Basic Laboratory Procedures in Clinical Bacteriology PDF 2nd Edition

BASIC LABORATORY PROCEDURES IN CLINICAL BACTERIOLOGY Communicable diseases are the most common cause of death in developing countries, and their diagnosis and treatment represents a significant challenge to the health services in those areas. To ensure accurate identification of causative micro-orga...

BASIC LABORATORY PROCEDURES IN CLINICAL BACTERIOLOGY Communicable diseases are the most common cause of death in developing countries, and their diagnosis and treatment represents a significant challenge to the health services in those areas. To ensure accurate identification of causative micro-organisms, laboratories need B A S I C to use standard procedures for microbiological investigations and susceptibility testing, and to implement an effective programme of quality assurance. L A B O R AT O R Y This 2nd edition of the Basic Laboratory Procedures in Clinical Bacteriology has been updated in many areas, including a greatly PROCEDURES enhanced section on stool specimens and a new section on serological tests. This manual is a practical guide, for use by laboratory workers in health centres and district hospitals, to the procedures to be followed in obtaining specimens, isolating and identifying bacteria, and assessing their resistance to antibiotics. It covers bacteriological investigation of IN CLINICAL blood, cerebrospinal fluid, urine, stool, sputum, pharyngeal and genital specimens, and purulent exudates. Particular attention is given to the need for quality control of all laboratory procedures. A list of media and reagents needed for the isolation and identification of the most common bacterial pathogens is included, together with an indication of their relative importance for the intermediary laboratory. This list is intended for adaptation to local circumstances. BACTERIOLOGY 2nd edition 2nd edition ISBN 92 4 154545 3 World Health Organization WHO Geneva The World Health Organization was established in 1948 as a specialized agency of the United Nations serving as the directing and coordinating authority for international health matters and public health. One of WHO’s constitutional func- tions is to provide objective and reliable information and advice in the field of human health, a responsibility that it fulfils in part through its extensive programme of publications. The Organization seeks through its publications to support national health strategies and address the most pressing public health concerns of populations around the world. To respond to the needs of Member States at all levels of devel- opment, WHO publishes practical manuals, handbooks and training material for specific categories of health workers; internationally applicable guidelines and standards; reviews and analyses of health policies, programmes and research; and state-of-the-art consensus reports that offer technical advice and recommendations for decision-makers. 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By helping to promote and protect health and prevent and control disease throughout the world, WHO’s books contribute to achieving the Organization’s principal objective—the attainment by all people of the highest possible level of health. Basic laboratory procedures in clinical bacteriology A Basic laboratory procedures in clinical bacteriology Second edition J. Vandepitte and J. Verhaegen Department of Microbiology St Rafaël Academic Hospital Leuven, Belgium K. Engbaek Department of Clinical Microbiology University of Copenhagen Herlev Hospital Herlev, Denmark P. Rohner Department of Clinical Microbiology Cantonal University Hospital Geneva, Switzerland P. Piot Joint United Nations Programme on HIV/AIDS Geneva, Switzerland C. C. Heuck World Health Organization Geneva, Switzerland World Health Organization Geneva 2003 A WHO Library Cataloguing-in-Publication Data Basic laboratory procedures in clinical bacteriology / J. Vandepitte... [et al.].—2nd ed. 1.Bacteriological techniques—standards 2.Laboratory techniques and procedures standards 3.Manuals I.Vandepitte, J. ISBN 92 4 154545 3 (NLM classification: QY 100) © World Health Organization 2003 All rights reserved. Publications of the World Health Organization can be obtained from Marketing and Dissemination, World Health Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland (tel: +41 22 791 2476; fax: +41 22 791 4857; email: [email protected]). Requests for permission to reproduce or translate WHO publications–whether for sale or for noncommercial distribution–should be addressed to Publications, at the above address (fax: +41 22 791 4806; email: [email protected]). 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Typeset in Hong Kong Printed in Singapore 2001/13712—SNPBest-set/SNPSprint—6000 Contents Preface viii Introduction 1 Quality assurance in bacteriology 2 Introduction 2 Definitions 2 Internal quality control 6 External quality assessment 16 PART I Bacteriological investigations 19 Blood 20 Introduction 20 When and where bacteraemia may occur 20 Blood collection 20 Blood-culture media 22 Processing of blood cultures 23 Cerebrospinal fluid 25 Introduction 25 Collection and transportation of specimens 25 Macroscopic inspection 26 Microscopic examination 26 Preliminary identification 28 Susceptibility testing 29 Urine 30 Introduction 30 Specimen collection 30 Culture and interpretation 32 Interpretation of quantitative urine culture results 34 Identification 35 Susceptibility tests 36 Stool 37 Introduction 37 Etiological agents and clinical features 37 Appropriate use of laboratory resources 39 Collection and transport of stool specimens 40 Visual examination of stool specimens 41 Enrichment and inoculation of stool specimens 41 Media for enteric pathogens 42 Primary isolation 42 Preliminary identification of isolates 44 A v CONTENTS Final microbiological identification 50 Serological identification 54 Upper respiratory tract infections 60 Introduction 60 Normal flora of the pharynx 60 Bacterial agents of pharyngitis 61 Collection and dispatch of specimens 62 Direct microscopy 62 Culture and identification 63 Susceptibility testing 65 Lower respiratory tract infections 66 Introduction 66 The most common infections 66 Collection of sputum specimens 68 Processing of sputum in the laboratory (for non-tuberculous infections) 68 Culture for Mycobacterium tuberculosis 72 Interpretation of cultures for M. tuberculosis 74 General note on safety 74 Sexually transmitted diseases 76 Introduction 76 Urethritis in men 