Specimen collection in Infectious diseases

1. Specimen CollectionIn Infectious Diseases Dr.T.V.Rao MD

2. MICROBIOLOGY Information derived from the results has impact on : Diagnosis of infectious diseases Antibiotic prescribing Formulation of local antibiotic policy Public health impact eg food handlers Infection Control measures eg MRSA,

3. Why specimen collection is Important in Microbiology Specimen collection in Microbiology to isolate and identify the causative agents forms back bone of the investigative procedures. In developing world, lack of awareness and casual attitude among junior staff hampers the definitive diagnosis. Specific procedures in collecting specimens will certainly improve the quality of services of Microbiology Departments

4. Successful laboratory investigations advance planning collection of appropriate and adequate specimens labeling and documentation of laboratory specimen storage, packaging and transport to appropriate laboratory biosafety and decontamination procedures to reduce the risk of further spread of the disease timely communication of results

5. Collection & transposition of Specimen Collection & transportation of Good quality specimen for microbiological examination is crucial

6. Some tips Laboratory investigation should start as early as possible Specimens obtained early, preferably prior to antimicrobial treatment likely to yield the infective pathogen Before doing anything, 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

7. Specimen collection: key issues Consider differential diagnoses Decide on test(s) to be conducted Decide on clinical samples to be collected to conduct these tests consultation between microbiologists, clinicians and epidemiologists highly helpful Dr.T.V.Rao MD

8. Important questions before collecting a specimen • Are you suspecting an Infection ? If so what is the Nature of infection, eg Bacterial, Viral, Mycological or Parasitological • Which tests are your priority ? • When to collect the specimen ? • How to collect the specimen ? • Am I choosing the correct container ? • Why to send the specimens promptly if not what I should do ?

9. Fishing for Diagnosis in Laboratories, Is it worth? • The physicians and Microbiologists should be aware of the clinical manifestations, before undertaking the test. • Microbiological tests are expensive and technically demanding • Causal testing of Microbiological tests are counterproductive.

10. Policies on Specimen Collection. Every laboratory should assist extra examinations,outwith the standard procedures may be required if specifically requested by the Physician or if the clinical information provided on the request form suggests that an unusual infection may be present.

11. Why Proper written Request • Your request is a legal document. • Identifies all the outcome of test. • No interchange of results. • Short forms are dangerous • Signature of the Doctor / Nurse is essential in legible form, can help to contact in case of results which can save a patient. Dr.T.V.Rao MD

12. An Ideal Request form • Name xxxx Age Sex • IP/ OP No xyz Time Date • Ward xx123 Urgent / Routine • Nature of specimen • Investigation needed Doctor/Staff Contact No 1234567

13. Tele contact is crucial in serious patients • When the patient is serious, write a Tele contact number which can help in prompt delivery of results

14. When one Expects the Results On sending the sample the Physician will be anticipating the early reports, the Microbiologists should promptly dispatch results in all life saving investigations. However the Doctors must be made aware limitation of the investigations and discuss the pros and cons of the Laboratory reports

15. When to Repeat Diagnostic Tests On many occasions less than ideal sample is received in laboratory. The rejection of clinical samples should be done with great care and wisdom of only senior staff who should take the responsibility. In the welfare of the patient samples can be repeatedly collected for better diagnosis, as we need repeated isolation to confirm uncommon pathogens.

16. When to Collect the Earliest Specimen • Start collection of specimens for all cultures before starting an Antibiotic. • The advice is ideal but may not be possible, as many prescribe Antibiotics before considers the Microbiological diagnostic options.

17. When to Request Transport Medium • When facilities are not available to perform the desired tests at the place of collection or laboratory located far away, request the Diagnostic laboratories to advice on transportation of specimens, and consider how to preserve and transport in ideal medium before it is processed

18. What containers to use • Containers must be leak proof, • Unbreakable • For cultures sterile containers a Must • Microbiology specimens should never be sent in formalin Dr.T.V.Rao MD

19. Label High risk Specimens • Sputum with suspected Tuberculosis • Fecal samples suspected with Cholera, Typhoid, • Anthrax ? • Serum when suspected with HIV/ HBV/HCV, infections

20. Request for Gram Staining

21. Cultures That Should Include a Gram Stain • CSF or sterile body fluid (cytospin) • Eye • Purulent discharge • Sputum or transtracheal aspirate • All surgical specimens • Tissue • Urethral exudates (male only, intracellular gonococcus)) • Vaginal specimens • Wounds

22. Blood for Culturing

23. Blood for cultures Collection Venous blood infants: 0.5 – 2 ml children: 2 – 5 ml adults: 5 – 10 ml Requires aseptic technique Collect within 10 minutes of fever if suspect bacterial endocarditis: 3 sets of blood culture

24. Blood Collection for Culturing • Most important investigation • An appropriate procedures in collection and processing, identifying and timely reporting can be Life saving Dr.T.V.Rao MD

25. Collection of Blood • A scientific approaches and dedicated staff participating in blood collection will eliminate the basic failure as Contamination • Improper handling of syringes increases chances of contamination • Contamination hampers the ideal reporting, • A valuable time is lost • The goal in blood collection is avoiding the contamination

26. Collecting the Blood for Culturing • Teach the staff how to collect the Blood. • The nurse are advised on principles of aseptic precautions by self as washing hands and wearing gloves • Proper areas of disinfection with good antiseptic solutions. Dr.T.V.Rao MD

27. Hygienic precautions will decrease contamination • The staff should be advised how to disinfect the skin over vein, to use a fresh sterile syringe for the venepuncture with fresh sterile needle before inoculating culture bottle • The staff should disinfect their hands before doing the procedure.

