NL2015553B1 - A method for detecting a marker for active tuberculosis. - Google Patents
A method for detecting a marker for active tuberculosis. Download PDFInfo
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- NL2015553B1 NL2015553B1 NL2015553A NL2015553A NL2015553B1 NL 2015553 B1 NL2015553 B1 NL 2015553B1 NL 2015553 A NL2015553 A NL 2015553A NL 2015553 A NL2015553 A NL 2015553A NL 2015553 B1 NL2015553 B1 NL 2015553B1
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Abstract
The present invention relates to a method of detecting a marker for active tuberculosis, a system for carrying out a method of detecting a marker for active tuberculosis, a method for pre-treating a sample stream 5 from a human or animal suspected of having active tuberculosis, a system for pre-treating a sample stream from a human or animal suspected of having active tuberculosis and kits for performing said methods.
Description
A method for detecting a marker for active tuberculosis
The present invention relates to a method of detecting a marker for active tuberculosis, a system for carrying out a method of detecting a marker for active tuberculosis, a method for pre-treating a sample stream from a human or animal suspected of having active tuberculosis, a system for pre-treating a sample stream from a human or animal suspected of having active tuberculosis and kits for performing said methods.
Introduction
Mycobacterium tuberculosis is a pathogenic bacterial species in the family Mycobacteriaceae and the causative agent of most cases of tuberculosis (TB).
Nine million people fell ill with TB in 2013, including 1.5 million cases among people with HIV. In 2013, 1.5 million people died from TB, including 360000 among people who were HIV-positive. TB is one of the top three killers of women worldwide, 510000 women died from TB in 2013. Of the TB deaths among HIV-positive people, 50% were among women. At least 550000 children became ill with TB and an estimated 80 000 children who were HIVnegative died of TB in 2013. Globally in 2013, an estimated 480000 people developed multidrug-resistant TB (MDR-TB) and there were an estimated 210000 deaths from MDR-TB. At least one case of extensively drug-resistant TB (XDR-TB) has been reported by 100 countries by the end of 2013. On average, an estimated 9% of MDR-TB cases have XDR-TB. A reliable and fast way of diagnosing tuberculosis is therefore of utmost importance.
Several methods of diagnosing tuberculosis have been developed, but all methods have their disadvantages. Diagnosing active tuberculosis based merely on signs and symptoms is difficult, as is diagnosing the disease in those who are immunosuppressed. The TB skin test (also called the Mantoux tuberculin skin test), TB blood tests (also called interferon-gamma release assays or IGRAs), and chest radiography (X-ray), and tests on the presence of acid-fast-bacilli (AFB) on a sputum smear, indicate some of the infected individuals in days. A definitive diagnosis of TB is made by identifying M. tuberculosis in a clinical sample (e.g. sputum, pus, or a tissue biopsy). However, the difficult culture process for this slow-growing organism can take two to six weeks for blood or sputum culture.
Humans or animals infected with the M. tuberculosis normally produce antibodies directed against the Mycobacterium. Presence of these antibodies in a sample taken from infected individuals indicates the infection. WO 2005/116654 describes a method based on this principle and discloses detecting a marker for active tuberculosis. The method of WO 2005/116654 involves obtaining first, second and third samples from a subject suspected of having active tuberculosis, diluting the first sample and exposing part of it to an immobilized mycolic acid antigen in a test vessel and part of it to an immobilized mycolic antigen in a control vessel. The second sample is exposed to mycolic acid antigen-containing liposomes and the third sample is exposed to liposomes not containing mycolic acid antigens. The second sample is added to the test vessel and the third to the control vessel and binding of antibodies to the mycolic acid and antigen in both the test and control vessel is detected. The degree of binding between the test and control vessels is compared and lesser binding in the test vessel is an indicator of the presence of antibodies to the mycolic acid antigen. A further development was described in WO 2013/186679, which discloses a method of detecting antigen specific biomarker antibodies for the diagnosis of active tuberculosis, the method including the steps of: providing a lipid antigen-presenting liposomal composition comprising liposomes comprising a sterol-modified lipid and a purified mycobacterial lipid cell wall component or analogue or derivative thereof; immobilizing the liposomes to produce immobilized mycolic acid antigens comprising the purified mycobacterial lipid cell wall component or analogue or derivative thereof; obtaining a first, a second and a third sample from a human or animal suspected of having active tuberculosis, wherein each sample may contain antibodies to the antigen, the first sample having a lower concentration by dilution than the second and third samples; exposing part of the first sample to the immobilized mycolic acid antigens in a test vessel; exposing part of the first sample to the immobilized mycolic acid antigens in a control vessel; exposing the second sample to the lipid antigen-presenting liposomal composition provided in the first step ; exposing the third sample to liposomes not containing mycolic acid antigen; adding the second sample, after exposure to the mycolic acid antigen-containing liposomal composition provided in the first step, to the test vessel; adding the third sample, after exposure to the liposomes not containing mycolic acid, to the control vessel; detecting binding of antibodies to the mycolic acid antigen in both the test and control vessels in real time; and comparing the degree or extent of binding between the test and the control vessels, the weaker binding in the test vessel being an indicator of the presence of antibodies to the mycolic acid antigen in the sample that indicates active tuberculosis in the human or animal from which the sample originated.
The methods described in WO 2005/116654 and WO 2013/186679 provide reliable methods for diagnosis of tuberculosis, but have several disadvantages. First, the methods described in WO 2005/116654 and WO 2013/186679 require provision of several samples from the same subject. Second, these methods require several dilution steps and several transfer steps of samples to different unconnected compartments, requiring a complicated and large set of instrumental parts. Third, these methods involve separate incubation steps of the sample and dilutions thereof and consequently several separate measurements. For these reasons the methods of WO 2005/116654 and WO 2013/186679 are complicated and time consuming. In fact, the total time from obtaining a sample from a subject suspected of having active tuberculosis to determining whether or not an individual is infected with active tuberculosis using the methods disclosed in WO 2005/116654 and WO 2013/186679 takes an estimated time of at least 2 hours per sample. Furthermore, because the above methods require a complicated and large set of instrumental parts, said methods may be useful in a hospital environment, but not in areas which are deprived from hospitals and well developed healthcare, for instance in developing countries. It is in particular in these countries where tuberculosis is most prevalent and where reliable and fast diagnosis is most desired.
