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Role of specimen collection in Microbiology


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Role of specimen collection in Microbiology

Role of specimen collection in Microbiology

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  • 1. Specimen Collection In Microbiology Dr.T.V.Rao MD Professor of Microbiology Dr.T.V.Rao MD 1
  • 2. NABH Standards for Hospital • The programme is made to fulfill major objectives of the AAC.7 Clause C on Policies and procedures guidecollection, identification, handling safe transportation, processing and disposal Dr.T.V.Rao MD 2
  • 3. MICROBIOLOGYInformation 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, Dr.T.V.Rao MD 3
  • 4. 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 andcasual attitude among junior staff hampers the definitive diagnosis.Specific procedures in collecting specimenswill certainly improve the quality of services of Microbiology Departments Dr.T.V.Rao MD 4
  • 5. 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 Dr.T.V.Rao MD 5
  • 6. Collection & transposition of Specimen Collection & transportation of Good quality specimen for microbiological examination is crucial Dr.T.V.Rao MD 6
  • 7. 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 Dr.T.V.Rao MD 7
  • 8. 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 Dr.T.V.Rao MD 8
  • 9. 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 ? Dr.T.V.Rao MD 9
  • 10. Fishing for Diagnosis inLaboratories, 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. Dr.T.V.Rao MD 10
  • 11. Policies on Specimen Collection. Every laboratory should assist extra examinations, outwith the standard procedures may be required ifspecifically requested by the Physicianor if the clinical information provided on the request form suggests that an unusual infection may be present. Dr.T.V.Rao MD 11
  • 12. 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 Dr.T.V.Rao MD 12
  • 13. 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 Dr.T.V.Rao MD 13
  • 14. 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 Dr.T.V.Rao MD 14
  • 15. 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 madeaware limitation of the investigations and discuss the pros and cons of the Laboratory reports Dr.T.V.Rao MD 15
  • 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. Dr.T.V.Rao MD 16
  • 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 Dr.T.V.Rao MD 17
  • 18. What containers to use Containers must be leak proof, Unbreakable For cultures sterile containers a MustMicrobiology specimens should never be sent in formalinDr.T.V.Rao MD Dr.T.V.Rao MD 18
  • 19. Label High risk Specimens Sputum with suspected Tuberculosis Fecal samples suspected with Cholera, Typhoid, Anthrax ? Serum when suspected with HIV/ HBV/HCV, infections Dr.T.V.Rao MD 19
  • 20. Request for Gram Staining Dr.T.V.Rao MD 20
  • 21. Cultures That Should Include a Gram Stain• CSF or sterile body fluid (cytospin)• Eye• Purulent discharge• Sputum or trans tracheal aspirate• All surgical specimens• Tissue• Urethral exudates (male only, intracellular gonococcus))• Vaginal specimens• Wounds Dr.T.V.Rao MD 21
  • 22. Blood for Culturing Dr.T.V.Rao MD 22
  • 23. Blood for culturesCollection 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 Dr.T.V.Rao MD 23
  • 24. Blood Collection for Culturing Most important investigation An appropriate procedures in collection and processing, identifying and timely reporting can be Life savingDr.T.V.Rao MD Dr.T.V.Rao MD 24
  • 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 Dr.T.V.Rao MD 25
  • 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 Dr.T.V.Rao MD 26
  • 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. Dr.T.V.Rao MD 27
  • 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 Dr.T.V.Rao MD 28
  • 29. Do not collect from existing or indwelling catheters The staff are warned thatcontamination is very likely if the specimen is collected from an indwelling peripheral venous catheter instead of from a fresh venepuncture. Dr.T.V.Rao MD 29
  • 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. Dr.T.V.Rao MD 30
  • 31. Collection Serum Venous blood in sterile tube • let clot for 30 minutes at ambient temperature • glass better than plasticHandling 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 Dr.T.V.Rao MD 31
  • 32. Transport Serum 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 Dr.T.V.Rao MD 32
  • 33. Cerebrospinal fluid examination Dr.T.V.Rao MD 33
  • 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. Dr.T.V.Rao MD 34
  • 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 Dr.T.V.Rao MD 35