77 Genital specimens from women 79 Specimens from genital ulcers 82 Purulent exudates, wounds and abscesses 86 Introduction 86 Commonly encountered clinical conditions and the most frequent etiological agents 86 Collection and transportation of specimens 89 Macroscopic evaluation 90 Microscopic examination 91 Culture 92 Identification 93 Susceptibility testing 97 Anaerobic bacteriology 98 Introduction 98 Description of bacteria in relation to oxygen requirement 98 Bacteriology 98 Antimicrobial susceptibility testing 103 Introduction 103 General principles of antimicrobial susceptibility testing 103 Clinical definition of terms “resistant” and “susceptible”: the three category system 104 Indications for routine susceptibility tests 106 vi CONTENTS Choice of drugs for routine susceptibility tests in the clinical laboratory 107 The modified Kirby–Bauer method 109 Direct versus indirect susceptibility tests 117 Technical factors influencing the size of the zone in the disc-diffusion method 118 Quality control 120 Serological tests 122 Introduction 122 Quality control measures 122 Serological reactions 125 Serological tests for syphilis 126 Febrile agglutinins tests 133 Antistreptolysin O test 135 Bacterial antigen tests 137 PART II Essential media and reagents 141 Introduction 142 Pathogens, media and diagnostic reagents 143 Blood 144 Cerebrospinal fluid 144 Urine 145 Stool 146 Upper respiratory tract 147 Lower respiratory tract 148 Urogenital specimens for exclusion of sexually transmitted diseases 149 Pus and exudates 149 List of recommended media and diagnostic reagents for the intermediate microbiological laboratory 150 Selected further reading 154 Index 155 A vii Preface Communicable diseases are the most common cause of death in developing countries, and their diagnosis and treatment represent a significant challenge to the health services in those areas. The World Health Organization has long been actively involved in developing and promoting standard techniques for laboratory investigations of such diseases, a first attempt to standardize sus- ceptibility testing of bacterial pathogens being made in 1960.1 Following on from this, in 1976, the WHO Expert Committee on Biological Standardization drew up requirements for antibiotic susceptibility testing using the disc method.2 At the same time, efforts were being made to introduce quality control into laboratory performance. In 1981, WHO established an International External Quality Assessment Scheme for Microbiology. The laboratories that are involved in this scheme are able to play a leading role in the implementation of national quality assessment schemes at all levels of the health care system. The present publication brings together and updates the various guidelines produced by WHO over the years on sampling of specimens for laboratory investigation, identification of bacteria, and testing of antimicrobial resistance. The information included is intended to lead to harmonization of microbio- logical investigations and susceptibility testing, and to improve the quality of laboratories at both central and intermediate levels. It concentrates on the pro- cedures to be followed, rather than the basic techniques of microscopy and staining, which have been described in detail in another WHO publication.3 1 The public health aspects of antibiotics in feedstuffs. Report on a Working Group, Bremen, 1–5 October 1973. Copenhagen, WHO Regional Office for Europe, 1973 (document no. EURO 3604 (2)). 2 WHO Expert Committee on Biological Standardization. Twenty-eighth report. Geneva, World Health Organization, 1977 (WHO Technical Report Series, No. 610). 3 Manual of basic techniques for a health laboratory, 2nd ed. Geneva, World Health Organiza- tion, 2003. viii BLMIN 1/17/04 2:08 PM Page 1 Introduction Communicable diseases continue to account for an unduly high proportion of the health budgets of developing countries. According to The world health report,1 acute diarrhoea is responsible for as many as 2.2 million deaths annu- ally. Acute respiratory infections (primarily pneumonia) are another impor- tant cause of death, resulting in an estimated 4 million deaths each year. Analysis of data on lung aspirates appears to indicate that, in developing countries, bacteria such as Haemophilus influenzae and Streptococcus pneumo- niae, rather than viruses, are the predominant pathogens in childhood pneu- monia. b-Lactamase-producing H. influenzae and S. pneumoniae with decreased sensitivity to benzylpenicillin have appeared in different parts of the world, making the surveillance of these pathogens increasingly important. Sexually transmitted diseases are on the increase. There are still threats of epidemics and pandemics of viral or bacterial origin, made more likely by inadequate epidemiological surveillance and deficient preventive measures. To prevent and control the main bacterial diseases, there is a need to develop simple tools for use in epidemiological surveillance and disease monitoring, as well as simplified and reliable diagnostic techniques. To meet the challenge that this situation represents, the health laboratory ser- vices must be based on a network of laboratories carrying out microbiologi- cal diagnostic work for health centres, hospital doctors, and epidemiologists. The complexity of the work will increase from the peripheral to the inter- mediate and central laboratories. Only in this way will it be possible to gather, quickly enough, sufficient relevant information to improve surveillance, and permit the early recognition of epidemics or unusual infections and the devel- opment, application, and evaluation of specific intervention measures. 