Therefore the invention aims to provide a method for determining whether or not an individual is infected with active tuberculosis (e.g. pulmonary or extra-pulmonary tuberculosis) which is fast and reliable, and which can be carried out outside of a professional medical environment, i.e. outside of a hospital, for instance on the streets.
Summary of the invention
In a first aspect the invention relates to a method of detecting a marker for active tuberculosis as defined in claim 1.
In a second aspect the invention relates to a method for pre-treating a sample from a human or animal suspected of having active tuberculosis as defined in claim 7.
In a third aspect the invention relates to a system for carrying out a method of detecting a marker for active tuberculosis as defined in claim 16 and claim 22.
In a fourth aspect the invention relates to a system for pre-treating a sample from a human or animal suspected of having active tuberculosis as defined in claims 19 and 21.
In a fourth aspect the invention relates to a kit for use in diagnosing tuberculosis as defined in claims 26 and 27 .
Description of the figures
Fig. 1 describes an exemplary embodiment of the system according to claim 19.
Description of the invention
The present invention relates in one aspect to a detection method for detecting antibodies against mycolic acid derivatives, such as cord factors antibodies or mycolic acid (MA) antibodies in a sample.
This method comprises the steps of: i) providing a sample from a human or animal suspected of having active tuberculosis; ii) contacting said sample to a sterol lipid; iii) obtaining at least two fractions of said sample either before or after exposing it to said sterol lipid; iv) exposing the first of said fractions to a substrate carrying an immobilised mycolic acid derived antigen; v) exposing the second of said fractions to a substrate not carrying an immobilised mycolic acid derived antigen; vi) exposing at least part of the sample fraction exposed in step iv) to a test substrate carrying an immobilised mycolic acid derived antigen and exposing at least part of the sample fraction exposed in step v) to a control substrate carrying an immobilised mycolic acid derived antigen; vii) detecting binding of antibodies to the antigen of step vi) in an end-point assay; and viii) comparing the degree or extent of antibody binding between the test and control substrates, any observed lesser binding to the test substrate being an indicator of the presence of antibodies to the antigen in the sample that indicates active tuberculosis in the human or animal from which the sample originated.
The pre-treatment steps i)-v) do no necessarily have to be followed directly by the detection steps vi-viii). For instance the samples fractions obtained after step v) can be stored until further use or for transport to the detection substrate. This way the practiser has great flexibility in planning his test. For instance, the fact that steps i) -iv) do not necessarily have to be followed by steps vi)-viii) allows pre-treated samples to be collected, so that they can be tested all at once on one substrate material, which improves reliability and reproducibility. This also makes it easier to test samples from multiple patients under the same conditions.
Therefore, in another aspect the invention also relates to a method for pre-treating a sample from a human or animal suspected of having active tuberculosis for detection: i) providing a sample from a human or animal suspected of having active tuberculosis; ii) exposing said sample to a sterol lipid; iii) obtaining at least two fractions of said sample either before or after exposing it to said sterol lipid; iv) exposing the first of said fractions to a substrate carrying an immobilised mycolic acid derived antigen; v) exposing the second of said fractions to a substrate not carrying an immobilised mycolic acid derived antigen; wherein after exposure in steps iv) and v) at least part of the sample fractions are stored for further use.
The detection method of the invention enables samples, preferably blood samples to be analyzed in a reliable and fast way. The subsequent pre-incubation steps i)-v) of the sample with a sterol lipid, followed by subjecting a fraction of the sample to a pre-incubation step with a mycolic acid derived antigen to obtain a test sample and another fraction of the sample to a preincubation without a mycolic acid derived antigen to obtain a control sample render the sample suitable for direct application in the eventual end-point detection step which entails binding of mycolic acid antigens in the pretreated sample to a substrate carrying a mycolic acid. The substrate therefore does not require pre-incubation or pretreatment with a dilution of the sample. Accordingly, the the several dilution and transfer steps required in methods of the prior art are not required.
Furthermore the detection method of the invention makes it possible to determine whether or not an individual is infected with active tuberculosis (e.g. pulmonary or extra-pulmonary tuberculosis) in fast and reliable way, allowing detection of tuberculosis in less than 15 to 25 minutes. This is in particular due to the pretreatment steps i)-v) of claims 1 and 7. Furthermore the method can be carried out outside of a professional medical environment. In particular the invention provides a quick method for determining whether or not an individual is infected with active tuberculosis as a point of care test i.e. as a test at or near the site of patient care. The test of the present invention provides simple medical tests which can be performed at the bedside.
Furthermore, because of the pre-treatment steps i)-v) taking one sample from a subject is sufficient, which sample can be used directly for detection without further treatment.
The principle of the invention will now be explained in more detail with reference to the steps of method claims 1 and 7.
Step i
The detection method of the invention is based on diagnostic testing of samples, in particular blood samples of a human or animal suspected of having active tuberculosis. For this purpose in step i) of a sample from a human or animal suspected of having active tuberculosis is provided. The sample is preferably a whole blood sample. The sample may be obtained by any regular means of obtaining blood from a subject. In order to be used in the methods of the invention samples may be used that have been collected at an earlier stage, stored until use under suitable conditions and provided at a suitable moment. Alternatively, a sample may be used in the detection method of the invention on the spot, i.e. as a point of care test. Another alternative is that the sample is pretreated on the spot in accordance with abovementioned steps i)-v), and that the resulting pre-treated samples are stored until further use and/or transferred to another location where detection takes place.
In case the sample is a whole blood sample, the sample may, depending on the way of detection of binding of antibodies to the antigen, be filtered or separated to plasma or serum before step ii) . In case the detection is carried out by means detecting mass differences, a filtering step may be preferred to filter out high mass components such as blood cells. In case binding is detected by fluorescence, one may choose not to filter the sample. This would even shorten the time required for pretreatment and diagnosis.
