Clinical role of the microbiology laboratory

Patient diagnosis and management

The microbiological processing of patient specimens and serodiagnostic analyses are fundamental to the everyday diagnosis, treatment and management of patients with infectious diseases.

a) Some infectious diseases can often be diagnosed clinically with the addition of some simple, rapid ward-based tests and treated empirically (e.g. the first attacks of acute cystitis in young sexually active women). However, it is still wise to also process specimens to confirm the diagnosis and the specific implicated pathogen, as well determining its antimicrobial susceptibility pattern (antibiogram). There are also some non-microbiological tests e.g. procalcitonin, C reactive protein that play important roles in diagnostics but are not examined here.

b) Some infections must be treated without delay after clinical diagnosis with empiric antimicrobials (e.g. acute meningitis, sepsis or severe pneumonia),but the clinician will also want the results of specimen analyses to establish the exact microbial aetiology.

c) However, in most infections, and especially in HAIs (see below), it is important to perform aetiological diagnosis and antimicrobial susceptibility testing.

d) Much research is being conducted currently to provide diagnostic laboratories with more rapid or point-of-care tests to prove the exact aetiology and correct therapy as quickly as possible, so that morbidity and mortality can be reduced, as well as enabling the switch to more targeted antimicrobial therapy to reduce the selection pressures that encourage the emergence of antimicrobial resistance (AMR).

Specific issues of HAIs

Microbial analyses are especially important in the diagnosis of HAIs as:

a) most are caused by microorganisms whose antimicrobial susceptibility is less predictable and more resistant than those causing community associated infections.

b) defined clinical HAIs syndromes e.g. surgical site infections can be caused by several microorganisms, as well as the same microorganism causing different HAIs clinical syndromes.

c) the correct aetiological diagnosis and more targeted antimicrobial therapy will also lead to the earlier eradication of the infecting microorganisms and help prevent the patient from continuing to be the source of microbial spread to other patients (i.e. the diagnostic role also comprises part of the laboratory’s public health/epidemiological role).

d) normal (commensal) microorganisms cannot necessarily be ignored as they can become opportunistic HAIs pathogens. The reasons for this lie in the interactions between the constituents of the classical pathogenesis triangle [the “seed” (microbe), the “soil” (patient) and the “climate” (environment)]. For example, an intravenous cannula (a “climatic” factor) can provide the portal of entry for skin flora e.g. coagulase negative staphylococci, to cause a bloodstream infection. More vulnerable patients e.g. the immunocompromised patient (a “soil” factor) are more prone to infections caused by commensal organisms such Candida spp.

The challenges of microbiological specimens

These are far more challenging than, for example, those taken for haematological or clinical chemistry investigations and careful attention needs to be paid to them, not least because it is often impossible to re-collect them. The second major issue is that microorganisms are living organisms and, if good specimen submission management is not in place, it can result in serious issues, such as false positive or negative laboratory results. These can arise if, for example, infecting microorganisms are allowed:

a) to multiply further (the numbers of organisms identified can often be important to the correct clinical diagnosis e.g. numbers of organisms in a urine specimen);

b) to lose viability (a particular issue with more delicate organisms such as Streptococcus pneumoniae);

c) be difficult to isolate because of the overgrowth of other microbes e.g. commensal flora in the specimen. The latter is a particular issue because, as we have already pointed out, such commensals can also cause infections especially HAIs).

Requirements of clinical specimens for microbiological analyses

a) specimens should be selected properly (the right indication, source e.g. mid-stream urine, timing of collection; e.g. for the clinical diagnosis of typhoid fever one should take blood in the acute stage during continuous fever, a week or so later a stool sample could become positive, later on a urine, and serology will be positive two weeks after infection, although blood will be negative at that time);

b) specimens should be collected ideally before antimicrobial therapy has been initiated;

c) specimens should be collected in sufficient volume, depending on test(s) ordered;

d) specimens should be collected in the correct container (e.g. for serology, or molecular diagnostics, or in transport medium);

e) specimen should be accompanied by information on the precise clinical site e.g. right leg wound; relevant procedure e.g. surgical site, time/date of onset, ward and other important patient clinical information e.g. what antimicrobial treatment the patient is receiving or starting;

f) specimens should be transported correctly (i.e. consideration needs to be given to timeliness, temperature, medium and atmosphere depending on many factors);

g) specimens of insufficient quality should not be processed. However, before declining these, the laboratory must communicate with the clinician in charge. For example, a wound swab sent for culture requesting a search for anaerobic bacteria, but unfortunately sent without insertion of the swab into an appropriate transport medium, should not be cultured as such organisms are unlikely to be still viable. If the specimen is unrepeatable, then culture should be agreed and the report covered with the appropriate caveats.

