Specimen collection for clinical microbiology laboratory
- 1. Specimen Collection for Clinical Microbiology Laboratory Dr.Siti Hawa binti Hamzah Microbiology Unit, Pathology Dept Hospital Seberang Jaya Pulau Pinang, MALAYSIA
- 2. Introduction • Clinical microbiology is a science of interpretive judgment • Microbes grow, multiply, and die very quickly. • If any of those events occur during the pre-analytical specimen management processes, the results of analysis will be compromised and interpretation could be misleading.
- 3. • To provide that level of quality, the laboratory requires that all microbiology specimens be properly selected, collected, and transported to optimize analysis and interpretation. • Result interpretation in microbiology depends entirely on the quality of the specimen submitted for analysis, specimen management cannot be left to chance
- 4. Specimen collection in Microbiology • To isolate and identify the causative agents forms back bone of the investigative procedures. • Specific procedures in collecting specimens will certainly improve the quality of results • In-house tests vs outsourced tests
- 6. Factors to consider prior specimen collection • Specimens should be taken start as early as possible • Specimens obtained early, preferably prior to antimicrobial treatment likely to yield the infective pathogen • Before the procedure of sample collection, explain the procedure to patient and relatives • When collecting the specimen, avoid contamination • Take a sufficient quantity of material • Follow the appropriate precautions for safety
- 10. GP ID GN ID
- 11. Vitek2 ID System API 20NE
- 12. Impact of specimen management • The impact of proper specimen management on patient care is enormous. • it is the key to accurate laboratory diagnosis and confirmation • it directly affects patient care and patient outcomes, it influences therapeutic decisions • it impacts hospital infection control, patient length of stay • hospital and laboratory costs • it influences antibiotic stewardship • it drives laboratory efficiency.
- 13. • Microbiology specimen selection and collection are the responsibility of the medical personnel • Clinicians and other medical personnel should consult the laboratory to ensure that selection, collection, transport, and storage of patient specimens they collect are managed properly
- 14. Request form • PER, PAT 301 • Patient’s identification and R/N ( 2 identifier needed) • Type of sample & site & time taken • Doctors name & signature • ALL are important
- 15. Patient education and preparation • Patient collected samples – clear instructions to the patients • For example: –Urine – “ mid stream” –Sputum – rinse the mouth, gargle with water, expectorate with deep cough & first morning specimen –Sputum….not saliva
- 16. Bacteriology lab
- 17. • Appropriate collection devices, specimen containers, & culture media must be used to ensure optimal recovery of microorganism
- 18. • Specimen container must be properly labeled, place in the biohazard plastic bag and accompanied laboratory request form • Specimen are best transported immediately to the laboratory
- 19. Blood culture • The diagnosis of bloodstream infections (BSIs) is one of the most critical functions of clinical microbiology laboratories. • For the great majority of etiologic agents of BSIs, • conventional blood culture methods provide positive results within 72 hours; • incubation for >5 days seldom is required when modern automated continuous-monitoring blood culture systems and media are used
- 20. • The volume of blood that is obtained for each blood culture request is the most important variable in recovering bacteria and fungi from adult and pediatric patients with bloodstream infections • For adults, 20–30 mL of blood per culture set is recommended and may require >2 culture bottles depending on the system. • For neonates and adolescents, an age- and weight- appropriate volume of blood should be cultured
- 23. • Skin contaminants in blood culture bottles are common, very costly to the healthcare system, and frequently confusing to clinicians. • To minimize the risk of contamination of the blood culture with commensal skin microbiota, meticulous care should be taken in skin preparation prior to venipuncture.
- 24. Key points for the laboratory diagnosis of bacteremia/fungemia: • Volume of blood collected, not timing, is most critical. • Disinfect the venipuncture site with chlorhexidine or 2% iodine tincture in adults and children >2 months old (chlorhexidine NOT recommended for children <2 months old), using povidone-iodine and alcohol).
- 25. • Draw blood for culture before initiating antimicrobial therapy. • Catheter-drawn blood cultures have a higher risk of contamination (false positives). • Never refrigerate blood prior to incubation. • Use a 2- to 3-bottle blood culture set for adults, at least 1 aerobic and 1 anaerobic; use 1–2 aerobic bottles for children and consider aerobic and anaerobic when clinically relevant.