The sample is preferably a blood derived sample. The sample may be a whole blood sample, a plasma sample or a serum sample. Blood serum is blood plasma without clotting factors and is preferred as plasma. The word "plasma" in this application may therefore as well refer to (blood) serum. The choice of blood plasma or blood serum depends on whether the device of the invention is designed to separate the whole blood into plasma or serum. Serum is preferred because it contains less different materials than blood plasma which may lead to aspecific interactions or unwanted biological activity. In addition, serum may have a lower viscosity than blood plasma. Using serum therefore may circumvent the need for diluting a sample, which saves time and materials.
About 55% of whole blood consists of plasma/serum. If a whole blood sample is not filtered perfectly or if the patient's physical situation necessitates it, it may be desired to dilute the whole blood sample or plasma or serum. The words plasma or serum in this application may therefore also refer to diluted plasma or serum. A dilution of the blood or plasma may be implemented in the methods of the invention. A dilution of 10 x to 250 x is preferred, in particular when the end-point assay applied is an immunogold filtration assay, such as a dot immunogold filtration assay. Depending on the viscosity of the sample, such dilution may take place before the separating the plasma from the blood step or after the separating step or alternative to the separating step. For instance the dilution step may take place after the separating step but before step iv) or v) or after step v)/vi) and before step vii).
Dilution may be performed with any suitable diluent, for example a PBS based buffer. Such buffer may for example be a PBS/AE buffer comprising NaCl, KC1, KH2PO4, Na2HPC>4 and EDTA in water at physiological pH. Such buffer may be a PBS based buffer consisting of 8.0 g NaCl, 0.2 g KC1, 0.2 g KH2PO4, and 1.05 g Na2HPC>4 per liter of double distilled, deionized water containing 1 mM EDTA and 0.025% (m/v) sodium azide which is adjusted to pH 7.4.
The whole blood sample or plasma or serum may be further diluted with agents that prevent blood clotting, such as EDTA, heparine or citrate.
Optionally a detergent may be added in low concentration to the blood/plasma/serum to avoid sticking of components to walls of tubings, vessels or containers.
Step ii
In step ii, the sample is exposed to, i.e. contacted with a sterol lipid.
Although the inventor does not wish to be bound by any theory, it is assumed that the sterol lipid scavenges away the anti-cholesterol antibodies from the sample that would otherwise cross react with the mycolic acid antigens on the substrate and lead to false positive diagnosis of tuberculosis .
The exposure time of the sample to the sterol lipid is preferably less than 10 minutes, such as 2 to 8, 3 to 7, 4 to 6 or about 5 minutes. The exposure time depends on the way the sample is brought into contact with the sterol lipid.
One exemplary way of exposing the sample to the sterol lipid is to lead the sample into a long spiral channel and pass it along the length of the channel, which is pre-coated with a sterol lipid, which is preferably cholesterol. At the end of the channel the lipid sterol exposed sample may pass a means for dividing the sample stream such as a passive valved branch point that leads the divided sample streams to the next substrate carrying an immobilised mycolic acid derived antigen of step v) and the substrate not carrying an immobilised mycolic acid derived antigen of step vi), i.e. a container comprising a substrate carrying an immobilised mycolic acid derived antigen, and a container comprising a substrate not carrying an immobilised mycolic acid derived antigen respectively.
An alternative exemplary way of exposing the sample to the sterol lipid is to inject it into a container coated with cholesterol, followed by incubation therein for less than 10 minutes.
Another alternative exemplary way is to expose the sample to the sterol lipid, wherein the sterol lipid is contained in a compartment of a column. In such a compartment the sterol lipid is preferably coated on beads .
Whole blood/plasma/serum is rich in hundreds different kind of molecules with hydrophilic to hydrophobic properties. Therefore substrate material will be used that is inert for non-specific binding of molecules of this sample. In this context the substrate should be understood to be a support material.
Steps iv and v
In the next step a fraction of the sterol lipid exposed sample is exposed to a substrate carrying an immobilised mycolic acid derived antigen (step iv) ) and another fraction of the sterol lipid exposed sample to a substrate not carrying an immobilised mycolic acid derived antigen (step v)).
To obtain at least two fractions, at a certain moment in the process the blood/plasma stream has to be divided (step iii). For sake of convenience and to optimize reliability of the method, the stream may be divided after exposure to the sterol lipid, i.e. that the sterol lipid exposed sample is divided into at least two fractions to provide a test stream and a control stream. It is preferred that the sample is divided in two equal or substantially equal fractions, because this enables a simple comparison of both fractions without the need for correction calculations. Dividing the sample stream may be carried out by any means for dividing the sample stream such as a passive valved branch point.
Alternatively the sample stream (which is filtered to plasma or serum) is divided into at least two fractions after the filtering step, but before exposure to the sterol lipid. Alternatively the blood stream is divided before filtering. This is less preferred because in this case two filters would be required.
In case columns are used the stream is in this case divided before exposure to the sterol lipid in step ii), so that steps ii) and iv) can take place in the same column and step ii) and v) can take place in another column .
The substrate carrying an immobilised mycolic acid derived antigen and the substrate not carrying an immobilised mycolic acid derived antigen of steps iv) and v) are preferably of the same material.
In addition, since sterol lipids, (e.g. cholesterol) and mycolic acid derivatives are both hydrophobic, the substrates for exposure in steps iv) and v) may be of the same material as the substrate that may be used to in step ii) . It will be understood that suitable substrate material is inert for non-specific binding of molecules of this sample.
The exposure time of the sample to the sterol lipid is preferably less than 10 minutes, such as 2 to 8, 3 to 7, 4 to 6 or about 5 minutes.
One exemplary way of exposing the sample to the substrates of step iv) and v) is to lead the divided sample streams into long spiral channels, preferably implemented in micro-chips, pre-coated either with a mycolic acid derivative (step iv) or without a mycolic acid derivative (step v) and pass it along the length of these channels.
An alternative exemplary way of exposing the divided lipid sterol exposed samples to a substrate coated either with a mycolic acid derivative or without a mycolic acid derivative is to inject the a first stream into a container comprising a substrate carrying an immobilised mycolic acid derived antigen (step iv), and a second sample stream into a container comprising a second substrate not carrying an immobilised mycolic acid derived antigen (step v) and incubate the samples for less than 10 minutes, such as 2 to 8, 3 to 7, 4 to 6 or about 5 minutes .
