Indian Journal of Medical Microbiology, (2004) 22 (2):81-86

Special Article

QUALITY ASSURANCE IN MICROBIOLOGY

DR Arora

Abstract
Quality assurance (QA) is the total process whereby the quality of laboratory reports can be guaranteed. The term quality control covers that part of QA, which primarily concerns the control of errors in the performance of tests and verification of test results. All materials, equipment and procedures must be adequately controlled. Culture media must be tested for sterility and performance. Each laboratory must have standard operating procedures (SOPs). QA of pre-analytical, analytical and post-analytical stages of microbiological procedures should be incorporated in SOPs. The laboratory must be well lit with dust-free air-conditioned environment. Environmental conditions should be monitored. Supervisory and technical personnel should be well qualified. The laboratory should participate in external and internal quality assurance schemes. Key words: Quality assurance, quality control, standard operating procedures, external quality assurance, internal quality assurance

Microbiological investigations are important in the diagnosis, treatment, and surveillance of infectious diseases and policies regarding the selection and use of antimicrobial drugs. It is, therefore, essential that test reports are relevant, reliable, timely, and interpreted correctly. High cost of culture media and reagents, lack of rational approach to the selection and use of microbiological investigations, and a shortage of trained technical staff and clinical microbiologists are important factors in preventing the establishment of essential microbiology services in developing countries. Quality assurance (QA) Quality assurance has been defined by WHO1 as the total process whereby the quality of laboratory reports can be guaranteed. It has been summarized as the:right result, at the right time, on the right specimen, from the right patient, with the result interpretation based on correct reference data, and at the right price. Quality control (QC) The term QC covers that part of QA, which primarily concerns the control of errors in the performance of tests and verification of test results. QC must cover all aspects of every procedure within the department. It must be practical, achievable, and affordable. All materials, equipment and procedures must be adequately controlled. Culture media must be tested for sterility and performance.2 *Corresponding author Department of Microbiology, Postgraduate Institute of Medical Sciences, Rohtak - 124 001, Haryana, India. Received : 27-02-2004 Accepted : 15-03-2004

Standard operating procedures (SOPs) Each laboratory must have SOPs, sometimes referred to as the local laboratory bench manual. It is required for the following reasons: a) to improve and maintain the quality of laboratory service to patients and identify problems associated with poor work performance. b) to provide laboratory staff with written instructions on how to perform tests consistently to an acceptable standard in the laboratory. c) to help avoid short-cuts being taken when performing tests. d) to provide written standardized techniques for use in the training of laboratory personnel. e) to facilitate the preparation of a list and inventory of essential reagents, chemicals and equipment. f) to promote safe laboratory practice.

Preparation of SOPs SOPs must be written and implemented by a qualified experienced laboratory officer, and followed exactly by all members of staff. Each SOP must be given a title and identification number and be dated and signed by an authorized person. Effective QA detects errors at an early stage before they lead to incorrect test results. Laboratory personnel need to be aware of the errors that can occur when collecting specimens (pre-analytical stage), reporting and interpreting test results (post-analytical). Following apply

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to the QA of the pre-analytical, analytical, and postanalytical stages of microbiological procedures and should be incorporated in SOPs for microbiology laboratory. Pre-analytical stage SOPs need to describe selection and appropriate use of microbiological investigations, proper filling of request form, collection and transport of specimens, and checks must be made when the specimen and request form reach the laboratory. Each of these can have a minor effect on accuracy of the result. Appropriate use of microbiological investigations This aspect of QA requires collaboration between laboratory personnel and clinicians. The fewer the resources the more important it is to establish priorities based on clinical needs. Clear guidelines should be provided on the use and value of specific microbiological investigations. Request form Each specimen must be accompanied by a request form which details: a) Patients name, age, gender, occupation, outpatient or inpatient number, ward or health center. b) Type and source of specimen, date and time of collection. c) Investigation(s) required. d) Clinical note summarizing the patients illness, suspected diagnosis and information on any antimicrobial treatment that may have been started at home or in the hospital. e) Name of medical officer requesting the investigation. Collection and transport of specimens It must be borne in mind that the quality of microbiological report is fundamentally dependent upon the quality of the specimen submitted, nature and timing of specimen, the suitability of sampling method and transport, use of transport media, and transit time and adequacy of information given to the laboratory. Specimens such as urine and sputum are best collected soon after a patient wakes up when organisms have had the opportunity to multiply over several hours. Blood for culture is usually best collected when a patients temperature begins to rise. The time of collection of most other specimens will depend on the condition of the patient, and the times agreed between