  • 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. Dr.T.V.Rao MD 36
  • 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. Dr.T.V.Rao MD 37
  • 38. Transportation to Laboratory The collected specimen of CSF to be dispatched promptly to Laboratory , delay may cause death of delicate pathogens, eg Meningococcal and disintegrate leukocytes Dr.T.V.Rao MD 38
  • 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. Dr.T.V.Rao MD 39
  • 40. Upper Respiratory Infections Dr.T.V.Rao MD 40
  • 41. What are UpperRespiratory 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. Dr.T.V.Rao MD 41
  • 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 Pneumococcus .Haemophilus influenza, Staphylococcus aureus, and Streptococcus pyogenes. Drug resistant coli form bacilli or yeasts may dominate the throat flora in patients receiving antibiotics. Dr.T.V.Rao MD 42
  • 43. Specimen collection in 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 Dr.T.V.Rao MD 43
  • 44. Cooperation of the patient and idealtechniques 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 Dr.T.V.Rao MD 44
  • 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. Dr.T.V.Rao MD 45
  • 47. Collection should be done caution• The following should be collected as soon as possible after illness onset: nasopharyngeal swab, nasal aspirate or a combined nasopharyngeal swab with oropharyngeal swab. If these specimens cannot be collected, a nasal swab or oropharyngeal swab is acceptable. For patients who are intubated, an endotracheal aspirate should also be collected. Bronchoalveloar lavage (BAL) and sputum specimens are also acceptable. Dr.T.V.Rao MD 47
  • 48. Sent in Transport Medium• Specimens should be placed into sterile viral transport media (VTM) and immediately placed on ice or cold packs or at 4°C (refrigerator) for transport to the laboratory. Recommended infection control guidance is available for persons collecting clinical specimens in clinics and other clinical settings and for laboratory personnel. Dr.T.V.Rao MD 48
  • 49. Specimens Stored at …..• All respiratory specimens should be kept at 4°C for no longer than 4 days. Dr.T.V.Rao MD 49
  • 50. Transportation of Throat SwabsThe swab should bereplaced in its tube withcare not to soil the rimIf it cannot betransportedimmediately tolaboratory it should beplaced in a refrigeratorat 4ºc until delivery orpreferably submitted ina tube of transportmedium Dr.T.V.Rao MD 50
  • 51. Shipping specimens…. • Clinical specimens should be shipped on wet ice or cold packs in appropriate packaging. All specimens should be labeled clearly and include information requested by your state public health laboratory. Dr.T.V.Rao MD 51
  • 52. 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. Dr.T.V.Rao MD 52
  • 53. Nasopharyngeal swabTilt head backwardsInsert flexible fine-shafted polyester swabinto nostril and back tonasopharynxLeave in place a fewsecondsWithdraw slowly; rotatingmotion WHO/CDS/EPR/ARO/2006.1 Dr.T.V.Rao MD 53
  • 54. Nasopharyngeal aspirateTilt head slightly backwardInstill 1-1.5 ml of VTM/sterile normal saline intoone nostrilUse aspiration mucus trapInsert silicon catheter innostril and aspirate thesecretion gently by suctionin each nostril Dr.T.V.Rao MD 54 WHO/CDS/EPR/ARO/2006.1
  • 55. 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. Dr.T.V.Rao MD 55
  • 56. Collection of Ear Swabs Acute otitis MediaAcute otitis Media – as long as eardrum remains intact, none of the infected exudates can be collected on an ear swab , though culture of the throat swab may give a provisional indication of casual organism Dr.T.V.Rao MD 56
  • 57. Chronic suppurative otitis media Swabs of the discharge in the external meatus should be cultured to guide the choice of antibiotics for systemic and topical therapy. Dr.T.V.Rao MD 57
  • 58. Otitis externa A swab should be taken from the meatus and cultured aerobically on blood agar and MacConkey agar plates for the bacteria. All specimens should also cultured on Sabouraud’s agar plate with Nystatin 50 units for Candida and Aspergillus. Dr.T.V.Rao MD 58
  • 59. Eye SwabsObtaining a adequate specimen is difficult.It is best to make smears and seed culture plates beside the patient immediately after collecting the material from the eye. Dr.T.V.Rao MD 59
  • 60. Collection of Eye swabs It is ideal to pick up the material with a loop or on the smoothly rounded tip of a thin glass rod or on the thin serum coated swab Clinical material from Conjunctiva, i.e. from everted eyelid, The margin of the eyelid should be avoided. Dr.T.V.Rao MD 60
  • 61. Specimens for Lower Respiratory Infections. Dr.T.V.Rao MD 61
