1.2 Impact of sample quality on testing and reporting of results

Specimen contamination

It is important to recognise that when samples are taken from most sites in the body, the environment is not sterile. This means that a mix of the following may be present:

• Normal commensal flora: Bacteria (or other microorganisms) that are usually found at that location in or on the body, e.g. skin, genital region, mouth, etc. For example, normal flora are likely to be found on superficial swabs, genital swabs and in respiratory specimens.
• Colonising organisms: Potentially pathogenic organisms not usually found at that part of the body, and may be displacing the commensal bacteria.
• Contaminants: Microorganisms collected during specimen collection, e.g. from the patient, healthcare provider taking the sample or on the collection tube.

Blood and CSF are normally sterile; therefore any microorganisms found in samples taken from these parts of the body are likely to be the cause of an infection. However, if there is contamination of the sample during the process of collection, commensal bacteria may be isolated as a contaminant. Similarly, urine samples should also be sterile but are easily contaminated at the time of collection. These contaminants can grow in the sample, making interpretation of the culture results difficult.

Reducing contamination

Clinical staff can take several measures to improve the quality of the specimens they send to laboratories, either by reducing contamination through aseptic techniques and/or increasing the yield of true pathogens. For example, sterile containers and equipment should be used for sample collection, to avoid the introduction of contaminating organisms. Specimens need to be large enough to handle effectively and to minimise changes such as pH, drying out, etc., that may affect the viability of the organism concerned.

Some types of sample may require special treatment or have other considerations.

• Sputum samples, for instance are very useful for diagnosing TB and are frequently sent to diagnose other respiratory tract infections. However, the results have to be considered carefully as the sample is likely to be contaminated with the commensal flora in the upper respiratory tract. Identifying all the organisms in a sputum sample will not help the diagnosis and takes up a lot of laboratory time and resources.
• Blood cultures, which are important in diagnosing severe and/or bloodstream infections, are another sample type that is easily contaminated by the commensal flora of the skin. Even though the blood is taken carefully to minimise contamination and inoculated directly into blood culture media by the person taking the samples, contaminants will still sometimes be found. A video showing safe and accurate blood culture collection using ANTT (The Hillingdon Hospitals NHS Foundation Trust, 2019), shows how careful decontamination of the skin and bottles, plus aseptic technique can minimise sample contamination. Video 1 illustrates some of the key features of aseptic technique.
• Urine samples need to be collected carefully in midstream, to help reduce contamination with the commensal flora in the genital region. The sample should be refrigerated if it cannot be processed immediately. Boric acid can be used as a preservative if this is not possible. Microscopy helps determine whether a urinary tract infection (UTI) is present before culturing by looking for pus cells which indicate inflammation/infection.

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Transcript: Video 1 Aseptic technique

Microbes are grown in the laboratory as cultures. Cultures can be grown from samples from a variety of sources including clinical specimens. In their natural environments, many species of bacteria grow together, but if we want to study a particular species, we need to grow it on its own in a pure culture. In a pure culture, all the cells present are of the same type. Pure cultures are maintained by sterilising media before use and by a practice called ‘aseptic technique’.

Aseptic technique prevents other microbes contaminating our culture and also prevents cultured microbes from escaping into the environment, which is very important when we’re dealing with pathogens.

To grow microbes, we must supply all their nutritional requirements by growing them in culture media. Media can be liquid, when they are called broths, or they can be solidified with a jelly-like substance derived from seaweed, called agar.

A wide range of different media are available as different bacteria have different nutritional requirements. In fact microbes can sometimes be identified by the media they can or cannot grow on.

Media must be sterilised before use and this is normally done in an autoclave, a type of industrial pressure cooker. Materials are sterilised by exposure to high pressure steam at a temperature of over 121 °C for 15 minutes. Sterilising media before use means that the only cultures that grow are the ones we put there.

To check whether sterility (the complete absence of living organisms) has been achieved, the materials are marked with special tape that changes colour under these conditions.

To prevent the spread of any pathogens into the environment, bacterial cultures that are not required any more are sterilised by autoclaving.

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Video 1 Aseptic technique

Laboratory staff also need to take care not to introduce contaminants into cultures. Good laboratory practice should be followed at all times.

Reporting results

After testing it is important to avoid reporting any additional organisms and instead focus only on the causative pathogen, if found. Correct reporting is needed to guide accurate treatment decisions and for surveillance purposes.