- 29. Cerebrospinal Fluid • Obtained by LP or through the reservoir of an indwelling ventricular catheter • Aseptic technique / experience MO • Adequate volume: 3-4 ml (Bijou bottles ) • 3 sterile tubes – microscopy , culture, biochemistry • Send immediately to the lab • Do not store in the refrigerator • NIA indicator
- 31. Key points for the laboratory diagnosis of CNS infections • Whenever possible, collect specimens prior to initiating antimicrobial therapy. • Blood culture should also be obtained if bacterial meningitis is suspected. • Do not refrigerate CSF - pathogen causing meningitis is cold sensitive eg. Neisseria meningitidis
- 32. • Inform the microbiology laboratory if unusual organisms are possible (eg, Nocardia, fungi, mycobacteria), for which special procedures are necessary. • CSF tubes #2 or #3, not #1, should be submitted for bacterial culture and molecular testing. • Attempt to collect as much sample as possible for multiple studies (minimum recommended is 1 mL); prioritize multiple test requests on small-volume samples.
- 33. Indian ink for Cryptococcus
- 34. Sterile body fluid • Sterile body fluid eg. peritoneal fluid, pleural fluid • Collected in the sterile container • Sent immediately to the lab
- 41. Tissue vs Pus vs Swab C+S • Clean the area around the wound • Clean the wound • Fresh culture material • Adequate quantity • Send tissue for culture in sterile container • Do not send slough – * surface wound is unsuitable for anaerobic culture
- 42. • Pus • Send in a sterile universal container • anaerobic organism – immediately sent to the lab
- 45. Pus swab • Never send dry swab • Amies Transport media • Material should be obtained from both middle and edge of the lesions • Sterile normal saline may be added to prevent drying • Never add formalin
- 51. •Respiratory specimens • Nasal swab –Screening for MRSA carrier • Pernasal swab –Whooping cough (Bordetella pertussis) • Throat swab –Streptococcus pyogenes (pharyngitis) –Amies Transport Media
- 53. Sputum • Expectorated / induced • TB – 3 consecutive specimens • Sterile universal container • Delay – store in the refrigerator • Bronchial alveolar lavage (BAL)/brushing/bx • Sterile container
- 57. Urogenital specimens • Vaginal swab –Suitable for diagnosis of candidiasis and other causes of vaginitis but NOT gonorrhoea • Endocervical swab –Best specimen for the diagnosis of gonorrhea & puerperal sepsis • Urethral Discharge –Insert the swab about 2 cm into the urethra
- 61. Urine specimen • mid stream urine in sterile container • Catheterized urine – aseptic puncture of the catheter conduit , not from the bag • Suprapubic aspiration / cyctoscopy • Urine need to be processed within 2h after collection
- 63. Key points for the laboratory diagnosis of urinary tract infections (UTIs): • Urine should not sit at room temperature for more than 30 minutes. • Hold at refrigerator temperatures if not cultured within 30 minutes, or use a urine transport device (boric acid or other preservative).
- 64. • Reflexing to culture after a positive pyuria screen should be a locally approved policy. • The presence of 3 or more species of bacteria in a urine specimen usually indicates contamination at the time of collection, and interpretation is fraught with error. • Do not ask the laboratory to report “everything that grows” without first consulting with the laboratory and providing documentation for interpretive criteria for culture that is not in the laboratory procedure manual.
- 66. Stool culture • Stool • Clean wide-mouth screw-capped plastic container • Liquid – one-third full • Enrichment media –Alkaline peptone water for Vibrios –Selenite F for Salmonella • Rectal swab • Stool clearance culture – typhoid (completion of therapy)
- 68. Key points for the laboratory diagnosis of blood and tissue parasites: • Microscopy is the cornerstone of laboratory identification • Proper specimen collection and transport are essential components of morphology and culture-based techniques. • NAATs are useful for detection of low parasitemia or in specifically identifying organisms that cannot be differentiated microscopically.