  • 62. SputumCollection Instruct patient to take a deep breath and cough up sputum directly into a wide-mouth sterile container –avoid saliva or postnasal discharge –1 ml minimum volume Dr.T.V.Rao MD 62
  • 63. Lower Respiratory Infections Sputum is the material from the lower respiratory infections most commonly submitted for bacteriological examination. The sputum is a mixture of bronchial secretions and inflammatory exudates coughed up into the mouth and expectorated There are several difficulties both in collecting a suitable sample and interpreting the results of the culture Busy and uninstructed staff may send collection of saliva to the laboratory. On several occasions repeat sample may be required to isolate the causative agent. Dr.T.V.Rao MD 63
  • 64. Instruction for collecting sputum Make the collection in a disposable and wide mouthed screw capped plastic container of 50 – 100 ml capacity. Collect sputum before antibiotics are given. Ideal to have when patient wakes up and with first cough. Dr.T.V.Rao MD 64
  • 65. Precautions in handling the specimens Avoid spilling the material over the rim.  Tightly screw on the cap of the container.  Wipe off any spilled material on its outside with tissue paper  Deliver the specimen quickly to laboratory Dr.T.V.Rao MD 65
  • 66. Sputum Examination for Tuberculosis Specimen should be collected with biosafety precautions. Several specimens should be collected before coming to negative conclusions. Dr.T.V.Rao MD 66
  • 67. Specimens forUrinary Tract Infections Dr.T.V.Rao MD 67
  • 68. Collecting Urine for examination Collect the Mid stream specimens of Urine Do not collect spontaneously passed urine without instructions, which can lead to contamination with commensals bacteria Colonized on urethral orifice and perineum Dr.T.V.Rao MD 68
  • 69. Specimen Collection The urine collected in a wide mouthed container from patients A mid stream specimen is the most ideal for processing Female patients passes urine with a labia separated and mid stream sample is collected Dr.T.V.Rao MD 69
  • 70. How Urine Specimens collected in young and infants  Non invasive methods are safe and ideal  Follow the Broom hall method, By tapping just above the pubis with two fingers placed on supra pubic region after 1 hour of feed, tapping on at the rate of 1 tap/second for a period of 1 minute, if not successful tapping is repeated once again. The child spontaneously pass the Urine and to be collected in a sterile container Dr.T.V.Rao MD 70
  • 71. Transport of Urine for Culturing All collected specimens of urine to be transported to laboratory with out delay Delay of 1 – 2 hour deter the quality of diagnostic evaluations. If the delay is anticipated the specimens are at preserved at 40c In field conditions Boric acid can be added at a concentration of 1.8 % Dr.T.V.Rao MD 71
  • 72. Genital Tract Infections Dr.T.V.Rao MD 72
  • 73. Genital Infections in womenGenital infections present with, urethritis, vaginitis, genital ulceration, cervicitis, uterine sepsis, salphingits, oophoritis, and pelvic inflammatory disease. Dr.T.V.Rao MD 73
  • 74. Collection of specimens The specimen commonly collected for the diagnosis of vaginitiss, vaginosis or uterine sepsis is high vaginal swab The swab is inserted into upper part of the vagina and rotated there before withdrawing it. Dr.T.V.Rao MD 74
  • 75. Specimen collection in Gonorrhoea An endocervical swab must be collected for examination for gonococci. A vaginal speculum must be used to provide a clear sight of the cervix and swab is rubbed in and around the introitus of the cervix and withdrawn without contamination from vaginal wall. Dr.T.V.Rao MD 75
  • 76. Specimens from other genital areas  Other swabs should be collected from any exudate discharged from the meatus of the urethra or a Bartholins gland.  Rectal or pharyngeal swabs should be considered depends on sexual habits of the patient Dr.T.V.Rao MD 76
  • 77. Transportation of specimensAll the swabs to be promptly transported to laboratory, in cases of delay or in cases of delicate microbes to be transported in Amies transport medium.If possible two swabs to be collected and submitted for each site. Dr.T.V.Rao MD 77
  • 78. Specimen collection in MenThe infection in men are mostly caused by the same organism as in women. Urethritis is commonest presentation may be caused by Gonococci or Non-gonococcal. May present with Genital Ulcers. Dr.T.V.Rao MD 78
  • 79. Gonococcus infection in MenThe specimen is collected by milking the urethra and urethral discharge is smeared on slides and inoculated on warmed plates of heated blood agar or selective medium for isolation of Gonococci Dr.T.V.Rao MD 79
