Ministry of Health & Family Welfare Government of India TRAINING MODULE ON QUALITY CONTROL LABS FOR LIFE PROJECT 2018 p Bias \ Linearity —wian re See \ a ernal » i) 4 ( “one ethod metuation G A1\ v 3 uality Sigma Q Metrics a Ca VOLUME - II INTERNAL QUALITY CONTROLS QUALITATIVEMinistry of Health & Family Welfare Government of India TRAINING MODULE ON QUALITY CONTROL LABS FOR LIFE PROJECT MARCH 2018 VOLUME II GUIDELINES FOR QUALITY ASSURANCE OF QUALITATIVE LABORATORY TESTSPrinted in March 2018 By Labs for Life Project Ministry of Health and Family Welfare, Government of India Labs for Life is a partnership project of the Ministry of Health and Family Welfare (MoHFW) and the U.S. Centers for Disease Control and Prevention (CDC), to strengthen the public health laboratories in the country through lab workforce development and implementation of Quality Management System. This training module is purely for educational purposes and must not be used for commercial purposes. Brand names or trademarks, ifany, are incidental and strictly not for promotional purposes. This module is meant for free distribution. Reproduction of any of the excerpts from this document does not require permission from the publisher so long asitis verbatim and the source is acknowledged. Development of this resource was supported by a cooperative agreement No- 5U2G GHO01103-02 with the U.S. Centers for Disease Control and Prevention (CDC). Its contents are solely the responsibility of the author and do not necessarily reflect the office views of the Department of Health and the Human Services and the Centers for Disease Control and Prevention.ARG BROT N&co sanea wa etary wear Fare 4 agta wee faa ess s Government of India Voila GAR, asl. Ministry of Health & Family Welfare ome afar ya Hericerp, aTeY National AIDS Control Organisation SANJEEVA KUMAR, 14s Additional Secretary & Director General, NACO Foreword The success of a healthcare organization is ultimately measured through patient health outcomes and effectiveness of healthcare. Diagnostic laboratory service with quality assured reports is a key enabler towards this effectiveness. Such reliable lab results, in turn, depend on the degree of quality assurance that the laboratory undertakes, Ministry of Health and Family Welfare, through the National Health Mission is investing great efforts in Quality Assurance in healthcare. Quality Controls, both internal and external, play a key role in Quality Assurance in laboratories. These concepts are being promoted and resources are being allocated for the same by the government. It is imperative that these resources be utilized optimally. Labs for Life in its endeavor to supplement these efforts of quality improvement, has developed these training modules on the technicalities of using these controlling mechanisms optimally. These modules have been designed to help doctors and technicians alike to understand and incorporate the quality procedures in their labs. | am sure these modules will be used extensively by laboratorians to give impetus to the efforts of the government towards improving the quality of diagnostic services, Nil 6th Floor, Chandralok Building, 36 Janpath, New Delhi - 110001 Telefax : 011-23325331/23351700 E-mail ::dgnaco@gmail.com. ome wasrgft oren orl, Prema wren srevara A qa were a ora oe Know your HIV status, go to the nearest Government Hospital for free Voluntary Counselling and Testingnfco wae we asia ge Freearr ast arte wa PIT OF RAT BT HATT SRT TTT Alok Saxena iV National AIDS Control Organisation Joint Secretary Ministry of Health & Family Welfare ‘Government of India Preface Ensuring quality in laboratory services is vital, given that results from tests significantly influence diagnosis and treatment of patients. It is mandatory that a well-managed laboratory should have a good quality system that ensures the provision of accurate and reliable test results. Usage of Quality Controls (QC) safeguards the analytical phase of laboratory operations by detecting errors, shifts and changes in the analytical system, thus ensuring reliability, accuracy, and quality of test results. This training module on Quality Controls addresses the mechanisins of safeguarding the analytical phase employing internal and external controls. The module comprises of two volumes where the volume one emphasizes on the explanation of tests results in yielding ‘numerical values and the second volume focuses on the interpretation, explanations, and evaluations of the tests results containing no numerical values. It has been mainly designed to help doctors and technicians alike to understand and incorporate the quality procedures in their labs, | hope that this training module will immensely help and enable laboratories to monitor and boost the quality of the performance of their diagnostic services. te ee (Alok Saxena) 9th Floor, Chandralok Building, 36 Janpath, New Delhi - 110001 Tele.: 011-23325343 Fax : 011 - 23325335 E-mail :js@naco.gov.in ort yang semen or, Areas ear scare A qua were @ vita ae Know your HIV status, go to the nearest Govemmment Hospital for free Voluntary Counselling and Testingata ad Government of India Ministry of Health & Family Welfare Department of Health & Family Welfare Naresh Goel Labs for Life Project 9 Floor, Chandralok Building. De pte General, NACO & (A Partnership Project with CDC) 36, Janpath, New Delhi-110001 Coordinator for LAL project. Tel. : 91-11-23351719, 43509917 Fax :91-11-23731746 E-mail : labsforlife mohfw@gmail.com Message A clinical laboratory is a place where a battery of processes work together. Each of these Processes has the potential to go wrong necessitating the need for Quality Assurance at each step. Labs for Life in its endeavor for Quality Improvement in labs, has been addressing each of the Quality System Essentials through module development and training. ‘The principles of Quality Management, Assurance and Control have become the foundation by which clinical laboratories are managed and operated. ISO 15189 in clause 5.6 elaborates the need for “Assuring the Quality of Examinations”. These modules on Quality Controls have been developed to address Quality Control Mechanisms of the analytical phase employing internal and external control mechanisms, Many tests in the lab yield numerical values and can be continuously evaluated through statistical methods. Aspects of these mechanisms are explained in the first volume. Some tests however contain no numerical data and have reports with interpretation, explanations and evaluations of probability. These are explained in the second volume. Whichever be the case, the analytical phase has to be closely monitored to avoid errors. Analytical measures overlap to some extent into the pre and post analytical sections. These are also discussed wherever relevant External Quality Assurance also needs interpretation of results to understand the proficiency of the lab. Details of these are explained with reference to the frequently used EQA Schemes. Labs for Life also is launching a statistical toolkit on its website to facilitate the use of techniques described herein. Using these two resources together can enable the labs to monitor and enhance the quality of their performance. 'hope these modules will be of great help to all the readers on their journey towards better quality in laboratories. Yeh (Or. Naresh Goel)Acknowledgement This module on “Quality Control” for Laboratory Quality Management System has been developed by the Labs for Life Project of Ministry of Health and Family Welfare, Government of India. Dr. Anu George is acknowledged for the technical contribution and collation of available information related to the subject. Dr. Sarika Mohan is acknowledged, especially for developing the microbiology section. Dr. Anna Marie Murphy, Technical Consultant from the American Society for Clinical Pathology and Dr. Katy Yao, SLMTA Global Program Director, CDC are acknowledged with gratitude for their selfless efforts in disseminating information on Statistical Quality Controls. Other experts who have put their best efforts in drafting and finalization of this module are also sincerely acknowledged. Their names are enlisted in the list of contributors. The material in this training module has also utilized multiple open sources such as Textbooks, Manuals, National and International Guidelines, Review Articles and related Websites. The sources have been duly acknowledged in the section on references and furtherreading. Sincere gratitude is expressed to the U.S. Centers for Disease Control and Prevention- Division of Global HIV/TB (CDC/DGHT), India Office, its implementing partner Christian Medical Association of India (CMAI) and Becton and Dickinson (BD), collaborating partner for Labs for Life project for providing technical assistance, guidance and support in finalization of this module.Technical Writing / Editorial Members Dr, Naresh Goel DDG,NACO & Project Coordinator L4L.MoHFW 2, | Dr Anu George Technical Manager, Labs for Life 3. | Dr Sarika Mohan Senior Scientific Advisor, CMAI 4, | Ms. Anna Marie Murphy Technical Consultant from the American Society for Clinical Pathology 5. | Dr Sunita Upadhyaya Senior Laboratory Advisor, CDC-DGHT 6. | Dr Paramjeet Singh Gill Professor, Dept. of Microbiology, PGIMS Rohtak 7. | Dr Jayashree. Bhattacharya Prof. & HOD of. Biochemistry. Lady Harding Medical. College and Assoc. Hospital, New Delhi 8. | Dr. Manoj Jais Director Professor Microbiology, LHMC. 9. | Mr Jaichand Junior Scientific Officer, State Public Health Laboratories, Trivandrum 10. | Dr. Aarti Tiwari Assistant Director Microbiology, NRLNCDC. 14. | Dr. Nikhil Prakash Senior Consultant, QA Division, NHSRC 12. | Dr. Deepika Consultant, QA Division, NHSRC 13. | Dr. Joy Mammen Faculty in the Dept. of Immunohematology & Transfusion Medicine at CMC Vellore 14, | Dr. Ramani Manoj Kumar MBBS, MD, FRC Path, Department of Pathology, Christian Medical College, Vellore 15. | Dr. Sunita Kapoor Histo-pathologist, Director Star Diagnostic 16. | Ms. Ranjana Upadhyay Quality Coordinator, Labs for Life Project 17. | Ms. Chhavi Garg Project Associate, Labs for LifeVolume I: Guidelines for Common Statistical Methods Used In Clinical Laboratories Table of Contents Chapter 1: Overview 11. 12 13 14 15 16 Quality Controls: Ongoing Performance Evaluation: Overview Method Evaluation Objectives of the Module Target Audience Method How to Use the Module Part 1: Ongoing Evaluation of Method Performance Chapter 2: Internal Controls: Quantitative 24 22 23 24 25 26 27 28 29 2.10 an 2.12 213 (Statistical Quality Controls) Internal Controls: Overview Quantitative SQCs: Basic Concepts Interpreting Quality Control Data: LJ Charts New Lot QC Total Error Total Allowable Error How Far Can The Mean Shift? QC Planning Uncertainty of Measurement (Mu) ‘Average of Normals (Aon) & Bull's Algorithm Radar/ Spider Charts Harmonization/ Comparability Tests Conclusion: SAC Chapter 3: Proficiency Testing or External 3.4 32 3.3 Quality Assurance 1SO Requirements Proficiency Testing or PT ‘Qualitative and Semi-Quantitative EQAS in India3.4 35 3.6 Inter Laboratory Comparison (ILC) Programs (Peer Group Comparisons) Split Sample Analysis: ‘Troubleshooting and Corrective Actions Part 2: Method Evaluation As per ISO: 15189 5.3.1.2 and 5.5.1 Chapter 4: Method Evaluation 4.1 Validation and Verification 4.2 Process for Introducing a New Method 4.3 Pre-purchase Assessment 4.4 Acceptance Testing/ Method Evaluation/Performance Verification 4.5 Verification Plan 4.6 Understanding Quality Requirements 4.7 Select Performance Characteristics considered under method evaluation 4.8 Precision 4.9 Accuracy [Trueness] (Measured as Bias) (“correlation studies") 4.10 Linearity 4.11 LoD/LoQ Limit of Detection (LoD) & Limit of Quantitation (LoQ) (sometimes referred to as “Analytical Sensitivity") 4.12 Interference /Specificity 413 Carryover 4.14 Reference Intervals 4.15 Carryover 4.16 Documentation of Method Evaluation Part 3: Continual Improvement ISO 1518: 2012 (Clauses 4.9 To 4.12) Chapter 5: General Concepts in Quality Assurance 5.4 52 53 5.4 55 5.6 57 Introduction PDCA (Plan, DO, Check, Act) The 5S Failure Modes and Effects Analysis (FMEA) Too! Pareto Principle Trend Analysis Root cause analysis (RCA) & Cause & Effect Analysis,Volume Il: Internal Quality Controls: Qualitative Part 4: On-going Evaluation of Method Performance: Semi Quantitative and Qualitative Controls Chapter 6: Internal Control: Semi-Quantitative and Qualitative QCs Overview 6.1 Qualitative Examinations 62 Semi-quantitative Examinations 63 Built-in Controls, Rapid Card Tests 6.4 