Quality assurance systems

Laboratories should be accredited for all of their services. To do this, appropriate standard operating procedures (SOPs) should be in place to validate all of their work, including the specimen processing and correct interpretation of any results.

SOPs should also extend to correct specimen selection, collection and transport and these should be agreed between the clinicians, clinical laboratory personnel and hospital management. It would include consideration of any equipment e.g. servicing of incubators for blood cultures if there is no on-site laboratory and that the materials required e.g. swabs, transport medium, are made readily available. The clinical microbiologists should lead the drafting of specimen collection/transmission instructions and pilot these with the relevant staff to ensure that they are unambiguous and easy-to-follow. These systems should be audited regularly and any issues which arise addressed; records of this should be available for accreditation bodies.

Here is not the place to cover this in any depth, but it is essential to be able to prove the quality of the results of every microbiology laboratory. All the necessary steps should be taken to ensure quality assurance (i.e. total quality management) is in place, including external quality controls/assurance (e.g. NEQAS). The laboratory should save all specimens until the result is finalised. It is desirable to save all relevant microbial strains (especially from invasive isolates) for possible further analysis (also for medico-legal needs); the duration of such storage needs to be agreed with the relevant national/legal bodies. This can be especially important for HAIs isolates.

The laboratory should have an SOP manual for all performed tests, records of all laboratory QA meetings and a record of all specimens collected and results. Where other laboratories are needed to perform more specialised tests, then there should be interactions with these laboratories and an SOP written as to how the specimen will be collected and how and where it will be sent, and records of these kept. This is especially important when one knows ab initio that these relate to medico-legal/terrorism cases.

Microbiological laboratory methods

To be most useful, microbiological diagnostic tests should be fast, and accurate (see below: high sensitivity and specificity), and the clinical relevance (or not) of the results explained.

Rules for interpretative reporting

There should be clear rules in the laboratory for interpretative reporting.

1. The laboratory must have a policy not to report every microbe isolated, but only ones relevant to infections, together with their antimicrobial susceptibility results (these should be agreed by the AS committee and stated in the hospital formulary/policy).
2. Where there is any doubt regarding the relevance of culture results, the clinical microbiologist should communicate with the clinician in agreed ways depending on the situation's urgency. Medical microbiologists should regularly contact clinicians to communicate the laboratory results and inform them of their significance.
3. Clinicians and medical microbiologists should be aware of any issues of sensitivity and specificity when such tests are ordered. Knowledge of the local rate of occurrence of the organisms causing the various infections is important, as this will affect the tests’ positive and negative predictive values. In this way, all can best judge the significance of any results.
4. Specimen processing for microbiological diagnosis progresses in various phases (see below: direct smear, culture, organism identifications, antimicrobial susceptibilities) each should be communicated with clinicians, especially for specimens from urgent/important e.g. severe infections. The medical microbiologist informs and advises the clinicians along this diagnostic pathway. Indeed, such communication is as important as any laboratory equipment or technique for accurately interpreting microbiological culture results.

Diagnostic Methods

Several diagnostic medical microbiological methods are utilised. These include:

1. direct smear of the specimen: microbial stains e.g. Gram stain can help with the interpretation of the culture results and provide a rapid clue as to the likely pathogen;
2. culture for bacteria and fungi followed by
3. identification (using classic biochemical/ antigen determination methods or newer methods like MALDI-TOF, SELDI-TOF, or other molecular methods) and

antimicrobial susceptibility testing results. This is one of the most important laboratory clinical functions. Semi-quantitative testing is performed routinely; test results in Europe should be interpreted using EUCAST standards. The laboratory will also advise on, and perform, quantitative susceptibility testing, and, where relevant, identify resistance genes;

