Human testing methods – a guide for patients
When testing a human (and many animals) for leptospirosis there are three basic techniques we can use:-
- Direct observation – looking for the bacteria under a microscope
- Antibody tests – looking for the antibodies you create when your immune system fights infection
- DNA tests – looking directly for the bacterial DNA signature
Lab tests are often called ‘assays’ by medical staff, and are usually based on a blood or urine sample taken from the patient. In very specific situations the tests may use other tissue samples (like a biopsy of an internal organ) or the fluid from the inside of the eyeball, but this is only when dealing with very unusual cases or at a post-mortem examination. For normal living humans a blood test is standard – or more particularly two. The problem is that for some of the tests – the antibody assays – we can get a false positive if the patient has been infected in the past and carries immunity, so by taking two samples about a week apart we can see if their test results are changing or not. An increase in the result between the two samples shows the patient is infected right now, and not just carrying immunity from something five years ago. DNA-based tests require only one sample as they are looking for the bacteria directly, and not just your immune response to them.
are often used at the very first stage, as they’re fast and can give a positive result before the other tests have completed. At the most basic level, a lab technician looks at the sample under a powerful microscope and tries to see the bacteria. It needs very careful preparation of the sample (usually a blood or urine sample) so that the bacteria are stained enough to show up (natural-colored they are incredibly hard to see) and often the lab technician misses the bacteria, so giving a false negative. The test is very cheap, so is often used to screen samples in the developing world or after natural disasters.
are also called “agglutination tests”, rely on the fact that the antibodies in your blood can cause a culture containing leptospira to change appearance – clumping together when viewed under a microscope. These tests require some operator skill, and are specific to certain serogroups, but use commercial test cultures and so may need to be ordered in from a manufacturer. Mostly live cultures of leptospira are used, which presents obvious risks to the lab staff, but testing with killed cultures is not as sensitive.
There are several types of agglutination test, each with strange names and each with some benefits and limitations. All of them report their results as a “titer” (titre in English) – a ratio of dilution for the sample. A high titer means that the sample was able to trigger the test even when diluted by a large amount, so there must have been a lot of bacteria in the sample to start with. Low titres mean than the sample had to be kept more concentrated, so when comparing two tests on a patient, it’s a bad sign if the second test is higher – something we call a ‘raising titer’ – as it means the number of bacteria in the sample have increased.
This means ‘microscopic agglutination test’ – the sample (taken from the patient’s blood) is applied to a series of very tiny plates, tubes or wells containing some live bacteria, and then allowed to react for a few hours. What we’re looking for is the antibodies in the sample (produced by the patient’s immune system) attacking the bacteria and causing them to clump together – so it’s specific to a serovar or group because the antibodies are too. The concentration that causes agglutination is decided by the operator viewing each reaction under a microscope. Some forms of MAT test are better than others – versions that use dried samples on slides are less sensitive but far easier to use, the versions that use tiny liquid samples of live bacteria are the most sensitive but also the most complex to perform, and can take several days if the lab has to first grow the bacterial solutions from scratch. MAT is still the ‘standard’ test for most human health in the world, and will probably be the test used on you if your blood is sent for testing by your doctor.
This means ‘enzyme-linked immunosorbent assay’ and is based on a chemical reaction rather than a sample of live bacteria. In a lab setting the sample (prepared from the patient’s blood or urine) is fixed to a special plate, then washed with a liquid containing antibodies that bind to the bacteria. The antibodies are linked to an enzyme that either changes color or fluoresces, so the effect can be detected by eye or using a lab instrument. Since the test doesn’t need lots of live bacteria to be grown, it’s far faster and easier to perform – ELISA tests are now available as color-changing dipsticks. The drawback is that it’s not as easy to tell the concentration in the original sample, but these ‘field screen kits’ are often used for testing after natural disasters or during outbreaks.
A form of ELISA test that only reacts to one type of antibody (IgM, IgG or IgA) is available that can help determine if the titer is from a current or historical origin.
There are many other tests available that show some success, but are less sensitive or more difficult to perform. These often only detect the bacteria as a species and so can’t show which serovar it is – MAT and DNA testing can do that and is more sensitive too.
This uses a method called PCR (polymerase chain reaction), to duplicate sections of the bacterial DNA and display the sequence on a glass slide (or in the latest equipment, on a computer readout). Made famous on TV forensic shows, PCR is extremely accurate and sensitive, but is also very expensive and takes a very long time (something they never show on TV!) so is often run as well as one of the more rapid tests. A positive PCR assay is diagnostic in that it can’t be caused by historical immunity, and it also shows the particular serovar causing the illness, but in many regions of the world the lab equipment is simply not available. One drawback of DNA testing is that it’s very good at telling what the sample is, but not how much there is – so there’s not as accurate a concentration figure as for the agglutination tests. PCR can however find bacteria at lower levels than other types of testing.
The LIC cannot assist labs in obtaining testing equipment or cultures as these are commercial products. Tests are widely available and all public health laboratories will be able to order consumables and samples from their usual suppliers.