Process Control: Overview Chapter 7: Quality Assurance in Microbiology 7.4 Introduction 7.2. Extended Responsibilities of a Microbiology Lab 7.3. Key points to ensure quality in the path of a Microbiology Sample 7.4 Issues Addressed Through Quality Assurance 7.5 Quality Assurance of Methods & Materials 7.6 Quality Assurance of Stains 7.7 Quality Assurance of Culture Media 7.7.1 In-house Media 7.7.2 Commercially Prepared Media 7.7.3 Quality Control of Blood Agar Medium 7.8 Quality Assurance of Organisms 7.8.1 Source of Quality Control Organisms 7.8.2 Storage of Quality Control Organisms 7.8.3 Processing of Quality Control Organisms 7.8.4 Frequency of testing (CLSI, 2015) 7.8.5 Inoculation of Quality Control Media 7.8.6 MoFarland Standard 7.8.7 Incubation Conditions 7.8.8 Interpretation of Results 7.8.9 Reasons for Lot Failure 7.9 Quality Assurance of Biochemical Reactions 7.10 Quality Assurance of Anaerobic Bacteria 01 01 02 02 02 03 05, 06 07 15 18 18 24 22 or 35, 36 36 37 37 38 38 39 40 40 40 a 44744 7.42 713 7.14 7.15 7.16 Quality Assurance for Mycobacterium tuberculosis Quality Assurance in Parasitology Quality Assurance in Medical Mycology 7.18.1 General QC procedures 7.13.2 QC for Stains 7.13.3 QC for Media 7.13.4 Storage of Quality Control Organisms: 7.13.5. QC in Techniques used in Common Mould Identification 7.13.6 Quality Control of Broth dilution Antifungal Susceptibility testing methods for yeasts 7.13.7 Antifungal Susceptibility Testing of Fungi 7.13.8 Batch of Medium and Lot of Plastic ware Control 7.13.9 QC Frequency 7.13.10 Other Control Procedures Quality Assurance for Antimicrobial Susceptibility Testing Procedures in Bacteriology 7.14.1 Quality Control Organisms 7.14.2. AST Methods 7.14.3 General comments in Performance standards for AST as per CLSI guidelines 7.14.4 Automated Systems for Identification of Bacteria and Fungi Quality Assurance in Serological tests for Infectious Diseases 7.15.1 Importance 7.15.2 Ensuring Quality of Serological Examination Test Results 7.18.3 Quality Assurance of Rapid Serological Tests Quality Assurance in Molecular Diagnostic Methods 7.16.1 Applications 7.16.2 Specimen Collection, transportation, Storage and processing 7.16.3 Utility of Molecular Diagnostic Tests for Infectious Diseases 7.16.4 Limitations 7.16.5 Detection of Inhibitors and Interfering Substances 7.16.6 Methods 7.16.7 Real-Time PCR Instruments 46 48 55 55 55 55 56 56 87 59 60 61 62 63 63 64 74 76 80 81 81 81 83 83 85 86 86 87 88 887.16.8 Quality Assurance (QA) 7.16.9 Laboratory Design and Practices 7.16.10 Instruments 7.16.11 Quality Assurance during Development of Molecular Diagnostic Tests 7.16.12 Control Materials 7.16.13. Selecting Organism Strains for Analytical Studies 7.16.14 Preparing Nucleic Acid Controls 7.16.15 Types of Testing During Assay Development 7.16.16 Qualification of Sample Preparation Methods (Cell Lysis and Extraction) 7.16.17 Specimen Type 7.16.18 Quality Assurance (QA) For Implementation of Molecular Diagnostic Tests 7.16.19 Viral load Testing 7.16.20 Reporting Of Results Chapter 8: IQC (Qualitative) in Hematology and at 82 83 84 85 Clinical Pathology Smears 81.1 Spreader 812 Slides 8.1.3 Washing the Slides 814 Smear Preparation 8.1.5 Staining 81.6 — Quality Control Measures in Smears Reticulocyte Count ESR Urinalysis 8.4.1 Effects of time lag on urine 8.4.2 Controlling Reagent Strips 8.4.3 Inter- Observer Variance Semen Analysis 92 94 95 100 100 101 102 102 104 104 105 106 106 107 107 107 107 108 108 1 112 113 113 114 114 114 115 11586 Cavity Fluids 86.1 CSF 8.6.2 Pleural Fluid 8.6.3 Peritoneal Fluid 8.6.4 Synovial Fluid Chapter 9: Quality Assurance in Cytopathology 94 92 9.3 94 95 and Histopathology Laboratory General Pre-Analytical Aspects Histopathology 9.2.1 Process Control of Histopathology Technique’ Paraffin embedded, H& E sections 92.2 Artefacts in Histologic Sections 9.2.3 Immunohistochemistry (IHC) 9.2.4 Frozen section or cryo-section Cytopathology 9.3.1 Sample Collection 9.3.2. Sample Transportation 9.3.3. Processing 9.3.4 — Staining 9.3.5 Cell Block 9.3.6 Reporting Inter Laboratory Comparison for Histopathology and Cytology Post-Analytical Aspects 9.5.1 Documents and Records 9.5.2 Paraffin Blocks Filing 9.5.3 Slide Filing 9.5.4 Report Retention 9.5.5 Sample Retention 9.5.6 Sample Discarding 116 116 117 117 118 119 120 120 421 132 133 133 133 133 134 134 135 136 137 136 139 139 140 140 144 141 14196 97 Continuous Quality Improvement 9.6.1 Process Flow Plan 9.6.2 Personnel Training, Training Evaluation 9.6.3 Patients and Clinicians Feedback and Complaints 9.6.4 Indicators and their Monitoring Conclusion References Glossary Abbreviations Annexure 141 141 141 142 142 142 143 147 149 151List of Tables & Figures Figure No. & Name Page No Figure Flaming of Loop 18 Figure Sample of a Stain Bottle Label 19 Figure The Right & Wrong Ways of Storing Stains 19 Figure Good Quality Wright stain 20 Figure Good Quality Gram stain 20 Figure Good Quality Gram Stain 20 Figure Poor Quality Gram Stain 20 Figure Broad Classification of Media; Microbiology 24 Figure MacConkey Agar QC 22 Figure McFarland Standards 40 Figure QCs of Biochemical Reactions 42 Figure Quality Monitoring and Quality Control in Mycobacteriology Laboratory 46 Figure ‘Some Helminthic Ova 48 Figure Antifungal Susceptibility Testing of Fungi 59 Figure Solvents and Diluents for Preparation of Stock Solutions of Antifungal Agents 62 Figure AST Methods 64 Figure Interpretation of Results 67 Figure E Test Antimicrobial Gradient Method 74 Figure Matrix Assisted Laser Desorption lonization - Time of Flight Mass Spectrometry (MALDI-TOF MS) 76 Figure MALDI-TOF MS 79 Figure Real time PCR 89 Figure Real-Time PCR Amplification Plot 90 Figure Cutting 127 Figure Correct way of spray fixation 134 Figure Correct screening of smears 137 Figure Marking slides under scanning lens 137List of Tables Table Equipment Routine Care, Monitoring and Technical Maintenance 12 Table Trouble Shooting Guide for Culture Media 26 Table Exempt & Non-Exempt categories for Media Included in CAP Surveys (1984,1988, 2001) 27 Table Processing of Exempt Media 30 Table Manufacturing Quality Control Requirements for Commercially Prepared Media 34 Table Performance check of nonexempt media 34 Table Quality control of commonly used media: Suggested ATCC control organisms and 36 expected reactions Table Storage of QC Organisms 37 Table Incubation Conditions for QC organisms 40 Table Gram Negative Biochemical Chart 43 Table PCR Cabinets 96 Table Errors of Staining: Romano sky stains 112 Table Troubleshooting tips for poor processing 125How to use this module VOLUME II: INTERNAL QUALITY CONTROL: QUALITATIVE Part 4: On-going Evaluation of Method Performance: Semi Quantitative and Qualitative Controls Chapter 6: Internal Control Semi Quantitative and Qualitative Controls: Overview Ageneral introduction to non-statistical methods of QCs are outlined in this chapter. Chapter 7: Qu All aspects of a microbiology lab including bacteriology, parasitology, and mycology are explained. Antibiotic susceptibility testing mechanisms are described. Outlines of serology and molecular diagnostics are also explainedin terms of Quality Assurance. Assurance in Microbiology Chapter 8: QC (Qualitative) in Hematology and Clinical Pathology This chapter describes a few points to keep in mind, where making blood and bone marrow films are concerned. Some general errors in doing ESR are pointed out. Control mechanisms including pre- analytical and post analytical are enumerated for cavity fluids, urine analysis and semen analysis. Chapter 9: Quality Assurance in Histopathology and Cytology The processes that happen in histopathology and cytology labs are several. Each step includes chances of potential error. These should be understood and avoided as part of the quality assurance process. To this end, each step in elaborated with suggestions of how to manage an error free histopathology and cytology lab.ON-GOING EVALUATION OF METHOD PERFORMANCE: SEMI QUANTITATIVE AND QUALITATIVE CONTROLSCHAPTER 6 INTERNAL CONTROL: SEMI-QUANTITATIVE AND QUALITATIVE QCs OVERVIEW Only a test result that is a numerical can be controlled using SQCs. Thus a whole realm of laboratory testing is out of the scope of Statistical Quality Controls. This does not mean that these tests do not require controlling. Specific processes must be builtinto the laboratory's Quality Control Protocols for controlling these tests. A brief overview of these mechanisms in discussed below. Further details are given in appropriate sections. 6.1 Qualitative Examinations It measures the presence or absence of a substance as reported accordingly. Methods that evaluate cellular characteristics such as morphology through a gamut of staining techniques are reported descriptively. The results are not expressed in numerical terms, but in qualitative terms such as “positive,” “negative,” “reactive,” “non-reactive,” “normal,” or “abnormal” or with detailed description ofthe findings. 6.2 Semi-quantitative Examinations These are similar to qualitative examinations; testing does not measure the precise quantity of substance. The difference is that results of these tests are expressed as a rough estimate of how much of a measured substance is present. This estimate is sometimes reported as a number. Therefore, test results for semi-quantitative tests may be shown as “trace amount", “1+, 2+, or 3+”, or positive at 60 (titer or dilution). Examples of semi-quantitative examinations are urine dipsticks, urine routine examination for Albumin, Glucose etc. Some microscopic examinations are considered semi-quantitative because results are reported as estimates of the number of cells seen per low power field or high power field. For example, a urine microscopic examination might report: 0-5 red blood cells seen per high power field 6.3 Built-in Controls, Rapid Card Tests ‘There are several different platforms commonly used to build rapid diagnostic tests. The vast majority of RDTS in use today are based on immunoassay technology due to its relative64 simplicity. These tests generally involve the interaction of a fixed reagent of either target antigen or antibody that is linked to some type of visible detector that then reacts with a patient sample. Built-in controls in RDTs are those that are integrated into the design of a test system such as a test kit device, Usually, the device is marked with designated areas where colored lines, bars, or dots should appear to indicate success or failure of positive and negative controls, and these controls are performed automatically with each test. The manufacturer's product instructions may also refer to these as procedural controls, on- board controls, or internal controls. Most builtin controls monitor only a portion of the examination phase, and they vary from one test to another as to what is being monitored. For example, bu indicate that all the reagents impregnated into the device are active and working properly, whereas builtin controls for other kits may only indicate that a sample was added and solutions flowed through the device correctly. Itis important to carefully read the instructions provided by the manufacturer to understand what the built-in controls monitor, and to determine whether additional controls may be needed. in controls for some kits may Even though these built-in controls give some degree of confidence, they do not monitor for all conditions that could affect test results. Thus an internal control mechanism of reevaluating the test kits periodically should be evolved using in-house control samples, both positive and negative, for added confidence in the accuracy and reliability of test results. Process Control: Overview As with quantitative procedures, it is important to verify that results of qualitative and semi- quantitative examinations are correct prior to reporting them. Conducting quality control for many of these tests is not as easily accomplished as with quantitative tests. Therefore, it becomes essential that other processes within the quality system are carefully conducted, in addition to traditional quality control methods. Following are some important over-arching concepts for quality that apply to qualitative and semi-quantitative tests. * Due to the inherent and unavoidable subjectivity involved in these processes, the laboratory should have robust raining programs for these staffreporting such tests. * In addition, robust interobserver variance detection programs must be built into the training evaluation programs so as to enable uniform reporting processes, especially in the case