MED-002-Microbiology-29-Mar-2019

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ACCREDITATION SCHEME FOR LABORATORIES

Technical Notes MED 002


Specific Criteria for Microbiology
Section

Technical Notes MED 002 - Microbiology, 29 March 2019


MED 002-Microbiology, 29
TheMarch 2019
SAC Accreditation Page 1 ofSingapore
Programme is managed by Enterprise 10
© All rights reserved
1. Introduction & Scope
1.1 a) This document describes the specific requirements for clinical
microbiology laboratory to be accredited.

b) The International Standard ‘ISO 15189 Medical laboratories –


Particular requirements for quality and competence’, other MEDICAL
Series Technical Notes published by SAC-SINGLAS shall be studied
in conjunction with this document.

1.2 Microbiology includes bacteriology, mycobacteriology, mycology,


parasitology, virology and serology. The tests may be performed in
subspecialty laboratories, in general microbiology laboratories, or as part
of a general or core laboratory. Laboratories shall be accredited
according to their scope of tests.

2. General Technical Note : Medical 001


2.1 Please refer General Technical Note: Medical - 001 for the following:

 Personnel
 Accommodation & Environmental Conditions
- Physical Facilities and Laboratory Safety
 Laboratory Equipment – Calibration & Maintenance
 Pre-examination Procedures
– Requisitions, Collection and Handling of Specimens
 Examination Procedures
- Test Methods and Method Validation
 Assuring Quality of Examination Procedures
- Quality Control, Proficiency Testing, Reagents and Reference
Materials
 Post-examination Procedures
- Retained Samples and Waste Disposal
 Reporting of Results

3. Personnel
3.1 Refer to Personnel in General Technical Note: Medical - 001.

4. Accommodation and Environmental Conditions


4.1 Refer to Accommodation & Environmental Conditions - Physical Facilities
and Laboratory Safety in General Technical Note: Medical - 001. In
addition to that the following is applicable to microbiology laboratory.

MED 002-Microbiology, 29 March 2019 Page 1 of 10


4.2 Safety
4.2.1 Biological safety cabinet:

a) A biological safety cabinet shall be provided for handling cultures.


b) Handling of cultures with mycelia growth during laboratory
investigations should be done in a biological safety cabinet.
c) The biological safety cabinet shall meet minimum requirements for
microbiological work. It shall be certified annually to assure that the
filters are functioning properly and the airflow rates meet
specifications.
d) There shall be written procedures for staff handling microbiological
cultures to ensure that they recognize potential risk groups 3 (RG3) or
biothreat agents, and take further action to ensure safety of personnel
and correct identification of the pathogen. There shall be procedures
for handling suspected RG3 agent and other agents with special
hazard of serious laboratory-acquired infection suspected e.g.
Burkholderia pseudomallei, Neisseria meningitidis, Brucella spp.

4.2.2 Centrifuge: Sealed buckets shall be used in centrifuges when infectious


organisms are present or are likely to be present. Where infection may
be acquired by aerosolisation, the bucket shall be unloaded in a
biological safety after waiting for a suitable time before opening the
sealed buckets.

4.2.3 Autoclave: All persons using the autoclave should have been trained and
demonstrate competence in its operation. Face shields and protective
aprons should be used for unloading liquids. Heat-proof gloves should
be available for unloading the autoclave.

4.2.4 There should be documented policies for handling spills of contaminated


materials.

4.3 Mycobacteriology
4.3.1 The laboratory shall be assessed according to the types of tests offered:
Acid-fast smear, Molecular diagnosis and Mycobacterial culture and
identification. A risk assessment should be done and safe work practices,
accommodation and environment provided to minimize risk of
transmission to staff. The use of a BSL-3 or equivalent facility for TB
culture work will need to be in compliance with requirements under the
Biological Agents and Toxins Act.

5. Equipment:

5.1 Refer to Laboratory Equipment – Calibration & Maintenance in General


Technical Note: Medical - 001. In addition to that the following is
applicable to microbiology laboratory.
MED 002-Microbiology, 29 March 2019 Page 2 of 10
5.2 Anaerobic jars should be checked with methylene blue strips, fastidious
anaerobes or other appropriate procedure.

5.3 Parasitology
5.3.1 A calibrated ocular micrometer is required for the measurement of eggs
cysts and larva.

6. Pre-examination Procedures
6.1 Refer to Pre-examination Procedures – Requisitions, Collection and
Handling of Specimens in General Technical Note: Medical - 001.