Another alternative way is that steps iv) and v) take place in a column. The stream is in this case divided before exposure to the sterol lipid in step ii) , so that step ii) and iv) can take place in one column and step ii) and v) can take place in another column.
Step vi
In this step the divided sterol lipid exposed sample streams, either exposed to mycolic acid derivatives in step iv(test stream) or not in step v (control stream) are passed to further substrates, test and control substrates respectively, carrying mycolic acid derivatives, preferably the same derivatives as in step iv.
The test and control substrates may be any substrate which can carry an immobilized mycolic acid derivative antigen and from which binding between antibodies contained in the sample to said antigens can be detected.
In a preferred embodiment detection takes place by means of an immunogold filtration assay. In such an assay the test and control substrate are a microporous membrane, preferably a nitrocellulose membrane, which is coated an immobilised mycolic acid derived antigen.
The test substrate and the control substrate may be separate entities. Alternatively the test substrate and the control substrate may be realized as different positions on one substrate entity. For instance, in case of a microporous membrane the test substrate and the control substrate may be formed by a first microporous (e.g. nitrocellulose) membrane and a second microporous (e.g. nitrocellulose) membrane. Alternatively the test substrate and control substrate are contained on one membrane. In such a case there is a test area where antibody/antigen interaction can take place (e.g. a spot) on one position of the membrane and a control area where antibody/antigen interaction can take place on another position of the membrane.
Steps vii and viii
In step vii) the binding of antibodies to the immobilised mycolic acid antigens is detected in an endpoint assay. At least part of the obtained pre-treated fractions is subjected to said end-point detection assay. It is possible to use the whole fractions for the endpoint assay. Alternatively part of the fractions are used to detect binding of antibodies to the immobilised mycolic acid antigens in the end-point assay and another part is stored for further testing depending on the results of the first part of the fractions.
The term "end-point assay" is to be understood as an assay wherein the outcome of interest is the end result after a fixed assay incubation period, in contrast to a so-called real-time assay. In the context of the detection method of the invention that means that the treated samples obtained after steps iv) and v) are taken and subjected to a test that provides an indication of the amount of antibodies that are present in the treated samples after steps iv) and v) .
An end-point detection assay may for instance detect changes to levels of color, fluorescence, absorbance or luminescence at the end of a test. In end-point assays detection may suitably be performed by means of techniques such as photospectroscopy, fluorescence microscopy, chemoluminescence or electrochemiluminescence detection techniques. This may involve use of for instance spectrophotometric/colorimetric plate readers, fluorescence plate readers or chemiluminescence readers.
Suitable end-point assays may involve enzyme-linked immunosorbent assay (ELISA), Western blotting, radioactive labelling assay, photospectrometric assay, immunofluorescence, immunoprecipitation, immunocytochemistry, immunohistochemistry etc. ELISA for instance involves at least one antibody with specificity for a particular antigen. In the context of the the invention the antigen is a mycolic acid derived antigen and the antibody is an antibody against mycolic acid derivative antigens.
In an end-point assay interaction of antibodies with the mycolic acid derived antigens may be carried out using secondary antibodies that bind the heavy chain of the antibodies against the mycolic acid derivatives. Many suitable secondary antibodies are commercially available. The secondary antibody may be coupled to beads, for instance gold beads, or associated with liposomes. Examples of secondary antibodies may be protein A or G, possibly conjugated with an enzyme that enables detection. A particular suitable technique or detecting the binding of antibodies to the immobilised mycolic acid antigens in step vii) is the so-called immunogold filtration assay (IGFA), and in particular the dot immunogold filtration assay (DIGFA).
Immunogold filtration assays are methods combining ELISA and immunogold technique and are methods in which a sample to be assayed is allowed to filtrate through a microporous membrane, preferably a nitrocellulose membrane, and is captured by a capture probe coated on the membrane. A colloidal gold labelled probe is allowed to filtrate through the microporous membrane in the same manner. By using a microporous membrane as the carrier for the capture probe and employing the capillary action and permeability of the membrane antigens and antibodies can easily react and may conveniently be subjected to optional washing and/or blocking steps. When the colloidal gold labelled probe binds to the capture probe the colloidal gold particles aggregate and a red dot appears which is visible with the naked eye.
Immunogold filtration assays are simple and rapid detection methods because no instruments are required except a membrane and the reagents and the results can be observed by the naked eye within a few minutes.
In an immunogold filtration assay the microporous membrane may be for example a nitrocellulose membrane, a cellulose acetate membrane or a PVDF membrane with a suitable pore diameter. Preferably nitrocellulose is used. A suitable pore diameter is 0,2 to 5 pm.
In the present method antibodies contained in a sample are detected by means of an end-point assay. In case the end-point assay is an immunogold filtration assay, the substrate mentioned in steps vi) and viii) of the detection method of the invention, is a microporous membrane, preferably a nitrocellulose membrane, which is coated an immobilised mycolic acid derived antigen. After immobilizing the mycolic acid derived antigen onto the microporous membrane, the sample fractions pre-treated in steps i) to v) can be applied to the membrane. After addition of the sample fractions and reaction of the immobilized mycolic acid derived antigens with the antibodies contained in the samples on the membrane, colloidal gold-labeled second antibodies are added onto the membrane to have gold particle aggregation in the antigen-antibody reaction place. In case of aggregation visible red or brown spots are formed. The intensity of the spot is proportional to the amount of reactions between antigen and antibody, i.e. to the amount of antibodies in the pre-treated sample. In case the control substrate shows a more intense signal than the test substrate this is indicative for active tuberculosis in the person from which the sample was derived. On the other hand, in case the control substrate shows no (or only insignificant) difference in color intensity signal compared the test substrate this indicates that the human or animal from which the sample was derived does not have active tuberculosis.