1 The world health report 2000. Geneva, World Health Organization, 2000. A 1 BLMIN 1/17/04 2:08 PM Page 2 Quality assurance in bacteriology Introduction Quality assurance programmes are an efficient way of maintaining the standards of performance of diagnostic laboratories, and of upgrading those standards where necessary. In microbiology, quality goes beyond technical perfection to take into account the speed, cost, and usefulness or clinical relevance of the test. Laboratory tests in general are expensive and, with progress in medicine, they tend to use up an increasing proportion of the health budget. Definitions To be of good quality, a diagnostic test must be clinically relevant, i.e. it must help in the prevention or treatment of disease. Other measures of quality in a diagnostic test are: Reliability: Is the result correct? Reproducibility: Is the same result obtained when the test is repeated? Speed: Is the test rapid enough to be of use to the doctor in prescribing treatment? Cost–benefit ratio: Is the cost of the test reasonable in relation to the benefit to the patient and the community? Factors that affect the reliability and reproducibility of laboratory results Sources of error may include the following: Personnel. The performance of the laboratory worker or technician is directly related to the quality of education and training received, the person’s experience, and the conditions of employment. Environmental factors. Inadequate working space, lighting, or ventilation, extreme temperatures, excessive noise levels, or unsafe working conditions may affect results. Specimens. The method and time of sampling and the source of the speci- men are often outside the direct control of the laboratory, but have a direct bearing on the ability of the laboratory to achieve reliable results. Other factors that the laboratory can control and that affect quality are the trans- port, identification, storage, and preparation (processing) of specimens. The laboratory therefore has a role in educating those taking and trans- porting specimens. Written instructions should be made available and regularly reviewed with the clinical and nursing staff. Laboratory materials. The quality of reagents, chemicals, glassware, stains, culture media, and laboratory animals all influence the reliability of test results. Test method. Some methods are more reliable than others. Equipment. Lack of equipment or the use of substandard or poorly main- tained instruments will give unreliable results. Examination and reading. Hurried reading of results, or failure to examine a sufficient number of microscope fields, can cause errors. Reporting. Transcription errors, or incomplete reports, cause problems. 2 BLMIN 1/17/04 2:08 PM Page 3 QUALITY ASSURANCE IN BACTERIOLOGY Quality of interpretation of test results Interpretation is of particular importance in microbiology. At each stage in the examination of a specimen, the results should be interpreted in order to select the optimum test, in terms of speed and reliability, for the next stage of the examination. Quality assurance in the microbiology laboratory Quality assurance is the sum of all those activities in which the laboratory is engaged to ensure that test results are of good quality. It must be: — comprehensive: to cover every step in the cycle from collecting the specimen to sending the final report to the doctor (Fig. 1); — rational: to concentrate on the most critical steps in the cycle; — regular: to provide continuous monitoring of test procedures; — frequent: to detect and correct errors as they occur. GOOD-QUALITY LABORATORY SERVICES MEAN GOOD-QUALITY MEDICINE Quality assurance helps to ensure that expensive tests are used as economi- cally as possible; it also determines whether new tests are valid or worthless, improves the performance of clinical and public health laboratories, and may help to make the results obtained in different laboratories comparable. Types of quality assurance There are two types of quality assurance: internal and external. Internal. This is called QUALITY CONTROL. Each laboratory has a pro- gramme to check the quality of its own tests. Fig. 1. Steps in laboratory investigation of an infected patient A 3 BLMIN 1/17/04 2:08 PM Page 4 BASIC LABORATORY PROCEDURES IN CLINICAL BACTERIOLOGY Internal quality control involves, ideally: — continuous monitoring of test quality; — comprehensive checking of all steps, from collecting the specimen (whenever possible) to sending the final report. Laboratories have an ethical responsibility to the patient to produce accurate, meaningful results. INTERNAL QUALITY CONTROL IS ABSOLUTELY ESSENTIAL FOR GOOD OPERATING PROCEDURE External. This is called QUALITY ASSESSMENT. Laboratory performance is controlled by an external agency. In some countries, participation is mandatory (regulated by the government) and required for licensure. External quality assessment involves: — periodic monitoring of test quality; — spot checking of identification tests, and sometimes of isolation techniques. Quality criteria in microbiology 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: 1. If a few colonies of Gram-negative rods are isolated from the sputum or throat swab of a hospitalized patient, further identification and an anti- biogram are of no clinical relevance, since neither procedure will have any effect on treatment of the patient. 2. If Streptococcus pyogenes is isolated, a full antibiogram has no clinical rele- vance, since benzylpenicillin is the drug of choice, and this is always active in vitro. 3. 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. 4. If a Gram-stained smear shows “mixed anaerobic flora”, routine identifi- cation of the anaerobes is of no clinical relevance. It would be costly in time and materials, and would not affect treatment of the patient. 5. If a yeast is isolated from a respiratory tract specimen, an identification test for Cryptococcus should be done. Further identification tests have no clin- ical relevance, since they would have no effect on patient management. 4 BLMIN 1/17/04 2:08 PM Page 5 QUALITY ASSURANCE IN BACTERIOLOGY In summary, a test of good quality is one that is accurate and gives useful results for the prevention or cure of infection. It is not necessary to isolate and identify all the different types of organism in the sample. Reliability 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: — antibiotic assay of serum; — measurement of minimal inhibitory concentration (MIC) values of anti- biotics 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. Inter- nationally 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 suscepti- bility tests should be performed with an internationally recognized technique, such as the modified Kirby–Bauer test (page 109). Reproducibility The reproducibility or precision of a microbiological test is 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 multi- ply or die at different rates. Repeat culturing may therefore isolate differ- ent 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 diag- nosis of a pathogen or a pathological condition. This is measured by two criteria: 1. Diagnostic sensitivity total number of positive results Sensitivity = total number of infected patients The greater the sensitivity of a test, the fewer the number of false-negative results. A 5 BLMIN 1/17/04 2:08 