  • 80. Other Genital infectionsWhen prostatitis is suspected and there is no spontaneous discharge from urethra, massage of the prostate per rectum may express some exudate for examination, and culture. Dr.T.V.Rao MD 80
  • 81. Collection of specimens in ChancresThe examination of chancre requires the careful collection of exudates and its preparation for dark ground microscopy.Many patients need clotted blood for specific serological investigation. Dr.T.V.Rao MD 81
  • 82. Wound, Skin, and Deep Sepsis Dr.T.V.Rao MD 82
  • 83. Collection of specimensPus or exudates is often submitted on a swab for laboratory investigation. The swabs are inefficient sampling device and tends to desiccate the specimen and trap the bacteria which are then not released on to culture plate Dr.T.V.Rao MD 83
  • 84. Ideal samples from wounds  The ideal sample is pus or exudates should be submitted in a small screw- capped bottle in firmly stoppered tube or syringe or a sealed capillary tube.  Fragments of excised tissue removed at wound toilet or curettings from infected sinuses and other tissues should be sent in a sterile container. Dr.T.V.Rao MD 84
  • 85. Gastrointestinal Infections Dr.T.V.Rao MD 85
  • 86. Stool samplesCollection: Freshly passed stool samples • avoid specimens from a bed pan Use sterile or clean container • do not clean with disinfectant During an outbreak - collect from 10-20 patients Dr.T.V.Rao MD 86
  • 87. Timing – within 48 hours of onsetSample amount – 5-10 ml fresh stool from patients (and controls)Methods – fresh stool unmixed with urine in clean, dry and sterile container Dr.T.V.Rao MD 87
  • 88. Storage – refrigerate at 4°C; do not freeze – store at -15°C - for Ag detection, polymerase chain reaction (PCR)Transport – 4°C (do not freeze); dry ice for (Ag detection and PCR) Dr.T.V.Rao MD 88
  • 89. Collection of specimensWhenever possible, a specimen of faeces should be collected.A rectal swab is unsatisfactory, unless it is heavily charged and visibly stained with faeces collected from rectum, not anus Dr.T.V.Rao MD 89
  • 90. Collection of FaecesThe specimen may be collected from faeces passed into a clean bed pan, not mixed with urine, or disinfectant or from the surface of heavily soiled toilet paper.The specimen is collected into 25 ml screw capped wide mouthed disposable container. Dr.T.V.Rao MD 90
  • 91. Stool samples for parasitesTiming – as soon as possible after onsetSample amount and size – at least 3 x 5-10 ml fresh stool from patients and controlsMethod – mix with 10% formalin or polyvinyl chloride, 3 parts stool to 1 part preservative – unpreserved samples for Ag detection and PCR Dr.T.V.Rao MD 91
  • 92. Stool samples for parasitesStorage – refrigerate at 4°C; store at -15°C for Ag detection and PCRTransport – 4°C (do not freeze); dry ice for antigen detection and PCR Dr.T.V.Rao MD 92
  • 93. Transportation of specimenCollect 1-2 ml of faeces, and apply the cap tightly.Take care not to soil the rim or outside of the bottle.Transmit the container quickly to laboratory.If delay is unavoidable and particularly when the weather is warm collect the specimens in a container holding 6 ml buffered glycerol saline transport medium Dr.T.V.Rao MD 93
  • 94. Specimen collection for Virological diseases Direct virus diagnosis depends on the detectionof virus particles, viral antigen or viral nucleic acid in specimen taken from the site of infection. Specimens should be delivered promptly to the laboratory so that no deterioration in the quality of the cells occur. However the specimen collection techniquesalter depending on the Aetiological agent and site of involvement. Dr.T.V.Rao MD 94
  • 95. MICROBIOLOGYInformation 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, – PfA target for reduction of C difficile, MSSA and MRSA bacteraemia Dr.T.V.Rao MD 95
  • 96. Lab reports: No sender’s address or not collected Dr.T.V.Rao MD 96
  • 97. When to Repeat Diagnostic TestsOn 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 berepeatedly collected for better diagnosis, as weneed repeated isolation to confirm uncommon pathogens. Dr.T.V.Rao MD 97
  • 98. Criteria for rejecting samplesMismatch of information on the label and the requestInappropriate transport temperatureExcessive delay in transportationInappropriate transport medium – specimen received in a fixative – dry specimen – sample with questionable relevanceInsufficient quantityLeakage Dr.T.V.Rao MD 98
  • 99. Biosafety Precaution • All theTechnical staff should follow the Universal and other Biosafety Precautions while handling and Disposing the Microbiology Specimens Dr.T.V.Rao MD 99
  • 100. Programme created by Dr.T.V.Rao MD for resourcesfor Laboratory Personal in the Developing World email Dr.T.V.Rao MD 100