Between the various steps of an immunogold filtration assay the membrane may be washed with a suitable buffer, for example a PBS based buffer. Such buffer may for example be a PBS/AE buffer comprising NaCl, KC1, KH2PO4, Na2HPC>4 and EDTA in water at physiological pH. Such buffer may be a PBS based buffer consisting of 8.0 g NaCl, 0.2 g KC1, 0.2 g KH2PO4, and 1.05 g Na2HPC>4 per liter of double distilled, deionized water containing 1 mM EDTA and 0.025% (m/v) sodium azide which is adjusted to pH 7.4.
In case DIGFA is used, the microporous membrane may be coated with said mycolic acid derived antigen in a dot wise manner. In a DIGFA assay the samples pre-treated in steps i) to vi) are applied to the membrane in the form of dots. Also the colloidal gold-labeled second antibodies are added in the form of dots. In such an embodiment, the control and test substrate are preferably the same microporous membrane with with dots of mycolic acid derived antigens immobilised thereon. The detection method of the invention makes it possible to detect both the control and test fractions on the same membrane. This improves reliability of the test and is cost effective. A DIGFA assay is particularly preferred because at different spots on several membranes various antigens deriving from various mycobacterial strains may be immobilized. This way it becomes possible to provide information on which mycobacterial strain a patient is infected with. Another advantage of using DIGFA is that samples derived from different persons suspected of having active tuberculosis can be compared in one test, because DIGFA enables fast and reliable detection of antibody-antigen interaction in an unlimited amount of spots, depending on the size of the membrane .
The detection of binding of antibodies and/or other material to the mycolic acid antigen, for instance the red staining in case a DIGFA assay is used as a detection method, may be carried out in an automated device. Various automated devices will be known to the person skilled in the art and the skilled person will be able to select suitable software means to make the comparison of step viii) of the degree or extent of binding between the test and control substrates, wherein any observed lesser binding to the test substrate is an indicator of the presence of antibodies to the antigen in the samples that relates to active tuberculosis in the human or animal from which the samples originated. In this context it should be understood that lesser can be interpreted qualitatively and quantitatively, i.e. lesser binding may be interpreted as having less binding events as well as having weaker bindings .
The detection of binding of antibodies and/or other material to the mycolic acid antigen may be performed by a visual detection technique or any other suitable detection technique.In a particular preferred embodiment, detection by means of the end-point assay takes place visually, preferably with the naked eye. This has the advantage of easy detection without the need for expensive and complicated detection technology.
In case DIGFA is used binding of antibody antibodies and/or other material to the mycolic acid antigen may be assessed by means of the naked eye. A visual signal, e.g. the red staining in case a DIGFA assay is applied as end-point assay, may also be detected with help of a mobile app, i.e. a computer program designed to run on mobile devices such as tablet computers or smart phones. For instance, an app can be used that is designed to compare the binding signal of the test and control substrate and which indicates whether the human or animal from which the sample originated has active tuberculosis.
System
In another aspect the invention relates to a system or device, preferably a portable system or device, for carrying out a method of detecting a marker for active tuberculosis. This system can be subdivided in 1) a system or device for pre-treating a sample stream from a human or animal suspected of having active tuberculosis in order to make the sample suitable for detection with the method of the invention; and 2) a detection means to enable detection of interaction of antibodies contained in the pre-treated sample.
In one embodiment the system comprises at least one container comprising a sterol lipid arranged and configured to receive a sample stream from a human or animal suspected of having active tuberculosis; means for dividing said sample stream into at least a first and a second sample stream; said means either connected upstream or downstream of said at least one container comprising a sterol lipid; a further container for receiving said first sample stream in downstream connection with a said container comprising a sterol lipid, and comprising a first substrate carrying an immobilised mycolic acid derived antigen; a still further container for receiving the second sample stream in parallel arrangement to said further container and in downstream connection with a said container comprising a sterol lipid; and comprising a second substrate not carrying an immobilised mycolic acid derived antigen, and a first detection substrate carrying an immobilised mycolic acid derived antigen to receive at least part of the first sample stream and a second detection substrate carrying an immobilised mycolic acid derived antigen to receive at least part of the second sample stream.
In a particular embodiment the system for carrying out a method of detecting a marker for active tuberculosis comprises at least one container comprising a sterol lipid arranged and configured to receive a sample stream from a human or animal suspected of having active tuberculosis; means for dividing said sample stream into at least a first and a second sample stream; said means either connected upstream or downstream of said at least one container comprising a sterol lipid; a further container for receiving said first sample stream in downstream connection with a said container comprising a sterol lipid, and comprising a first substrate carrying an immobilised mycolic acid derived antigen; a still further container for receiving the second sample stream in parallel arrangement to said further container and in downstream connection with a said container comprising a sterol lipid; and comprising a second substrate not carrying an immobilised mycolic acid derived antigen, and a first detection substrate carrying an immobilised mycolic acid derived antigen to receive at least part of the first sample stream from said further container and a second detection substrate carrying an immobilised mycolic acid derived antigen to receive at least part of the second sample stream from said still further container, wherein the first and second detection substrate are a microporous membrane.
The first and second detection substrates are preferably a microporous membrane, preferably a nitrocellulose membrane. The first and second detection substrates may in this case have separate spots, i.e. first and second detection spots, on the same membrane. This way one and the same membrane can be used for detection of antibody/antigen interaction. This increases reliability of the detection and makes it easier to compare the interaction detected on the test and control substrate. Moreover, this enables multiple samples to be tested at the same time.
The detection substrates do not have to be in physical connection with the other components of the system. For instance the samples derived after passing said further and still further containers may be stored until further use or for transport to the detection substrate. This way the practicer has great flexibility in planning his test. For instance, this allows pre-treated samples from a number of subjects to be collected, so that they can be tested all at once on one substrate material. This also makes it easier to test samples from multiple patients under the same conditions.