PM Page 6 BASIC LABORATORY PROCEDURES IN CLINICAL BACTERIOLOGY 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 total number of negative results Specificity = total number of unifected patients The greater the specificity of a test, the fewer the number of false-positive results. For example: Ziehl–Neelsen staining of sputum is highly specific for diagnosing tuberculosis, because it 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 reduc- ing 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 popula- tion under investigation. Internal quality control Requirements An internal quality control programme should be practical, realistic, and economical. An internal quality control programme should not attempt to evaluate every procedure, reagent, and culture medium on every working day. It should eval- uate each procedure, reagent, and culture medium according to a practical schedule, based on the importance of each item to the quality of the test as a whole. Procedures Internal quality control begins with proper laboratory operation. Laboratory operations manual Each laboratory should have an operations manual that includes the follow- ing subjects: — cleaning of the working space, — personal hygiene, — safety precautions, — designated eating and smoking areas located outside the laboratory, — handling and disposal of infected material, 6 BLMIN 1/17/04 2:08 PM Page 7 QUALITY ASSURANCE IN BACTERIOLOGY — appropriate vaccinations for workers, e.g. hepatitis B, — care of equipment, — collection of specimens, — registration of specimens, — elimination of unsuitable specimens, — processing of specimens, — recording of results, — reporting of results. The operations manual should be carefully followed, and regularly revised and updated. Care of equipment It is particularly important to take good care of laboratory equipment. Good quality tests cannot be performed if the equipment used is either of poor quality or poorly maintained. Table 1 is a schedule for the routine care and maintenance of essential equip- ment. Equipment operating temperatures may be recorded on a form such as the one shown in Fig. 2. Culture media Culture media may be prepared in the laboratory from the basic ingredients or from commercially available dehydrated powders, or they may be pur- chased ready for use. Commercial dehydrated powders are recommended because they are economical to transport and store, and their quality is likely to be higher than media prepared in the laboratory. For best results, careful attention is required to the points itemized below. Selection of media An efficient laboratory stocks the smallest possible range of media consistent with the types of test performed. For example, a good agar base can be used as an all-purpose medium for preparing blood agar, chocolate agar, and several selective media. One highly selective medium (Salmonella–Shigella agar or deoxycholate citrate agar) and one less selective medium (MacConkey agar) are necessary for the isolation of pathogenic Enterobacteriaceae from stools. A special culture medium should be added for the recovery of Campylobacter spp. Ordering and storage of dehydrated media 1. Order quantities that will be used up in 6 months, or at most 1 year. 2. The overall quantity should be packed in containers that will be used up in 1–2 months. 3. On receipt, tighten caps of all containers securely. Dehydrated media absorb water from the atmosphere. In a humid climate, seal the tops of containers of dehydrated media with paraffin wax (fill the space between the lid and container with molten wax, and let it harden). A 7 BLMIN 1/17/04 2:08 PM Page 8 BASIC LABORATORY PROCEDURES IN CLINICAL BACTERIOLOGY Table 1. Quality control of equipment Equipment Routine care Monitoring Technical maintenance and inspection Anaerobic jar Clean inside of jar each week Use methylene blue indicator Inspect gasket Reactivate catalyst after each strip with each run sealing in the run (160 ∞C, 2 h) Note and record decolorization lid weekly Replace catalyst every 3 months time of indicator each week Autoclave Clean and change water Check and adjust water level Every 6 months monthly before each run Record time and temperature or pressure for each run Record performance with spore-strips weekly Centrifuge Wipe inner walls with antiseptic Replace brushes solution weekly or after annually breakage of glass tubes or spillage Hot-air oven Clean inside monthly Record time and Every 6 months for sterilization temperature for each run of glassware Incubator Clean inside walls and Record temperature at the Every 6 months shelves monthly start of each working day (allowance 35 ± 1 ∞C) Microscope Wipe lenses with tissue or lens Check alignment of Annually paper after each day’s work condenser monthly Clean and lubricate mechanical Place a dish of blue silica stage weekly with the microscope under Protect with dust cover when the dust cover to prevent not in use fungal growth in humid climates Refrigerator Clean and defrost every 2 Record temperature every Every 6 months months and after power failure morning (allowance 2–8 ∞C) Water-bath Wipe inside walls and change Check water level daily Every 6 months water monthly Record temperature on first day of each week (allowance 55–57 ∞C) 4. Write the date of receipt on each container. 5. Store in a dark, cool, well-ventilated place. 6. Rotate the stock so that the older materials are used first. 7. When a container is opened, write the date of opening on it. 8. Discard all dehydrated media that are either caked or darkened. 9. Keep written records of media in stock. Preparation of media 1. Follow strictly the manufacturer’s instructions for preparation. 2. Prepare a quantity that will be used up before the shelf-life expires (see below). 8 BLMIN 1/17/04 2:08 PM Page 9 Fig. 2. Record of equipment operating temperature A 9 BLMIN 1/17/04 2:08 PM Page 10 BASIC LABORATORY PROCEDURES IN CLINICAL BACTERIOLOGY Storage of prepared media 1. Protect against sunlight. 2. Protect against heat. Media containing blood, other organic additives, or antibiotics should be stored in the refrigerator. 3. The shelf-life of prepared media, when stored in a cool, dark place, will depend on the type of container used. Typical shelf-lives are: — tubes with cotton-wool plugs, 3 weeks; — tubes with loose caps, 2 weeks; — containers with screw-caps, 3 months; — Petri dishes, if sealed in plastic bags, 4 weeks. Quality control of prepared media 1. pH testing. The pH of the prepared medium need not be checked routinely when it is correctly prepared from dehydrated powder. If the medium is prepared from basic ingredients, it should be allowed to cool before the pH is tested. Solid media should be tested with a surface electrode or after maceration in distilled water. If the pH differs by more than 0.2 units from the specification, adjust with acid or alkali or prepare a new batch. 