6.2 There must be procedures/policies for appropriate collection transport


and rejection of samples including how to deal with samples submitted
after office hours.

Clients should be instructed to send microbiological specimens in


appropriate leak-proof containment. Specimens sent to a referral
laboratory should also be packaged and labelled with proper
containment.

7. Examination Procedures
7.1 Bacteriology

Suitable control strains, for identification and antibiotic susceptibility


testing, should be traceable to type collections and the passage history
documented. There should be procedures for maintenance of the control
strains including when to change aliquots or re-characterize the controls.

7.1.1 Respiratory cultures: All sputa shall be assessed for adequacy of


specimen (i.e. whether good quality sputum was obtained). Laboratories
handling throat swabs should have facilities to identify C. diphtheriae in-
house or via a referral laboratory, when that is requested by the
physician.

7.1.2 Urine cultures: The laboratory should perform and report quantitative
cultures and use media and procedures that permit isolation of clinically-
relevant Gram positive and negative bacteria. Specimens should be
processed in a timely manner so that bacterial overgrowth does not occur
in the urine. Where delay in plating is expected, provision should be
made to prevent overgrowth: viz. storing at 4 deg C, or by adding
preservative to the container, and or using dip slide cultures. For dip
slide cultures, there must be evidence that the estimated counts correlate
with actual colony counts.

7.1.3 Urethral and Cervical cultures: Transport and culture conditions should
be satisfactory for the isolation of N.gonorrhoeae.

MED 002-Microbiology, 29 March 2019 Page 3 of 10


7.1.4 Stool cultures: The procedure should permit isolation and identification of
enteric pathogens in patients with diarrhoea (using appropriate selective
media and enrichment media). The range of pathogens detected by the
culture should be indicated to lab users, and procedures in place to cater
to requests for gastroenteritis agents not routinely detected. The policy
for reporting susceptibility test results should not lead to inappropriate
antibiotic use.

7.1.5 Cerebrospinal fluid: Specimens should be processed and cultured


immediately on receipt. A Gram stain should be performed routinely on
sediments and results reported directly to the physician. There should be
procedures to check and resolve discrepancies between Gram stain and
culture. Media and incubation conditions must permit recovery of
fastidious bacteria (e.g. Neisseria meningitidis, H. influenzae) and
cultures should be performed on both smear positive and negative CSF
specimens.

7.1.6 Blood cultures: Sterile technique for drawing and handling blood cultures
should be defined. The blood culture system shall be designed to recover
both aerobic and anaerobic organisms. Sub-cultures and/or stains need
not be done on blood cultures performed by automated methods if the
bottles are monitored as recommended by the vendors. There shall be a
policy for immediate notification of positive blood results

7.1.7 Wound cultures: When indicated, Gram stain of direct smears should be
examined and reported. Both aerobic and anaerobic cultures should be
performed on specimens from appropriate sites.

7.1.8 Anaerobic cultures: Specimens and cultures should be placed in an


anaerobic atmosphere as soon as practicable.

7.2 Mycobacteriology
7.2.1 Rapid and reliable methods shall be used for microscopic examination,
isolation, identification and antimycobacterial susceptibility testing of
Mycobacterium tuberculosis complex.

7.2.2 Certain specimens (e.g. sputum) should be concentrated before AFB


smear examination and culture.

7.3 Mycology

7.3.1 Technical procedures for isolation and identification of fungi directly from
specimens and cultures shall be available.

7.3.2 These include the use of microscopy, stains, selective media,


biochemical tests, serological tests and nucleic acid tests.

MED 002-Microbiology, 29 March 2019 Page 4 of 10


7.4 Parasitology

7.4.1 A concentration method and a permanent mount should be used to


examine stools for optimal detection of parasites. The method should fit
the purpose according to clinical indication. If a wet mount is performed,
the limitation should be communicated in the report.

7.4.2 Both thick and thin blood films should be used in the examination for
malarial parasites in suspected cases of malaria.

7.4.3 Stained films should be washed with a buffer of known pH (6.8 - 7.2).
Thick film examination should include at least 100 oil immersion fields
(approximately 10-15 mins).

7.4.4 The physician shall be informed immediately of a blood film positive for
malaria. Where infection by P. knowlesi is suspected, there should be a
process for identification.