of semi-quantitative assays. * For indeterminate results in RDTs the Lab must adopt a uniform policy of retesting or referring to more conclusive tests. The policy must be understood and followed by the testing staff and authorized signatories. + Sample management: Examinations that are dependent on a viable organism in the ‘sample may need closer monitoring and better communication with non-laboratory staf.Dedicated, professional staff who understand the principles of QC. Appropriate storage of kits and reagents is warranted. Specific instructions of these should be available at the lab Problems arising from wrong storage, wrong processes at each and every step should be made available in the form of special instructions. Incubators, refrigerators, microscopes, autoclaves, and other equipment must be maintained and monitored carefully. Positive and negative controls must be used to monitor the effectiveness of test procedures that use special stains or reagents, tests with endpoints such as agglutination, color change, or other non-numeric results. Reagents should be stored according to manufacturer's instructions, labeled with the date they are opened and putinto use, and discarded at the expiration date. Keeping records of all quality control processes and corrective actions is necessary for continualimprovement of the laboratory quality system, When problems ocour, investigate, correct, and repeat patient sample.CHAPTER 7 QUALITY ASSURANCE IN MICROBIOLOGY 7.1 Learning Objectives Atthe end of this chapter the learners will be able to understand: + The importance of Quality Assurance (QA) in general microbiology procedures. + The basic practical quality control measures which should be adopted for appropriate pre-analytical, analytical and post-analytical processes in a Clinical Microbiology laboratory. + The practical quality control procedures in appropriate selection and maintenance of stains, media, QC organisms, serological materials, antimicrobial discs / solutions or molecular reagents by various levels of staff. + Themeasures to followin diagnostic systems to generate quality testresults. Introduction These Quality Assurance guidelines cover mainly the analytical factors affecting quality of microbiology report. Most of the pre-and post-analytical factors have already been discussed in previous modules of Labs for Life Project. However, some overlapping concepts are briefly addressed. The main work of a clinical microbiology laboratory is to examine specimens from patients for the presence of potentially pathogenic microorganisms, to detect antibodies to such organisms, to determine the antimicrobial sensitivities of infecting organisms, and to assess the infective potential of environmental materials. The purpose is to quickly and economically obtain information that will: Help clinicians to treat their patients. Assist public health experts to prevent spread of infection in the community. It is, therefore, essential that appropriate quality program is in place in microbiology laboratory where 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. To address the concerns comprehensively a quality management system is to be established in a microbiology lab. Designating a Quality Manager for the following functions is the first step towards building a QMS. Setting up and organizing QC measures throughout the laboratory. Preparation, implementation, update and review of all methods and procedures. p___] [ABS FOR LIFE PROJECT7.2 + Regular monitoring of all quality control records to ensure compliance and satisfactory maintenance of standards. + Preparation andissue ofinternal quality assessment specimens slides. + Analysis ofresults = Issue and return of all EQAspecimens on time. + Analysis ofresults. Trend analysis. + Analysis ofall relevant QC data to ensure satisfactory performance, + Identification of problems/non-conformities. = Root cause analysis, corrective and preventive action, = Reporting on a regular basis to management on QC performance and all matters related to quality. = Communication to all staff and users on quality issues, + Recommendations regarding changes in materials, equipment, human resource training, or procedures for continual improvement. Extended Responsibilities of a Microbiology Lab In addition to the main task of providing helpful reports on submitted specimens, the microbiology staff should keep the following in mind: a. Advocacy: Take steps to inform all potential users of the service about the range of investigations available, the supply of specimen containers, the procedures for collecting specimens and the arrangements for transmitting them to the laboratory. This may be done through a leafletibooklet distributed to hospital units (e.g. wards, OPDs, ICUs), medical staff, family doctors and public health department. These may include address and telephone number of laboratory, the times for normal receipt of specimens and the arrangements for “emergency” specimens and the supply of specimen containers and request forms. It may outline the range of examinations and the correct procedures for collecting each kind of specimens to the laboratory, including safety precautions to be observed with specimens likely to contain especially dangerous pathogens. b. PublicHealth Functions: + Identify findings that require urgent referral E.g. by telephone, to the clinician or notifying agency. + Advice on investigation of outbreaks of infection, preventive measures to be taken, and procedures to control hospital acquired infections and procedures for sterilization and disinfection + Participate in education and knowledge programs of various categories of health service personnel in matters relating to the occurrence, diagnosis and prevention of infection = [ABS FOR LIFE PROJECT¢. Infection prevention and control: Methodologies for appropriate infection control, hand hygiene, biomedical waste management and good housekeeping cleanliness may be adopted and disseminated in other areas of the healthcare facility Please refer to “Facility management and safety" modules of Labs for Life Project for details. The reader may also refer to 5S techniques described in Volume | of this QC module under Continual Improvement. 7.3. Key points to ensure quality in the path of a Microbiology Sample a. Request forms: The request form or an electronic equivalent should contain at least the following: + Date of sampling + Time of sampling + Name ofsampler + Type ofsample + Name ofthe patient + IDnumber ofthe patient + Sexofthe patient + Age ofthe patient + Medication taken by the patient + Address ofthe patient + Temperature / clinical details of the patient primary diagnosis /date-time of symptoms onset + Tests requested b, Collection & Transportation: There must be clearly defined arrangements for the collection of specimens from users of service and their safe delivery to the laboratory. Suitable transport boxes should be provided for safe transport of specimens. Follow triple layer packaging and transportation and as perlocal, state, national and international regulations. + Special collection protocols must be observed (for example, in case of filariasis, without DEC provocation, blood sample needs to be collected 10 pm & 2am). Please refer to “Pre-analytical and sample collection manual” ¢. Primary Sample Collection Manual: Every microbiology laboratory should have a sample collection manual distributed even at the site of collection of samples. This manual must contain all the following relevantinformation:1) Listoftests available 2) Instructions for pre-collection a i. Instructions for preparation of patient for sample collection (e.g. in case of venipuncture) ii, Type and amount of the primary sample to be collected with descriptions of the primary sample containers (e.g. blood samples in plan evacuated tubes to be collected for serology, blood samples for CD4 testing to be collected in K2-EDTA evacuated tubes, samples for blood culture to be collected in blood culture bottles). ili, Special timing of collection, where needed (e.g. Specimens such as urine and sputum are best collected soon after a patient wakes up in the morning when organisms have had the opportunity to multiply over several hours) iv. Clinical information relevant to or affecting sample collection, examination performance or resultinterpretation (e.g. history of administration of drugs). 3) Instructions for Collection activities i. Determination of the identity of the patient from whom a primary sample is collected (to avoid errors) ii. Verification that the patient meets pre-examination requirements (to obtain a good quality sample) ili, Instructions for collection of primary blood and non-blood samples, with descriptions of the primary sample containers and any necessary additives (to preserve the integrity of samples). iv. Instructions for labeling of primary samples in a manner that provides an unequivocal link with the patients from whom they are collected (to ensure traceability) v. Recording of the identity of the person collecting the primary sample and the collection date, and, when needed, recording of the collection time (to understand TAT and training needs). vi. Instructions for proper storage conditions before collected samples are delivered to the laboratory (to maintain integrity of samples despite storage). 4) Instructions fortransport of specimens. i, Within a time frame appropriate to the nature of the requested examinations and the laboratory discipline concemed. ii, Within a temperature interval specified for sample collection Inamannerthat ensures th egrity ofthe sample. Avoidance of a delay in processing and the maintenance of a cold chain during transportation must be ensured particularly for urine samples / BAL / endotracheal aspirates that are to be cultured since these tests are semi-quantitative Reception of specimens: For safety, the reception of specimens should be done in a room separate from the processing / reporting area. The reception staff should be trained in the appropriate safety precautions and well-versed with specimen acceptance / rejection criteriaFew specimen rejection criteria which may be used: + Wrong conservation temperature + Samples in inadequatelinappropriate transport media / anticoagulant (e.g, specimens for anaerobic bacteria submitted in aerobic transport media) + Incompletely filed request form + Wrong specimen identification + Specimen volume quantity inadequate/insufficient + Delay when bringing specimen / expired sample (e.g. urine specimen transport time exceeds 2 hours postcollection or it is not preserved) + Sample is haemolysed + Deteriorated samples/damaged package + The specimen was received in a fixative (formalin), which, in essence, Kills any microorganism present 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 notin transport media or faecal specimen containing blood and mucus, etc. It is important to inform the requesting physician or patient before discarding unacceptable specimens. Ifan unsuitable sample is accepted (e.g. biopsy samples/ post-surgery samples), then a note of caution must be given with the report mentioning the quality of sample affecting the test result Maintain registers, log books, test requisition forms, consent forms and other documents and records in the sample reception and accessioning area. This is essential for traceability of specimens, easy and faster retrieval of relevant information. Referred Specimens: In case of specimens received from other labs/hospitals, following points should be considered + Do the samples always come along with a standardized request forms to order lab test? Look at the day specimens + Approximate percentage of samples accompanied with standardized request form + Approximate percentage of these completely filed Please refer to “Documentation and record management” module for details. e. _ Laboratory processing after reception: 1, All processing should begin with a gross examination of specimen. Areas of blood or mucus should be located and sampled for culture and direct examination. Notationsshould be made on the working register regarding the status of the specimen (e.g. bloody, clotted, turbid) so that if more than one person works on the sample, the results of gross examination are available for consultation Allappropriate specimens should have a direct microscopic examination. It serves several purposes: + Assesses the quality of specimen e.g. sputum can be rejected that contains only saliva and not lower respiratory tract secretions by quantitation of WBCs or squamous epithelial cells presentin the