2. Sterility testing. Carry out routine sterility tests on media to which blood or other components have been added after autoclaving. Take 3–5% of each batch and incubate at 35 ∞C for 2 days. Refrigerate the rest. If more than two colonies per plate are seen, discard the whole batch. 3. Performance testing. The laboratory should keep a set of stock strains for monitoring the performance of media. A suggested list of stock strains is give in Table 2. These strains can be obtained through routine work, or from commercial or official sources. Recommendations for the mainte- nance and use of stock strains are given on page 14. A list of performance tests for commonly used media is given in Table 3. Table 2. Suggested stock strains for quality controla Gram-positive cocci Enterobacteriaceae Enterococcus faecalis (ATCC 29212 Citrobacter freundii or 33186) Enterobacter cloacae Staphylococcus aureus (ATCC 25923) Escherichia coli (ATCC 25922) Staphylococcus epidermidis Klebsiella pneumoniae Streptococcus agalactiae Proteus mirabilis Streptococcus mitis Salmonella typhimurium Streptococcus pneumoniae Serratia marcescens Streptococcus pyogenes Shigella flexneri Gram-negative fastidious organisms Yersinia enterocolitica Moraxella catarrhalis Other Gram-negative rods Haemophilus influenzae type b Acinetobacter lwoffi b-lactamase-negative Pseudomonas aeruginosa (ATCC 27853) b-lactamase-positive Vibrio cholerae (non-01) Haemophilus parainfluenzae Fungi Neisseria gonorrhoeae Candida albicans Neisseria meningitidis Anaerobes Bacteroides fragilis Clostridium perfringens a The strains most relevant to the needs of the laboratory should be selected. 10 BLMIN 1/17/04 2:08 PM Page 11 QUALITY ASSURANCE IN BACTERIOLOGY Table 3. Performance tests on commonly used media Medium Incubation Control organism Expected result Bile–aesculin agar 24 h Enterococcus faecalis Growth and blackening a-Haemolytic No growth, with haemolysis Streptococcus Blood agar 24 h, CO2 Streptococcus Growth and b-haemolysis pyogenes S. pneumoniae Growth and a-haemolysis Chocolate agar 24 h, CO2 Haemophilus influenzae Growth Decarboxylase (cover with sterile oil) — lysine 48 h Shigella typhimurium Positive Shigella flexneri Negative — ornithine 48 h S. typhimurium Positive Klebsiella pneumoniae Negative Dihydrolase — arginine 48 h S. typhimurium Positive Proteus mirabilis Negative Gelatinase (rapid tests) 24 h Escherichia coli Negative Serratia marcescens Positive Kligler iron agar (see Triple sugar iron agar) MacConkey agar with crystal 24 h E. coli Red colonies violet P. mirabilis Colourless colonies (no swarming) E. faecalis No growth Malonate broth 24 h E. coli Negative (green) K. pneumoniae Positive (blue) Mannitol salt agar 24 h Staphylococcus aureus Yellow colonies Staphylococcus Rose colonies epidermidis E. coli No growth Methyl red/Voges–Proskauer 48 h E. coli Positive/negative K. pneumoniae Negative/positive Mueller–Hinton agar 24 h E. coli ATCC 25922 S. aureus ATCC 25923 Acceptable zone sizes Pseudomonas (Table 24, p. 110) aeruginosa ATCC 27853 Nitrate broth 24 h E. coli Positive Acinetobacter lwoffi Negative Oxidation/fermentation 24 h P. aeruginosa Oxidation at the suface dextrose (without oil) A. lwoffi No change Peptone water (indole) 24 h E. coli Positive K. pneumoniae Negative Phenylalanine deaminase/ 24 h E. coli Negative ferrichloride P. mirabilis Positive Salmonella–Shigella agar or 24 h E. coli No growth deoxycholate citrate agar S. typhimurium Colourless colonies Yersina enterocolitica Colourless colonies S. flexneri Colourless colonies Selenite broth 24 h S. typhimurium Growth after subculture E. coli No growth after subculture Simmons citrate agar (incubate 48 h E. coli No growth with loose screw-cap) K. pneumoniae Growth, blue colour A 11 BLMIN 1/17/04 2:08 PM Page 12 BASIC LABORATORY PROCEDURES IN CLINICAL BACTERIOLOGY Table 3 (continued) Medium Incubation Control organism Expected result Thiosulfate citrate bile salts 24 h Vibrio spp. (non- Yellow colonies agar agglutinating) Thayer–Martin agar 24 h, CO2 Neisseria meningitidis Growth Neisseria gonorrhoeae Growth Staphylococcus spp. No growth E. coli No growth C. albicans No growth Thioglycollate broth 24 h Bacteroides fragilis Growth Triple sugar iron agar (depth of 24 h Citrobacter freundii A/A gasa + H2S butt should be at least 2.5 cm; S. typhimurium K/A gasa + H2S incubate with loose screw-cap) S. flexneri K/A gasa A. lwoffi No change Urea medium 24 h E. coli Negative P. mirabilis Positive (pink) Voges–Proskauer (see Methyl red/Voges–Proskauer) a A/A: acid slant; K/A: alkaline slant. The procedures to be followed when carrying out performance tests on new batches of media are: 1. Prepare a suspension of the stock strain with a barely visible turbidity, equivalent to that of the barium sulfate standard used in the modified Kirby–Bauer method (McFarland 0.5) (see page 109) and use 1 loopful as inoculum. 2. Incubate for the length of time used routinely. Read the plates in the usual way. 3. Keep proper records of results. Stains and reagents Recommendations for testing a number of reagents are given in Table 4. Testing should be carried out: — each time a new batch of working solution is prepared; — every week (this is critical for the cold Ziehl–Neelsen stain: the classical stain has a shelf-life of several months). Stains and reagents should be discarded when: — the manufacturer’s expiry date is reached; — visible signs of deterioration appear (turbidity, precipitate, discoloration). Diagnostic antigens and antisera In order to obtain the best results from antigens and antisera: Always follow the manufacturer’s instructions. Store at the recommended temperature. Some serological reagents do not tolerate freezing. 