Appropriate use of the microbiology laboratory

Background
The Microbiology Laboratory has an important role to play in both the diagnosis of infection and
the control and prevention of infection. It is important therefore, that users of a laboratory such as
doctors, understand how to use the laboratory effectively and enhance patient care whilst at the
same time not wasting resources. However, infection is a clinical diagnosis in the first instance,
laboratory investigations should only follow when an infection has been suspected and the
possible anatomical location and aetiology considered. Treatment is often required before the
result of laboratory investigations are available and in some patients, no aetiological cause is
ever confirmed.
It is essential that the prescriber of antibiotics interpret the results from the Microbiology
Laboratory in the light of the patient's clinical condition. Patients, not laboratory reports
are treated with antibiotics!

The role and functions of the microbiology laboratory can be

briefly enumerated as follows:
Diagnosis or confirmation of diagnosis of infection
Provision of antimicrobial susceptibility results to guide treatment
Control of hospital infection by identifying multi-antibiotic resistant bacteria
Provision of important epidemiological information necessary for community-based prevention
strategies such as vaccination

What are the qualities of the ideal microbiology laboratory service?

1. The results should be available rapidly.
2. The results should be accurate.
3. The test repertoire should be comprehensive.
4. There should be interpretation of the results.
How rapid are microbiological investigations available?

Unlike biochemistry or haematological investigations, most microbiological investigations require

18 hours or more. There is a balance to be made between providing as rapid a result as possible
and a detailed or exhaustive analysis of a specimen, which if significantly delayed, may not be
very relevant clinically.

Why is a comprehensive repertoire of tests important?

The laboratory service should consider all the common and important causes of infection but this
is sometimes dependant upon clinical details supplied. For example as culture for diphtheria is
no longer routine, this must be requested in an unvaccinated patient with a severe sore throat
who may be at risk, e.g. somebody returning from at risk countries such as Russia.

Why are accurate results important?

The result should reflect the organism or organisms causing infection. This is more difficult in
specimens where there may be commensal flora present such as faeces. Where infection is not
present, a false positive result may be very misleading.

Why is interpretation important?

The isolation of bacteria from a specimen does not always imply infection and the corollary is that
the failure to isolate an organism does not exclude possible infection. Most microbiology
laboratories attempt to interpret the results in the light of the patient group, the clinical information
supplied with the request, and knowledge of the local epidemiological patterns of infection.

What different categories of laboratories exist?

Most conventional hospital-based microbiology laboratories provide a comprehensive
bacteriological and mycological service. Other investigations such as virology or specialised
parasitology may be carried out elsewhere. For example, the National Virus Reference
Laboratory in University College Dublin provides much of the virology service to Dublin
Hospitals. Additional reference or specialist tests such as PCR for meningococcal disease or
tuberculosis or a specialised serology, e.g. schistosoma, may need to be sent to other Irish
hospitals or abroad.

What laboratory techniques are available in microbiology

laboratories?

Microscopy

Culture

Susceptibility testing

Antigen detection

Serology

Molecular approaches

Other approaches

What is Microscopy and how is it useful?

The correct answer is: Gram stain, Ziehl-Neelsen or auramine stain and wet preparations enable
the microscopist to observe possible pathogens. This results in a very rapid result but positive
microscopy requires large numbers of organisms to be present. Hence, "Open" or infectious
tuberculosis is microscopy positive. In other less "infectious" patients the tubercle bacillus is
subsequently cultured but not seen on initial microscopy

What is the role of culture and how long does it take?

Culture is a laboratory-based technique which is commonly used in most laboratories,
especially in bacteriology. Most bacteria will grow within 24-48 hours and hence the definitive
results should be available at this time. However, some bacteria such as nocardia, an
opportunist pathogen, and most fungi such as aspergillosis, may take longer. Full clinical details
with the request are essential to ensure that the laboratory seeks to isolate those pathogens
which take longer to grow. Although there have been developments in recent years, culture of
viruses still takes a week or longer.

What is susceptibility testing and what methods are used?

The basis of antimicrobial susceptibility testing is a comparison between the inhibition by a range
of antibiotics of the growth of the pathogen compared with antimicrobial susceptible control
strains. Zones of inhibition around paper disks containing antibiotics are the commonest form of
antimicrobial susceptibility but others such as the minimum inhibitory concentrations (MICs) as
determined by the E test are increasingly used. Many laboratories now use an automated
sensitivity machine, which like conventional susceptibility testing, requires overnight incubation
but allows the laboratory to test a larger range of antibiotics against the target pathogen. Whilst a
range of antibiotics may be tested, a limited number are often released to facilitate sensible and
appropriate antibiotic prescribing.

What is antigen detection?

Like microscopy, antigen detection provides a rapid presumptive diagnosis, e.g. Hepatitis B
antigen in serum or the presence of pneumococcal antigen in urine indicating pneumonia. A
variety of techniques are used such as fluorescent microscopy or latex agglutination. This is
only appropriate and available, however, in a minority of infections.