specimen + The microbiologist and clinician can be given an early indication of what may be wrong with patient (e.g. 4+ gram-positive cocci in clusters in an exudate) + The workup of the specimen can be guided by comparing what grows in culture to what was seen on the original smear. Primary culture media are divided into several categories. + The first are nutritive media e.g. blood or chocolate agars. Nutritive media support growth of a wide range of microorganisms and are considered nonselective. Nutritive media can also be differential, in that microorganisms can be distinguished on the basis of certain growth characteristics evident on the medium. Blood agar is considered nutritive as well as differential + Selective media supports the growth of one group of microorganisms but not other, by adding antimicrobials, dyes or alcohol to medium. E.g. MacConkey agar contains crystal violet which inhibits gram positive organism. It is also a differential media by differentiating lactose and non-lactose fermenters. + In some cases (sterile body fluids, tissues, or deep abscesses in a patient with antimicrobial therapy), enrichment broth medium is inoculated along with primary solid media, so small numbers of organisms present may be detected e.g. Thioglycollate broth, BHI broth and trypticase soy broth. Selection of media is usually based on the organisms most likely to be involved in the disease process. E.g. in determining whatto set up fora CSF specimen, one considers the most likely pathogens that cause meningitis (e.g. Neisseria meningitides, S. pneumoniae, E.coli etc) and select media which will support the growth of all these organisms (blood and chocolate agar at the minimum). Likewise, if a specimen is collected from a source likely to be contaminated by normal flora, e.g. stool/anal fistula, then a selective medium is usually added e.g, DCA/XLD to inhibit the growth of normal flora. ‘Specimens can be inoculated onto solid media either quantitatively by a dilution procedure ‘or by means of a quantitative loop, or semiquantitatively using an ordinary inoculating loop. Urine cultures and tissues from burn victims are plated quantitatively. Some laboratories choose to process urine samples semi-quantitatively with interpretations on significant bacteriuria Inoculated media are incubated under various temperatures and environmental conditions, depending on the organisms suspected e.g. 28°C to 30°C for fungi and 35-37°C for bacteria, viruses and acid-fast bacillus. Aerobes grow in ambient air, which contains 21% oxygen (O,) anda small amount (0.03%) of carbon dioxide (CO,). Anaerobes cannot grow in the presence of oxygen, and the atmosphere in anaerobe jars, bags or chambers is composed of 5% to 10% hydrogen (H,), 5-10% CO*, 80-90% N, and 0% O,,. Capnophilese.g, Haemophilus influenza and Neisseria gonorrhoeae, require increased concentrations of CO, (5-10%) and approximately 15%O,, This atmosphere can be achieved by a candle jar (3% CO,) or a CO, incubator, jar or bag. Microaerophiles (Campylobacter jejuni, Helicobacter pylori) grow under reduced O, (5-10%) and increased CO, (8-10%) which can be obtainedin specially designed jars or bags. 7. Microbiology testing should be 4 |. EVELS OF BIOSAFETY separated from other testing areas. Incompatible activities should be performed in separate areas e.g , High risk microbes area for media preparation should be different from that of specimen processing in Microbiology. EA 8. Diagnostic and healthcare laboratories should be designed for at least BSL-2. Desirable equipment for a BSL-2 facility includes a biological safety cabinet and an autoclave (Refer to NABL 112 and WHO lab biosafety manual). Appropriate practices include biohazard waming signs, use of PPE, “airborne” and “sharps” precautions and a biosafety manual, defining Biomedical waste managementand infection control practices as per local, state or national regulations. Low risk, microbes f. Standard Operating procedures: The following should be included in the microbiological SOPs covering the analytical stage incorporating quality control measures at every step’ 1. Aseptic techniques and safe handling of infectious material Detailed procedure for examining different specimens. Preparation and QC of culture media and preservation of stock strains. Inoculation of liquid and solid media (putting up controls along with the specimens) Staining techniques and QC of stains. Techniques used to identify pathogens. Reading and interpretation of cultures. Antimicrobial sensitivity testing and QC of procedures and antibiotic discs. © erneaeren Immunologic techniques and QC of antigen and antibody reagents. 10. Cleaning and QC of equipment use ‘obiology laboratory. 11. Safe working practices. 12. Disposal of specimens and cultures. 13. Cleaning of glassware and plastic ware. 14. Steriliz: ion procedures and their control.Laboratory equipment, reagents and consumables: All clinical samples, standard stains and media should be segregated from each other during storage. Autoclaves, hot air ovens and anaerobic jars should be checked by physical, chemical and biological controls. Chemical controls must be used with each batch, whereas a biological control should be used at least once a week. Records of physical parameters (e.g, temperature, pressure) should be maintained by operating staff Table: Equipment Routine Care, Monitoring and Technical Maintenance Technical Equipment Routine care Monitoring maintenance and inspection Clean inside of jar each week | Use methylene blue reactivate catalyst after each | indicatorstrip with eachrun | inspect gasket Anaerobic jar run (160°C, 2h) nm za. | Sealing in the lid lote and record decoloriza- Replace ctaystevery | fon me afte ofindestor | WO8KY each week Check and adjust water level before each run Clean and change water | Record time and tempera- Autoclave monthly ture orpressureforeachrun | Every 6 months Record performance with spore-strips weekly Wipe inner walls with antiseptic solution weekly or Replace brushes, Centrifuge | after breakage of glass tubes annually orspillage Hot-air oven forsteriization || Clean inside monthly | Recordtime and Every 6 months of glassware temperature for each run ; Record temperature at the Incubator Clean inside walls and | start of each working day| Every 6 months shelves monthly i {allowance 35+ 1°C) Wipe lenses with tissue orlens | Check alignment of paperatter eachday'swork | Condenser monthly Clean and lubricate | Place a dish of blue silica Microscope | mechanical stage weekly with the microscope under Annually the dust cover to prevent Protect with dust cover when | fungal growth in humid notinuse climates Clean and defrost every 2 | Record temperature every Rofigrator | months and after power | rig aliowancea-8c) | E¥rY 6 months Check water level daily Wipe inside walls and change Water-bath | water monthly Record temperature on fst | Every 6 months day of each week (allowance 58-57°C) Please refer to “Equipment Management” module for details.h. Media / Biochemical tests: Laboratory should ensure that commercial or in-house prepared media are sterile, able to support growth and are appropriately reactive biochemically. For this, the laboratory maintains stocks of characterized organisms (procured from a standard source/PT provider/NABL accredited laboratory). These are to be used to test the media and biochemical tests. Blood agar should contain 5-10% sheep blood agar. i Stains / reagents / kits: Stains, reagents and kits must be labelled, dated and stored properly. They should not be used beyond their expiry date or if they show signs of deterioration such as abnormal turbidity and/or deterioration. Appropriate controls should be used, as per standard references, for stains and reagents. Control smears should be stained whenever anew batch of stainis prepared. Culture: Enrichment and selective media should be used for isolation of organisms from stool, sputum, throat, urethral and cervical swabs etc., wherever indicated. For urine samples, the laboratory should perform, report semi-quantitative cultures and use media and procedures that permit isolation of Gram-positive cocci, Gram-negative bacilli and Candida. k. Antibiotic susceptibility testing (AST): The laboratory should follow CLSI (formerly NCCLS) / EUCAST/BSAC guidelines for AST. The number of antibiotic discs applied on the Petri dishto test antibiotic sensitivity should be as per these recommendations. To prevent misuse of antibiotics, the list of antibiotics for which susceptibility tests to be reported should be decided based on standard guidelines, consultation meetings with referring clinicians, antibiotic policy (in hospital settings) and clinical and therapeutic status of the patient. Standard strains for antibiotic susceptibility testing: Each lot of antibiotic sensitivity discs must be checked for activity and potency using standard reference strains (e.g. ATCC) before being placedin service andat least weekly thereafter. |. Mycobacteriology and Mycology: Laboratories that only manipulate specimens for direct smear microscopy or for the Xpert MTBIRIF assay, are considered “low-risk TB laboratories’, In such laboratories, sample processing may be carried out on open bench in an adequately ventilated area (with 6-12 air ‘changes per hour, with directional airflow, away from technician, across the work area with potentially infectious materials, away from occupied areas of the room and outside). Ventilated workstations/Class II BSC are options when adequate natural or mechanical ventilation are not practical. However, when a concentration method precedes staining or if mycobacterial/mycological culture is included in the scope of laboratory work, the work should be carried out ina Class I BSC. Zieh! Neelson sti 1g: Staining for acid-fast bacilli (AFB) should follow the guidelines of the Revised National Tuberculosis Control Programme (RNTCP) of the Govt. of India, including staining of direct (un-concentrated) specimens with grading of smears that are AFB positive. Subsequent concentration and decontamination procedures should follow standard references and appropriate guidelines.Serology: 1. Dengue serology: Guidelines of the national Vector Borne Disease Control Programme (NVBDCP), Govt. of India, should be followed. Dengue rapid test reports should contain a disclaimer stating that the report is based on a screening test and is provisional and that the diagnosis of acute dengue infection should be confirmed by an IgM capture ELISA. 2, Tuberculosis serology: The Quantiferon test should be reported with the disclaimer that itcannot differentiate between latent infection and active tuberculosis. 3. HIV testing: Laboratory should follow HIV testing as per National AIDS Control Organization (NACO) guidelines which mandate pre- and posttest counseling by a counselor. Choice of tests: The laboratory should have a policy for the selection of stains or special microscopy, culture procedures, biochemical tests, serological tests and antibiotic sensitivity tests to be used in the examination of each kind of specimen and microbial isolate. Note: The greatest effort should be made to diagnose the more serious infections with epidemic potential. The degree of precision for microbial characterization should be determined by the likely clinical or epidemiological value of a precise identification, When commensal bacteria with potential pathogenicity are isolated from a hospital patient, the clinician generally requires no more detailed information than that a potential pathogen is present, and the range of its antibiotic sensitivities. Full characterization should be done based on need. This selective approach avoids the waste of resources incurred in “fully” characterizing the majority ofisolates, for which precise species identification is unnecessary. Reading of results: The results of bacteriological examinations usually become available in stages on successive days. Microscopic observations on stained films may be obtained on the day of receipt of the specimen and if significant, be given in a preliminary report to the clinician. Taken in conjunction with the clinical information on the request form, they may help to guide the choice of culture media on which specimen is to be inoculated. The growths in primary cultures are usually observed after overnight incubation i.e. on the second day, when the findings help to determine what further identification tests and what antimicrobial sensitivity tests are to be done on subcultures. The results of these later tests are generally available on the third day, when the content of final report can be decided. 