Antimicrobial Chemotherapy, 4th Edition
Laboratory aspects of antimicrobial therapy
Use of the laboratory
- C. B. Slack
This book is about antimicrobial therapy and is not intended as a treatise on clinical laboratory microbiology. However, basic principles must be understood in order to appreciate the scope and limitations of laboratory control of antimicrobial therapy in the individual patient, and the object of this chapter is to fill in a little of this background. The examples given refer to bacteriological practice, but the principles apply to the investigation of any infection. It is increasingly recognised that accurate laboratory diagnosis is essential for the specific use of antiviral, antifungal, or antiparasitic agents.
There is still more art to the science of microbiology than to other branches of clinical pathology. There is, for example, more scope for singular methods of processing specimens and for individual interpretation of results in examining a specimen of sputum than in measuring plasma urea. Very often the methods used depend on the experience and preferences of the individual microbiologist. In some countries attempts have been made to bring about more standardization of microbiological methods (in particular antibiotic susceptibility testing), but standardization does not guarantee that the correct result is always obtained!
Clinical laboratories rely on the quality of the specimens they receive; none more so than microbiology departments where the ‘result’ of culture may depend on the degree of care observed in taking the specimen. A single extraneous bacterium introduced into a blood culture during collection may contaminate the culture, resulting in a false positive result. This is one of the many slips that can totally alter laboratory results and may, on occasion, be detrimental to the patient.
A few of the more common problems are listed.
- Inappropriate specimen. Saliva is submitted instead of sputum; a superficial skin swab is taken instead of a swab of pus (a specimen of pus in a sterile bottle is always preferable to a swab when possible).
- Inadequate specimen. The specimen may be too small (especially fluids for culture for tubercle bacilli); rectal swabs are no substitute for faeces.
- Wrong timing. Specimens taken after the start of chemotherapy, when the causative organism may no longer be demonstrable, or after the patient has recovered—it is not uncommon for the laboratory to receive rock-hard faeces from patients with ‘diarrhoea’.
- Wrong container. Blood for culture put in a plain (sometimes unsterile) bottle instead of the correct culture fluid; biopsies put into bactericidal fixatives.
- Clerical errors. Incorrect labelling; incomplete or misleading information on request forms.
Not only must the specimen be collected properly, but it must also be received in the laboratory in good condition. Prompt transport to the laboratory is essential. Material submitted for culture is alive; any delay in reaching the optimal cultural conditions will result in loss of viability. With fastidious organisms such as gonococci or viruses this may result in failure to isolate the organism. The converse problem—overgrowth of pathogens by fast-growing commensals—also commonly occurs during the period between collection of the specimen and processing in the laboratory.
The ideal would be to eliminate transport problems by inoculating appropriate culture media at the bedside and incubating them immediately. This may be achieved in special units with laboratories attached, but is not practicable in most situations. An exception is blood culture, where the counsel of perfection should apply.
The nearest approach to ‘culture in transit’ that has been widely adopted is the dip-culture method for culture of urine (p. 237). For swabs, most laborato- ries recommend a form of suspended animation in which the specimen is placed in a special transport medium comprising soft buffered agar containing charcoal to inactivate any toxic substances.
Specimens from potential medical emergencies, such as bacterial meningitis or malaria, should be delivered to the laboratory immediately (and it should not be below the dignity of a doctor to do this!) and brought to the attention of a senior member of the laboratory staff.
The flow diagram (Fig. 10.1) outlines the three main steps which occur for every bacteriological request: microscopy, culture, identification, and sensitivity testing. Even with the most rapidly growing bacteria and with improved methods, results of culture and sensitivity often take 48 h. This may be further delayed if there is a mixture of organisms or if slow-growing pathogens, such as Mycobacterium tuberculosis or viruses, are involved. Not all micro-organisms are readily cultivable and a report of ‘sterile’ or ‘no growth’ does not definitively mean that the specimen contained no organisms, but that the laboratory was unable to isolate any from the specimen.
Fig. 10.1 The various steps between obtaining a specimen from a patient and the issue of the final report. Note the importance of the period before the specimen arrives in the laboratory: unless the specimen is properly taken and transported it may be useless, and unless the request card is properly completed wrong tests may be done.