the medical, nursing, and laboratory staff for delivery of specimens to the laboratory. Every effort must be made to collect specimens for microbiological investigation before antimicrobial treatment. The laboratory must issue written instructions to all those responsible for collecting microbiological specimens. Checking of specimen and request form When the specimens reach the laboratory these should be checked to ensure that correct specimen has been sent and the specimen is the same as that on the request form. Also included should be the comment that the specimen requires immediate attention, e.g., CSF, urine, swabs not in transport media or faecal specimen containing blood and mucus, etc. Dry faecal swab, saliva instead of sputum, eye swab that has not been freshly collected, and a leaking specimen, are not acceptable. Analytical stage The following should be incorporated in the microbiological SOPs covering the analytical stage: a) Detailed procedure for examining different specimens. b) Staining techniques and QC of stains. c) Aseptic techniques and safe handling of infectious material. d) Preparation and QC of culture media and preservation of stock strains. e) Inoculation of liquid and solid media. f) Reading and interpretation of cultures. g) Techniques used to identify pathogens. h) Antimicrobial sensitivity testing and QC of procedures and antibiotic discs. i) j) Cleaning and QC of equipment used in microbiology laboratory. Immunologic techniques and QC of antigen and antibody reagents. Disposal of specimens and cultures.

k) Safe working practices. l) m) Cleaning of glassware, plasticware, etc. n) Sterilization procedures and their control. Control of stains and reagents All stains and reagents must be clearly labelled, dated, and stored correctly. These should not be used

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beyond their expiry date or when they show signs of deterioration, such as, abnormal turbidity and decolouration. At regular intervals and whenever a new stain is prepared, control smears should be stained. Control smear for Ziehl-Neelsen stain should include smears with few to moderate number of AFB. Control smear for Gram stain can be prepared from mixed culture of staphylococci and Escherichia coli. Smear should not be too thick. When a smear is too thick, the decolourization is often incomplete which can result in gram negative organisms being reported as gram positive.

Control of Equipment All equipment used for tests, having a significant effect on the accuracy of result of the test should be calibrated before being put into service and on regular intervals thereafter. For each item of equipment there should be clear operating and cleaning instructions, and service sheets. Regular cleaning, servicing and maintenance are essential if the equipment is to remain in good working order and safe to use. A brief list of some of the equipment, the monitoring procedures to be carried out, and the frequency and tolerance limit is given in table 1. Specimen containers should be inspected regularly, especially the caps of bottles and tubes for missing or worn liners.

Table 1 : Quality control surveillance procedures of commonly used microbiology equipment Equipment Refrigerators Freezers Incubators Water baths Autoclaves Anaerobic jars Serology rotator Centrifuges Safety hoods Procedures Recording of temperature Recording of temperature Recording of temperature Recording of temperature Test with spore strip (Bacillus stearothermophilus) Methylene blue indicator strip Count revolutions per minute Check revolutions with tachometer Measure air velocity across face opening Schedule Daily Daily Daily Daily At least weekly With each use With each use Monthly Semiannually or quarterly Tolerance limits 2C to 8C 8C to 20C 60C to 75C 35.5C 1C 36C to 38C 55C to 57C No growth of spores in subcultures indicate sterile run Conversion of strip from blue to white indicates low O2 tension 180 rpm 10 rpm Within 5% of dial indicator setting 50 feet airflow per minute 5 feet per minute