- 70. Serology Testing •Tests available • HIV AgAb • HBsAg and Anti-HBs • HCV Ab • HIV PA • VDRL and TPPA • Rheumatoid factor • ASOT
- 75. Transport of specimen • Specimens should reach the lab as soon as possible or else transport medium should be used • Amies transport medium is widely used for specimens collected on swabs
- 76. Safety • Protection of transporter and laboratory personals • All specimens must be protected in leak proof container • Plastic bags and separate pouches on the outside are recommended • Precious sample : transport individually and handover personally to the lab personal
- 77. User manual • HSJ website • Perkhidmatan Klinikal Diagnostik • Patologi • User manual • Specimen collection • List of outsourced tests
- 78. Preservation, storage and transport of specimen • H1N1 • MERS-CoV • Zika Virus • SARS-CoV2
- 81. Viral specimen collection • Dacron swab • Viral transport media • 3 layer packaging
- 83. Why dacron swab for viral specimen? • Dacron is a registered trademark for polyester fibre filament. • With the increasing use of PCR, swabs stick need to be free of extraneous DNA and DNAse and inhibitors that were present in cotton and alginate swab
- 84. Dacron vs cotton swab
- 85. Molecular testing • Test for H1N1 – Flu A • Test for Mers CoV – Flu A, Flu B and Mers CoV • Once Flu A – will be send to IMR for H1N1 • SARS-CoV2 • Rapid molecular – GeneXpert and ID NOW • RT-PCR
- 86. Rejection criteria • Leaking • Wrong container • Request not stated / not clearly written • Test requested not performed in unit • Specimen not labeled / mismatch from request form • Dry swab • Lysed blood
- 87. Interpretations • Don’t treat the lab result, treat the patient • Correlate clinically • Infection? Colonization? Contamination?
- 88. Quality-in, Quality-out • What you give, you get back ! • Ensure to follow guidelines before sending specimen • Best result… started from taking proper specimen in the wards • YOU..yes you are responsible
- 89. A little help from everybody • Communication is very important • Call the lab before sending specimen you are not sure • Write proper identification and history
- 91. Ten important points for good culture results • 1. Specimens of poor quality must be rejected. • Microbiologists act correctly and responsibly when they call physicians to clarify and resolve problems with specimen submissions. • 2. Physicians should not demand that the laboratory report “everything that grows.” • This can provide irrelevant information that could result in inaccurate diagnosis and inappropriate therapy.
- 92. • 3. “Background noise” of commensal microbiota must be avoided where possible. • Many body sites have normal, commensal microbiota that can easily contaminate the inappropriately collected specimen and complicate interpretation. • Specimens from sites such as lower respiratory tract (sputum), nasal sinuses, superficial wounds, fistulae, and others require care in collection.
- 93. • 4. The laboratory requires a specimen, not a swab of a specimen. Actual tissue, aspirates, and fluids are always specimens of choice, especially from surgery. • A swab is not the specimen of choice for many specimens • Swabs pick up extraneous microbes, hold extremely small volumes of the specimen (0.05 mL), and make it difficult to get bacteria or fungi away from the swab fibers and onto media, and the inoculum from the swab is often not uniform across several different agar plates. • Swabs are expected from the nasopharynx and to diagnose most viral respiratory infections
- 94. • 5. The laboratory must follow its procedure manual or face legal challenges. The procedures in the manuals should be supported by the literature, especially evidence-based literature. • To request the laboratory to provide testing apart from the procedure manual places everyone at legal risk.
- 95. • 6. A specimen should be collected prior to administration of antibiotics. Once antibiotics have been started, the microbiota changes and etiologic agents are impacted, leading topotentially misleading culture results. • 7. Susceptibility testing should be done only on clinically significant isolates, not on all microorganisms recovered in culture.
- 96. • 8. Microbiology laboratory results that are reported should be accurate, significant, and clinically relevant. • 9. The laboratory should set technical policy; this is not the purview of the medical staff. Good communication and mutual respect will lead to collaborative policies. • 10. Specimens must be labelled accurately and completely so that interpretation of results will be reliable. • Labels such as “eye” and “wound” are not helpful to the interpretation of results without more specific site and clinical information (eg, dog bite wound right forefinger).