1. microbial antigen testing e.g. for Legionella spp;
2. serology; as a proof of immune response to infectious agent (this usually requires specimens to be taken 10-14 days after the infection, when antibodies first appear).
3. nucleic acid (DNA and RNA) detection in the specimen (i.e. molecular microbiology techniques). This is a field in which there is continuing progress:
4. advantages of nucleic acid detection methods, these comprise:
   1. speed, as the process does not require microbial culture,
   2. sensitivity (i.e. low false negativity as, in theory, one organisms can be detected although, in practice, at least 10-100 organisms/mL are usually required)
   3. specificity (i.e. low false positives; in theory every microbial species/diagnostic toxin has amplifiable and identifiable specific gene[s]);
   4. disadvantages of nucleic acid detection methods, these comprise:
   5. one can find only what one is searching for (known: especially important where there are new AMR genes emerging)
   6. the presence of DNA does not mean it is functioning (also important for AMR, although more complicated RNA amplification techniques can solve such issues),

biological samples can often contain inhibitors to DNA/RNA amplification methods

1. 1. they need trained staff (easier if the same equipment is used for all such tests)
   2. they may not be cost effective (this will depend on the organization of the hospital services, costs of reagents, equipment rental/servicing balanced against the savings from improved testing). This is another example of how the patient diagnostic and public health/epidemiological roles of the laboratory need to be considered. Incremental cost effectiveness and other econometric methodologies are used to inform decision making and will depend crucially on the current incidence of the infection/AMR problem and prevention and control interventions being proposed.

The challenges of consolidated microbiology laboratory

Increasingly there is a global tendency to centralise medical microbiology laboratory services often outside any of the “customer” hospitals. Reasons for this include financial rationalisation, enabling more sophisticated, otherwise prohibitively expensive or low volume tests to be performed and ease of training and succession planning/sustaining skilled services. However, unless such centralisation is well thought through it can lower the quality and timeliness of results. Education of the laboratory staff in hospital matters is also vital to ensure the delivery of a clinically relevant service. The hospitals served should ideally all be involved in the designing and improving all the relevant systems. Transport of specimens can be prolonged unless co-ordinated and other mechanisms are in place e.g. availability of rapid motorised transport for urgent specimens, availability of several assay runs during the day so, for example, important tests are readily available e.g. toxin testing, ability to add an urgent specimen in the middle of assay runs or where this is an issue, availability of single specimen rapid molecular analysis, the means to incubate blood cultures en-route or overnight, lower temperature storage to avoid overgrowth of microbes etc. Sometimes the medical microbiologists are only available at certain hospitals or just centrally, thus threatening communication. Other systems thus need to be explored e.g. telecommunication. These threats are also applicable to the second role of the microbiology in IC of HAIs and AS as well as in the role of microbiology laboratory in education of the staff.

Future developments

Many newer diagnostic approaches are being further developed or invented and have an enormous potential to further revolutionize microbiological diagnostics. There are many reviews of these and we do not intend to repeat these here. We would mention the development of multiplex DNA amplification tests, however, that enable, in theory at least, the investigation of causative agents of various syndromes (e.g. a syndromic test for respiratory viruses in nasopharyngeal secretions). Some multiplex tests, such as those for the causative agents of bacteraemia, whilst promising, are not yet sufficiently reliable. The main challenges are that they cannot yet identify all the possible pathogens. If strategies are employed e.g. to broaden the targets with different primers or to use existing primers under less stringent conditions, they cannot then distinguish between common pathogens and contaminants. This is even more complicated, especially in HAIs diagnostics, where contaminants may in fact be opportunistic pathogens, as we have already mentioned.

Point of care (POC) tests

Attempts are underway to move diagnosis nearer to the patient, so that less skilled staff can use simpler/automated equipment that will enable faster unambiguous diagnosis at the bedside, so reducing morbidity, mortality and reducing the need for empirical antimicrobials. Such tests are developed on different platforms, but nucleic acid technologies are providing the greatest potential, especially in multiplex format, to provide rapid, sensitive and specific results.

Several such tests are already in use for the POC diagnosis of e.g. malaria, HIV, HCV, syphilis, measles, respiratory viruses (especially influenza), norovirus and tuberculosis.

Much more remains to be done and the question continues to be posed for the clinical setting if it would be better to do it in the laboratory, or to do it at the ward where you need to educate more staff to do it. Interactions with the laboratory are important and some systems do this automatically. In this way quality assurance of results is more feasible.

Some of these tests are very useful, not only for the management of individual patients, but also for IC in hospital (influenza, respiratory syncytial virus, norovirus, Group B streptococci, AMR genes).

_____

References are in the 8.1 The role of the clinical microbiology laboratory in infection prevention and control.

Original contribution from: Smilja Kalenic,University of Zagreb School of Medicine