Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/56911—Bacteria
  • G01N33/5695—Mycobacteria
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
  • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
  • G01N27/416—Systems
  • G01N27/447—Systems using electrophoresis
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/483—Physical analysis of biological material
  • G01N33/487—Physical analysis of biological material of liquid biological material
  • G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
  • G01N33/48714—Physical analysis of biological material of liquid biological material by electrical means for determining substances foreign to the organism, e.g. drugs or heavy metals

Definitions

• the present invention relates to a method of detecting the presence of a mycobacterial infection in a subject.
• the invention also relates to a method for use in the diagnosis of disease.
• Mycobacterium is a genus of Actinobacteria with its own family, the Mycobacteriaceae. Infection with Mycobacterium is known to cause serious diseases in humans including tuberculosis (Mycobacterium tuberculosis) and leprosy (Mycobacterium leprae).
• Tuberculosis is a bacterial infectious disease caused by Mycobacteria related to
• Mycobacterium tuberculosis complex mainly Mycobacteria tuber culosituberculosis. It is a pathogenic, aerobic and rod-shaped bacterium.
• Tuberculosis (TB) is one of the largest and most widespread infectious diseases in the world. Infection affects the lungs (pulmonary TB). In humans the disease is transmitted from person to person through droplets produced while coughing or sneezing. Droplets of an infected person are propelled by coughing and sneezing into the air and are deposited on the mouth or nose of people nearby.
• the active disease can also affect e.g. lymphatic nodes, bones, brain and stomach etc. Such manifestations of disease are known as extra-pulmonary TB.
• An infection with M. tuberculosis may remain latent for years in healthy people and only become active when the immune system is compromised. Active tuberculosis is, however, treatable with antibiotics, such as a 6 month course.
• Tuberculosis should be suspected in subjects suffering unexplained weight loss, loss of appetite, night sweats, fever and fatigue. Symptoms of pulmonary tuberculosis (i.e.
• tuberculosis in the lungs include coughing for longer than three weeks, hemoptysis (coughing up blood) and chest pain.
• Methods of detecting the presence of a dormant mycobacterial infection in a subject currently include the Mantoux tuberculin skin test (TST) or TB blood tests can be used to test for sensitisation to M. tuberculosis antigens. These tests are called interferon gamma release assays (IGRA).
• TST Mantoux tuberculin skin test
• IGRA interferon gamma release assays
• the Mantoux tuberculin skin test is performed by injecting a small amount of fluid called tuberculin into the skin in the lower part of the arm. The test is read within 48 to 72 hours by a trained health care worker, who looks for a reaction (induration) on the arm.
• the TB blood test measures the patient's immune system reactivity to M. tuberculosis specific antigens.
• Mantoux tuberculin skin test and the IGRA tests are not primarily designed to identify persons with active TB but they may be used in some instances as supplementary tests in addition to e.g. culture, sputum staining and nucleic acid amplification techniques
• Chest radiographs are used to detect chest abnormalities. Lesions may appear anywhere in the lungs and may differ in size, shape, density, and cavitation. These abnormalities may suggest active TB, but cannot be used to definitively diagnose TB. However, a chest radiograph may be used to rule out the possibility of pulmonary TB in a person who has had a positive reaction to a TST or TB blood test and no symptoms of disease. The presence of acid-fast-bacilli (AFB) on a sputum smear or other specimen often indicates TB disease. Acid-fast microscopy is easy and quick, but it does not confirm a diagnosis of TB because some acid-fast-bacilli are not M. tuberculosis.
• AFB acid-fast-bacilli
• the sensitivity of sputum staining is at its best 50%. Therefore, a culture is carried out on all initial samples to confirm the diagnosis. This procedure is the gold standard to diagnose mycobacterial infections. A positive culture for M. tuberculosis confirms the diagnosis of TB disease. Culture examinations should be completed on all specimens, regardless of AFB smear results.
• Bovine tuberculosis is caused by Mycobacterium bovis (M. bovis) a bacterium that is closely related to M. tuberculosis.
• Bovine tuberculosis and indeed tuberculosis of animals in general is listed in the World Organisation for Animal Health (OIE) Terrestrial Animal Health Code, and must be reported to the OIE as detailed in the OIE Terrestrial Animal Health Code.