12 BLMIN 1/17/04 2:08 PM Page 13 QUALITY ASSURANCE IN BACTERIOLOGY Table 4. Performance tests on commonly used reagents Reagent or stain Species suitable for testing Medium Positive Negative Bacitracin disc S. pyogenes (zone) E. faecalis Blood agar Catalase S. aureus E. faecalis Tryptic soy agar Coagulase plasma S. aureus S. epidermidis Tryptic soy agar b-Glucuronidase (PGUA)a E. coli K. pneumoniae Tryptic soy agar Gram stain Staphylococcus spp E. coli Mixed in smear ONPGb E. coli S. typhimurium Triple sugar iron agar or Kligler iron agar Optochin disc S. pneumoniae (zone) Streptococcus mitis Blood agar Oxidase Pseudomonas aeruginosa E. coli Tryptic soy agar Tellurite disc E. faecalis (no zone) Streptococcus Blood agar agalactiae (zone) V-factor (disc or strips) Haemophilus parainfluenzae Haemophilus influenzae Tryptic soy agar XV-factor (disc or strips) H. influenzae Tryptic soy agar Ziehl–Neelsen stain Mycobacterium tuberculosis Mixed non-acid-fast Sputum smear c flora a 4-Nitrophenyl-b-D-glucopyranosiduronic acid. (PGUA) b o-Nitrophenyl-b-D-galactopyranoside. c Prepare a number of smears from known positive and negative patients. Fix by heat, wrap individually in paper, and store in the refrigerator. Avoid repeated freezing and thawing. Before freezing, divide antiserum into aliquot portions sufficient for a few tests. Discard when the manufacturer’s expiry date is reached. To test agglutinating antisera, always use fresh pure cultures of known reactivity. Always include a serum control of known reactivity in each batch of tests. The serum may be from a patient, or from a commercial source. If possible, the potency of the control serum should be expressed in Inter- national Units per millilitre. Paired sera from the same patient, taken during the acute and convales- cent phases of the disease, should be tested with the same batch of reagents. For the serological diagnosis of syphilis, only nationally or internationally recognized procedures should be used. Each batch of serological tests should include: — a negative serum (specificity control); — a weakly reactive serum (sensitivity control); — a strongly reactive serum (titration control), which should read within one dilution of its titre when last tested. Always record all control serum titres. Antibiotic susceptibility tests The routine use of the modified Kirby–Bauer method is recommended (page 109). To avoid errors, the following guidelines should be used: Discs should be of correct diameter (6.35 mm). Discs should be of correct potency (Table 24, page 110). The stock supply should be stored frozen (-20 ∞C). A 13 BLMIN 1/17/04 2:08 PM Page 14 BASIC LABORATORY PROCEDURES IN CLINICAL BACTERIOLOGY The working supply should be kept no longer than 1 month in a refriger- ator (2–8 ∞C). Only Mueller–Hinton agar of performance-tested quality should be used. Correct pH (7.2–7.4) of the finished medium is essential for some anti- biotics. The inoculum should be standardized against the prescribed turbidity standard (page 111). Zone sizes should be measured exactly. Zone sizes should be interpreted by referring to a table of critical diame- ters. Zone diameters for each organism should fall within the limits given in Table 24 (page 110). The three standard control strains are:1 — Staphylococcus aureus (ATCC 25923; NCTC 6571); — Escherichia coli (ATCC 25922; NCTC 10418); — Pseudomonas aeruginosa (ATCC 27853; NCTC 10622). Tests should be carried out with the three standard strains: — when a new batch of discs is put into use; — when a new batch of medium is put into use; — once a week, in parallel with the routine antibiograms. Use the quality control chart shown in Fig. 16 (page 121) for recording and evaluating performance tests. Maintenance and use of stock cultures Selection and origin Select the strains so that the maximum number of morphological, metabolic, and serological characteristics can be tested with the minimum number of cul- tures; a suggested list is given in Table 2. These strains can be obtained from a combination of the following sources: — properly documented isolates from clinical specimens; — official culture collections; — commercial producers; — external quality assessment surveys; — reference laboratories. Preservation Long-term preservation Long-term preservation methods permit intervals of months or even years between subcultures. The best methods are lyophilization (freeze-drying), or storage at -70 ∞C or below, in an electric freezer or in liquid nitrogen. Alter- native methods are described below. Glycerol at -20 ∞C 1. Grow a pure culture on an appropriate solid medium. 2. When the culture is fully developed, scrape it off with a loop. 3. Suspend small clumps of the culture in sterile neutral glycerol. 1 These strains can be obtained from: American Type Culture Collection (ATCC), 10801 Univer- sity Boulevard, Manassas, VA 20110, USA; or National Collection of Type Cultures (NCTC), PHLS Central Public Health Laboratory, 61 Colindale Avenue, London NW9 5HT, England. 14 BLMIN 1/17/04 2:08 PM Page 15 QUALITY ASSURANCE IN BACTERIOLOGY 4. Distribute in quantities of 1–2 ml in screw-capped tubes or vials. 5. Store at -20 ∞C. Avoid repeated freezing and thawing. Transfer after 12–18 months. Mineral oil at room temperature1 1. Prepare tubes of heart infusion agar with a short slant. For fastidious organisms, add fresh native or heated blood. 2. Sterilize mineral oil (liquid petrolatum) in hot air (170 ∞C for 1 hour). 3. Grow a pure culture on the agar slant. 4. When good growth is seen, add sterile mineral oil to about 1 cm above the tip of the slant. 5. Subculture when needed by scraping growth from under the oil. 6. Store at room temperature. Transfer after 6–12 months. Stab cultures at room temperature (use for non-fastidious organisms only, such as staphylococci and Enterobacteriaceae) 1. Prepare tubes with a deep butt of carbohydrate-free agar. Tryptic soy agar (soybean casein digest agar) is recommended. 2. Stab the organism into the agar. 3. Incubate overnight at 35 ∞C. 4. Close tube with screw-cap or cork. Dip cap or cork into molten paraffin wax to seal. 5. Store at room temperature. Transfer after 1 year. Stab cultures in cystine trypticase agar (CTA) (for Neisseria and streptococci) 1. Prepare tubes of CTA basal medium. 2. Stab the organism into the medium. 3. Incubate overnight at 35 ∞C. 4. Close tube with screw-cap or cork. Dip cap or cork into molten paraffin wax to seal. 5. For Neisseria, store at 35 ∞C, and transfer every 2 weeks. For streptococci, store at room temperature, and transfer every month. Cooked-meat medium for anaerobes 1. Inoculate tubes. 2. Incubate overnight at 35 ∞C. 3. Close tube with screw-cap or cork. 4. Store at room temperature. Transfer every 2 months. Short-term preservation Working cultures for daily routine tests can be prepared in the following ways. Rapid-growing organisms 1. Inoculate on tryptic soy agar slants in screw-capped tubes. 