Post-analytical stage SOP needs to include reporting and verifying of microbiological test results, interpreting test reports correctly, taking appropriate action when a result has serious implications for a patient or public health. Reporting results The terminology and format used in reporting should be standardized and agreed between laboratory personnel and clinicians. Any preliminary report must be followed by a full written report. All reports must be checked for correctness and clarity and signed by head of the department. Report distribution and delivery systems must be efficient and urgent reports should be telephoned at all the significant stages of the

investigation. Appropriate steps must be taken to ensure confidentiality of reports both in the laboratory and during transfer. Those receiving the reports should consult the laboratory when any part of the report is not clear. There must be effective communication between those requesting tests and laboratory staff. Microbiologist should be prepared to give advice on the type of investigations that might be helpful in the diagnosis and be prepared to advise on antibiotic treatment. A patients interest is generally better served by an early report of the provisional identification of a potential pathogen, and its possible antibiotic sensitivities, than by a delayed report with a precise and confirmed identification.

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Facilities Each laboratory must possess space for sample collection, sample analysis, storage of samples, reagents, chemicals, stationary, record, etc., washing, media preparation, and autoclaving, seminar room and library, staff room and toilets.3 Laboratory must be well lit with dust-free, airconditioned environment and uninterrupted power supply. The laboratory must monitor, control and record environmental conditions like biological sterility, electromagnetic disturbances, radiation, humidity and temperature. Types of laboratories Small laboratory: deals with up to 50 patients per day. Medium laboratory: deals with 51 to 500 patients per day. Large laboratory: deals with >500 patients per day. Superspeciality laboratory: restricting to one or two disciplines. Staff and qualifications Supervisory personnel Small and medium laboratory may be manned by an MBBS or an MSc in concerned speciality with at least 5 years experience in laboratory medicine. Large and superspeciality laboratory shall be manned by a medical person with postgraduate qualification in Pathology, Microbiology or Biochemistry/PhD in the respective discipline. Technical personnel The technical person performing the test and reporting results should have one of the following qualifications: a) Graduate in medical laboratory technology. b) Science graduate with one-year experience in a medium-sized laboratory. c) Diploma in medical laboratory technology with 2 years experience in a medium-sized laboratory. d) A laboratory may appoint up to 25% of staff without experience but with requisite qualifications or with more than 10 years of laboratory experience with at least matriculation in science.

Quality control of culture media4,5 Sterility Sterility control is a fundamental check for any medium. The level of control depends upon the type of medium. A medium that is prepared, dispensed then autoclaved, will require only one unit to be incubated overnight as a check of sterility. Media consisting of various non-sterile products, i.e., blood, serum, glucose, antibiotics, any other heat-labile or non-filterable solutions that may then require aseptic dispensing, would require strict sterility control up to a level of incubation of the entire batch for 3 days; if detected contamination exceeds 10%, the whole batch should be discarded. Performance A list of suggested organisms and acceptable results for the culture media most commonly used in clinical laboratories is given in table 2. Quality control of antimicrobial susceptibility discs Antimicrobial sensitivity discs must be tested at weekly intervals with standard control organisms of known susceptibility. QC of personnel There should be continuing education programme and in-service training. The staff should be encouraged to participate as often as possible in local, regional and national seminars and workshops. Blind unknown samples for laboratory testing should be included in test runs and source of error, if any, should be pin-pointed and corrected. External quality assessment (EQA) An EQA scheme should include testing for major pathogens.5 It should not be too complicated, costly, or time consuming. Although steps may be taken in a laboratory to ensure test results are reliable, a system of assessing a laboratory to do this to a satisfactory standard is recommended i.e. an EQA scheme. Participation in EQA schemes should always be regarded as additional to internal QC because it can assess only past performance when test results have already been reported and acted on.