1. General Principles: For specimens where the clinical significance of isolate needs to be assessed in relation to the clinical information of the patient, it is preferable that the reading of primary cultures and the determination of reports should be done by senior laboratory staff in close liaison with the responsible medically qualified bacteriologist 2, Wording of reports: The aim of clinical microbiologist is to provide clinicians and health care providers with reports that are understandable, instructive, relevant and reliable, The laboratory should have a policy for the wording of reports and interpretative comments. It should be communicated to all staffExample: Finding of coagulase-negative staphylococci in a blood culture may be reported with the comment “probably a contaminant from the skin” and without giving its antibiotic sensitivities. However, in an immunocompromised patient, the same organism isreported as “possible clinical significance” and the antibiotic sensitivities given. 3, Issue of reports: All levels of staff should be trained on their roles and responsibilities on transcription of reports in and out of working registers and reports. All the completed reports should be scrutinized for credibility by senior staff before final authorization and release of reports. 4. Release of reports: Automatic generation and release of reports in Microbiology should be considered only for specimens of blood and sterile body fluids which are negative by automated culture systems (e.g. Bactec). These are reported as "No growth after hours, Final reportto follow at days" as per defined policy of the laboratory. Issues Addressed Through Quality Assurance Clinical relevance ‘An important criterion of quality for a microbiological test is how much it contributes to the prevention or cure of infectious diseases; this is called its clinical relevance. Clinical relevance can only be ensured when there is good communication between the clinician and the laboratory. To illustrate clinical relevance, here are some examples: = Ifa few colonies of Gram-negative rods are isolated from the sputum or throat swab of a hospitalized patient, further identification and an antibiograms are of no clinical relevance, since neither procedure will have any effect on treatment of the patient. = If Streptococcus pyogenes is isolated, a full antibiogram has no clinical relevance, since benzylpenicilinis the drug of choice, and this is always active in vitro. "If Escherichia coli is isolated from a sporadic case of non-bloody diarrhoea, identification of the serotype is of no clinical relevance, since there is no clearly established correlation between serotype and pathogenicity. = Ifa Gram-stained smear shows “mixed anaerobic flora", routine identification of the anaerobes is of no clinical relevance, It would be costly in time and materials, and would Notaffect treatment of the patient. = If a yeast is isolated from a respiratory tract specimen, an identification test for Cryptococcus should be done, Further identification tests have no clinical relevance, since they would have no effect on patient management. In summary, a test of good quality is one that is accurate and gives useful results for the prevention or cure of infection. Itis notnecessary to isolate and identify al the different types of organism in the sample.Reliat ity For tests that give quantitative results, reliability is measured by how close the results are to the true value. Some examples of tests of this kind are: + antibioticassay of serum + measurement of minimal inhibitory concentration (MIC) values of antibiotics in vitro + Serum antibody titrations For tests that give qualitative results, reliability is measured by whether the result is correct. Some examples of tests of this kind are: + Identification of pathogens + Antibiotic susceptibility testing of isolates by the disc method Standard terminology for microorganisms is essential to reliability. Internationally recognized nomenclature should always be used. For example: Staphylococcus aureus, NOT “pathogenic staphylococci’; Streptococcus pyogenes, NOT “haemolytic streptococci Use of uniform, approved methods is essential. For example, disc susceptibility tests should be performed with an internationally recognized technique, such as the modified Kirby-Bauertest. Reproducibility The reproducibility or precision of amicrobiological testis reduced by two things: 1. Lack of homogeneity. A single sample from a patient may contain more than one organism. Repeat culturing may therefore isolate different organisms. 2, Lack of stability. As time passes, the microorganisms in a specimen multiply or die at different rates. Repeat culturing may therefore isolate different organisms. To improve precision, therefore, specimens should be tested as soon as possible after collection. Efficiency The efficiency of a microbiological test is its ability to give the correct diagnosis of a pathogen ora pathological condition. This is measured by two criteria’ 1, Diagnostic Sensitivity The greater the sensitivity of a test, the fewer the number of false-negative results. Sensitivity =total number of positive results/total number of infected patients. For example, the sensitivity of MacConkey agar is poor for the isolation of Salmonella typhi from stool. This important enteric pathogen is often missed because of overgrowth by nonpathogenic intestinal bacteria 2, Diagnostic Specificity The greater the specificity of test, the fewer the number of false-positive results. For example: + ZiehLNeelsen staining of sputum is highly specific for diagnosing tuberculosis, becauseit gives only a few false-positive results. + Ziehl-Neelsen staining of urine is much less specific, because it gives many false- positive results (as a result of atypical mycobacteria).+ The Widal test has a very low specificity for the diagnosis of typhoid fever, because cross-agglutinating antibodies remaining from past infections with related salmonella serotypes give false-positive results. The sensitivity and specificity of a test are interrelated. By lowering the level of discrimination, the sensitivity of a test can be increased at the cost of reducing its specificity, and vice versa. The diagnostic sensitivity and specificity of a test are also related to the prevalence of the given infection in the population under investigation, Quality Audits and Benchmarks The scheduled regular audits and striving towards set benchmarks gives a measurable tool for achieving consistent quality. For example, in blood cultures: + The most commonly studied documented and reported quality metric regarding blood cultures is the contamination rate, The blood culture contamination rate should be kept at or below 3% for hospitalized patients. + Another common assessment of quality is the number of blood cultures drawn per septic episode. + A third important measurement is the adequacy of fill of blood culture bottles. Weighing filled bottles and comparing weights against those of a known standard most readily achieve this goal In case of non-conformance to standards and benchmarks, appropriate corrective and preventive action should be taken CT ea Cet All Materials Used in Microbiology Laboratory Require Quality Assurance!! p___] [ABS FOR LIFE PROJECT 1775 76 Quality Assurance of Methods & Materials Many materials produced within laboratory will be made up entirely of constituents which have been quality controlled by the manufacturer, but end products must be treated as in- house products and quality controlled accordingly. This is required because they will have been processed to some extent in the laboratory, this does not invariably guarantee that an end product will perform to the standard expected. General practices HOLD LOOP / NEEDLE AT 60" AND PASS THE LOOP THROUGH + Flaming ofloop THE FLAME SO AS TO HEAT THE WIRE RED-HOT ALONG ENTIRE LENGTH + Smear preparation for staining - Most initial observations of micro- organisms are made with stained preparations. - Inthe beginning, slide is made grease free by passing emply slide few times through flame quickly. COOL FOR 10SEC = Label the slide and area marked on the BEFORE USE opposite side ofthe smear. - For CSF/body fluids/urine, no need to Figure: Flaming of Loop spread on slide, Place loop-full of desired fluid three times on the same spotonslide. = Airdry/blot dry the smear. = Before staining, the microorganisms mustbe fixed (attached) to the microscope slide. ~ Fixing simultaneously kills microorganisms and fixes them to slide. It also preserves the morphology ofthe organisms and tissue/other cells with minimal distortion Quality Assurance of Stains Stains are used for microscopic procedures that provide information for either preliminary or definitive diagnosis. These are frequent in bacteriology, parasitology, mycobacteriology, mycology, virology and other laboratory areas. Gram stains are used for identification of bacteria and yeast from colonies and samples. Acid-fast stains are particularly important for preliminary diagnosis of mycobacteria. Permanent stains such as acridine orange, trichrome and iron-hematoxylin for faecal parasites, and Giemsa stain for malaria, are frequently used in microbiology. For wet mounts, iodine solutions are used to detect cysts and eggs in faecal samples, and potassium hydroxide preparations are used to detect fungal elements. Some stains can be purchased commercially, but others must be prepared by the laboratory following an established procedure.Once stains are made, their bottles should be labeled with the following information: Figure: Sample ofa Stan Bottle Label Important points: 1 It may be useful to keep a logbook for recording information on each stain in Use, including the lotnumber and date received, 2. Stains should be stored at the correct temperature at all times and in an appropriate staining bottle. Some stains must be protected from light. In some cases, working solutions can be made from stock solutions. If so, storage of working solutions should be carefully monitored. Figure: The Right & Wrong Ways of Storing Stains pam) wy LABS FOR LIFE PROJECT‘Stain Management a) _use established procedure for preparation or reconstitution b) label: content, concentration, date prepared and placed in service, expiration, initials ©) store appropriately Quality Control of Stains ‘As appropriate for particular stain: a) check with known organisms or cells b) examine for crystal shards or for precipit ©) examine for contaminants such as bacteria and fungi Gram Stain All stains and reagents should be stored according to manufacturer's recommendations and tested with positive and negative controls before use. Name of stain Positive control Negative control Gram stain Staphylococcus aureus | Escherichia coli Acid-fast stain Mycobacteruim ‘Non-acid fast organism (e.g. E. coli) Figure: Good Qualty Wright stain Figure: Good Quaity Gram stain Figure: Good Quality Gram Stan Figure: Poor Quaty Gram Stain = LABS FOR LIFE PROJECT77 Quality Assurance of Culture Me: General Microbiological laboratories acquire media in one of two ways, either purchasing the media pre-made from a manufacturer (Commercially prepared), or making the media (either in whole orin part) in-house Clearly, ifthe mediais purchased from the vendor there is little opportunity to control the preparation beyond having confidence in the supplier. Media prepared in-house offers several opportunities for quality control. The raw materials (either the dehydrated complete media or the components) must be stored under appropriate and controlled conditions and used within established expiry dates. Broad Classifications of culture media in microbiology: 1, Selective media: These media contain antimicrobial or chemical agents that inhibit the growth of certain microorganisms. 