IQC in Microbiology Dr Bansidhar Tarai Senior consultant Microbiology Max Super Speciality Hospital

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1 IQC in Microbiology Dr Bansidhar Tarai Senior consultant Microbiology Max Super Speciality Hospital 12/25/2017 IQC in Microbiology

2 Microbiology Lab design Microbiological testing has to be separated from other testing areas. The area for media preparation should be different sample processing in Microbiology. from that of Microbiology lab must all be designed for at least bio-safety level 2 (BSL-2). Desirable equipment for a BSL-2 facility includes a biological safety cabinet and an autoclave The autoclave for both sterile articles and decontamination should be placed separately with proper exhaust. Gas cylinders shall be kept secure to prevent unintended movement

3 TB - Lab area/ process flow TB Lab that only manipulate specimens for direct smear microscopy, or Xpert assay, are considered low-risk TB laboratories. In low risk lab, sample processing may be carried out on an open bench in an adequately ventilated area (with 6-12 air changes per hour) (Tuberculosis Laboratory Biosafety Manual, 2012, World Health Organization, p20). Concentration method AFB staining, or TB culture - should be carried out in a Class II biological safety cabinet.

4 Fungal Lab area/ process flow For filamentous fungi separate class II biological safety cabinet is necessary, which shall not be shared for other culture work. A laboratory performing fungus culture shall be equipped with a biological oxygen demand (BOD, heating and cooling) incubator to meet the environmental conditions for the isolation of dimorphic fungi that require incubation at 25ºC.

5 Molecular Lab area/ process flow PCR testing should be in physically separated areas, with the complete and effective segregation of post-pcr steps Contamination of samples or master mix should not occur Class II biosafety cabinet should be available for sample preparation and nucleic acid extraction. All equipment in the post-pcr area, e.g., micropipettes, microfuge, vortex mixer, personal protective gear etc. should be dedicated only for this area and should not be moved outside.

6 LAB PERSONNEL Employment of sufficient, qualified personnel proportional to volume & complexity of work performed E.g. 1 technologist per year per specimens Documentation of technical training on-the-job Documentation of continuing education programs Assessment of competence at periodic interval

7 Calibration / Preventive maintainace Autoclave 1 year Weight balance 1 year Biological safety cabinet- 1 year Laminar flow 1 year Centrifuge 1 year Thermometer 1 year Calibration should be done earlier too if the workload of the lab is substantial All equipment before being put into service for the first time. ELISA reader 1year Microscope calibration not required, only cleaning PCR machines/ Automated culture & Vitek2 as per manufacture

8 EQUIPMENT Quality Assurance in microbiology, DR Arora; ijmm april 2004

9 Microbiology Quality Control Quality assurance Internal quality assessment External quality assessment Quality control Preanalytical Analytical Post - analytical Stains Culture media Sensitivity Equipment Immunologic techniques Sterilization Raw material Sterilization Physical Microbiologic Gel strength Contaminati on

10 Pre-analytical stage Transport Specimen collection Specimen storage Request form SOP Checking & rejection Approp. investigation Pt evaluation Sample processing

11 Pre-analytic Informed consent - HIV testing Sample collection in sterile container Skin antiseptics for blood culture collection Transport as earliest, cold chain to be maintained (urine, BAL, ET etc), appropriate transport medium Specimens should be processed immediately If delay in processing, refrigerate except for CSF and pus / aspirate of liver abscess for suspected Entamoeba trophozoites

12 Blood culture Volume (Max)

13 5.94

14 Pre-analytic molecular EDTA (Plasma) sample is the preferred for molecular test methods Heparin is a PCR inhibitor Blood for DNA analysis can be stored at room temperature for up to 24 hours or at 2 to 8 C for up to 72 hours prior to DNA extraction As RNA degrades rapidly, the laboratory shall have a policy about the receipt of transported samples for RNA based assays

15 Analytical stage Staining sterilisatn Culture media Disposal SOP Inoculatn Safe lab practices Interpretation Equipmnt Sensitivity

16 CULTURE MEDIA wrong media Sterilisation Dispensing water SOURCE OF ERROR Glassware weighing

17 STERILISATION PARAMETER Autoclaving- time & quantity regulated Optimization of heating process Nutrient destruction by heat Volume of media in one batch kept small- 2 litres Monitoring of temperature & pressure Indicators Bowie Dick test Spores of Bacillus stearothermophilus

18 PHYSICAL PARAMETERS Excessive bubbles or pits Unequal filling of plates Thickness measured at 4 points Mean thickness must be 4+/- 0.2mm Optimum amount poured ml for 150 mm plates ml for 100 mm plates ml for 90 mm plates Cracked medium Freezing or crystallisation ph checked before & after autoclaving

19 MICROBIOLOGICAL PARAMETERS Ability to support growth Ability to yield the appropriate biochemical results Reflect the use of medium & range of organisms Selective media- control organisms for growth as well as inhibition Enteric media- growth of pathogen & suppression of normal flora Differential media- controls with all possible outcomes

20 PACKAGING, TRANSPORT AND STORAGE OF PREPARED MEDIA PACKAGING: Minimise moisture loss Provide protection against physical, thermal, light-induced damage and microbial contamination STORAGE: Prepared media stored in unopened /resealable packages at 2-8 C Maximum period of 7-10 days TRANSPORT: Ensure against exposure to potentially detrimental conditions.

