TR TECHNICAL REQUIREMENTS FOR THE ACCREDITATION OF MICROBIOLOGY IN MEDICAL LABORATORIES. Approved By: Acting Chief Executive Officer: Ron Josias

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1 TECHNICAL REQUIREMENTS FOR THE ACCREDITATION OF MICROBIOLOGY IN MEDICAL LABORATORIES Approved By: Acting Chief Executive Officer: Ron Josias Senior Manager: Christinah Leballo Date of Approval: Date of Implementation: SANAS Page 1 of 11

2 CONTENTS: 1. Purpose and Scope 2. Definitions and References 3. List of General Requirements 3.1 Quality Management System; Examination by Referral Laboratories/ Pre- Examination Procedures & Examination Procedures 3.2 Examination Procedures Bacteriology Mycology Mycobacteriology Parasitology Microbiological Serology 3.3 Reporting of Results 3.4 Laboratory Equipment 3.5 Laboratory Safety and Accommodation & Environmental Conditions 4. Authorship ADDENDUM 1: Amendment Record SANAS Page 2 of 11

3 1. Purpose and Scope This document describes the managerial and technical accreditation requirements of a Microbiology Laboratory. It applies to all Medical Laboratories requiring accreditation to ISO or ISO/IEC Definitions and References SANAS PM SANAS A01 ISO ISO/IEC 17025:2005 SANAS Policy Manual References, Acronyms and Definitions Medical Laboratories Particular requirements for quality and competence General requirements for the competence of testing and calibration laboratories 3. List of General Requirements All assessments are done in accordance with the relevant ISO or ISO/IEC Standards and SANAS requirements. SANAS documents are available on the SANAS Website. 3.1 Quality Management System; Examination by Referral Laboratories; and Pre- Examination Procedures The scope of activities of the laboratory shall indicate which of the following services are provided and which are not provided on site: a) Bacteriology b) Mycobacteriology c) Mycology d) Parasitology e) Serology f) Drug susceptibility testing g) Therapeutic drug monitoring For the procedures carried out on site the laboratory shall indicate the extent to which such examination procedures are carried out: a) Direct examinations b) Isolation c) Culture d) Identification: limited or definitive e) Screening methods used f) Confirmatory methods used g) Any other relevant information Where the samples are referred to another laboratory for complete or part examination procedures, such information shall be provided by the laboratory. 3.2 Examination Procedures Note: detailed procedures shall be available for the examination, isolation, culture and identification of each species / organism tested for by the laboratory Bacteriology Where sputum cultures are performed the following shall apply: a) All steps shall be taken to obtain a "true" sputum sample. b) Appropriate procedures shall be available for the identification of Streptococcus pneumoniae and Haemophilus influenzae. c) Where appropriate other gram negative bacilli shall be identified. SANAS Page 3 of 11

4 d) Appropriate procedures shall be available for the isolation and identification of other pathogens i.e. Cryptococcus; Candida albicans The following shall apply to urine microscopy and culture: a) Urine samples should be examined within 2 hours of voiding or refrigerated if this is not possible. b) If the conditions in (a) are not met the urine should be received in the laboratory with the appropriate preservative added. c) Quantitative or qualitative cell counts, culture and colony counts should be performed. d) The media used shall allow for the identification of both gram positive and gram negative organisms. e) The relevant urine samples shall be tested for antibacterial activity Urethral Cervical Cultures for Neisseria gonorrhoeae: a) The urethral / cervical cultures shall be inoculated directly or received in transport media. b) The media and means of inoculation shall be optimum for the isolation of Neisseria gonorrhoeae The following shall apply for Faeces Cultures, Rectal Swabs Etc: a) Routine procedures shall allow for both rapid isolation and identification of enteric pathogens in patients with diarrhoea. b) Routine procedures shall allow for recovery of small numbers of enteric pathogens in asymptomatic carriers. c) When indicated, procedures shall be available for the recovery of the following bacteria: Neisseria gonorrhoeae Campylobacter species Vibrio species Yersinia enterocolitica Aeromonas species d) When the laboratory does not have a suitable parasitology section, microscopic methods should be used to detect common enteric parasites such as Giardia and Cryptosporidium The following shall apply to Cerebrospinal Fluid Cultures: a) The CSF samples shall be processed immediately on receipt. b) The macroscopic appearance of both the specimen and the supernatant shall be recorded. c) The differential cell count shall be performed. d) All samples shall be centrifuged and followed by gram stain. e) The routine primary culture procedure shall allow for the recovery of common disease producing bacteria with fastidious requirements (e.g. Neisseria meningitidis, Haemophilis influenza). f) All isolates shall be identified. g) Rapid testing, such as the Latex agglutination test, may be used for detecting bacterial antigens The following are applicable for Blood Cultures: a) The blood culture medium shall be warmed to room temperature before inoculation, and then incubated at 37 ºC. b) Blood cultures shall be incubated immediately to recover: Aerobic organisms CO 2 dependant organisms SANAS Page 4 of 11