2. Non-selective (nutritive): hese media promote the growth ofall organisms, Both selective and non-selective media can be solid (agar-based) or liquid (broth-based). Solid (Agar-based) eres ueret ro Perey ey eri} Figure: Broad Classification of Mea; MicrobiologyAll media should be carefully checked for: + Physical characteristics: Check for turbidity, dryness, evenness of layer, abnormal color + Chemical characteristics: pH + Sterility: To verity freedom from contamination = Incubate overnight before use + Performance: To demonstrate correct performance of the medium when used in the usual or widely accepted manner and also * Ability to support growth—using stock organisms = Ability to yield the appropriate biochemical results—using stock organisms Quality Control of Media: + Verify performance of all media + In-house prepared: all batches + Commercially prepared: new lot only Non-lactose fermenter Lactose fermenter Figure: MacConkey Agar QC 7.7.1 In-house Media Most of the laboratories usually prepare their own media for routine diagnostics as well as research purposes. However, to ensure that the media is of good quality and capable of giving satisfactory results, proper quality management system is essential. For that purpose certain parameters of media prepared should be thoroughly checked and then passed for laboratory use: 1) Raw Material Parameters a) Water quality: The quality of the media depends directly upon the quality of the raw materials used for their preparation. Water is the most important raw material used for the preparation of culture media. The parameters to be checked are presence of p___] [ABS FOR LIFE PROJECTcopper ions, conductivity and pH. Ideally there should be no copper ions present in water because it is inhibitory for the growth of microorganisms. The conductivity should be less than 15 1S (microsiemens). The pH of the water should be slightly on the acidic side, but should notbeless than 5.5. b) Petri dish: The quality of petri dishes used for pouring of media is also an important factor. The plates should be even and not cracked or broken. There are various types of petri dishes available with manufacturers. Most common are the sterile disposable, plastic autoclavable ones and glass petri dishes. ¢) Additives: There are various additives used in preparation of media. Bloodis the most important one of them. Hence the quality of the blood plays an important role in the performance of the blood containing media. The sterility, homogeneity, viscosity and colour of the blood should be scrupulously checked before it is used for media preparation. For other additives the certificate of analysis and sterility conditions should be considered. Human blood should not be used because: i. toomuch batchto batch variation ii, mayinclude inhibitory substances, including antimicrobials Iii, maybe bio hazardous (.g., hepatitis virus, HIV) 2) Sterility Sterility controlis 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-flterable solutions that may then require aseptic dispensing, would require strict sterility control up to a level of incubation at 37°C for 24 hours of the entire batch for 3 days. If detected contamination exceeds 10%, the whole batch should be discarded Sterility testing of media is generally carried out at 37°C, which is logical since this is the temperature most commonly employed in clinical microbiology; however, it is often worthwhile to incubate units of media at lower temperatures. The most convenient way to do this is, after overnight incubation at 37°C and examination of the media, to incubate ata lower temperature such as room temperature for a further 24 hours. Allfiuid media should also be sub-cultured after incubation to demonstrate sterility. 3) Performance The performance of a prepared medium must also be assessed before it is released for use. Testing must reflect the use of the medium, and the test organisms must reflect the likely encountered in actual use. Incubation temperatures, times and atmosphericconditions must also be the same as in routine usage. This means in many cases that more than one unit of medium will require testing, The selection of organisms for performance testing will aim to demonstrate that the medium does what it is expected to do. Thus, blood agars will be seeded with a variety of test organisms, both aerobic and anaerobic, which will not only exhibit satisfactory growth but also their typical colonial morphology and typical characters such as haemolysis; some of the test strains should be extremely sensitive to antimicrobial substances so that any contamination or carry over of such substances is detected. Selective media must be tested for the satisfactory inhibition of normally encountered flora that requires suppression; for example, enteric media will require testing to show growth of the normal bowel flora. In this case, a mixture of coliform organisms, in the form of negative faecal sample, and a selection of indicator organisms may be the test inocula. Enteric media, especially selective enrichment media, should also be tested for the ability to enrich the pathogens selectively in the presence of contaminating flora. Incase of identification media, positive and negative controls will be required. In the case of media used for combined tests (e.g. acid/gas), all the combinations of outcomes must berepresented. Antibiotic sensitivity test media are difficult to control. The wide variety of organisms and antimicrobials to be tested gives rise to a virtually limitless number of combinations. They are best tested for their ability to sustain growth, only, in the first instance, and their satisfactory performance for testing of sensitivities can then be done by controlling all tests according to the “Stokes criteria". When the breakpoint sensitivities are used, the media must be tested after preparation to ensure correct performance before routine use; any failure will affect a batch of specimens. Finally, a daily test is carried out on a panel of organisms that exhibit sensitivity and resistance to all of the antibiotics in use. All findings with the performance strains must be satisfactory and should not vary for different batches of the same medium. Adequate records are essential for analysis of the performance of any medium. These must include’ a) Batchnumbers ofall constituents b) Date ofpreparation ©) Details of the dispensing equipmentused d) Laboratory operators concerned e) Batchnumber of containers f) Sterility and quality control results,4) 5) Physical parameters a) The gross physical appearance of media often suggests the quality, Media prepared should be screened for physical characteristics such as excessive bubbles or pits, unequal filling of plates (uniform leveling), cracked medium in plate and freezing or crystallization. ») Allthe above mentioned characters can be checked visually by naked eye. However, for unequal filling of plates, thickness of medium can be checked at four points. These four points are the two ends of the two diameters of the plate, which are at right angles to each other. Thus all the four sides can be simultaneously checked. The thickness at the four points is noted down and the mean thickness is determined and reported as mean thickness of the medium in the plate, which must be 4.0 + 0.2mm. ©) The pH value of the medium is also one of the important physical characters, which must be checked. It can be measured while preparation of the medium before and after autoclaving by using the standard pH meter after proper calibration with standard buffers. Visually inspect in-house prepared media plates: + Agardetached from the petri plates + Unequal filing of the plates + Insufficient agar in the plates (<3mm) + Hemolysis of blood containing media cS + Change in the expected color of the media (possible pH problem) + Excessive bubbles or rough surfaces + Excessive moisture or dehydration + Obvious contamination + Presence of precipitatesTroubleshooting Guide Information about the various problems found in culture media and their probable causes areas follows: Table: Troubleshooting Guide for Culture Media Contaminated glassware, impure water, overheating, chemical contamination, pH taken at wrong temperature, pH measuring equipment faulty or poorly standardized, deterioration of dehydrated medium | parkering | Exeeshoatdteratonot hydrated modu Excess heat (scorching or burning), deterioration of Toxicity dehydrated medium Contaminated water or glassware, incorrect weighing and Poor selective | mixing, differential deterioration of dehydrated medium, excess heat properties It is important to keep careful records for media that is prepared in the laboratory. A logbook should be maintained thatrecords: + Dateand preparer's name + Name ofthe medium, the lot number and manufacturer + Number of prepared plates, tubes, bottles or flasks + Assigned lotand batch numbers + Color, consistency and appearance + Number of plates used for qo + Sterility test results at 24 and 48 hours + Growth test(s) + pH = LABS FOR LIFE PROJECT7.7.2 Commercially Prepared Media Commercial Media are divided into two additional categories (determined by QC performance data collected by CAP): i. An exempt medium (maintain consistent user performance with minimum variation and requires minimum quality control). ii, Nonexempt (documented by user QC performance data collected by CAP to vary in performance from lot to lot, or any media prepared by the user). Table: Exempt & Non-Exempt categories for Media Included in CAP Surveys (1984,1988, 2001) General bacteriologic | Blood agar Nutrient broth media Chocolate agar” Thioglycolate broth Urease agar Blood culture media® | Brainheartinfusion (BHI) blood culture broth Biphasic blood culture bottle medium Centritugationvisolation rubes (adult) Thiol blood culture broth ‘Trypticase soy blood culture broth Peptonebroth ‘Media for gram-positive | Columbia (CNA) agar Todd-Hewitt broth Cos Selective media for enterococci with or without | Desoxycholate broth’ fade ‘Trans-vaginal broth" cbr ‘Chocolate agar with Mannitol satagar phridoxal’ Phenylethylalcohol (PEA) agar Selective agarfor Group A Streptococcus ‘Sheep blood agar with sulfamethoxazole / timethoprim (SXT) Enterococcus (Streptococcus) feacalts broth Media for gram- Cofsuledinirgasannovobiocin (CIN) agar MacConkey sorbitol regative bacteria Citrate agar agar Cystine lactose electrolyte deficient (CLED) agar | Chocolate agar with ‘gram-negative (GN) broth Paciracin Hektoen (HEK) agar MacConkey agar Salmonella Shigella (SS) agar Selenite broth Thiosuifate citrate bile salts sucrose (TCBS) agar ‘Triple sugariron (TSI) agar = 27 LABS FOR LIFE PROJECTBordetella pertussis media Reagan-Loweagar Border Gengou agar’ Legionella media Legionella selective (CYE/BCYE) agar “* Seletive Legionella agar with DGVP Campylobacter media ‘Charcoal selective agar with CVC" Campylobacter blood agar (Blaser) Campylobacter agar with ova" ‘Anaerobic media Anaerobic blood agar ‘Anaerobic phenylethy| alcohol (PEA) agar Bacteroides bile esculin (BBE) agar Brucella agar Brucella agar w / hemin / Vitamin K Brucella laked blood agar with KV* CDC anaerobe laked blood agar with Kv" CDC anaerobic 5% sheep blood with KV * Egg yolk (modified) agar Kanamycin laked blood agar CDC anaerobe 5% sheep blood agarwith PEA AFB briphasie bottio medium* Middlebrook 7H9 broth Lowenstein-Jensen media Middlebrook agar ‘Automated AFB bottle broths? Middlebrook 7H10 agar Middlebrook 7H1 1 agar ‘American Trudeau Society (ATS) agar* Mitchison’s agar” Petragnani medium* Fungal media Commeal agar Inhibitory mould agar Inhibitory mould agar with gentamicin Soy peptone agar with CC without pH indicators” Potato dextrose agar Brain heart infusion agar with 59% sheep blood/CG* ‘Sabouraud's dextrose agar ‘Sabouraud's dextrose agar with CG" Commeal agar with Tween Brain heart infusion agar with 6% sheep blood/C BIGGY agar Birdseed agar® Brain heart infusion agar with 5% sheep blood /PS. Dermatophyte test ‘medium* Potato flakes agar with or without co** * emp: Berapolte Faure Rate of 0.5%; Nonexempt: Exvapolsted Faure Rate f >0'5%; media wt insutcint da for catagorzatonisconsderednonexemotend QCisrequred * Represents formulations ftom 8D Diagnostic Systems (Sparks, MO) or BioMerieux (RaleighvDurhar, NC. Aeter to ‘manutactuterspackageinsertor spect OCinermaton. = LABS FOR LIFE PROJECTGeneral Quality Control Requirements for both Exempt and Nonexempt Media a Commonly used media and bases obtained from commercial suppliers should be ordered in bulk so that their consistency is ensured over long periods of time - provided that they are known to be stable that their shelf fe is assured, and that local facilities for storage are satisfactory. On receipt, conform that a bulk supply is the entire one batch. Before itis used, do an extended quality control check to ensure that itis of an acceptable standard or to detect any variation or deficiency. Be aware that QC reports and assurances supplied by manufacturer may not meet the standards that the laboratory wish to set and should not be accepted as an excuse for not doing adequate in-house testing Check the following procedures when a media shipment arrives in the laboratory - Verify delivery of the ordered amount. Check each medium type for multiple lot numbers and/or impending