafts are suitable for collecting most upper respiratory tract microorganisms. One swab can be used for both anterior nares Procedures Wash your hands well with soap and water or use alcohol that comes in the form of a gel before and after taking the sample.

31. Nasal Swab Collection of specimens Procedures Explain the procedure to the patient Moist a swab in sterile saline Insert a swab, approx. 1–2 cm into the nares. Gently rotate the swab at 360° against the nasal mucosa for 3–5 seconds to swab any pus or exudates. Repeat the procedure with the same swab in the other nostril.

32. Nasal Swab Transportation of specimens Nasal swabs must be transported directly to the laboratory within half an hour, and in case of delay or distance from the laboratory, Stuart's transport medium must be used, with the previous information recorded on the medium. To detect viruses, nasopharyngeal specimens are placed into an appropriate transport medium, with or without antibiotics, and transported promptly to the laboratory or stored briefly in the refrigerator and packed in ice for transport as soon as possible.

33. Nasal Swab First day Microscopic examination Culture Routinely Additionally Gram smear Dilute carbol fuchsin Routinely Additionally

34. Nasal Swab First day Culture Routinely Additionally Blood agar Chocolate agar MacConkey agar Incubate in CO2 for 18-24hrs Incubate aerobically for 18-24hrs Thayer-Martin Agar Blood agar with X and V factors Incubate in CO2 for 18-24hrs Mannitol salt agar with NaCl and methicillin

35. Nasal Swab Second day Colonial growth description Gram stain from the colonies for differentiating the bacteria Biochemical for identification isolated bacteria Antimicrobial sensitivity testing based on colony morphology and gram staining for the isolated bacteria.

36. Nasal Swab Second day Antibiotic sensitivity test on Muller Hinton Agar with or without blood according to isolated bacteria. Third day Read the result of biochemical reaction Read the result of antibiotic sensitivity test Write the final result

37. Nasal Swab Others Polymerase chain reaction (PCR) for rapid detection of bacteria and viruses Antigen detection directly from specimens. Cultivation of viruses in tissue culture

38. Terms: 0.5 MacFarland standard Disc diffusion MIC MBC E-test Detection of β- lactamases Sensitive Intermediate Resistant D test Detection of ESBL Detection of MRSA

39. Antibiotic sensitivity test Media used for each bacteria Antibiotics used for each bacteria Antibiotics gram positive bacteria Antibiotics gram negative bacteria

40. Antimicrobial susceptibility pattern of nasal swab isolates Antibiotics Method Disc or MIC Disc content S I R                                                                                    

41. Thank you