5 Anaerobic organisms (When clinically indicated.) Fastidious organisms c) All blood cultures shall be examined early each day, for the appropriate number of days, if not using an automated system. d) All isolates should be identified. e) In non automated blood culture monitoring systems: i) Negative cultures shall be sub cultured within 24 hours and also at 48 hours. ii) For optimal growth of fastidious organisms, agitation of the blood cultures is very important within the first 24 hrs Wound Cultures and Aspirates: a) Special procedures shall be established to minimise the loss of anaerobes in specimens. b) Gram stains shall be performed and reported routinely. c) Both aerobic and anaerobic media shall be inoculated routinely. d) Selective methods shall be used to detect and recover strict anaerobes, when appropriate Beta-lactamase Production: Procedures shall be available to detect beta-lactamase production by Neisseria gonorrhoeae, Haemophilus and other relevant organisms Anaerobic Isolations: a) For specimens to be examined for anaerobes adequate arrangements shall be made for preventing or minimising their exposure to oxygen. b) All specimens for anaerobes shall be transported to the laboratory in suitable transport media. c) The method shall specify whether direct gram stains are done on all such specimens. d) The method shall specify the means by which an anaerobic environment is obtained i.e. anaerobic jar, anaerobic glove box, Hungate roll tubes, other. e) The use of pre-reduced media for anaerobic isolation shall be stated. f) Samples for anaerobes should be inoculated directly onto solid media. g) Cultures on solid media shall be placed into an anaerobic environment immediately. h) The liquid media used for specimens for anaerobes shall be specified i.e. thioglycollate media, Cooked meat broth, other. i) When a palladium catalyst is used the catalyst should be replaced after incubation of H 2 S producing organisms. j) The means used for assessing anaerobiosis during and after incubation shall be stated (Methylene blue indicator, Resazurine indicator, Control cultures of a strict aerobe or anaerobe.) k) Only the following methods shall be used for susceptibility tests: i) Agar dilution ii) Disc diffusion (Not accepted by the CLSI Sensitivities are not normally done as there are no guidelines available.) iii) E-Test iv) Broth dilution v) Gas liquid chromatography (where applicable) l) Control organisms shall be included with each batch of susceptibility tests performed. SANAS Page 5 of 11

6 Antibiotic Testing Mycology a) Susceptibility Testing i) Control strains of known susceptibility shall be tested at least once a week according to CLSI recommendations. ii) The inoculum size shall be standardized. iii) Tests to predict meca-mediated resistance to oxacillin shall be carried out and the specific method shall be specified. b) Type of Susceptibility Testing: i) If discs are used, the zone sizes of controls shall be measured and recorded. ii) The zone sizes of antimicrobials measured shall be used for recording sensitivity or resistance. iii) Strict control of the strength and identity of all diluted drugs shall be enforced. iv) Kirby Bauer disc susceptibility testing. c) Blood Levels: (May also be performed in a chemistry laboratory) i) Blood samples shall be collected at proper times, relative to dose and route of administration; details of all antibiotics administered shall be available. ii) When cytotoxic drugs are assayed, suitable personnel safety measures shall apply Isolation and Identification: a) The laboratory shall perform preliminary screening procedures such as direct preparation and stains b) Suitable selective media shall be used for the growth and isolation of Dermatophytes and Systemic fungi. c) Antibiotics shall be added to media to suppress the growth of contaminants. d) The temperatures for the growth and isolation of dermatophytes and systemic fungi as defined under the requirements of the culture Differential Tests: Mycobacteriology a) Appropriate procedures for the differentiation and identification of fungi used in respect of the type of work undertaken by the laboratory shall be used. b) Differential procedures shall include: i) Chlamydiospore formation ii) Temperature growth requirements (e.g.: O C) iii) Biochemical tests iv) Slide cultures Diagnostic Procedures: a) All specimens including urine and gastric aspirates shall be examined microscopically. b) Ziehl-Neelsen, Fluorescence and/or Kinyoun staining methods shall be used for microscopy. SANAS Page 6 of 11