• Tuberculosis is an important disease in cattle and wild animals and is a significant zoonosis. The disease in cattle is passed on through contact, but the disease can also be passed on from cow to calf through milk, and similarly can be passed to humans through milk.
• Lipoarabinomannan in the following abbreviated "LAM" is a heterogeneous,
• phosphatidylinositol anchored lipoglycan component of the mycobacterial cell wall This molecule contains an arabinan backbone and mannose caps which are species specific.
• Mannose capped LAM mediates the binding of Mycobacteria to the C-type lectins of dendritic cells and macrophages, and has a key role in mediating host immune response during infection.
• a method for the detection of LAM is disclosed in European Patent 1 710 584.
• the method disclosed therein comprises a step of allowing a Limulus reagent to contact with the LAM containing sample. On contact, LAM in the sample activates factor C in the Limulus reagent and a Limulus reaction is induced.
• the Limulus reaction is detected and measured by conventionally known methods e.g. chromogenic assay. The Limulus reaction is, thus, indicative of the presence of LAM in the sample.
• International Patent Application WO 98/29132 relates to a method for the early detection of the presence of a mycobacterial disease or infection in which a biological fluid sample is taken from a subject and the sample is assayed for the presence of antibodies specific for LAM.
• US Patent 6,599,691 relates to a method for detecting antibodies in human saliva, specific to LAM.
• polyclonal antibodies in immunological methods suffers from the in-built problems of cross-reactivity with as yet non-defined molecules (e.g. in urinary tract infection disease control subjects that were caused by a variety of gram-negative or gram-positive bacteria (Savolainen et al. 2013 Clin Vaccine Immunol).
• Lipoarabinomannan also called LAM
• LAM is a glycolipid, and a virulence factor associated with Mycobacterium tuberculosis (M. tuberculosis), the bacteria responsible for tuberculosis. Its primary function is to inactivate macrophages and scavenge oxidative radicals.
• LAM is also an antigen of the cell wall of replicating Mycobacteria that is present in the samples of urine of subjects infected with M. tuberculosis in tiny amounts.
• a non-immunological method i.e. by carrying out capillary electrophoresis on a sample that has been concentrated in the capillary by subjecting the concentrated sample to an electric voltage across the capillary. The presence of LAM in the sample is indicative of a mycobacterial infection.
• the method can be used in the diagnosis of an infectious disease as defined in claim 12.
• Advantageous Effects of Invention are provided in the diagnosis of an infectious disease as defined in claim 12.
• the invention provides several advantages.
• the presence of LAM is detected in a biological sample.
• the method is robust and fast, and provides a result within about 30 minutes i.e. the method is faster than ELISA assays.
• the method is non-labour intensive and can be automated. Only a small amount of sample is required and the cost of the analysis is very low compared with other commercially available methods.
• the method also provides an ecological advantage in that no non-renewable waste is produced.
• LAM is detected by a non-immunological method, thus avoiding all of the drawbacks associated therewith, e.g. cross reactivity of LAM with other substances and LAM being a poor immunogen, i.e. obtaining specific monoclonal antibodies is extremely challenging.
• Other features and advantages will become apparent from the following description.
• Figure 1 shows an electropherogram of LAM standard without concentration.
• Figure 2 shows an electropherogram of LAM with online concentration.
• Figure 3 shows an electropherogram of LAM showing online concentration effect.
• Figure 4 shows detection of LAM with capillary electrophoresis.
• Figure 5 shows electropherograms of LAM without online concentration.
• Figure 6 shows electropherograms of LAM with online concentration.
• Figure 7 shows electropherograms with a chromatographic standard.
• Figure 8 shows electropherograms of LAM, LAM in urine and urine matrix.
• Figure 9 shows an electropherogram of LAM with stacking.
• Figure 10 shows an electropherogram of 20 ppb LAM with online concentration.
• Figure 1 1 shows an electropherogram of 100 ppb LAM standard with online concentration.
• Figure 12 shows an electropherogram of 1 ppb LAM standard with online concentration.