Therefore, in another aspect the invention relates to a system or device, preferably a portable system or device, for pretreating a sample stream from a human or animal suspected of having active tuberculosis in order to make the sample suitable for detection in a detection method, comprising at least one container comprising a sterol lipid arranged and configured to receive a sample stream from a human or animal suspected of having active tuberculosis; means for dividing said sample stream into at least a first and a second sample stream; said means either connected upstream or downstream of said at least one container comprising a sterol lipid; a further container for receiving said first sample stream in downstream connection with a said container comprising a sterol lipid, and comprising a first substrate carrying an immobilised mycolic acid derived antigen; a still further container for receiving the second sample stream in parallel arrangement to said further container and in downstream connection with a said container comprising a sterol lipid; and comprising a second substrate not carrying an immobilised mycolic acid derived antigen, and a means for storage or transport of the treated samples. This or these means do not have to be in physical connection with the containers comprising said first and second substrates respectively and are located downstream of these containers when the system is in use in order to receive the pre-treated sample fractions.
In one embodiment the system or device for pretreating a sample stream from a human or animal suspected of having active tuberculosis in order to make the sample suitable for detection with the method of the invention may comprise two columns. Each column comprises two compartments or containers. An upper compartment or container comprises a substrate coated with a sterol lipid, for instance beads coated with cholesterol. A lower compartment of one of the columns comprises a first substrate carrying an immobilised mycolic acid derived antigen and a lower compartment of another column comprises a substrate not carrying an immobilised mycolic acid derived antigen.
In this respect the invention also relates to a system for pre-treating a sample stream derived from a human or animal suspected of having active tuberculosis, which comprises means for dividing said sample stream into at least a first and a second sample stream; at least a first and a second column, configured to receive said first and a second sample stream respectively; said first column comprising a compartment comprising a sterol lipid and a further compartment comprising a first substrate carrying an immobilised mycolic acid derived antigen downstream of said compartment comprising a sterol lipid; and said second column comprising a compartment comprising a sterol lipid and a still further compartment comprising a second substrate not carrying an immobilised mycolic acid derived antigen downstream of said compartment comprising a sterol lipid; and means for storage or transport of the treated samples passed through said columns. This or these means for storage and transport do not have to be in physical connection with the containers comprising said first and second substrates respectively and are located downstream of the columns when the system is in use in order to receive the pre-treated sample fractions .
This principle can also be applied to a system for carrying out a method of detecting a marker for active tuberculosis. In this respect the invention also relates to a system for carrying out a method of detecting a marker for active tuberculosis in a sample derived from a human or animal suspected of having active tuberculosis, which comprises means for dividing said sample stream into at least a first and a second sample stream; at least a first and a second column, configured to receive said first and a second sample stream respectively; said first column comprising a compartment comprising a sterol lipid and a further compartment comprising a first substrate carrying an immobilised mycolic acid derived antigen downstream of said compartment comprising a sterol lipid; and said second column comprising a compartment comprising a sterol lipid and a still further compartment comprising a second substrate not carrying an immobilised mycolic acid derived antigen downstream of said compartment comprising a sterol lipid and a first detection substrate carrying an immobilised mycolic acid derived antigen to receive at least part of the first sample stream and a second detection substrate carrying an immobilised mycolic acid derived antigen to receive at least part of the second sample stream.
Preferably the system or device comprises a skin penetrating end connected to a first tubing, which is arranged and configured receive a blood sample from the skin penetrating end and to carry said blood sample away from the skin site to said container/compartment comprising a sterol lipid.
In another preferred embodiment the system or device comprises a filter unit in connection with said first tubing for receiving said blood sample and configured to separate plasma from said whole blood sample.
In a further embodiment the system or device comprises a skin penetrating end connected to a first tubing, which is arranged and configured receive a blood sample from the skin penetrating end and to carry said blood sample away from the skin site; a filter unit in connection with said first tubing for receiving said blood sample and configured to separate plasma from said whole blood sample; a first container for receiving said sample, said first container comprising a sterol lipid; means for dividing said sample into at least a first and a second sample stream; a second container for receiving said first sample stream, said second container comprising a first substrate carrying an immobilised mycolic acid derived antigen; a third container for receiving the second sample stream, said third container comprising a second substrate not carrying an immobilised mycolic acid derived antigen; and a means for storage or transport of the treated s amp1e s.
The stream through the system or device is preferably driven by a pump, preferably a continuous flow pump, for example a peristaltic pump or a diaphragm pump. In case columns are applied, the stream may be driven by gravity or capillary action
The skin penetrating means may be any suitable means to obtain a blood sample from a human or animal, such as a needle syringe or the like.
The first tubing has dimensions that are suitable for purpose of the device, i.e. it needs to be well adaptable to the skin penetrating means and the first container.
The components of the the system or device, i.e. the containers, means for diluting and filter unit may be interconnected by suitable interlinking tubings. Alternatively the components of the device may be designed such that they can be connected directly to each other, and separated by for instance filters through which the sample but not the substrate can selectively pass. The material of the tubings used in the invention can be any suitable material which is known to the person skilled in the field of testing blood samples. Suitable materials are inert to blood/plasma/serum components and include polytetrafluorethylene (e.g. Teflon®), polypropylene, polyetherketone (PEEK) and polyethylene.
Further components may be connected between the components of the device. Such further components may be connected in tubings interlinking the components are directly attached to the components.
The system or sub-device for performing steps i-v of the methods of the invention may also comprise a means for dilution of the blood or plasma. Such means may be implemented before the sterol lipid containing container, before the filter unit, between the filter unit and the sterol lipid containing container, between the sterol containing container and the other (further and still further or (second and third) containers or between the further/still further or second /third containers and the means for storage and transport. For instance a 10 ml container may be implemented at the outlet of the further or second and still further or third containers. A suitable buffer may already be present in the container or be added into this container to provide the desired dilution. This way the volume of the sample is kept as low as possible during most of the steps of the methods of the invention, which is beneficial to the speed of the process and the compactness of the device of the invention.
The filter unit may comprise a filter matrix. Preferably the filter is implemented on a filter microchip for the sake of compactness.
