Diagnosis of infection

It can be very difficult to decide whether or not a patient is suffering from an Aspergillus infection. It is one thing to be faced with an X-ray that clearly shows evidence of invasive fungal infection of the lungs for example, but it is quite another to decide whether a patient who is seriously asthmatic has ABPA (Allergic BronchoPulmonary Aspergillosis) and therefore has Aspergillus growing within their lungs or whether it is a quite different infection which is causing the asthma flare-up.

Doctors accumulate clues to help them make this judgement - clues such as;

• What are the symptoms - are they consistent with a fungal infection?
• Is the patient immunocompromised?
• Any other factors which may make the patient at high risk of fungal infection?
• Is infection unresponsive to antibiotics?

If a fungal infection is still suspected the doctor can order X-ray/CT scan; for examples of these go to the image section of the Aspergillus website. However a fungal infection may not show up.

Attempts are made to obtain a culture of the infecting organism - this is easy if the infection is easy to 'get at' e.g. an ear canal or nail infection, but more difficult if it is a lung infection that is suspected. Sputum samples are easy to obtain but these can be contaminated by spores in the air passages as the sputum is 'brought up'! (such spores are frequently present in most peoples' air passages but they do not go on to cause disease). Samples from other tissues e.g. brain are even harder to obtain. Biopsies can often be carried out and these provide very good proof if a fungal culture can be obtained from tissue brought from deep within the body - but these are difficult to do and are laborious requiring skilled personnel to perform them. Such cultures do have the advantage of providing a means to accurately identify which species of fungus is involved.

How much easier it would be if a simple blood sample could be taken and the presence of a specific fungus detected in that sample! There are currently two processes being developed so that it will be possible to do just that; immunological detection and PCR (Polymerase Chain Reaction) detection.

Immunological detection

There are two targets for this method of diagnosis, both of which act on very small components of all living things called proteins.
Different proteins can be detected using antibodies - this is how our immune systems specifically detect any foreign proteins which have got inside us (e.g. fungi, bacteria & viruses - i.e. germs!) - we produce our own antibodies to help kill the germs using our immune systems.
Scientists have learned how to manufacture antibodies in the laboratory and we can now use them to detect proteins with a high degree of specificity. NB Specificity is important - when an antibody is used it would be no good if it detected more than the few proteins that it was designed to detect:

• Detection and identification of fungal proteins.
• Detection and identification of the antibodies produced by the patient as a response to infection.

Both methods are in active development.
Fungal protein detection is interesting as this could provide the basis for the development of a vaccine against fungal infection.
Such tests are also useful to rapidly identify a particular fungal species in a small biopsy sample - this takes only a day or so by this method rather than the two weeks it can take to identify by growing up a culture.

There are already commercial kits available for the detection of several fungal proteins and these are proving useful in some circumstances e.g. galacomannan detection for invasive Aspergillus - this approach is potentially most useful when a patient is quite heavily immuno-supressed and thus cannot react to the infection.

Detection of antibodies produced by the patient has a number of potential advantages. In some infections there may be very little fungal protein in the blood of the patient, but there may be quite a large immune response by the patient's immune system. Detection of this response is thus much easier than trying to detect fungal protein itself.
Measurement of the response of a patient to infection, quite apart from identifying what is causing the infection also can be used to find out how the infection is progressing and how the patient is responding to the infection and/or treatment, especially if blood samples are taken at intervals over several days.
ABPA sufferers are familiar with this process - their disease is characterised by long-term contact with Aspergillus and so it is inevitable that sufferers will be producing lots of antibodies to the fungus. The presence of Aspergillus is often detected by taking a blood sample which is then tested for levels of IgE antibodies which are particularly associated with the allergic response.

Antibody production by our immune systems is a complex process which involves five different groups of antibody; these are called IgA, IgD, IgE, IgG and IgM (Ig = Immunoglubulin).

• IgG and IgM are part of the main response to infection and play key roles in eliminating the invading organism.
• IgA is found mainly in secreted fluids such as saliva, tears, sweat and secretions of the lung and gut lining but is not found in high concentrations in blood.
• IgE is normally found in very low levels in the blood and is responsible for allergic responses such as asthma & hay fever, triggering many of the unpleasant symptoms experienced by the sufferers of those disorders - specifically inflammation of the affected tissue. This usually helps the body to fight infection as it increases bloood supply to the affected part and thus speeds the healing process. Sometimes this process goes too far and for unknown reasons causes a hypersensitive reaction. Allergic asthma is one such reaction.

Detection of high levels of IgG or IgM in the blood are thus indicative that a body is fighting an infection - useful for invasive diseases - while detection of high levels of IgE indicate an allergic response and is thus particularly useful for ABPA and other allergic diseases.

PCR detection

The Polymerase Chain Reaction (PCR) was invented some 15 years ago and involves the analysis of a different set of tiny components of all living cells. Immunology works with proteins while PCR utilises strands of DeoxyriboNucleic Acid (DNA). This chemical is used by all living cells in order to replicate themselves and takes the form of a very long thread rather like a zipper in that there are two sides which interlock and zip-up together in a very precise way.

PCR uses very short pieces of this zipper (called primers) which stick only to particular parts of it and then zip-up to give new strands of DNA which are easily detectable. The same primers give DNA strands of different sizes (or none at all) depending on whether they stick (or not) to human DNA or fungal DNA, so it is easy to see if a blood sample contains any fungal DNA by comparing the sizes of the DNA strands.
If fungal DNA is detected then it could be said that the patient who gave that sample has a fungal infection. It may even be possible to say which fungal species is causing the infection.

PCR is very rapid, giving results in a few hours rather than weeks, and has the potential to be very precise in identification and very sensitive - only requiring a few molecules of fungal DNA to give a positive result.

Limitations

If the ultimate aim of PCR and immunology is to detect fungal infections in blood samples then there are significant problems and shortcomings of each technique.
Immunological techniques suffer because many infections do not provok an immune response or release fungal proteins which are detectable in the blood. Such infections are thus not detectable using these methods.
PCR is more sensitive and should be able to detect very small amounts of fungal material in the blood, but the very sensitivity of this techique can prove its downfall as it can detect extremely small amounts of contaminating material and thus potentially give false positive results.

The answer to these problems is two-fold:

• Continue to refine these methods to improve sensitivity and reliability - this is ongoing and improvements are being made continually. Some immunological tests are already in use in the clinic.
• Use several available techniques and sources of information (e.g. symptoms) in combination to get the complete picture before diagnosing e.g. a positive result for PCR combined with negative CT scan and negative immunological test result could be taken to mean a negative result (no infection) but a follow-up series of tests should be done a few days later.
  In other words PCR and immunology add more tools to the armoury of the clinician but each by themselves may not be diagnostic.