Article Category: article
Publicado en línea: 15 oct 2019
Páginas: 205 - 211
Recibido: 01 mar 2019
Aceptado: 01 may 2019
DOI: https://doi.org/10.21307/PM-2019.58.2.205
© 2019 Ewa Młodzińska et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
Quality assurance in microbiological diagnostics is the sum of all activities in a laboratory in order to guarantee the reliability of the obtained results. The World Health Organization (WHO) has defined quality assurance as “the total process thanks to which the quality of laboratory results may be guaranteed.” This means the right result, at the right time, on the right and properly collected material, from the right patient, interpreted in accordance with the current guidelines and obtained at the right price [20].
In accordance with Good Laboratory Practice (GLP), all activities in a laboratory should be performed in the best possible way, so that the results are as accurate as possible. Conducting a microbiological test serves to determine an infection’s aetiological factor and allows for the selection of optimal treatment. It also enables the collection and subsequent analysis of data for epidemiological purposes. Obtaining reliable, reproducible results of diagnostic tests carried out in the shortest possible time also allows one to make appropriate decisions in the field of targeted therapy and prevention, in addition to contributing to the optimization of empirical therapy (microbiological map). Providing high-quality laboratory services guarantees good quality medicine.
Guidelines on quality standards for medical diagnostic and microbiological laboratories contained in Regulation of the Minister of Health from 23 March 2006 (Journal of Laws of 2016, item 1665, as amended) in Annex no. 2 to the Regulation outline quality standards in the field of microbiological laboratory tests, the assessment of their quality and diagnostic value, as well as laboratory interpretation and authorization of test results. Point 7 of Annex no. 2 contains requirements with regard to ensuring the quality of laboratory tests [18, 19]. A laboratory is required to constantly conduct internal quality control in relation to the quality of tests in accordance with knowledge based on scientific evidence, using modern control tools for all the types of tests performed in the laboratory. In accordance with the provisions, a laboratory is also obliged to participate in an external assessment programme at all times as part of inter-laboratory quality control with regard to tests.
Laboratories performing microbiological tests may apply for accreditation. In accordance with the PN-EN ISO 15189:2012 standard, accreditation is a procedure as a result of which an authorized body formally confirms that an organization possesses the competencies to perform specific tasks [12]. Aim of accreditation is to build and strengthen confidence in the results of calibrations, tests, certified products and services, the qualifications of certified individuals, and certified management systems. The Polish Centre for Accreditation (PCA) is the national accreditation body. PCA leads accreditation processes and supervises various institutions, including research and calibration laboratories, as well as medical laboratories [9, 17].
The basis for accrediting medical diagnostic laboratories are two major standards: PN-EN ISO 17025: “General requirements for the competence of testing and calibration laboratories” – a standard for testing and calibration laboratories (regardless of their profile) and PN-EN ISO 15189:2012 “Medical laboratories – Requirements for quality and competence” – a standard for medical laboratories [11, 12]. One of the key requirements of the standards is to ensure the quality of test results in a laboratory by prosecute internal quality control programme and participation in extra-laboratory assessment programmes. Laboratories applying for accreditation must fulfil all the requirements of a standard. According to the PCA document “Akredytacja Laboratoriów Medycznych” (Accreditation of Medical Laboratories) DAM-01 3rd edition from 16.07.2015, the assessment of a laboratory in the accreditation process consists of a review of the documentation and an on-site assessment of the laboratory’s competencies to carry out the activities submitted for accreditation. Observation of the activities submitted for accreditation concerns the performance of tests conducted on real objects and in actual conditions, as well as the completion of pre-analytical processes. The laboratory’s competencies in the scope of completing pre-analytical processes are also verified during the assessment [9, 16].
A laboratory should develop an appropriate quality assurance programme in order to obtain accurate and reliable test results and meet the requirements of quality standards in the scope of microbiological laboratory tests. A quality assurance programme is an essential element of a laboratory’s management system. According to the PN-EN ISO 9000 standard, quality management means: “coordinated activities with regard to managing an organization and supervising it, which determine the quality policy, objectives and responsibilities, as well as their achievement by means such as: quality planning, quality control, quality assurance and quality improvement” [15]. A quality management system is an individual solution for a given laboratory. It comprises many components, but it is particularly important to determine the responsibility of the staff, to develop a code of conduct and to validate diagnostic procedures and their performance, as well as to observe the principles of health and safety at work. A laboratory must comply with the requirements of normative and legal acts, guidelines and recommendations, as well as the requirements and needs of the laboratory’s clients. Ensuring the quality of tests also means leading internal quality control with regard to tests at a laboratory and participating in external quality control programmes which enable the constant monitoring of the laboratory’s work and maintaining the high quality of routine diagnostics [1, 8].