7 c) Positive and negative controls shall be included in all batches performing microscopic, culture, identification and susceptibility testing. d) The procedure shall indicate clearly which samples are to be cultured. e) A decontamination procedure shall be used to prepare specimens for microscopy and culture; the type of decontaminant used shall be stated. f) The concentration method used for the culturing of Mycobacteria in sputum, urine and gastric washings shall be stated. It should only allow a contamination rate of 5% per batch. g) The use of a specific culture medium for primary isolation shall be indicated. h) The temperature for the incubation of primary cultures shall be 37 O C. i) The length of incubation of the culture, before a negative report is issued, shall be clearly stated Identification of Isolates: Parasitology All suspected mycobacterial isolates shall be checked for acid fastness Species identification shall be done, and the method(s) used shall be specified The panel of reference cultures shall be maintained as controls for identification tests Sensitivity tests shall be carried out on all relevant isolates of Mycobacterium tuberculosis using an agreed method for M.tuberculosis and slow growing non tuberculous Mycobacteria A method for rapid growing non tuberculous mycobacteria should be available All samples shall be examined in a fresh state. When specimens are not examined immediately, any of the following steps shall be taken to preserve the sample: a) Unstained and iodine stained direct wet mounts b) A concentration procedure c) A permanent stain preparation d) Use of modified Ziehl Neelsen (Cryptosporidia) Where an ocular micrometer is available for determining the size of ova, larva, cysts or trophozoites, the ocular micrometer shall be calibrated The following reference materials should be available at the work bench: a) Permanent mounts b) Slides c) Printed atlases d) Similar illustrations and descriptions A procedure manual for parasitology shall be available in the work area, and shall include: a) Instruction for the proper collection and handling of specimens. b) Methods c) Criteria for the identification of ova and parasites Zinc sulphate shall be checked for specific gravity, and the zinc flotation dilution fluid stored in a tightly stoppered bottle Haematoxilin stain shall be prepared freshly on each day of use, and routinely checked with control specimens. SANAS Page 7 of 11

8 Polyvinyl alcohol fixative shall be checked for the adhesive properties and lack of gelling The following shall apply to blood films for malarial parasites: a) Both thick and thin films shall be made and examined. b) The preference of capillary or venous blood shall be clearly stated. c) When anti-coagulated venous blood is used, the films shall be made immediately. d) The slides shall be adequately identified. e) The following appropriate staining techniques shall be used: i) Field s stain for thick films ii) Wright, Giemsa stains for thin films iii) Appropriate buffers at ph 7.2 f) For thick films, are at least 100 fields shall be examined before a report "No parasites seen" is given Microbiological Serology 3.3 Reporting of Results Relevant steps shall be taken to exclude prozone reactions in manual syphilis serology testing Where appropriate, antigen controls, complement heterophile rheumatoid factor and auto-antibody controls shall be included Bacteriology: Final reports shall normally be available within hours Preliminary reports shall be issued when final reports are not available within 48 hours Where appropriate, reports shall indicate the safe trough and therapeutic levels. An interpretation should be offered by the reporting pathologist/scientist Mycology: Preliminary reports of the microscopic examination of specimens shall be made available to the requesting clinician Final reports shall be available within 4 to 6 weeks Mycobacteriology: If results are entered into a separate register, the record system shall allow for the easy and timeous retrieval of all results obtained for a particular patient Microscopy reports shall indicate the quantity of AFB in the smear, according to IUAT Microscopy reports shall be issued within 24 hrs of receipt of the specimen Interim reports shall be issued for specimens, which are culture positive Final reports which are awaiting identification and susceptibility testing shall be issued within a maximum period of 8 weeks. Exceptions (e.g.: Non tuberculous mycobacteria) may take longer. SANAS Page 8 of 11