7.5 Serology for Infectious Agents

7.5.1 In addition to the general requirements described in the “IMMUNOLOGY”


section, the following points apply to infectious disease serology.

7.5.2 The method used should be appropriate to the clinical indication.


Relevant interpretive comments should be inserted, and request for a
follow up specimen made where necessary.

7.5.3 Qualified personnel shall be available to provide consultation regarding


the interpretation of results.

8. Assuring Quality of Examination Procedures –

8.1 Quality Control and Proficiency Testing


Refer to Quality control and proficiency testing in General Technical
Note: Medical - 001. In addition to that the following is applicable to
microbiology laboratory.

8.2 Results of control tests shall be reviewed before reporting patient results.

8.3 There shall be records of at least weekly review of quality control results
by the head or designee.

8.4 External quality assurance (proficiency testing) specimens shall be tested


in the same manner as patient specimens. Follow up action, where
applicable, should be documented.

8.5 Corrective actions taken when errors or unacceptable results are


detected or when tolerance limits are exceeded shall be documented.
Any impact on test methods or reporting procedure should be addressed.

MED 002-Microbiology, 29 March 2019 Page 5 of 10


8.6 For areas where proficiency testing programme is not available, test
performance shall be checked at least semi-annually with appropriate
procedures like inter-laboratory testing. The microbiology laboratory head
or designee on receipt shall review results of proficiency testing
programme/s and prompt corrective actions taken in response to
unacceptable results on the survey report form shall be documented.

8.7 Reagents / Stains / Media / Kits / Antimicrobials

8.7.1 All reagents, stains, media, kits and antimicrobials should be stored as
recommended by the manufacturers and used within their indicated
expiry dates.

8.7.2 They should be labelled, as applicable and appropriate, with the content
and quantity, concentration or titre date received or prepared, date placed
in service, storage requirements and expiry date. If there are multiple
components of a reagent kit, the laboratory must use components of
reagent kits only with other kits that are of the same lot number unless
otherwise specified by the manufacturer.

8.7.3 New reagent lots shall be checked against old reagent lots or with
suitable reference material before being placed in service.

8.7.4 The use of commercial reagents and controls shall comply with
manufacturer’s instructions.

8.7.5 The laboratory shall have documented records of quality control results of
test procedures, reagents, stains, media, kits, antimicrobials, etc. These
should be checked prior to being placed in service and subsequently be
monitored at specified intervals for performance or limits of acceptability.
Corrective actions should be documented when such results are
unacceptable.

8.7.6 The laboratory shall perform and record results with positive and negative
controls at specified periodic intervals. Recording of qualitative
observations e.g. colour change, should be unambiguous with respect to
the expected result.

8.8 Media

8.8.1 The laboratory shall ensure that all media prepared in-house are sterile,
able to support growth and are appropriately reactive biochemically. This
will require that the laboratory maintains stock reference organisms and
tests the media before or concurrent with use.

8.8.2 For purchased media, the manufacturer shall document to the user that
their quality control activities meet the criteria described as above, 8.9.1
The laboratory should test media that are known to show significant
variability in performance e.g. chocolate agar (for H. influenzae),
campylobacter agar, Thayer-Martin medium
MED 002-Microbiology, 29 March 2019 Page 6 of 10
8.8.3 The user shall visually examine each batch of media for breakage,
contamination, appearance, or evidence of freezing or overheating.

8.8.4 All the quality control procedures that are carried out in the laboratory
shall be documented.

8.8.5 A record should be kept of all lot numbers and expiration dates of the
media received for the past two years.

8.8.6 Reference cultures and sera should be maintained for the proper control
of stains, media, reagents, antimicrobial susceptibility tests and
serological tests.

8.9 Bacteriology

8.9.1 Each new batch of stains (Gram stain, special stains, and fluorescent
stains) should be checked at least weekly with known positive and
negative control organisms for intended reactivity and results recorded.

8.9.2 Guidelines shall be established for the number and type of antibiotics to
be reported for organisms isolated from different sites of infection. They
should be clinically relevant and include suppressing the results of
selected antibiotics to encourage prudent antibiotic use.

8.9.3 Only single isolates or pure cultures shall be used for the final
performance of antibiotic susceptibility testing. Each new lot of antibiotic
discs should be checked for activity before being placed in service and at
least weekly thereafter with reference cultures.