28. Proper handling of Syringe is essential to obtain a blood specimen • The staff should hold the needle by its butt, not shaft. Either with sterile forceps or with fingers covered with a dry sterile rubber glove, and protect self with potentially infective pathogens

29. Do not collect from existing or indwelling catheters The staff are warned that contamination is very likely if the specimen is collected from an indwelling peripheral venous catheter instead of from a fresh venepuncture.

30. Always collect the Blood specimens in Hygienic areas • All procedures in relation to processing of the samples should be done in a sterile environment, or bacteria free areas. • Despite insistence on aseptic precautions, most laboratories report finding contamination in 1-5% of the blood cultures.

31. Serum Collection Venous blood in sterile tube let clot for 30 minutes at ambient temperature glass better than plastic Handling Place at 4-8°C for clot retraction for at least 1-2 hours Centrifuge at 1 500 RPM for 5-10 min separates serum from the clot

32. Serum Transport 4-8oC if transport lasts less than 10 days Freeze at -20°C if storage for weeks or months before processing and shipment to reference laboratory Avoid repeated freeze-thaw cycles destroys IgM To avoid hemolysis: do not freeze unseparated blood

33. Cerebrospinal fluid examination

34. Specimen collection for CSF Examination • Lumbar puncture to collect the CSF for examination to be collected by Physician trained in procedure with aseptic precautions to prevent introduction of Infection.

35. Procedure to collect CSF • The trained physician will collect only 3-5 ml into a labeled sterile container • Removal of large volume of CSF lead to headache, • The fluid to be collected at the rate of 4-5 drops per second. • If sudden removal of fluid is allowed may draw down cerebellum into the Foramen magnum and compress the Medulla of the Brain

36. CSF needs a New and Sterile container • Fresh sterile screw capped container to be used. • Reused containers, not to be used, contamination from the previous specimens misrepresent the present specimen.

37. Lumbar puncture for CSF collection • The best site for puncture is inter space between 3 and 4 lumbar vertebrae ( Corresponds to highest point of iliac crest )‏ The Physician should wear sterile gloves and conduct the procedure with sterile precautions, The site of procedure should be disinfected and sterile occlusive dressing applied to the puncture site after the procedure.

38. Transportation to Laboratory • The collected specimen of CSF to be dispatched promptly to Laboratory , delay may cause death of delicate pathogens, eg Meningococci and disintegrate leukocytes

39. Preservation of CSF • It is important when there is delay in transportation of specimens to Laboratory do not keep in Refrigerator, which tends to kill H. Influenza • If delay is anticipated leave at Room Temperature.

40. Upper Respiratory Infections

41. What are Upper Respiratory Infections • The commonest respiratory infections are localised in Oropharynx, Nasopharynx, and nasal cavity, • Causes Sore thraot,nasal discharge and often fever. Infect larynx,otitis media,sinusitis,conjunctivitis or keratitis. • May present with serious diseases whooping cough, influenza , measles and infectious mononucleosis.

42. Aetiological agents in Upper Respiratory Infections • In most cases the primary infections are caused by virus, difficult to isolate. • But many infections are caused by concomitant carriage or secondary infection with one of the potential pathogens present in the Nasopharynx 1 Pneumococcus .Haemophilus influenza Staphylococcus aureus, and Streptococcus pyogenes. • Drug resistant coli form bacilli or yeasts may dominate the throat flora in patients receiving antibiotics.

43. Specimen collectionin Throat Infections • A plain cotton wool swab should be used to collect as much exudates as possible from tonsils, posterior pharyngeal wall and other area that is inflamed or bears exudates

44. Cooperation of the patient and ideal techniques contributes better results • If cooperated by patient, the swab should be rubbed with rotation over one tonsillar area of the soft palate and uvula, the other tonsillar area and finally the posterior pharynx

45. Collecting the Swab • An adequate view of throat should be ensured by good lighting conditions and the use of a disposable wooden spatula or a tongue depressor to pull outwards and so depress the tongue.

46. Transportation of Throat Swabs • The swab should be replaced in its tube with care not to soil the rim • If it cannot be transported immediately to laboratory it should be placed in a refrigerator at 4ºc until delivery or preferably submitted in a tube of transport medium

47. Nasal specimens • A deep nasal swab generally yields the same information as throat swab. • Nasal swabs are taken to detect healthy carriers than diagnose deep infection • Deep nasal are taken to diagnose S.pyogenes and Diphtheria bacillus.

48. Nasopharyngeal swab Tilt head backwards Insert flexible fine-shafted polyester swab into nostril and back to nasopharynx Leave in place a few seconds Withdraw slowly; rotating motion WHO/CDS/EPR/ARO/2006.1

49. Nasopharyngeal aspirate Tilt head slightly backward Instill 1-1.5 ml of VTM /sterile normal saline into one nostril Use aspiration mucus trap Insert silicon catheter in nostril and aspirate the secretion gently by suction in each nostril WHO/CDS/EPR/ARO/2006.1

50. Specimens in sinusitis • Pus collected or aspirated from sinus, or a saline wash out should be examined in a Gram film and by culture on aerobic and anaerobic blood agar plates.