A reliable diagnostics of infections enables making appropriate decisions with regard to the therapy and actions related to controlling infections and constitutes an important element in ensuring the safety of patients. All the tests performed in a laboratory should be subject to permanent, daily control aimed at assessing their quality. This is achieved, among others, by implementing a quality assurance programme at a laboratory. Characterizing particular stages of a test is an important element: from collecting the sample, conducting the test, interpreting the result to issuing the test report [7]. The regulation on quality standards for microbiological laboratory tests contains specific requirements for the development, implementation and application of procedures, which have been collected in Table I [18].
Requirements for the preparation, implementation and application of procedures included in the regulation on quality standards
| Procedure | Stage of the test |
|---|---|
Ordering a laboratory test Collecting the material for laboratory tests Transporting the material for laboratory tests Accepting the material for laboratory tests Storing the material for laboratory tests | Pre-laboratory phase |
Testing methods The laboratory uses testing methods which are in line with the current state of medical knowledge The testing methods used at the laboratory have been validated The laboratory compiles a list the performed tests and makes it available to the ordering entity The laboratory develops, implements and observes the procedures pursuant to the testing methods used Storing strains of microorganisms as well as model strains and other ones treated as reference strains upon the completion of testing Ensuring the quality of laboratory tests The laboratory conducts a permanent internal control of the tests’ quality The laboratory is permanently enrolled in an inter-laboratory control of the tests’ quality | Laboratory phase |
Presenting and issuing reports from laboratory tests | Post-laboratory phase |
The pre-laboratory phase encompasses the responsibility of the laboratory for the preparation of appropriate procedures regarding the methods of collecting, marking and transporting samples to the laboratory, as well as training the staff tasked with collecting samples and transporting clinical material, in accordance with the principles of safely handling infectious material.
In the laboratory phase, the laboratory checks whether the test complies with the order, i.e. the type of sample and its marking, evaluates the suitability of the material for testing, selects the appropriate testing method, develops detailed testing procedures for various materials, including materials ordered in the “CITO” mode (as promptly as possible), which enable the achievement of a reliable result. The laboratory also prepares procedures for the internal quality control of tests, including staff training and the latest diagnostic recommendations.
The post-laboratory phase covers the interpretation of the results, drafting the test report, preparing the test report in the correct form, which must be understandable to the client (a doctor, a hospital infection team etc.). It is also important that the test result is released at a time that allows for it to be used optimally.
Pre-laboratory, laboratory and post-laboratory activities affect the quality of test results and should be subject to regular quality control both an internal one, as well as an external one. Leading internal quality control allows for the quality of markings to be monitored constantly and it is complemented by an external control, which makes it possible to assess the quality of test routinely performed in the laboratory.
The fundamental element of a quality assurance programme consists of the intra-laboratory test control. When creating an internal quality control programme, a laboratory should take into account all the stages of tests which together form microbiological diagnostics, namely, the isolation and identification of microorganisms, testing the microorganisms’ susceptibility to antimicrobials, as well as detecting and interpreting their resistance mechanisms. For this purpose, appropriate control materials as well as control strain (reference strains and model strains), originating from respectable national and international culture collections, should be used. In the guidelines of the European Committee on Susceptibility Testing (EUCAST), in addition to control strains originating from the American Type Culture Collection (ATCC), strains from other collections are also listed:
the ATCC collection (American Type Culture Collection)
the English collection – NCTC (National Collection of Type Cultures)
the French collection – CIP (Collection de Institut Pasteur)
the German collection – DSM (Bacterial cultures from Deutsche Sammlung von Mikroorganismen und Zellkulturen DSMZ)
the Swedish collection – CCUG (Culture Collection University of Gothenburg)
the Spanish collection – CECT (Colección Espaῆola de Cultivos Tipo)
The set of control strains for internal quality control should match the scope of diagnostics carried out by a laboratory, it should enable control of all stages of a laboratory’s routine work. The laboratory should have reference strains for quality control of identification tests, microscopic slides, microbiological media, antimicrobial susceptibility testing, identification and detection of resistance mechanisms, automatic systems for the identification and microorganisms’ antimicrobial susceptibility testing. The preparation of procedures specifying the rules of handling quality control strains, appointing people responsible for the observance of these rules and keeping records which serve as evidence of supervising them are also vital.