21 SHELF LIFE OF PREPARED MEDIA Expiry date- validated under the conditions of packaging, storage and transportation Date of manufacture should be indicated Validations of expiry should include simulated transportation phase(s) in the storage/testing protocol Such simulated transportation phases should reflect the least favourable conditions likely to be encountered during transportation

22 QC Organism Preparation, Inoculation and Reading Saline suspension McFarland standard Media inoculated using calibrated 1 ul loop Incubated & cultures inspected at 24 & 48 hrs If unacceptable, remove media from the refrigerator shelf ; Record; inform supplier

23 STAINING NABL document 112

24 BIOCHEMICAL REACTIONS PROCEDURE/ TEST CONTROL ORGANISM EXPECTED RESULT EXPECTED REACTION

25 BIOCHEMICAL REACTIONS PROCEDURE/ TEST CONTROL ORGANISM EXPECTED RESULT EXPECTED REACTION NABL document 112 WHO QA in bacteriology & immunology

26 Quality control of commonly used media MEDIUM CONTROL ORGANISM EXPECTED REACTION

27 Quality control of commonly used media.. MEDIUM CONTROL ORGANISM EXPECTED RESULT

28 Quality control of commonly used media.. MEDIUM CONTROL ORGANISM EXPECTED REACTION Eosin-Methylene blue agar E.coli K.pneumoniae Shigella flexneri Good growth, green metallic sheen Good growth, purple colonies, no sheen Good growth, transparent colonies Hektoen enteric agar Salmonella typhimurium S. flexneri E. coli Green colonies, black centres Green transparent centres Growth slightly inhibited, orange colonies Kligler s iron agar E.coli S.flexneri Salmonella Typhimurium Pseudomonas aeruginosa Acid/acid Alkaline/acid Alkaline/black Alkaline/alkaline Nitrate broth Lowenstein-Jensen medium E. coli Acinetobacter lwoffi E. coli Mycobacterium tuberculosis M. kansasii M. scrofulaceum Red Partial inhibition Good growth Good growth Good growth

29 Lot to lot verification The laboratory shall check each lot of control and reagent against an earlier tested in-use control / reagent lot. Records of comparative data shall be maintained.

30 Antibiotic susceptibility testing The laboratory shall follow CLSI (formerly NCCLS) / EUCAST / BSAC guidelines for antibiotic susceptibility testing. Each lot of antibiotic sensitivity discs must be checked for activity and potency using standard reference strains before being placed in service and at least weekly thereafter Some antibiotics that cannot be reported on the basis of disc diffusion method shall be reported on the basis of MIC testing Susceptibility testing against Mycobacterium, a standard strain of M. tuberculosis with known resistance pattern to different drugs shall be used with each batch of tests as a check on procedures.

31 QUALITY CONTROL ANTIMICROBIAL DISK SUSCEPTIBILITY Testing of QC strains with known susceptibility to the antimicrobial agents QC strains: Tested regularly(daily or weekly) Supplemental QC strains: Used to assess a particular characteristic of a test or test system May represent alternative QC strains Not necessary to include in routine

32 ATCC Each new lot of Mueller-Hinton agar tested with a control strain of Enterococcus faecalis (ATCC or 33l86) disc of co-trimoxazole Distinct inhibition zone of 20 mm or more Stocks of antibiotic discs- kept at -20ºC

33 ATCC strain stock Stock cultures maintained at -60ºC or in liq. N2 or lyophilised Suitable stabiliser is used 50% FCS in broth 10-15% glycerol in tryptic soy broth Defibrinated sheep blood Skim milk Subcultures stored at 2-8ºC

34 QC strain maintenance Rehydrate new stock culture/ obtain from frozen stock Subculture to approp media; incubate (primary subculture) Subculture, incubate & store. Use isolated colonies from days 1-7 as working cultures for testing Prepare new subculture every 7 days Fresh working cultures each test day After 4 weeks, discard subculture; new stock

35 Internal Quality Controls for ELISA Negative and positive (internal) controls are available with the kit and shall be used with each run. In addition, laboratory shall use a commercial control or an (external) control with a value close to the cut-off for the test. In-house external control sera should be stored as multiple aliquots frozen at 20 C to prevent repeated freezing and thawing

36 RT-PCR RT-PCR controls shall include controls for positive, normal, and negative (no DNA) reaction controls. A normal control for the specific region of the gene to be analyzed shall be included in each assay. For quantitative PCR, a low and high level positive control, preferably close to the levels of clinical decision making shall be used. Data from repetitive runs shall be used to monitor the %CV of the assay.

37 Control of records - Microbiology NABL requires the following minimum retention period for ensuring quality service and patient care: Microbiology and Infectious disease serology - 1 year Molecular testing gel images, Real time PCR raw data - 2 years (infectious diseases) The records can be maintained as physical copies (instrument printouts or as photocopies) or electronically

38 Evaluation and audits pre-analytical and post-analytical processes are also covered during its internal audit. The laboratory shall incorporate salient quality indicators for monitoring its performance. This shall describe the evaluation of various aspects sample collection and identification transportation and processing analysis and reporting of results turnaround time complaints equipment downtime uncertainty of measurements Performance in PT / EQA scheme

39 Critical call outs - Microbiology Critical results shall be communicated urgently to the clinician and / or collection centre for timely management. positive blood culture CSF positive (gram, culture) urethral discharge, gram stain - Gonococcus positive Eye culture positive Clostridium difficile AFB positive (stain, culture, GeneXpert) KOH positive Septate / Aseptate Hyphae India ink positive for Cryptococcus Mucormycosis MRSA/CRE / VRE All notifiable disease Shigella/Vibrio/ eneteric Salmonella HSV encephalitis (PCR) H1N1

40 Thank You