expiration dates. Report recurring problems to the manufacturer or distributor. - Record the lot number, expiration date and the received date (optional) for each medium type. - Store the media as specified by the manufacturer (generally 2-8°C) pending quality control. Separate exempt media from nonexempt media Visual Inspection Record the numbers of plates per lot that exhibit any of the characteristics listed below. If excessive, notify the manufacturer or distributor. Visually inspect each medium lot for obvious problems suchas’ + Cracked or damaged plates + Agardetached from the petri plates + Frozen ormeltedagar + Unequal filing of the plates + Insufficient agarin the plates (<3mm) + Hemolysis of blood containing media + Change in the expected color of the media (possible pH problem) + Excessive bubbles or rough surfaces + Excessive moisture or dehydration + Obvious contamination + Presence of precipitates Checking for contamination Because contamination testing is routinely performed by manufacturers, commercially prepared media need not be retested for sterlty by the end user. Instead, careful inspection for contamination should take place immediately before inoculation with patient specimens.h. Quality Assurance Laboratories are strongly recommended to confirm the ability to support growth for all media used for the recovery of fastidious organisms such as 1) 2) 3) 4) 5) 8) 7 Anaerobes Bordetella pertussis Burkholderia cepacia Campylobacter /Helicobacter Legionella Neisseria gonorrhea Any isolate with fastidious or unique growth requirements Laboratories must use media for primary culture which will support the growth of a wide variety of organisms e.g. blood agar and chocolate agar. i. UserQC requirements fornonexempt media only Perform visual and contamination checks Verify acceptable growth and/or inhibitory proper fungal control organisms. s with appropriate bacterial or Tabl rocessing of Exempt Media Media | Record the amount of media | Boxes properly labeled. | Boxes unlabeled. Required Arrival | received, the arrival date | Dates, lot numbers, and | information not recorded in QC (optional, lot numbers, and | amount received are | records. Failure to store media expiration dates in the QC | recorded in QC records. | atrecommended temperatures. records. Sequesterthelots(s) | Mediaare stored at2-8°C. pending QC testing, Lable boxes with the received date Visual_| Remove media packages | 1. Intact petri dish 4. Cracked/damaged petri Inspection | from outer boxes. Examine | > Agar attached to dish the packages for obvious |” bottom of dish 2, Agardisoldgedorloose damage. For every 10-20 3, Blood containing 3. Hemolysis of blood in plates in the lot, remove 1 | > Petter ie opacue regan. plate. Perform visual |, 1 ae | 4. te lation of Paspection, Recordrexc ledium is appropri ccessive accumulation inspection. Record resutts. Mec Recessive 5, Minimal moisture in| 5. Evidence of freezing or package medium desiccated 6 Not frozen or 7. Grosscontamination desiccated 8 Uneven or underfilled 7. No visible plates / agar desiccation contamination 9. Excessive bubbles / rough 8. Agar has even depth surface of atleast 8 mm 10, Presence of precipitates 9. Smooth agar surface 10. No precipitatesTable: Manufacturing Quality Control Requirements for Commercially Prepared Media Anaerobic sheep blood and | Anaerobic, 24-48 | B. fragilis (25285) Growth laked blood agar hase ©. Pertringens Growth, beta hemolysis (13128) Growth F ruclatum (25586) | Growth P. anaorobius apacroe Growth P. melaninogenica (25045) Anaerobic broths - see thiolycolate medium Blood agar - nonselective sheep | Anaerobic or CO, | S. pyogenes (19615) | Growth, beta hemolysis, bloodagarmedia 1824n,35'C | s, penoumoniae _| Grow, alpha hemolysis (6305) Growth ‘S. aureus (25923) Growth E, Coll (25922) Blood agar - CAMP test | Anaerobic, 18-24]. aureus (33863) or | Growth (trypticase soy agar [TSA] with | h, 95°C (25928) Positive reaction (Arrowhead blood only) 8. agalactiae area of clearing) (12986) Negative reaction (No 'S. pyogenes (19615) | arrowhead formation) Blood agar - Selective sheep | cna, co, 24-48 | S. pyogenes (19615) | Growth, beta hemolysis, blood agar media (Colmbia |p, a5°¢ ‘8. pneumoniae Growth, alpha hemolysis [CNA] agar, phenethyl alcoho! (6305) [Pealaon Growth 'S. aureus (25923) | Inhibition (partial) P mirabilis (12453) PEA, CO, 24-48 |S. pyogenes (19615) | Growth +h, 35°C 'S. aureus (25923) Growth P mirabilis (12453) | Inhibition (partial Bloodculturemedia. Tis apolies | Anaerobic, |B. fragilis (25285) | Growth to brain hear infusion, trypticase | days, 35°C Seyerotnvandink Heasedmedia. | aerobic, § days, | s, pneumoniae Growth or mocia or blood culture are | Sere (6305) ‘exempt from user performance testing provided organisms ‘appropriate or theirintended use aretested Campylobacter agar (user quality | Reduced, O, with | C. jejuni (33291) Growth control required) CO, 48h, 42°C |. coil (25922) Inhibition (parti) Chocolate agar ‘Go, 24 and 48 h, | N. gonorrhoeae Growth 35°C (43069) Growth E. influenzae (10211), = LABS FOR LIFE PROJECTCefsulodin irgasan novobiocin (CIN) agar ‘Aerobic, 24-48 h, 25°C NV. enterocolitica (9610) E. coil (25922) P. aeruginosa (27853) E. faecalis (29212) Growth; deep red center transparent border (to eye) Inhibition (partial to complete) Inhibition (partial to complete) Inhibition (partial to complete) Buffered charcoal yeast extract ‘Aerobic, 48-72 h, L. pneumophila Growth; yellow-green (BCVE) (CYE/BCYE) agar asc (3152) fluorescence under lon UV light L. bozemani Growth; blue-white (93217) {orescence L, micdadet (39204) | Growth Enrichmant broths for entories | Aerobic, 18-28, |. typhimurium | Growth on subouture (gram-negative [GN] broth, | 35°C (14028) Growth on subculture (may selenite broths) S, sonnei (9290) | be inhibited by media) E. coll (25922) Inhibition (partial to complete) on subeuture Growth on subculture from GN broth Eosin methylene blue media | Aerobic, 18-24h, | L. typhimurium Growth, colorless to amber (Levine EMB agar; EMB agar, | 35°C (14028) colonies modiied) E, col (25822) Growth, blue-black colonies F teccals (20212) | wih green metal sheen Inhibition (partial) Hektosnenteric(HEK)agar | Aerobie, 18-24, |. phimurium | Growth, colorless blue to 35°C (14028) green-bluewith black centers E, flexneri (12022) Growth, colonies green to blue- E, faecalis (29212) | reer col (25822) inhibition (partial); colonies| yellow Inhibition (partial to complete); colonies yellow salmon colored| MacConkey agar ‘Aerobie, 18-24, | E. coll (25922) Growth, pinkcolonies 38C P. mirabilis (12453) | Growth, colorless colonies, | L. typhimurium | Patialinhibtion swarming (14028) E. faecalis (29212) Growth, colorless colonies Inhibition (partial) Mannitol saltagar Aerobic, 24 and 48h, 35°C S. aureus (25923) S. epidermiais (12228) P. mirabilis (12453) Growth, colonies have yellow zones at 48h Growth, colonies have red zones at 48 h Inhibition (partial) Mycobacteria media (Lowenstein Jensen agar and Middlebrook) broth medium used for recovery of AFB are exempt from user quality control provided that manufactures certify acceptable performance using the QC isolates listed. Co, <2t days 35°C M.tuboroulosis H97Ra (2517) M. Kansasil Group | (12878) M. Serotulaceum Group Il (19981) M.intraceltulare Group ll (13950) M. fortuitum Group (341) E. colt (25922) Growth Growth Growth - May be inhibited on selective media Growth - May be inhibited on selective media Growth Inhibition (partial to complete onselective media) 32 p___] LABS FOR LIFE PROJECTPC (Burkholderia copacia) agar | Aerobic, 48-72 h, | B. cepacia (25416) | Growth with red zone 30°C E. coli (25922) Inhibition (partial to complete) P. acruginosa Inhibition (partial to complete) (27853) nh tal ete) aureus (25923) hibition (partial to complete) Nonselectivemycologymedia | Aerobie, <72h, | C. albicans (60193 | Growth 25-35°C or 10231) Growth . mentagrophytes (9533) ‘Salmonella-Shigella(SS)agar__| Aerobic, 24h, | S. typhimurium Growth, colonies colorless with 35°C (14028) ‘orwithout black centers SS. flexneri (12022) | Growth, colorless colonies E. feecalis (29212) | ition (complete) E. coli (25922) Inhibition (partial to complete; Colores pink to rose-red with precipitate Selectvemycologymedia | Aerobie, <7days, | A niger (16408) | Ibi (partial o complete) 2s 6 albicans (to2st) | on media containing T ertaraenm | efbhexiiae ‘mentagropnytes. |G (9533) Grown £. col (@s02a) | Grown Inhibition (partial to complete) con media containing chloramphenicol Selective media for pathogenic |Co, 24h, |N.gonomhoeae | Growin Neisseria app. 35°C (43069) Gr N menngits | pion pari wooo media containing trimethoprim mirabilis (48071) Inhibition (partial) E coll (25922) Inhibition (partial) N. sicca (9913) Inhibition (partial) . albicans (60193)" (12228) with azide 35°C . pyogenes (19615) E. coli (25922) Inhibition (partialto complete) Inhibition (partial) - Colorless colonies anbile esculin agar Thiolycolate broth, with or | Aerobic, 48h, |. fragilis (25285) | Growth withoutindicator (tightened cap) | s. aureus (25923) | Growth 35°C ‘Thiolycolate broth, enriched with | Aerobic, 48h, | P. anaorobius Growth vitamin Kandhemin (tightened cap) | (27837) Growth asic B. wulgatus (8482) | Growth ©. perfringens (13124) Tubed media (rain heartinlusion | Aerobic, 18-24, | E. col (25922) | Growth and tryptic soy broth) 350 . aureus (25923) | Growth = 33 LABS FOR LIFE PROJECTXylose lysine desoxycholate (XLD) agar Aerobic, 24h, 35°C S. typhimurium (14028) S. flexneri (12022) faecalis (29212) E. coli (25922) Growth - colonies red with black centers Growth - colonies red Inhibition partial Inhibition (partial to complete; colonies yellow to yellow - red) Table: Performance check of nonexempt media Nonselective | Preparea 1:10 dilution of the media PS in sterile saline Inoculate medium with 10 pl. toachiove 10°-10'cfulplate, Colonies of sufficient size and characteristic colony morphology to allow ination of routine testing within 24-48 hours incubation, No growth within 48 hours or insufficient colony size for observation of typical characteristics or initiation of additional testing procedures. Selective media | Prepare a 1:10 dilution ofthe PS in sterile saline Inoculate medium with 10 pL. toachieve 10°-10'cfu/plate, Colonies of sufficient size and characteristic colony ‘morphology to allow ination cof routine testing within 24-48 hours incubation of QC organisms used to test selective properties. 'No growth within 48 hours or insufficient colony size for observation of typical characteristics or ination of | additional testing procedures, Inadequate inhibition of QC organisms sedtotestselectivepropertes. Mycobacterial | Emulsily the growth in sterile media diluent containing bovine serum albumin (0.2%) and Tween 80 (0.002%). Add 812 sterile glass beads and mic vigorously for 10 min. Let stand for hours to allow large particles to settle, Transfer the supernatant to a stetile vil. ‘Adjust the call suspension to rmatcha05 McFatand standad. Proceed as described for preparation of dilutions and inoculation of nonselective and selective media, Colonies of sufficient size and characteristic colony ‘morphology to allow ination. of routine testing within after appropriate incubation of QC organisms used to test selective properties. No growth insufficient colony size for observation oftypical characteristics or initiation of | additional testing procedures, Inadequate inhibition of QC organisms used to test selective properties. as forbacteria Fungal media | Yeast: Procedureisthe same Moduls: Transfer a portion fo the colony to the plate Yeast: Colonies of sufficient size and characteristic, colony morphology within 72h, Moulds: Colonies of sufficient size and characteristic colony morphology within 3 - 5 ‘days. Total to partial inhibition of QC organisms used to test selective properties, No growth or insufficient colony size after recommended incubation period. Inadequate inhibition of QC organisms used to test selective properties. andar. (Prnary Suspension (PS). Reterto Table 2orepectictecommendatons * inoculum propa er asobicfanaerbie bacteria Propar a suspension fo a 28-hour culture in stare eaine to match a 0.§ MeFaland * Acceptable pedormance should general allow inision of routine esting wn 24.48 fr nonlastious, more rapily owing bate. 34 = LABS FOR LIFE PROJECTQuality Control of Media (CLSI, 2015) Mueller Hinton agar or the various other broth and agar media not containing antimicrobials should always be kept overnight at 370C for sterility checking prior to inoculation. Each batch or new lot of media should be checked with reference ATCC strains. Zone sizes and MICs obtained must be within acceptable CLS! limits. Else the batch should be rejected and any patient results obtained not reported. An example of measures of QC of blood agar medium is given below. Similar parameters must be checked for all types of culture media as per recommendations (CLSI/EUCAST). 