8.9.4 Inoculum density should be controlled using a turbidity standard or other


acceptable method and tolerance limits for potency of antimicrobials
(criteria for out of control) should be established.

8.9.5 Written criteria shall be available for interpretation of the end point or
zone size.

8.10 Mycology

8.10.1 All stains shall be checked with appropriate positive and negative controls
for each new batch of preparation and at least daily. (For stains like
Gomori’s methenamine silver, the slide itself serves as the negative
control).

8.10.2 Serological (antibody and antigen) and nucleic acid tests should be run
with known positive and negative control organisms or sera with each
new batch of preparation and when appropriate.

MED 002-Microbiology, 29 March 2019 Page 7 of 10


8.11 Mycobacteriology

8.11.1 Stains

8.11.1.1 Stains for acid-fast bacilli should be checked with known positive and
negative control organisms and the results recorded for each new batch
and thereafter at least weekly or on each day of use (whichever is less
frequent)

8.11.2 Identification

8.11.2.1 A known strain of M. tuberculosis should be run whenever identification of


M. tuberculosis complex is performed.

8.11.2.2 Biochemical tests used for identification should be checked each day of
use with appropriate positive controls.

8.11.2.3 Nucleic acid probes or nucleic acid amplification technique for


mycobacterial identification should be accompanied by appropriate
positive and negative controls on each day of use.

8.11.2.4 Temperature growth requirements and photo-reactivity studies shall be


done when appropriate if complete identification of mycobacterial
organisms cultured is performed by conventional methods. Alternative
methods for species identification e.g. HPLC, Maldi-TOF, DNA
sequencing, should have been verified for accuracy.

8.11.3 Susceptibility Testing

8.11.3.1 A control strain of M. tuberculosis, which is sensitive to all


antimycobacterial agents, should be included with each run.

8.12 Parasitology

8.12.1 Quality control checks of the specific gravity of concentrating solution


(e.g. Zinc sulphate) should be done periodically.

8.12.2 All permanent stains shall be checked, together with controls, for
intended staining results at least monthly, or with each test if test is
performed less frequently. Special stains used to detect specific
organisms (e.g. acid-fast, fluorescent stains) shall be checked with
appropriate control organisms each time the stain is used.

8.13 Virology

8.13.1 This section applies to any laboratory providing facilities for the diagnosis
of viral infections, which include cell culture, antigen detection, serology
or molecular diagnosis. (Refer to “MOLECULAR PATHOLOGY” section).

MED 002-Microbiology, 29 March 2019 Page 8 of 10


8.13.2 Sterility of all culture media shall be ensured following the addition of
ingredients post sterilization.

8.13.3 All cell cultures shall be tested for mycoplasma contamination


immediately upon receipt, after recovery from the deep freezer and at
regular intervals as the cultures are maintained in the laboratory.

8.13.4 Animal sera for use in culture media shall be tested to exclude toxicity to
cells.

8.13.5 Appropriate cell lines should be available to support the services offered
by the laboratory.

8.13.6 Tube monolayer cultures should be incubated for a sufficient time to


recover the relevant viruses. Tube cultures should be checked for
cytopathic effect every other day for the first two weeks, unless other
additional diagnostic methods are used (e.g. shell vials), in which case
the observation schedule may be modified as appropriate.

8.13.7 Media and diluents shall be checked for sterility and pH.

8.13.8 There shall be documentation of cell types, source, passages and media
used in their propagation.

8.13.9 Worksheets or records shall indicate titres, when known, of reagents and
control sera.

8.13.10 There shall be known positive and negative controls with each run of test
specimens for all direct antigen tests on patient specimens.

9. Post-examination Procedures

9.1 Sample Storage

There should be suitable facilities for storage of samples that may need
re-testing. There should be a policy on the duration of storage, to allow
retrieval of samples or significant isolates for re-testing or further testing.

9.2 Serology for Infectious Agents

Serum shall be stored for an appropriate length of time when paired titres
are expected to be performed.

10. Reporting of Results

10.1 Reports with abnormal results shall be reviewed by senior personnel and
evaluated for conformity with the clinical information available

10.2 Reports should be available in a timely manner, and preliminary reports


issued if specimens take a longer time to work up.
MED 002-Microbiology, 29 March 2019 Page 9 of 10
10.3 Results of control tests shall be reviewed before reporting patient results.
Tests are not to be released if controls are unacceptable. Unacceptable
control results shall be reported to the supervisor and corrective action
documented.

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