2. Incubate overnight at 35 ∞C. 3. Store in a refrigerator. Transfer every 2 weeks. 1 Morton HE, Pulaski EJ. The preservation of bacterial cultures. Journal of Bacteriology, 1938, 38:163–183. A 15 BLMIN 1/17/04 2:08 PM Page 16 BASIC LABORATORY PROCEDURES IN CLINICAL BACTERIOLOGY Streptococci 1. Inoculate on blood agar slants in screw-capped tubes. 2. Incubate overnight at 35 ∞C. 3. Store in a refrigerator. Transfer every 2 weeks. Meningococci and Haemophilus 1. Inoculate on chocolate agar slants or plates. 2. Incubate overnight at 35 ∞C. 3. Store at room temperature. Transfer twice a week. Gonococci 1. Inoculate on chocolate agar. 2. Incubate and store at 35 ∞C. Transfer every 2 days. 3. Replace the quality control strain by each new clinical isolate. Use of reference laboratories The following categories of specimen should be submitted to a regional or central reference laboratory: — specimens for infrequently requested or highly specialized tests (e.g. virol- ogy, serodiagnosis of parasitic infections); — occasional duplicate specimens, as a check on the submitting laboratory’s own results; — specimens needing further confirmation, specification, grouping, or typing of pathogens of great public health importance (e.g. Salmonella, Shigella, Vibrio cholerae, Brucella, meningococci, and pneumococci). Reference laboratories should be able to supply reference cultures for quality control and training needs, and standard sera and reagents for comparison with those in use in the referring laboratory. If no external quality assessment programme exists, the reference laboratory should be asked to supply blind, coded specimens and cultures so that the referring laboratory may test its own proficiency in isolation and identification. External quality assessment This section gives information on what is involved in participation in an exter- nal quality assessment scheme (sometimes known as a “proficiency testing scheme”). Purposes The purposes of a quality assessment programme are: — to provide assurance to both physicians and the general public that labo- ratory diagnosis is of good quality; — to assess and compare the reliability of laboratory performance on a national scale; — to identify common errors; — to encourage the use of uniform procedures; 16 BLMIN 1/17/04 2:08 PM Page 17 QUALITY ASSURANCE IN BACTERIOLOGY — to encourage the use of standard reagents; — to take administrative measures (which may include revocation of the operating licence) against substandard laboratories; — to stimulate the implementation of internal quality control programmes. Organization A quality assessment programme consists of a number of surveys in which coded specimens are distributed by mail to participating laboratories. These specimens should be incorporated into the laboratory routine, and handled and tested in exactly the same way as routine clinical specimens. The surveys should be conducted in accordance with the following recommendations: — surveys should be carried out at least 4 times a year; — a minimum of 3 specimens should be included in each survey; — the reporting period should be short, for example 2 weeks following receipt of the specimens; — instructions and report forms should be included with each survey and the report sheet should be in duplicate, with a clearly stated deadline. Cultures Cultures should be included for identification and for susceptibility testing against a limited range of antibiotics; they may be pure cultures or mixtures of two or more cultures. Cultures should represent at least the first 3 of the following 6 categories: 1. Bacterial species that are of great public health potential, but which are not often seen in routine practice, for example Corynebacterium diphtheriae, Salmonella paratyphi A. NOTE: Brucella and Salmonella typhi should not be used for quality assess- ment schemes, since they may give rise to serious accidental infections. 2. Abnormal biotypes that are often misidentified, for example H2S-positive Escherichia coli, lactose-negative E. coli, urease-negative Proteus. 3. Newly recognized or opportunistic pathogens, for example Yersinia ente- rocolitica, Vibrio parahaemolyticus, Burkholderia, Pseudomonas cepacia. 4. A mixture of Shigella, Citrobacter, E. coli, and Klebsiella may be used to test the skill of a laboratory in isolating pathogenic microorganisms from a number of commensal organisms. 5. A mixture of nonpathogenic organisms may be used to test for ability to recognize negative specimens. 6. Bacteria with special resistance patterns, for example meticillin-resistant S. aureus (MRSA). Sera Serological tests for the following infections should be part of an external quality assessment programme in bacteriology: — syphilis — rubella — brucellosis A 17 BLMIN 1/17/04 2:08 PM Page 18 BASIC LABORATORY PROCEDURES IN CLINICAL BACTERIOLOGY — streptococcal infections — typhoid fever. Rating and reporting of results As soon as all reports of results are received from participating, the correct answers should be sent to the laboratories. Within one month after that, a final report should be sent to the laboratories with an analysis of the results. A per- formance score is given to each laboratory. Each laboratory should have a code number known only to itself. Thus it can recognize its own performance in relation to others, but the other laboratories remain anonymous. 18 BLM1 1/17/04 2:01 PM Page 19 Part I Bacteriological investigations A BLM1 1/17/04 2:01 PM Page 20 Blood Introduction Blood is cultured to detect and identify bacteria or other cultivable microor- ganisms (yeasts, filamentous fungi). The presence of such organisms in the blood is called bacteraemia or fungaemia, and is usually pathological. In healthy subjects, the blood is sterile. However, there are a few exceptions: tran- sient bacteraemia often occurs shortly after a tooth extraction or other dental or surgical manipulation of contaminated mucous membranes, bronchoscopy, or urethral catheterization. This type of transient bacteraemia is generally due to commensal bacteria and usually resolves spontaneously through phagocy- tosis of the bacteria in the liver and spleen. Septicaemia is a clinical term used to describe bacteraemia with clinical manifestations of a severe infection, including chills, fever, malaise, toxicity, and hypotension, the extreme form being shock. Shock can be caused by toxins produced by Gram-negative rods or Gram-positive cocci. When and where bacteraemia may occur Bacteraemia is a feature of some infectious