Preparing a universal programme of the internal control of identification tests is not an easy task. Identification procedures may vary significantly between laboratories and their scope depends on the needs, capacity and experience of a laboratory in terms of diagnostics. Identification patterns may contain various available manual methods (isolation and selection plates, chromogenic media, biochemical tests), microscopic tests, biochemical examinations, serological tests, standardized commercial sets, automatic identification methods, as well as modern identification technologies (mass spectrometry) or advanced techniques using molecular methods.
In the identification of microorganisms, basic tests for particular properties may be used as screening tests (e.g. urea hydrolysis, catalase, indole, oxidase) or as key tests (e.g. coagulase). In order to confirm the reliability of a single test result, positive and negative control should be used. The microorganisms recommended for quality control of particular tests should be indicated by their manufacturer. The condition that is most difficult to be met is the simultaneous use of the appropriate organisms for the purposes of positive and negative control along with the tested strains. The lack of quality control of the performed marking poses a threat to obtaining the correct identification result.
Antimicrobial susceptibility testing is an important stage in routine microbiological diagnostics and it is usually carried out in order to identify the etiological factors of infections; one exception is the recently approved method of marking antimicrobial susceptibility directly from positive blood cultures [4]. The result of this method always has to be verified using the test’s settings for the isolated strain (see below). Testing susceptibility to antimicrobials necessitates the observance of standardized procedures. Various methods are used when testing antimicrobial susceptibility – both quantitative and qualitative ones. The fundamental and commonly used qualitative method is the disk diffusion method, developed at the beginning of the 1960s. This method takes advantage of a concentration gradient forming in the process of diffusing the substance in the medium and the end result is the determination whether a strain is susceptible or resistant to a given antimicrobial agent. The disk-diffusion method, along with the quality control requirements, is presented on the EUCAST website, in the document “Antimicrobial susceptibility testing EUCAST disk diffusion method” (Version 7.0, 2019) [2, 10].
Quantitative methods allow to determine the minimal inhibitory concentration (MIC) of a antimicrobial agent (mg/L). These are dilution methods in stock or in agar, where a bacterial suspension of a specific density is poured onto an antibiotic medium in incrementally smaller concentrations. The microdilution method in a liquid medium is a reference method for determining the activity of antibacterial agents
S – Susceptible. A microorganism is categorised as susceptible, standard dosing regimen, when there is a high likelihood of therapeutic success using a standard dosing regimen of the agent.
I – Susceptible, increased exposure. A microorganism is categorised as “susceptible, increased exposure” when there is a high likelihood of therapeutic success because exposure to the agent is increased by adjusting the dosing regimen or by its concentration at the site of infection.
R – Resistant. A microorganism is categorised as resistant when there is a high likelihood of therapeutic failure even when there is increased exposure [3].
Therefore, the proper assessment of a microorganism’s susceptibility is of vital importance, and obtaining reliable results is impossible without an internal quality control with regard to the markings used in a laboratory. In accordance with EUCAST recommendations, quality control should be performed for each test of a bacterial strain’s susceptibility to antimicrobial agent. Its basis lies in marking the susceptibility of the appropriate reference strains every day when the test was performed.