The containers may be vessels or a channel or tubing, such as a spiral channel or spiral tube. A spiral channel or tube is preferred because such structure takes little space whilst maintaining a long flow path. A spiral channel or tube may be advantageously implemented on a micro-chip. This contributes to the compactness of the device of the invention. The term "container" is to be interpreted in the broad sense, i.e. it can be seen as defined area in another unit, i.e. a compartiment, for instance a compartment of a column. The terms "container" and "compartment" may thus be used interchangeably in the context of the present invention. What matters is that sterol lipid containing container and the other (further and still further or (second and third) containers are spatially separated. In this respect a sterol containing container may be included in a column together with said further (or second) container while another sterol containing container is included in a column together with a still further (or third) container. In the context of the invention a column for preparing a stream to be tested on a test substrate may comprise an upper (or upstream) substrate coated with a sterol lipid which is separated by a barrier from, but in fluid connection with a lower (or downstream) substrate coated with mycolic acid derived antigens. A column for preparing a stream to be tested on a control substrate may comprise an upper (or upstream) substrate coated with a sterol lipid which is separated by a barrier from, but in fluid connection with a lower (or downstream substrate not coated with mycolic acid derived antigens. It is to be understood that in connection in this context means that the sample to be tested can pass through said barrier, while the barrier does not allow passing of the substrates. Suitable substrates for such columns are beads. In this respect an embodiment of the system of the invention for pre-treating a sample stream from a human or animal suspected of having active tuberculosis in order to make the sample suitable for detection with the method of the invention comprises which comprises at least a first and a second column, configured to receive said first and a second sample stream respectively, said first column comprising a container comprising a sterol lipid and said further container comprising a first substrate carrying an immobilised mycolic acid derived antigen, and said second column comprising a container comprising a sterol lipid and said still further container comprising a second substrate not carrying an immobilised mycolic acid derived antigen.
The material of the containers is preferably the same and suitable materials may be bk-7 glass, polytetrafluorethylene, polypropylene, polyether ketone or polyethylene. A boundary between two containers may be a filter that does not allow substrate material to pass, but which does allow passage of a sample stream. The substrates of the containers have to be material that is inert for non-specific binding of molecules of the sample, for instance bk-7 glass, polytetrafluorethylene, polypropylene, polystyrene polyether ketone or polyethylene. The substrates may be in the form of beads, optionally crosslinked beads, for instance polystyrene beads .
The systems of the invention comprise a container comprising a substrate carrying an immobilised mycolic acid derived antigen (second container) and a container comprising a substrate not carrying an immobilised mycolic acid derived antigen. Instead of an immobilised mycolic acid derived antigen, the latter container may an inert coating or no coating, as long as no aspecific binding takes place.
The means for storage or transport of the treated samples may be small vials wherein the samples are stored for further analysis, for example by means of a dot immunogold filtration assay. Alternatively, the means may be formed by a pipette which can be applied to transfer the sample fractions treated in steps i)-v) of the methods of the invention to the substrates with immobilised mycolic acid derived antigens of the end-point assay, for instance to spot fractions as dots in a dot immunogold filtration assay.
The device may be connected to any suitable automated analysis means, such as a computer with suitable software programs to carry out the comparison of binding of mycolic acid antibodies to the immobilised antigens on the substrate .
An exemplary embodiment of the system according to claim 19 is shown in Fig. 1.
Fig. 1 shows a skin penetrating needle 1 connected to a first tubing 2, which is arranged and configured receive blood from the needle 1 and to carry the amount of blood away from the skin site. Tubing 1 is connected to a filter unit 3. This filter unit serves to separating plasma from said whole blood sample. Filter unit 3 is connected via tubing 13 to a first container 4, which is in the form of a spiral channel of which the inner wall is coated with a sterol lipid. The spiral channel is connected via tubing 14 with a means for dividing plasma 5 in the form of a passive valved branch point. One branch of the branch point is connected via tubing 16 with a second container 6 for receiving a first plasma stream, which comprises a first substrate carrying an immobilised mycolic acid derived antigen. The other branch of the branch point is connected via tubing 15 with a third container 7 for receiving a second plasma stream, which comprises a second substrate not carrying an immobilised mycolic acid derived antigen. Containers 6 and 7 in this embodiment are in the of a spiral channel of which the inner wall is either coated with a mycolic acid derived antigen (container 6) or not coated with a mycolic acid derived antigen (container 7). Containers 6 and 7 are connecter via respectively tubings 17 and 18 with dilution containers 11 and 12 respectively. These containers may be prefilled with a suitable dilution buffer of comprise inlets 21, 22 for introducing a dilution buffer. The dilution containers 21 and 22 can be connected via respective tubings 19 and 20 to means for storage or transport 8 and 9 respectively. Alternatively, the dilution containers 21 and 22 can be connected via respective tubings 19 and 20 to test and control substrates with immobilized mycolic acid derived antigens that are tested in an end-point assay.
Kit
In another aspect the invention relates to a kit for use in diagnosing tuberculosis or pretreatment of samples for diagnosing tuberculosis, comprising one or more skin penetrating means; one or more tubings; one or more containers coated with a sterol lipid and optionally phosphatidyl choline, pectin, amphothericin B or β-cyclodextrin; one or more containers comprising a substrate with and without an immobilised mycolic acid derived antigen; one or more containers comprising a substrate without an immobilised mycolic acid derived antigen; optionally one or more containers for transport and storage of samples; optionally means for diluting blood or plasma and/or a filter unit; at least one microporous membrane with mycolic acid derived antigen immobilised thereon, and optionally secondary antibodies binding to the heavy chain of antibodies against mycolic acid derived antigens contained in a sample derived from a human or animal suspected of having active tuberculosis, preferably colloidal gold-labeled second antibodies.
In a preferred embodiment the kit comprises: one or more skin penetrating means; one or more tubings; one or more columns, comprising a compartment comprising a substrate coated with a sterol lipid and optionally phosphatidyl choline, pectin, amphothericin B or β-cyclodextrin; and a compartment comprising a substrate coated with an immobilised mycolic acid derived antigen below said compartment comprising a substrate coated with a sterol lipid; one or more columns, comprising a compartment comprising a substrate coated with a sterol lipid and optionally phosphatidyl choline, pectin, amphothericin B or β-cyclodextrin; and a compartment comprising a substrate coated without an immobilised mycolic acid derived antigen below said compartment comprising a substrate coated with a sterol lipid, optionally one or more containers for transport and storage of samples; optionally means for diluting blood or plasma and/or a filter unit; optionally at least one microporous membrane with mycolic acid derived antigen immobilised thereon, and optionally secondary antibodies binding to the heavy chain of antibodies against mycolic acid derived antigens contained in a sample derived from a human or animal suspected of having active tuberculosis.