9 Where appropriate, the report shall make reference to insufficiency of quality and quantity regarding the sample Serology: Where appropriate, the diagnostic values shall be indicated; a comment on the significance of the titres shall be made The need for a further sample, from the patient, for confirmatory or other testing shall be clearly indicated on the report. 3.4 Laboratory Equipment The size of autoclaves used shall be appropriate for the workload When liquid nitrogen is used as a facility, the levels shall be checked often to establish the liquid phase level The laboratory shall indicate clearly if the following microscopes are in use: A dark ground microscope; A phase contrast microscope; A polarised light microscope; A fluorescent light microscope The number of hours of use of high-energy light sources should be recorded (if not done automatically). 3.5 Laboratory Safety and Accommodation Environmental Conditions Laboratory Safety: All open specimens and cultures involving Class III and IV organisms shall always be handled within a biological safety cabinet Gloves shall be worn A specified and appropriate disinfectant, with stated appropriate concentration shall be used for swabbing benches Only centrifuges that are capable of containing biohazardous aerosols and spillages shall be used The following shall apply to the use of ether: All work, involving ether, shall be performed under well-ventilated conditions, in the absence of any source of ignition The ether disposed of in a safe manner Records of the disposal of ether shall be kept The following personal protective equipment (PPE) shall be available for the loading and unloading of autoclaves: Heatproof gloves Face shields Protective aprons All persons shall be trained in their operation. SANAS Page 9 of 11

10 4. Authorship Environment and accommodation Sufficient space shall be provided to enable all laboratory procedures to be carried out safely and competently A biological safety cabinet shall be used routinely for the handling of cultures of systemic fungal pathogens (e.g. histoplasma) The area used for mycobacteriological examination procedures shall contain a biohazard class 1 cabinet for TB microscopy, and a biohazard class 2 cabinet shall be used for TB cultures and preparation of TB microscopy slides T.B Laboratories When performing cultures using a microbiological safety cabinet, protective clothing that ties at the back shall be worn at all times Gloves shall be worn at all times. This Technical Requirement document has been prepared and revised by the Medical Specialist Technical Committee members. SANAS is indebted to the contribution made by the committee members in the preparation of this document. SANAS Page 10 of 11

11 ADDENDUM 1. AMENDMENT RECORD Proposed Section Change By: SM All Changed from a technical guidance (TG 34) document back to a Technical Requirement (TR 34) document. QM, FM All Removed The Bold sections in this document apply to small microbiology laboratories.. Unbolded all the Bold sections for small laboratories. STC Section 1 Deleted and shall be used in conjunction with the requirements listed in the ISO Standard. For ISO/IEC 17025:2005 clauses, refer to the equivalent sections in the standard. Deleted: SANAS accredited Added: requiring accreditation to ISO or ISO/IEC Section 3.1 Deleted: (Clauses 4.1; 4.5; 5.4) Section 3.2 Deleted: (Clause 5.5) Section Deleted: 1 Added: 2 Section (e) i Deleted: Apparently Section (a) i Deleted: included with each batch, and results recorded. (Standard practise to do it once a week.) Section (b) Added: iv) Kirby Bauer disc susceptibility testing. Section Deleted: Being used Added: in manual syphilis serology testing. Section 3.3 Deleted: (Clause 5.8) Section Added: -72 Section Deleted: All slides, blocks, reports and all records shall be maintained according to the guidelines of the Royal College of Pathologists Feb The Retention and Storage of Pathological Records and Archives. Section 3.4 Deleted: (Clause 5.8) Section Deleted: shall Added: should Section 3.5 Deleted: (Clause 5.2) Section Added: shall be used and preparation of TB microscopy slides.. SANAS Page 11 of 11

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