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