• Figure 13 shows electropherograms of standard LAM and urine spiked with 50ppb LAM without online concentration Description of Embodiments
• Capillary electrophoresis is a family of related techniques that employ narrow-bore (20-200 ⁇ internal diameter.) capillaries, in an embodiment of the invention 30 - 80 ⁇ internal diameter capillaries, to perform high efficiency separations of both large and small molecules. These separations are facilitated by the use of high voltages which may generate electroosmotic and electrophoretic flow of buffer solutions and ionic species, respectively, within the capillary. High voltages means in the kV range e.g. 5 kV to 30kV.
• the capillaries are any typical commercially available capillaries for use in CE, e.g. fused silica capillaries.
• the capillaries are coated with a polymer e.g. polyimide or Teflon.
• the capillary has a detection window, which must be transparent.
• the polyimide is burned or scraped away from a suitable portion of the capillary to provide a detection window that is typically several millimetres long.
• Capillaries with transparent coatings are also possible, thus negating the need for scraping a detection window and improving the stability of the capillary.
• the inner surface of the capillary may be coated to reduce electroosmotic flow to very low levels and to restore the normal direction of migration of ions, i.e. anions towards the anode and cations towards the cathode.
• the present invention relates to a method of detecting the presence of a
• the method comprises the steps of obtaining a sample from the subject, inactivating the sample, e.g. by boiling, and carrying out capillary electrophoresis on the sample by subjecting it to an electric voltage across a capillary to detect the presence of lipoarabinomannan.
• the sample is concentrated in the capillary while being subjected to electric voltage.
• the presence of lipoarabinomannan in the sample is indicative of mycobacterial infection.
• the present invention comprises various embodiments relating to a method of detecting the presence of a mycobacterial infection in a subject.
• the method comprises the steps of obtaining a sample from the subject.
• the sample After the sample has been obtained it is inactivated before the sample is provided to a sample vial.
• the sample is then introduced into a capillary having an inlet and an outlet by placing the inlet of the capillary into the sample vial containing the sample.
• the sample is introduced to the capillary by a method selected from capillary action, pressure, siphoning or electrokinetics before the capillary is returned to a source vial containing an electrolyte e.g. an aqueous buffer solution and an electrode.
• the outlet of the capillary is in a destination vial containing an electrolyte e.g. an aqueous buffer solution and an electrode of opposite polarity to the electrode in the source vial.
• Capillary electrophoresis is carried out on the sample by subjecting said sample to an electric voltage across the capillary to detect the presence of lipoarabinomannan, said presence of lipoarabinomannan being indicative of said mycobacterial infection, and said sample being concentrated in the capillary while being subjected to electric voltage.
• the presence of various mycobacterial infections can be detected by the method.
• the mycobacterial infection is a M. tuberculosis infection. M.
• tuberculosis is a pathogenic bacterial species in the family Mycobacteriaceae and the causative agent of most cases of tuberculosis.
• the sample is a liquid sample particularly a biological liquid sample selected from the group of urine, exudates and cerebral spinal fluid.
• urine is easy to collect from all subjects, even children, and no skills or special preparation of the subject are required to collect the sample. Further urine is easily transportable e.g. in liquid form or the sample can be adsorbed onto a solid support, or even collected into a capillary. For analysis itself, negligible amounts of samples are sufficient.
• inactivation means ridding the sample of infective agents by killing them with heat, in particular by boiling the sample.
• the inactivation comprises the steps of boiling the sample e.g. heating the sample to a temperature in excess of 60 °C, preferably in excess of 80 °C, most suitably in excess of 90 °C, particularly 100 °C for a time period of 1 - 60 minutes, preferably 20 - 40 minutes, suitably 30 - 35 minutes.
• the sample was boiled for 30 minutes.
• the method of the present invention provides excellent resolution of molecules in the sample to be analysed on the basis of both the molecular weight and the charge of the molecules. This allows the detection of a mixture of lipoarabinomannans, which are polymers with variable molecular weights.
• the method provides 100% specificity in the detection of molecules of interest. It is, however, to be noted that the presence of LAM in biological fluids is not restricted only to infections caused by Mycobacterium tuberculosis. These molecules are also a part of the cell wall of the bacteria of the genera of Actinomycetes. The diseases caused by Actinomycetes, however, are not clinically comparable to the clinical picture of TB, e.g. actinomycosis is a disease
• the capillary electrophoresis is carried out in a capillary electrophoresis apparatus comprising a silica capillary having an internal diameter of 30 - 80 ⁇ , preferably 40 - 65 ⁇ , most suitably 50 ⁇ .