What is the value and the weaknesses of serology?

Detection of a significantly raised antibody titre, e.g. 1 in 256, or alternatively a four-fold rise in
titre over a 10 -14 day period is presumptive evidence of infection. However, serological
diagnosis is a retrospective one and the result may not be available in time to influence
treatment. Nonetheless serology is important in confirming a diagnosis and providing
epidemiological information, which is particularly important for vaccine-preventable illnesses such
as measles.

What is the role of molecular diagnostics?

This area will be covered in another lecture but molecular biology such as the use of PCR is
making significant inroads particularly in virology. Indeed viral PCR is increasingly replacing
conventional viral culture in virus reference laboratories. Molecular tests.are also increasingly
available for bacteriological (e.g.Clostridium difficile, MRSA, Neisseria meningitides,
tuberculosis) and fungal diagnosis.

What other approaches can be used?

So called "non-microbiological approaches" are occasionally required such as blood films for the
diagnosis of malaria and histological examination for the presence of hyphae in tissues indicating
invasive fungal infection.
What measures need to be taken when collecting specimens from sterile sites such as
cerebrospinal fluid, blood, pleural fluid etc.?
The specimen should be collected aseptically and should be carefully and rapidly transported to
the laboratory. The laboratory should be informed such specimens are being sent to the
laboratory in advance of its arrival. Loss of such specimens is a serious problem as they cannot
be easily repeated due either to their invasive nature or because antibiotics may have already
been started.

What problems are there with collection of urines, sputum, faeces and wound swabs?
These specimens can be repeated although antibiotics may be administered which affects the
subsequent detection of organisms. Also, the result must be interpreted in the light of the
presence of comensal flora which are commonly isolated from such specimens.

How is CSF collected and processed?

The correct answer is: CSF is obtained under sterile conditions by means of a lumbar puncture
or per operatively if a neurosurgical infection is suspected. CSF is important in the diagnosis of
meningitis but it may not be possible to obtain a specimen if raised intracranial pressure is
present or suspected. CSFs should be processed immediately and the laboratory should be
notified in advance of its arrival. In addition to microscopy and culture, biochemical assessment
is also important particularly in distinguishing viral from bacterial meningitis.

When should blood cultures be collected?

One or two sets of blood cultures (three if endocarditis is suspected) should be taken in any
seriously ill febrile patient. Sometimes organisms may be isolated from blood but not from the
anatomical site, e.g. pneumococcal bacteraemia in a patient without a productive sputum.

Why are fluid aspirates and surgical specimens important?

These are obtained either from normally sterile sites (e.g. pleural fluid) or from collections of pus
(e.g. hepatic abscess) and provide important initial information from microscopy to guide therapy.
Most abscesses usually require drainage and culture of abscess fluid helps optimise therapy.
Repeated aspirations are not required if the patient continues to improve and fluid from a
drainage bag which has been present for some hours should never be sent as this may harbour
the growth of many organisms due to multiplication at room temperature.

What types of urine specimens can be sent in and which is the preferred specimen?
The correct answer is: A mid stream sample of urine (MSU) is the preferred specimen as
otherwise urine may be contaminated with urethral or skin flora. In infants a supra-pubic aspirate
may be required. Catheter specimens of urine should not be sent unless there is some clinical
evidence of infection in the form of symptoms (e.g. pain) or signs (e.g. fever) - because of the
presence of bio-material, most catheter specimens of urine will be culture positive but this does
not imply that the patient requires treatment. Treatment of such patients requires assessment of
clinical features of infection, e.g. elevated temperature.

What problems are there with examining specimens of faeces?

Because faecal samples contain a range of facultative aerobic and anaerobic bacteria, the
laboratory uses a number of selective artificial agar media to isolate pathogens such as
Salmonella, Shigella and Campylobacter. In general, hard non-diarrhoeal specimens should not
be sent to the laboratory for processing, as the yield from these is poor.

What is the importance of skin and wound swabs?

Together with urines, swabs represent the most frequent specimen received in the Microbiology
Laboratory. Swabs are never as reliable as pus/fluid or tissue in diagnosing infection, especially
in the diagnosis of surgical-related infection as anaerobes and more fastidious
organisms may not survive on a swab or be present in sufficient numbers. The location
of the swab should be clearly identified and repeated swabs from the same surgical site are not
necessary. Swabs are also used when screening for some resistant bacteria such as MRSA.
Separate specialised swabs with appropriate transport medium should be used to diagnose
viral or chlamydial infections.
How should serum for antibody testing be collected and what techniques can be used?

Blood for serological investigation should be taken as soon as the diagnosis is suspected and
repeated 7-10 days later. In practice, a second specimen is regrettably often not taken,
especially if the patient has improved, and therefore a diagnosis may never be confirmed. There
are a number of methodologies used for the detection of antibodies in patient's serum. These
include:

Immunofluorescence

Enyyme - immunoassay (including indirect or competitive EIA)

Latex agglutination