Diagnostic test

Details Diagnostic test Diagnostic test

2002-01-18

2003-08-19

Leary, Louise N. (Department: 1654)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving viable micro-organism

C435S004000, C435S968000, C435S975000, C435S805000, C435S810000

Reexamination Certificate

active

06607896

ABSTRACT:

This invention relates to a method for the detection of a disease, a device for use in such a method and a kit comprising such a device.
Bacterial vaginosis (BV) is a condition in which the most numerous micro-inhabitants of the vagina (Lactobacilli) are overwhelmed by other micro-organisms such as
Gardnerella vaginalis
. BV is associated with preterm labour (PTL). The mechanism behind this association is thought to be due to BV-related flora gaining access to the uterus and causing the release of inflammatory mediators that initiate labour.
It has previously been thought that in BV, the bacterial infection itself gives rise to PTL. Antibiotics are commonly used to treat BV, for example to prevent PTL, but such tratment is rarely successful. Moreover antibiotics can remove non-pathogenic bacteria, including beneficial strains such as Lactobacilli.
At present, the presence of BV is detected on the basis of a clinical examination and Gram staining for the bacteria characteristic of the disease. It takes a few days in order to get a result using Gram staining so the patient cannot be told the result at the time of visiting the clinic.
During pregnancy, there is no membranous barrier between the vagina and the uterus to prevent an ascending bacterial infection. However, the cervical mucus plug is thought to act as a physical barrier to in-utero infection.
The mucus plug is built on an infrastructure of carbohydrate-rich glycoproteins called mucins. Mucins are composed of oligosaccharide side chains joined to a central protein core. Sialic acid residues are present at the terminal ends of the carbohydrate side-chains.
High levels of bacterial sialidase activity are found in the vaginal secretions of women with BV as opposed to those without BV(MacGregor et al (1994) Am. J. Obstet. Gynaecol. 170 1048-1060; Briselden et al (1992) J. Clin. Microbiol. 30 663-666).
Bacterial sialidases are enzymes which cleave sialic acid residues of glycolipids and glycoproteins. To date, sialidase activity has been assayed by colorimetric, fluorimetric and radioactive techniques using a variety of natural and synthetic sialoglycoconjugate substrates. Substrates which have previously been used in assays for sialidase activity include: bovine submandibular gland mucin and human &agr;-1 acid glycoprotein (Howe et al (1999) in press); 2′-(4-methylumbelliferyl)-&agr;-D-N-acetylneuraminic acid (Paupermpoonsiri et al (1996) Clin. Inf. Dis. Vol. 23 748-752; Brieselden et al (1992) supra); 4-nitrophenyl-&agr;-sialic acid and 2-(3-methoxyphenyl)-N-acetyl-D-neuraminic acid (MacGregor et al (1994) supra). The sensitivity of many of the known sialidase substrates is low.
The sialidase substrates described to date are used in a variety of assays. For example, colorimetric or fluorimetric assays may be used with synthetic substrates, which detect the non-sialic moiety of the substrate (for example 4-methyl umbelliferone or 4-nitrophenol). Alternatively, the amount of sialic acid released from a sialoglycoconjugate substrate can be deteced by HPLC or a colorimetric assay. Also, radioactive assays are sometimes used which involve the introduction of a radiolabel into the sialoconjugate substrate and detection of the released sialic acid by liquid scintillation counting.
The present inventors have discovered that the link between PTL and BV is due to bacterial sialidase activity rather than bacterial infection itself. It is believed that sialidase activity is involved in the degradation of the mucus plug, which results in increased bacterial access into the upper reproductive tract.
The carboxyl groups of the sialic acids at the ends of the carbohydrate side-chains of the mucin molecules confer a negative ionic charge causing rigidity of the sugar side chains. Cleavage of sialic acid destroys the mutual repulsive charge between the mucin molecules causing a loss of viscosity of the mucus plug. Bacterial access into the upper reproductive tract is increased as a result of this action for two reasons. Firstly, mucin and therefore mucus matrix organisation is lost after degradation and the mechanical and bacteriostatic properties of the mucus matrix are rendered less effective as barrier mechanisms. Secondly, disintegration of the mucus layer facilitates bacterial adherence to the underlying epithelium which alters immunological recognition responses and reduces the likelihood that bacteria will be washed out by the movement of the vaginal fluid.
It follows from this discovery that sialidase inhibitors should be useful to treat and prevent conditions such as bacterial vaginosis, and will be useful in therapies to prevent PTL. It also follows that assays for sialidase activity will be useful to detect diseases such as BV.
The present inventors have also developed a diagnostic test for diseases such as BV which detects the presence of sialidase activity. A result in this test has been found to correlate significantly with the incidence of BV using a conventional Gram-stain diagnosis. The test method involves taking a sample from a patient and contacting the sample with a sialidase substrate. After incubation, detectable change such as a colour change, is observed if sialidase activity is detected. In a preferred embodiment, the test is a spot test, that is to say that the sample is applied to the sialidase substrate which is itself supported by a suitable solid medium, such as a filter paper.
The test makes use of substrates which have been found to have a very high sensitivity in the detection of BV, indicating possibly an optimum environment for the binding of sialidases from BV flora. The preferred substrate used in the present test is a salt of 5-bromo-4-chloro-3-indoyl &agr;-D-N-acetyl neuraminic acid, in particular the cyclohexyl amine (X-&agr;-NANA), which is available for Rose Scientific Ltd., Edmonton, Canada.
The test method of the present invention for detecting sialidase activity is highly sensitive, inexpensive to run and quick and simple to use. The test is especially useful to test for sialidase activity as an indicator of BV and therefore a predictor of the likelihood of preterm birth. The assay can be carried out in the same room as the patient or in a clinic or GP surgery, since the method of the invention does not require the use of items of expensive equipment or materials other than those which would normally be available. For example, in contrast to conventional methods, there is no need for a Gram staining kit or a light microscope. Moreover, the method of the invention is less labour intensive than a Gram stain.
Also, the sialidase spot test has an advantage over the calorimetric, fluorimetric or radioactivetests which are currently used to detect BV in that, since sialidase activity is not always present in BV or in all BV flora, its presence may be an indicator that the condition is more detrimental to the host in some cases than in others.
According to a first aspect of the invention there is provided a method for the detection of a disease comprising the following steps:
(i) obtaining a biological sample from a human subject;
(ii) applying the biological sample to a sialidase substrate which has been immobilised on a solid support medium; and
(iii)detecting a change in the immobilised sialidase substrate.
According to a second aspect there is provided a device for use in the detection of a disease, which device comprises a sialidase-specific substrate immobilised on a solid medium.
The device may be used in a method according to the first aspect of the invention, which comprises the following steps:
i) obtaining a biological sample from a human subject;
ii) applying the biological sample to the device; and
iii) detecting the presence or absence of a colour change in the immobilised sialidase substrate
According to a third aspect of the invention there is provided a kit for the diagnosis of a disease which comprises a device according to the second aspect of the invention.
The kit of this third aspect of the invention may also comprise an incubation medium, for example a Tris/HCl buffer an

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