Control strains recommended for an internal quality control of susceptibility to antimicrobial agents, i.e. the routine and the extended one, along with the expected results and ranges (growth inhibition zones diameter and MIC values) are included in EUCAST guidelines (Version 9.0, 2019). In an extended quality control, the following control strains for detecting resistance mechanisms (ESBL, MRSA, VRE or HLGR) are recommended:
The above-mentioned method of quickly marking the susceptibility to antimicrobial agent directly from positive blood cultures is also available – RAST (rapid antimicrobial susceptibility testing) EUCAST “Methodology – The European Committee on Antimicrobial Susceptibility Testing – rapid antimicrobial susceptibility testing (RAST) directly from positive blood culture bottles” [4]. This method is based on the disk diffusion method and has been validated for blood culture bottles in the BACTEC (Becton Dickinson), BacT/ALERT (bioMerieux) and VersaTREK (Thermo Fisher) systems. In the procedure, the way of preparing the suspension has been modified (100–150 μl of undiluted blood is collected from a positive bottle and poured onto an antibiogram medium), the incubation time has been shortened – a reading may be made after 4, 6 or 8 hours at most, principles of interpreting the results have been changed and specific breakpoints along with quality control criteria for the RAST method have been prepared. “The European Committee on Antimicrobial Susceptibility Testing. Zone diameter breakpoints for rapid antimicrobial susceptibility testing (RAST) directly from blood culture bottles” [6]. Three quality control strains have been prepared for verifying the RAST procedure in EUCAST tables:
Manufacturers of automatic systems are responsible for establishing and maintaining adequate quality control to ensure the safety and effectiveness of the identification system. This includes checking positive and negative results for each reagent or substrate in order to ensure the appropriate activity of all the components of a card or a panel for the identification of microorganisms. However, this does not absolve the user of the system from conducting an internal quality control. The manual for a system, panels or cards should contain information regarding quality control, along with an indication of the reference strains and the expected results of quality control, as well as recommendations to the frequency of performing controls.
The reference strains for a quality control of applying susceptibility to antimicrobial agents using automatic methods should be used in accordance with the manufacturer’s guidelines, who is the entity responsible for the preparation and performance of an appropriate quality control – by checking the results for every antimicrobial agent in order to ensure the proper activity of all the components of a panel or an antibiogram card. The strains recommended for quality control are mostly common to all the methods of antimicrobial susceptibility testing. Additionally, kits for control panels/antibiogram cards may include other control microorganisms, whose properties enable fully controlling the antibiogram. A laboratory regardless makes the decision on the frequency of performing internal quality controls, quality control of a new batch or tests from a new delivery to the laboratory is most often recommended.
Issues regarding internal quality control at a microbiological laboratory are not limited exclusively to using control strains and the ways of handling them in order to maintain and confirm their characteristic features. Internal quality control also means assessing the effectiveness of laboratory procedures, supervising the methods, equipment, tests and reagents used in a laboratory, ensuring storage conditions for reagents and tests, using them as intended in the manner recommended by the manufacturer. Supervising all the records kept at a laboratory with respect to the conducted tests, the applied media, the density of the suspension, the time and conditions of incubation and checking compliance with the current recommendations are also part of intra-laboratory control.
External quality control programmes enable checking the effectiveness of internal control procedures and maintaining the appropriate quality of a laboratory’s work. All the tests performed at a laboratory should be assessed in an external quality control programme (the assessment should cover every stage of a test). Participation in an external control makes it possible to compare the diagnostic level of participants to the other laboratories participating in a given programme. The purpose of an external quality assessment is also to promote high standards of good laboratory practices, encourage the use of standardized methods, staff training, providing repair mechanisms for found errors and preventing them.
The quality standards for medical and microbiological diagnostic laboratories stipulate that it is the duty of a laboratory to regularly participate in interlaboratory quality control programmes organized by, among others, Centre of Quality Control in Microbiology (CQCM) (
A laboratory should handle the received sample in the same way as it would handle routinely tested material received from patients, without using special methods, reagents, additional tests or entrusting it to more qualified staff. When handling the sample, a laboratory should take note of the instructions received from the centre that organizes the programme.
The results of an external laboratory control should always be analysed by a laboratory and discussed with the staff and every incorrect result should be investigated in order to verify what actions need to be taken to prevent discrepancies. In order to eliminate discrepancies a laboratory should introduce corrective actions.
The information included in the present work indicates that microbiological laboratories are obliged to prepare and implement a quality assurance programme. The legal acts in force, current recommendations and guidelines clearly outline the duties of medical laboratories in the scope of ensuring the quality of microbiological tests while taking all the diagnostic stages into account, namely the pre-laboratory phase, the laboratory phase and the post-laboratory phase. The requirements apply to all laboratories not only the ones applying for accreditation.
Systematic quality control by means of observing the procedures of an internal quality control of tests routinely performed at a laboratory and assessment of the tests’ quality by participating in external control programmes are the basis for obtaining reliable results of microbiological tests.