Because of the inevitable delay in obtaining culture results there is a need to inform clinicians of important findings before the complete results are known. Microscopical findings are usually available on the same day as the specimen is received and in urgent cases can be reported within 1 h or less. For example, a Gram-film of cerebrospinal fluid (CSF) can be done very quickly and may give the physician a reliable guide to primary therapy (which may be life saving) while waiting for cultural confirmation of the result. Similarly, positive blood cultures and other findings serious to the individual patient or his immediate contacts are usually telephoned directly to the doctor. Where the antibiotic sensitivity is predictable (e.g. Streptococcus pyogenes is always sensitive to penicillin) this advice may be given with the initial report. With many bacteria the report ‘sensitivity to follow’ is all that can be imparted before sensitivity testing, although a ‘best guess’ based on known patterns of resistance in the hospital or community may be suggested.
Although many diseases have a well-defined microbial aetiology, the information obtained about patients and the data accrued from the laboratory examination of their specimens are often too sparse to form a complete opinion as to the microbial cause in an individual case. Most patients with infection survive and many improve so rapidly that the significance of microbes isolated is never known. Thus although we know from historical and epidemiological evidence that Str. pyogenes causes tonsillitis and is involved in the aetiology of rheumatic fever, when an individual patient presents with joint pains following a sore throat we cannot be absolutely sure that the Str. pyogenes in the throat is the cause of the illness, since we know, also on epidemiological evidence, that these bacteria are carried normally in the pharynx of about 5 per cent of the community, and sore throat is often due to other causes. In such situations, additional information (such as raised antistreptolysin O antibodies in the serum in this case) may be needed in order to establish a causal relationship.
The greatest difficulty is encountered with specimens from areas of the body which have a resident microbial flora that may sometimes assume a pathogenic role. Isolations from respiratory sources are often the most difficult to interpret. Demonstration of M. tuberculosis is always a significant finding and even in the absence of clinical disease the patient must be further examined and treated. This organism, a recognized cause of tuberculosis from the time of Koch, is a true human pathogen and is high on anyone's list of ‘wanted bacteria’. On the other hand, Candida albicans is often thought of as a harmless commensal, although it can cause serious disease in immunocompromised patients. The ability of some organisms to strike while the host defences are down has given them the title opportunist pathogens. However, merely by looking at cultures of these opportunists, it is impossible to say in a particular case whether or not they are adopting a pathogenic role and laboratory personnel have to make an informed guess as to their significance based on ancillary findings (e.g. presence or absence of pus; numbers of organisms isolated) and clinical information provided on the request card. If the latter is absent, non-contributory, or misleading, as it frequently is, the report may be valueless.
In the absence of adequate guidance, the laboratory can adopt one of two approaches: report any microbe isolated regardless of any possible significance, or report only common pathogens and dismiss all the others as ‘normal flora’.
The importance of this to antimicrobial therapy is that if the isolate is not considered ‘significant’, no further work—including sensitivity tests—will be carried out. The corollary is that many sensitivities may be tested and reported on organisms that have no role in the patient's disease. This occurs more commonly than is usually admitted. Many patients receive potentially toxic antimicrobials because commensal bacteria isolated from a badly taken specimen were considered significant and sensitivity tests reported. Sometimes a great deal of effort and expense is put into treating colonizing organisms that are merely filling a vacuum left by the normal flora and which would quietly disappear if antimicrobial chemotherapy were withheld.
Rapid advances in immunological and molecular techniques have provided an array of antigen and DNA detection methods, which are increasingly being used especially to make a viral diagnosis. If the only way to confirm a diagnosis is by finding non-viable microbial products, it will not be possible to perform antimicrobial susceptibility tests. However, the sequences of many genes conferring antibiotic resistance in human pathogens have been characterized and ‘probes’ made which can recognize, for example, the different TEM β-lactamases (see p. 148). New or unusual mechanisms of resistance cannot be detected by these methods; nor is it possible to be certain that any gene found is expressed in vivo.
What the laboratory reports
The final report reaching the clinician must be self-explanatory, even dogmatic. It is not practicable, or desirable, to test each organism isolated against all antibiotics. What usually happens is that a restricted range of antimicrobial agents is tested against isolates considered significant, with a different selection for Gram-positive and Gram-negative bacteria. Primary testing can often be restricted to a few old and well-tried agents that are perfectly adequate for most common infections (Table 10.1). More extensive testing, particularly of expensive, broad-spectrum agents, should be reserved for resistant isolates or bacteria from patients with serious infections that are presenting problems of management. Usually, only two or three of the sensitivities tested are reported even if more are performed. Restricted reporting has the important function of reinforcing local antibiotic policies and of
discouraging clinicians from using inappropriate agents. Additional tests carried out, but not reported, are often useful if the patient falls to respond to the chosen agent or is hypersensitive to it. Testing many agents also provides useful epidemi-ological information of trends of antimicrobial susceptibility in the community and of clusters of multiply resistant bacteria, indicating cross-infection or spread from a common source.