7.7.3 Quality Assurance of Blood Agar Medium (a) Sterility Check Frequency: Each new batch of prepared medium. Controls: 1-3% of total number of plate's prepared, incubated at 35-37°C for 72 hrs, Acceptable results: No growth on any plate. Corrective Actions’ + If growthis seen on any plate, repeat sterility check using 10 additional plates. - If >1 plate is contaminated upon repeat testing, notify supervisor immediately, and discard entire batch. = _ Investigate and resolve problems, then prepare new media. Documentation: Record results. If contaminationis seen, documentthe corrective action, (b) Performance Check Frequency: Each new batch of prepared medium. Controls: 1-3% of total number of plates prepared, tested with diluted suspension of E. colior Staphylococcus aureus. Acceptable results: Growth of typical colonies within 48 hours. Corrective Actions: - Ifo growthis seen, repeat performance check. - If colonies are not typical of the bacteria used or the growth is mixed, repeat performance check with afresh culture of the stock strain. - If performance still not as expected upon repeat testing, notify supervisor immediately, and discard entire batch - Investigate and resolve problems, then prepare newmedia. Documentation: Record results. if QC results are not acceptable, document the corrective action.78 7.8.1 Table: Quality control of commonly used media: suggested ATCC control organisms and expected actions Anaerobic sheep | Anaerobic, 24-48 hr, | B. fragilis (25286) Growth blood agar and 35°C ©. portringens (13124) | Growth, betahaemolysis, laked blood agar nucleatum (25586) Growth P. anacrobius (27837) | Growth P_ melaninogenica 25845) | Growth Blood agar Aerobic or CO, , 18-24 |S. pyogenes (19615) Growth, betahaemolysis nonselective hy, 35°C ‘S. pneumoniae (6305) Growth, alpha haemolysis sheep blood agar ‘S. aureus (26923) Growth E. oll (25922) Growth ‘Mac Conkey Agar | Aerobic, 95°C E. coll (25922) Growth, pink-colored colonies (Lactose fermenter) Blood agar-CAMP | Aerobic, 18-240n,35°C | S. aureus (38862}0r(25923)] Growth test (trypticase S. agalactiae (12386) Positive reaction (Arrowhead soy agar with s area ofclearing) pyogenes (19615) sheep blood only) Negative reaction (No arrowhead formation) Blood agar media | Anaerobic, Sdays,95°C | B. fragilis (25285) Growth Aerobic, ,Sdays,36°C | S. pneumoniae (6308) | Growth Chocolate agar CO, 24-48 hy, 35°C 'N.gonorrhosae (43068) | Growth A. influenza (10211) Growth Cystine lactose Aerobic, 24-48hr,95°C | E. coli (25922) Growth: yellow centers electrolyte P. vulgaris (8427) Growth; bluish, spreading deficiont (GLED) S. aureus (25923, Inhibited (partial) growth; agar Uniform deep yellow Enrichment broths | Aerobic, 18-24h,35°C | S. typhimurium (14028) | Growth on subculture for entercs (Gram - S. sonnei (9290) Growth on subculture (may negative broth, be inhibited by selenite selenite broth) media) Quality Assurance of Organisms Source of Quality Control Organisms Quality Control Organisms are Available from the American Type Culture Collection (ATCC), ATCC- derived cultures are also available form commercial sources. Previously characterized organisms isolated from patients and shown to be phenotypically stable are also appropriate for media Documentation records of biochemical characterizations and identification of quality control. these isolates should be maintained. = 36 LABS FOR LIFE PROJECT7.8.2 7.8.3 Reference Strains for QC: + S.aureus ATCC 27923 + Escherichia coli ATCC 25922 + Escherichia coli ATCC 36218-ESBL detection + Klebsiella pneumoniae ATCC 700603-ESBL detection + Pseudomonas aeruginosa ATCC 27853 Storage of Quality Control Organisms Please refer to the table below for storage conditions and duration. For prolonged storage and to obtain isolated colonies, freeze-dried or frozen cultures should be sub-cultured twice prior to testing. Working cultures should be stored on trypticase soya agar or Chocolate agar at 2- 8°C. Before testing, strains should be subcultured inherent susceptibility, a new culture of the control strainis obtained Storage of QC Organisms ai Per Lyopizes Unilentatoncao rranvactuer Rapidly growing 28°C Working QC cultures: ‘Aweeks en ee Cc ec | ase stock cutes =ramenits = ae | Sapenseninayepesenaive | Table: Gram Negative Biochemical Chart Biochemical Reactions Testisubstrate" Species tac |mot | gas| ind | ve | cit | PDA | ure] ws | H,S | inos | ONPG Escherichia coli +{+f+]+]-|-| -[|-[+]-]- [+ Shigella groupsa,B,c|- |- | -|+#|-|-|-|-|-|-|- | - Sh. sonnei -[-|-|[-[-|-[|-[-[-]-]-]- ‘Salmonella typhi -[+]+]-]-]+]-]-[+] +] 4] - Salmonella paratyphiA|- | + | +|-|-|-| -]-|-]-]-] - Citrobacter freundi =| | + | +|-|-|+|-/#/-|2#]-/ + ©. koseri z[+f+l+[-[+[-]/e[-[-][- [+ Klebsiella pneumoniae |+ | - | ++] -|+]+] -]+]/+]-]+] + K. oxytoca +{-[*#l+l+l-+[-]*l+]- Te - Enterobacter aerogenes} + | + | ++|-| +/+] -|-[+]-]+ | + Ent. cloacae +*f+f+]-l-l+|-le|/-|-|-T- Hafnia alvei -l+f+f-[+/-|-/-[-|f-][-][- ‘Serratia marcescens’ |- |+ |a}-|+|+|- |-l+]- 12] + Proteus mirabilis ~f+f+l—lalel+ fel- fe [-f - vulgaris: -[+/[+l-[-[- [+ [el [+ [- 7 - Morganellamorganii |- | + | +|+|- |-| + |r{- [+ | - | - Providenica rettgei |- | + | -|+|- [+ | + |+#{- [- [+ | - Prov. stuart -[+]-]+]-]+]+ fe f-7- [+f - Prov. alcalifaciens ~l+l+lel- [+ [+ [-f-7- [- 7 - Yersinia enterocolitica’ |- |- | -| + ]- |-|- |#|- [- || + ¥. pestis ~f-T-T-]-|-/- [-[-/- /-T¢ ¥. psoudotubercuiosis |- |- | -|-|- |-]|- |+]- ]- |- | # * Lag, nos, fermentation oflactose, inositol; mot, moti; gas, gas from glucose; ind, indole production; VP, Voges- Proskauer, cit, citrate utilization (Simmons') PDA, phenylalanine deaminase; ure, urease; ys, lysine decareboxylase; H.S, H.S produced in TSlagar; ONPG, metabolism ofo-nitrophenyi-f-D -galactopyranoside, * Some strains of Serratia marcescens may produce ared pigment * Yersinia are motile at 22°C7.10 Quality Assurance of Anaerobic Bacteria Obligate anaerobes include Prevotella sp., Fusobacterium sp., and Bacteroides sp., which are killed upon brief exposure to atmospheric oxygen. Aerotolerant organisms (5% O,) include Actinomyces sp.. Bifidobacterium sp., and Clostridium sp. The importance of proper collection and transport of specimens for anaerobic culture cannot be overemphasized. Because indigenous anaerobes are often present in large numbers as normal flora on mucosal surfaces, even minimal contamination of a specimen can produce misleading results. In general, material for anaerobic culture is best obtained by tissue biopsy or by aspiration using a needle and syringe. Use os swabs is a poor alternative because of excessive exposure of the specimen to the deleterious effects of drying, the possibility of contamination during collection, and the easy retention of microorganisms in the fibres of the swab. Ifa swab must beused, it should be from an oxygen-free transport system. Clinical specimen suitable for anaerobic cutture: a) Bile b) Biopsy of endometrial tissue obtained with an endometrial suction curette ©) Blood d) Bonemarrow ©) Bronchial washings obtained with a double-lumen plugged catheter ) CSF 9) Culdocentesis aspirate h) Decubitus ulcer (if obtained from base of the lesion after thorough debridement of the ulcer's surface) i) Fluid form a normally sterile site (e,9, joint) i) Peritoneal fluid k) Percutaneous lung aspirate or biopsy )Sulfurgranules froma drai g fistula m) Suprapubic bladder aspirate n)Thoracocentesis (pleural) fluid 0) Tissue obtained at biopsy or autopsy p) Transtracheal aspirate q) Uterine contents Clinical specimens Unsuitable for anaerobic culture: a) Bronchial washings (unless obtained with a double-lumen plugged catheter) b) Coughed (expectorated) sputum °c) Faecesa) °) f) Gastric or small-bowel contents lleostomy or colostomy drainage Nasopharyngeal swab Rectal swab Secretions obtained by nasotracheal or orotracheal suction ‘Swab of superficial (open) skin lesion Throat swab Urethral swab Vaginal or cervical swab Voided or catheterized urine A crucial factor in obtaining valid test results with anaerobic cultures is the transport of specimen; the lethal effect of atmospheric oxygen must be nullified until specimen can be processedin the laboratory. Recapping a syringes no longer acceptable. Various kinds of anaerobic transport systems are available. E.g a) Anaerobic transport systems for liquid specimens b) Anaerobic transport systems for swab specimens (e.g. Vacutainer anaerobic specimen collector by BD) ©) Anaerobic transport systems for tissue specimens e.g. using GasPak pouch Blood culture — inoculate Thioglycollate broth (TB) and Robertson's cooked meat medium (RCM) Granules / gauze dressings from cases of suspected actinomycosis Necrotic tissue (muscle) if suspected gas gangrene Prompttransport- mandatory Promptinoculation into media- mandatory Culture - Use fresh media—T8, RCM, BA, NBA ~ Provide anaerobic conditions as directed in SOPs QC for effective anaerobiosis - Pseudomonas aeruginosa, astrict aerobe, is used as an indicator. If grown-QC failure = Thioglycollate broth: B. fragilis, C. perfringens; look for growth. General QC measures: are similar as enlisted above in bacteriology section7.11 Quality Assurance for Mycobacterium tuberculosis As per RNTCP guidelines, a patient is confirmed to have drug resistant TB only by an RNTCP quality assured Culture & DST Laboratory. Optimal Management of MDR-TB requires both mycobacterial and clinical laboratory services. The mycobacteriology reference laboratory, which at the state level is an intermediate reference laboratory (IRL) or any other RNTCP accredited Culture & DST laboratory, should provide: 1. Culture; 2. Confirmation of the species as M. tuberculosis or non-tuberculous mycobacteria (NTM); 3. Testing for susceptibility to at leastisoniazid and rifampicin. Clinical laboratory services are required for the proper evaluation and monitoring of patients, including basic hematology, biochemistry, serology, and urine analysis as would be available at the DOTS Plus sites identified by the state. A comprehensive, routine system of internal quality control and external quality assurance is mandatory. For the national reference laboratories, formal links should be made with the WHO network of Supra-National Reference Laboratories for provision of quality assurance through validation of drug susceptibility data. Quality assurance goes beyond the relationship with the SNRL and includes good infection control measures and internal quality assurance methods to documentthat the results are valid. RNTCP has a three tier laboratory network based on the designated microscopy centers (OMCs) covering 1 lakh populations and providing sputum smear microscopy services, and IRLs (undertaking training, external quality assessment [EQA] of sputum smear microscopy networkin the districts and DMCs, and culture and DST for first line drugs for M. tuberculosis), and NALs (undertaking training, EQA of sputum smear microscopy network in the states allotted to them, and culture and DST for first and second line drugs for M. tuberculosis) Please refer to RNTCP Website for details: http://health.bih.nic.in/Docs/Guidelines/Guidelines-DOTS-Plus.paf AILQC results should be documented in the same manner as patient specimens, ¢.g., MGIT and LJ culture results, colony counts, etc. Mycobacteriology laboratory QM/QC Schedule (Ref: WHO Mycobacteriology Manual) Refrigerator, freezer incubator, | Posie and negative | Internal qualty assessment | Positve and negative controls for Tooms and ceniituge | controls for MGIT and L! | to imprave microscopy — | new staining regents for AFB semars temperatures cultures results Positive and negative controls | Reference stain fr drug | MGIT Time to detection QC | Stenity and pertarmance testing ot for AFB smears susceptibility testing | for MTB reference strain | culture mesia- MGI L,7H9 Both, BAP MGIT Maintenance (See | Positive, negative and Complete Monthly Data | Reference strain testing for new kts Section 10: LiguitCuture- | reagent controls for Monitors form and other ant-TB drugs or drug kts Mir) identtiction kts 'MGIT OC Report (See Section | Postve, negative and reagent conrls 10: Liguid Cuture MIT) | fornewros of dentiicaion is Figure: Qualty Monitoring and Qualty Control in Mycobacteriology Laboratory