• the capillary has an effective length of 25 - 100 cm, preferably 50 - 90 cm, most suitably 86 cm.
• the presence of lipoarabinomannan is detected on an absorbance detector using UV or UV-Vis absorbance, such as e.g. a photodiode array detector, on a fluorescence detector such as e.g. a laser induced fluorescence detector, on an amperometric detector or on a conductivity detector, preferably on an absorbance detector.
• UV or UV-Vis absorbance such as e.g. a photodiode array detector
• a fluorescence detector such as e.g. a laser induced fluorescence detector
• amperometric detector e.g. a laser induced fluorescence detector
• amperometric detector e.g. a laser induced fluorescence detector
• the term 'concentrating online' and variations thereof means that sample compounds are separated into zones in the capillary. Thereafter the zones are focused by voltage, electrolyte composition and pH to more narrowed zones (the widths of the zones are in the range of about 200 milliseconds to 500 milliseconds).
• the process is carried out in the capillary during analysis before the zones move to a detector which is placed near to the outlet end of the capillary. Detection is carried out in in-line mode.
• the signal to noise ratio of the standard is 2 calculated with the intensity of the standard and the background value of the detector.
• the voltage is alternated, in an embodiment it remains at a fixed value throughout the analysis. In a further embodiment the voltage remains fixed and current and resistance are varied. In a still further embodiment the voltage applied across the capillary is in the range of 5 - 30 kV, preferably 10 - 25 kV, particularly 16 - 20 kV.
• a further embodiment relates to a method for use in the diagnosis of an infectious disease.
• the detection of LAM is indicative of the presence of an infectious disease.
• a method for use in the diagnosis of an infectious disease selected from the group of pulmonary tuberculosis, extrapulmonary tuberculosis, tuberculosis meningitis and tuberculosis pleurisy is disclosed
• a method to diagnose tuberculosis in a subject comprising
• the sample is a liquid sample selected from the group of urine, exudates and cerebral spinal fluid.
• CE capillary electrophoresis
• PDA UV absorbance detector
• CE Capillary Electrophoresis
• the CE methods were tested with standards, like monosaccharides, organic acids, oligosaccharides, and various LAM standards, which were isolated/purified from human matrices.
• the samples were LAM Japan, LAM Colorado and LAM Nicola. The suitability of the analyses were studied with human urine (pooled urine) spiked with the LAM standards.
• the electrolyte solution was prepared by weighting the chemicals (purity more than 98%) to non-ionic, purified water.
• the accurate pH of the solution was adjusted by using a
• Injection volume of sample or standard was 5.3 nL (containing 50-265 pg of the standard).
• Sample introduction was made with pressure-assisted injection at 0.5 p.s.i. for 4 s.
• the separation voltage during the analysis was either +16 kV, +20 kV, or +25 kV. No polarity changes were made during the separation (the applied field was uniform from the inlet to the outlet of the capillary).
• the UV detection was at either 200, 240, 255, 270 or 280 nm for LAM.
• the electrolyte solution was prepared by weighting the chemicals (purity more than 98%) to non-ionic, purified water.
• the accurate pH of the solution was adjusted by using a combination electrode and a pH meter. Calibration of the pH range was done with standards of pH 7.00, 10.01, and 12.00.
• the final electrolyte solution was mixed in ultra-sonication bath for 20 min to get a homogenous solution.
• the electrolyte solution used for the separations was kept in an ultra-sonic bath for 20 minutes before use.
• the untreated fused-silica capillaries of 86 cm (effective length) and 96 cm (total length) with 50 ⁇ internal diameter were used.
• the capillaries were conditioned by rinsing with 0.1 M sodium hydroxide, ultra-high purity water, and the electrolyte solution for 30 min each. Between each run, the capillary was flushed with electrolyte solution for 5 min. Both the capillary and samples were thermostatted at 15 °C.
• Various injections were used: Examples 0.7 p.s.i. for 19 s; 1.5 p.s.i. for 30 s and 40 s.
• the analysis temperature and the voltage were 30 °C and 20 kV, respectively.
• 2,3-PDC is 2,3-pyrrolidine dicarboxylic acid
• OFM-OH is Ion Select OFM Hydroxide Concentrate [WAT049387]
• CaCl 2 is calcium chloride.