diseases, e.g. brucellosis, lep- tospirosis and typhoid fever. Persistent bacteraemia is a feature of endovas- cular infections, e.g. endocarditis, infected aneurysm and thrombophlebitis. Transient bacteraemia often accompanies localized infections such as arthri- tis, bed sores, cholecystitis, enterocolitis, meningitis, osteomyelitis, peritoni- tis, pneumonia, pyelonephritis, and traumatic or surgical wound infections. It can arise from various surgical manipulations, but usually resolves spontaneously in healthy subjects. Bacteraemia and fungaemia may result from the iatrogenic introduction of microorganisms by the intravenous route: through contaminated intravenous fluids, catheters, or needle-puncture sites. Both types of infection may develop in users of intravenous drugs and in immunosuppressed subjects, including those with human immunodeficiency virus/the acquired immunodeficiency syndrome (HIV/AIDS). They are often caused by “opportunistic” microor- ganisms and may have serious consequences. Table 5 shows the most common causes of bacteraemia or fungaemia. Blood collection Timing of blood collection Whenever possible, blood should be taken before antibiotics are administered. The best time is when the patient is expected to have chills or a temperature spike. It is recommended that two or preferably three blood cultures be obtained, separated by intervals of approximately 1 hour (or less if treatment cannot be delayed). More than three blood cultures are rarely indicated. The advantages of repeated cultures are as follows: — the chance of missing a transient bacteraemia is reduced; — the pathogenic role of “saprophytic” isolates (e.g. Staphylococcus epider- midis) is confirmed if they are recovered from multiple venepunctures. 20 BLM1 1/17/04 2:01 PM Page 21 BACTERIOLOGICAL INVESTIGATIONS Table 5. Common causes of bacteraemia or fungaemia Gram-negative organisms Gram-positive organisms Escherichia coli Staphylococcus aureus Klebsiella spp. S. epidermidis Enterobacter spp. a-Haemolytic (viridans) streptococci Proteus spp. Streptococcus pneumoniae Salmonella typhi E. faecalis (group D) Salmonella spp. other than S. typhi S. pyogenes (group A) Pseudomonas aeruginosa S. agalactiae (group B) Neisseria meningitidis Listeria monocytogenes Haemophilus influenzae Clostridium perfringens Bacteroides fragilis (anaerobe) Peptostreptococcus spp. (anaerobes) Brucella spp. Candida albicans and other yeast- Burkholderia (Pseudomonas) pseudomallei like fungi (e.g. Cryptococcus (in certain areas) neoformans) It is important that blood specimens for culture are collected before initiating empirical antimicrobial therapy. If necessary, the choice of antimicrobial can be adjusted when the results of susceptibility tests become available. Quantity of blood Because the number of bacteria per millilitre of blood is usually low, it is important to take a reasonable quantity of blood: 10 ml per venepuncture for adults; 2–5 ml may suffice for children, who usually have higher levels of bac- teraemia; for infants and neonates, 1–2 ml is often the most that can be obtained. Two tubes should be used for each venepuncture: the first a vented tube for optimal recovery of strictly aerobic microorganisms, the second a non-vented tube for anaerobic culture. Skin disinfection The skin at the venepuncture site must be meticulously prepared using a bac- tericidal disinfectant: 2% tincture of iodine, 10% polyvidone iodine, 70% alcohol, or 0.5% chlorhexidine in 70% alcohol. The disinfectant should be allowed to evaporate on the skin surface before blood is withdrawn. If tinc- ture of iodine is used, it should be wiped off with 70% alcohol to avoid pos- sible skin irritation. Even after careful skin preparation, some bacteria persist in the deeper skin layers and may gain access to the blood, e.g. S. epidermidis, Propionibacterium acnes, and even spores of Clostridium. Pseudobacteraemia (false-positive blood culture) may result from the use of contaminated antiseptic solutions, syringes, or needles. The repeated isolation of an unusual organism (e.g. Burk- holderia (Pseudomonas) cepacia, Pantoea (Enterobacter) agglomerans, or Serratia spp.) in the same hospital must raise suspicion of a nosocomial infection and promote an investigation. Another source of contamination is contact of the needle with non-sterile vials (or solutions), if the same syringe is first used to provide blood for chemical analysis or measurement of the erythrocyte sedi- mentation rate. A 21 BLM1 1/17/04 2:01 PM Page 22 BLOOD Anticoagulant The use of sodium polyanethol sulfonate (SPS) as an anticoagulant is recom- mended because it also inhibits the antibacterial effect of serum and phago- cytes. If the blood is immediately added to a sufficient volume (50 ml) of broth and thoroughly mixed to prevent clotting, no anticoagulant is needed. It is recommended that blood-culture bottles be available at all hospitals and major health centres. If blood-culture bottles are not available, blood may be trans- ported to the laboratory in a tube containing a sterile anticoagulant solution (citrate, heparin, or SPS). Upon receipt in the laboratory, such blood samples must be transferred immediately to blood-culture bottles using a strict aseptic technique. Where blood is taken without anticoagulant, the clot can be asep- tically transferred to broth in the laboratory and the serum used for certain serological tests (e.g. Widal). Blood-culture media Choice of broth medium The blood-culture broth and tryptic soy broth (TSB) should be able to support growth of all clinically significant bacteria. Quantity of broth Ideally, the blood should be mixed with 10 times its volume of broth (5 ml of blood in 50 ml of broth) to dilute any antibiotic present and to reduce the bactericidal effect of human serum. Blood-culture bottles Blood-culture bottles (125 ml) with a pre-perforated screw-cap and a rubber diaphragm must be used. Fill the bottle with 50 ml of medium and then loosen the screw-cap half a turn. Cover the cap with a square piece of aluminium foil, and autoclave the bottle for 20 minutes at 120 ∞C. Immediately after autoclaving, while the bottle and the medium are still hot, securely tighten the cap without removing the aluminium foil (otherwise the cap will not be sterile). As the medium cools, a partial vacuum will be created in the bottle, which will facilitate injection of a blood specimen through the diaphragm. The top of the cap must be carefully disinfected just before the bottle is inoculated. Prior to distributi