Table 10.1 Examples of a restricted range of antimicrobial agents selected for primary sensitivity testing of some common pathogens
Most infections are caused by a single organism, often a well-known pathogen. In these cases there is usually no problem in deciding what to test and report. However, some specimens (e.g. those from abdominal wounds) are often infected with mixtures of organisms and each must be individually identified and tested against appropriate antimicrobial agents.
The choice of antibiotic tested varies with the site of infection and the pharmacological properties of the drug. Some agents, such as nitrofurantoin and nalidixic acid, achieve therapeutic concentrations only in urine and are of no value in other infections. Information about the individual patient may alter drug testing:
- if the patient is allergic to penicillins alternatives will be sought
- in pregnancy, sulphonamides and trimethoprim should be avoided if possible because of the risk of folate deficiency
- tetracyclines should not be used in late pregnancy or in young children owing to deposition in teeth.
These limitations of sensitivity testing can be taken into account by the laboratory only if the appropriate information is given on the request card.
Restrictions imposed by the large number of available antibiotics may be approached in various ways. Some groups of agents, such as sulphonamides or tetracyclines, are so similar in terms of their antibacterial spectrum that only one representative of each needs to be tested. With other drugs where there is differential susceptibility of bacteria to different members of the group, such a decision is less easy. An organism sensitive to cephalexin, one of the earliest cephalo-sporins, is also usually sensitive to all subsequent members of that group and this is often used as a screen for cephalosporin-sensitive bacteria. However, the converse is not true; an organism resistant to cephalexin may be sensitive to later cephalosporins and a definitive statement in this regard can be made only by testing the appropriate compound. The same principle applies to nalidixic acid and newer, more active quinolones.
Interpreting sensitivity reports
A report to the clinician on pus from an abscess might indicate that Staphylococcus aureus had been isolated and that the organism was resistant to penicillin, but sensitive to erythromycin and cloxacillin. The statement ‘this organism is resistant to penicillin’ means that penicillin would not influence the outcome. The infection may well improve due to host defences or to adequate drainage
of pus, but since penicillin-resistant staphylococci are invariably β-lactamase producers any penicillin which reached the wound would be rapidly destroyed. Such a statement is based on sound laboratory and clinical evidence. On the other hand, with bacteria of relatively low-level resistance (i.e. where disc testing shows inhibition of growth which is less than that produced using a sensitive control organism) the statement of resistance is more difficult to determine and depends on such factors as site of infection and drug penetration. The laboratory will try to weigh up the evidence and score the result as ‘sensitive’ or ‘resistant’, or may play safe by using the rather unsatisfactory phrase ‘reduced (or intermediate) sensitivity’.
The statement ‘this organism is sensitive to cloxacillin’ implies that use of this antibiotic (or a related β-lactamase-stable penicillin) would influence the outcome. This is more difficult to support than a statement about resistance. Treatment with the antibiotic may elicit little response in the patient because insufficient drug may have penetrated into a large collection of pus. More importantly (although unlikely in the present example, since Staph. aureus is commonly incriminated in infected wounds) the wrong organism may have been tested. One crucial limitation of the report to the clinician is that the innocent bystander was picked from a collection of bacteria isolated.
Assuming the correct organism and antimicrobial agents were tested and the results as good as possible in the laboratory, there is still a large gap between saying the isolate is sensitive and saying that the patient will recover from the infection. Many of the host factors influencing the outcome are described in Chapter 15.
The use of the laboratory requires a brain at both ends: thought in the request and in the collection of specimens and thought in the processing of the specimens and in the production of the end report in the laboratory. The final synthesis of interpretation and action based on the report depends on co-operation between clinicians and microbiologists, so that each knows what the other requires. Microbiological expertise is available in most large centres to advise on antimicrobial therapy and it is in the interests of the patients to use it. Many laboratories have virology departments and many of the points on specimen collection also apply to virus investigations. When in doubt, the laboratory should be consulted. For unusual diseases and problem cases it is often possible to seek help from specialized units such as tropical hospitals and institutes. In some countries reference laboratories are available which provide expertise in particular areas. In the UK many of these operate under the aegis of the Public Health Laboratory Service at Colindale. Worldwide, the Centers for Disease Control and Prevention, Atlanta, Georgia, USA, offer a service for the diagnosis and therapy of unusual infectious diseases.