• the electrolyte contained 0.3362 g of 2,3-PDC, 0.0220 g of CaCl 2 *2H 2 0, and either 150 ⁇ or 300 ⁇ OFM-OH " .
• the pH of the solution was adjusted with ammonia by using a combination electrode and a pH meter. Calibration of the pH range was done with standards of pH 7.00, 10.01, and 12.00.
• the final electrolyte solution was mixed in ultra-sonication bath for 20 min to get a homogenous solution.
• the volume of sample and standards in analyses were 19.79 nL, meaning the amount of 100- 1000 pg sample (depends on the original concentration of LAM in the sample). Separation voltage was -25 kV. The samples were kept at 15 °C during the separation.
• results of the capillary electrophoresis experiments are shown in the electropherograms provided.
• the electropherograms show the effects of concentration, electrolyte composition and need for sample cleaning/separation before quantification of LAM.
• the data provide results on concentration calibration.
• Figure 1 shows an electropherogram of lOOpg/nL LAM standard without online concentration. Electrolyte 1 , described above, was used with a separation voltage of 1 OkV. Injection of the sample was carried out at a pressure of 0.5 psi for 30s. The peaks are wide and no focussing (stacking) has taken place.
• Figure 2 shows an electropherogram of lOOpg/nL LAM with online concentration. Electrolyte 1 was used and a voltage of 1 OkV was applied. Injection of the sample was carried out at a pressure of 2 psi for 30s at. The present peaks are wide because of the high concentration in stacking.
• Figure 3 shows an electropherogram of LAM at 1.3 ng/10 nL concentration. This figure shows the suitability of the method for detecting low LAM quantities and to show the effect of online concentration under the applied electric field.
• Figure 4 shows detection of LAM with online concentration effect. In Figure 4 the peaks at 3.0 min and 4.2 min belong to LAM. The upper graph shows a 100 ppb LAM solution spiked with a 10,000 ppb solution. The lower graph shows a 10 ppb LAM solution.
• Capillary Electrophoresis was carried out using electrolyte 4 at a voltage of 25kV. Injection time was 10 s at a pressure of 2 psi.
• Figure 5 shows electropherograms of LAM without online concentration with electrolyte 4. It is clear from Figure 5 that LAM at 1 ppb cannot be seen in the electropherograms. Thus, online concentration in the capillary is needed.
• Figure 6 shows Electropherograms of LAM with online concentration.
• the second compound from LAM at 4.2 min is detected linearly at 1 ppb to 100 ppb level.
• the first LAM peak is seen at 20 ppb quantities and more.
• FIG. 1 Electropherograms in electrolyte solution 4 with LAM in concentrations of 10, 1 and 0.5 ppm. Also shown is an electropherogram of the chromatographic standard (formic acid).
• FIG 8 shows Electropherograms of LAM Colorado (black), LAM in urine (blue) and urine matrix (pink). The urine sample was not cleaned for CE analysis. Electrolyte 4 was used. Online concentration in the capillary gives good signals for 100 ppb LAM.
• Figure 10 shows an electropherogram of 20 ppb LAM in Electrolyte 4. Detection above limit of determination. This means that the LAM concentration was above the LOQ value needed for quantification i.e. meaning the peak height was reliable. Peak signal was two times higher than the height of the background signal. Online concentration effect was used.
• Figure 11 shows an electropherogram of 100 ppb LAM standard in electrolyte 4. Sensitivity is good. Online concentration was used.
• Figure 12 shows an electropherogram of 1 ppb LAM standard in electrolyte 4. Online concentration effect was used to calculate the limit of detection at a signal-to-ratio value of 2.
• Figure 13 shows two electropherograms of LAM Colorado. Standard LAM (above, black) and in urine (not purified, LAM Colorado spiked at 50 ppb, blue). No online concentration was carried out.
• the present invention finds application in diagnostics and in healthcare in general, and in veterinary science.
• the method of the present invention can be used to detect the presence of LAM in samples from any mammalian subject, the presence of LAM being indicative of a mycobacterial infection.

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• 2015
  • 2015-02-27 FI FI20155136A patent/FI125928B/fi not_active IP Right Cessation
• 2016
  • 2016-02-23 WO PCT/FI2016/050115 patent/WO2016135384A1/en active Application Filing

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