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Table 2 : Quality control of commonly used media: suggested control organisms and expected reactions Medium Blood agar Bile-esculin agar Control organism Gp. A Streptococci S. pneumoniae Enterococcus species, -haemolytic Streptococcus, not Group D H. Influenzae N. gonorrhoeae Proteus mirabilis Klebsiella pneumoniae Escherichia coli K. pneumoniae E. coli Serratia marcescens E. cloacae P. mirabilis K. pneumoniae E. coli P. mirabilis E. coli N. gonorrhoeae Salmonella species N. gonorrhoeae N. lactamicus N. gonorrhoeae K. pneumoniae Enterobacter sakazakii E. cloacae P. mirabilis P. mirabilis K. pneumoniae Serratia marcescens S. Typhimurium P. mirabilis E. coli S. Typhimurium E. coli K. pneumoniae E. coli Salmonella species E. coli Shigella species Expected reactions Good growth, -haemolytic Good growth, -haemolytic Good growth, black No growth Good growth Good growth Pink throughout (positive) Pink slant (partial positive) Yellow (negative) Growth or blue colour (positive) No growth, remains green (negative) Zone of clearing (add 1N HCl) No zone of clearing Media cloudy (positive) No feather edge on streak line (negative) Pink colonies (lactose positive) Colourless colonies, no spreading Yellow (positive) No colour change (negative) Yellow (positive) No colour change (negative) Yellow (positive) No colour change (negative) Bluish (positive) Yellow (negative) Bluish (positive) Yellow (negative) Bluish (positive) Yellow (negative) Yellow (positive) Colourless (negative) Green (add 10% FeCl3) No colour change (negative) Colourless colonies, black centre No growth Red (add reagents) No development (negative) Red colonies (positive lysine) Yellow colonies (positive sugars) Transparent colonies (negative)

Chocolate agar Christensen urea agar

Simmons citrate agar Deoxyribonuclease Motility (semisolid agar) MacConkey agar Sucrose Maltose Lactose Lysine Arginine Ornithine o-Nitrophenol- p-D galactopyranoside (ONPG) Phenylalanine deaminase Salmonella-Shigella (SS) agar Voges-Prauskauer Xylose-Lysine-Dextrose (XLD) agar

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The main objectives of an EQA scheme are to confirm that a laboratorys SOPs and internal QC procedures are working satisfactorily. EQA schemes help to identify errors, to improve the quality of work, stimulate staff motivation, and assure clients that the laboratory is performing to the standard required and to provide reliable results. According to WHO guidelines, EQA scheme should operate monthly or at least four times a year. Nodal laboratories Normally, accredited laboratories should be selected as nodal laboratories to conduct this programme. In case the accredited laboratories are not available for a particular test, then non-accredited laboratories with sufficient infrastructure and resources may be considered to act as nodal laboratories. In EQA programme, participating laboratories are provided, by nodal laboratory, with sub-samples. Sub-samples provided to each participating laboratory should be homogenous so that any wrong result should not be attributed to sample variability. Instructions and a report

form, to be returned with results after one week, should be sent with the specimens to each participating laboratory. Each specimen should be examined in the same way as routine clinical samples (not as a QC specimen) and the results returned to the nodal laboratory. Nodal laboratory should assist the poorly performing laboratory, and arrange for training of staff. Refresher courses should be held periodically to introduce new tests. Internal quality assessment (IQA) Each laboratory should have an internal quality assessment scheme. This can be carried out by regular use of certified reference material, replicate testing, retesting of retained items and correlation of results of different characteristics of an item. Assurance When comprehensive control measures and relevant assessments are in place, a laboratory can claim a level of assurance. QA can be seen as the sum of QC, IQA and EQA, i.e., QA = QC + IQA + EQA.

References
1. Practice of quality assurance in laboratory medicine in developing countries. In Health laboratory services in support of primary health care in developing countries. World Health Organization, New Delhi, 1994:77-137. Cheesbrough, M. District laboratory practice in tropical countries. Part 1. (Cambridge University Press, New York). 1st edition, 1998:3-37. Specific guidelines for accreditation of clinical laboratories. National Accreditation Board for Testing and Calibration Laboratories 112,1998:8. 4. Koneman EW, Allen SD, Janda WM, Schreckenberger PC, Winn Jr. WC. Color atlas and textbook of diagnostic microbiology. LippincottRaven Publishers, 5th edition, 1997:133-120. Cheesbrough, M. District laboratory practice in tropical countries. Part 2. (Cambridge University Press, New York). 1st edition, 2000:1-9.

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