The kit may also comprise one or more means to transfer sample fractions pre-treated in accordance with steps i) to v) of the methods of the invention to the microporous membrane, for instance a pipette and pipette accessories, such as pipette tips.
The kit may also comprise one or more means to store sample fractions pre-treated in accordance with steps i) to v) for further use, such as vials or tubes.
The kit may also comprise tools to assemble the components, such as screws, clamps, glue, tape, screwdrivers etc.
The kit may also comprise means to dilute the blood or plasma during blood analysis, such as containers (for instance of 10 ml) which can be implemented at the outlet of the second and third containers. A suitable buffer may already be present in the container or be added into this container to provide the desired dilution.
The components of the kit enable the person that is to perform a tuberculosis diagnosis test to assemble the systems of the invention on the spot, i.e. at the point of care. It is therefore to be understood that the components of the kit have the same characteristics and preferred properties as explained above for the systems of the invention. The systems of the invention are easy to be assembled by means of the components of the kit of the invention, i.e. no specialist technical background is required. The kit may be provided conveniently with instructions for assembly and use.
Sterol lipids
The sterol lipid used in the context of the invention preferably is cholesterol or a derivative thereof. The sterol lipid may also be a sterol modified phospholipid. Such sterol-modified lipid may a sterol-modified phospholipid, for instance a sterol-modified phosphatidylcholine lipid or glycerophospholipid. In such sterol modified lipid the sterol is preferably cholesterol. A good example of a sterol-modified lipid suitable for the purposes of the invention is 1-palmitoyl-2-cholesteryl carbonoyl-sn-glycero-3-phosphocholine.
The sterol lipid is preferably immobilized on a surface. An example is a substrate having a coating containing cholesterol or cholesterol ester wherein the cholesterol ester is cholesterol linoleate, wherein a weight ratio of linoleic acid to cholesterol is in the range from 1:3 to 1:20. A substrate may also be coated with a sterol lipid, preferably cholesterol, in combination with other molecules .
Preferably said sterol lipid is cholesterol immobilized on a substrate together with phosphatidyl choline. The sterol lipid scavenges away the anti cholesterol antibodies from the blood/plasma/serum that would otherwise cross react with the mycolic acid antigens on the substrate and lead to false positive diagnosis of tuberculosis. Phosphatidyl choline will bind to hydrophobic materials in the blood sample, rendering the sample more hydrophilic after exposure. The resulting hydrophilic sample will easier to be handled in the subsequent method steps and be less prone to clotting.
In one embodiment a sterol lipid, preferably cholesterol is immobilised in together with pectin on a substrate, e.g. the inner wall of a tubing. In this embodiment the sterol lipid scavenges away the anticholesterol antibodies from the blood/plasma/serum that would otherwise cross react with the mycolic acid antigens on the substrate and lead to false positive diagnosis of tuberculosis. In addition pectin scavenges away cholesterol in the sample and therewith also cholesterol antibodies. This leads to an even higher reliability of detection .
Alternatively a sterol lipid, preferably cholesterol is immobilised together with a compound binding to cholesterol in the blood derived sample, such as cholesterol binding heteropolysaccharide, such as β-cyclodextrin, pectin, amphothericin B or dextrin. Such molecules scavenge away cholesterol in the sample and therewith also cholesterol antibodies. This leads to an even higher reliability of detection.
For instance a sterol lipid, preferably cholesterol is immobilised with β-cyclodextrin or pectin, amphothericin B on a substrate, such as hollow fiber polypropylene membranes or glass.
Many methods are known to immobilise a sterol lipid, in particularly cholesterol on a substrate. The skilled person will be able to select the protocol suitable for his particular coating. For example a coating may be applied by initially dissolving cholesterol in an organic solvent and further diluting the dissolved cholesterol in an ethanol solution, permitting the solution to evaporate in place within a container, rinsing the coated container with buffered saline, air-drying the container, and sealing the container in vapor-proof pouches with desiccant.
Mycolic acid derived antigens
The mycolic acid derived antigen may be derived from mycobacteria selected from virulent and pathogenic mycobacteria. Preferably, the mycolic acid antigen is derived from Mycobacterium tuberculosis. Said mycolic acid derived antigen is at least one selected from the group of mycolic acid, cord factor, chemically modified mycolic acid, chemically modified cord factor, a synthetic mycolic acid derivative, a synthetic cord factor derivative.
Natural sources of mycolic acid derivatives include the cell walls of mycobacteria such as Mycobacterium tuberculosis include mixtures of different classes of compounds and different mycolic acid homologues, often as derivatives in which they are bonded to the wall of the cell.
In addition to the mycolic acids themselves, the cells of mycobacteria also contain compounds derived from the acid, such as sugar esters of mycolic acids. Naturally occurring sugar esters comprise for instance trehalose- 6, 6'-dimycolate, commonly referred to as TDM, also known as "cord factors"; and trehalose monomycolates (often referred to as TMM) . These sugar esters occur in nature as complex mixtures of different classes of mycolic acids and of different homologues within each class.
Because it is difficult to establish the identity of cord factors present in natural products and to separate individual molecular species it is preferred to use semisynthetic or more preferably synthetic mycolic acid derivatives for the purposes of the invention. Further, it is known that the structure of the mycolic acid unit affects the biological activity of the cord factor. Therefore when natural mycolic acid derivatives would be used in the present invention the difference in biological activity between these derivatives and thus the detection of binding of antibodies of the antigen in the sample may impart a factor of unpredictability and uncertainty to the outcome of the detection. In addition, deviations in the preparation of the natural mycolic acid derivatives may result in problems regarding reproducibility of different test batches.
Therefore in order to be able to provide a method with high reliability and reproducible results it is preferred to use semi-synthetic or even more preferred synthetic mycolic acid derivatives which are identical or closely analogous to single compounds found in natural mixtures .
Suitable semi-synthetic derivatives include semisynthetic cord-factors which may be prepared by attaching mycolic acids to the sugar group. These semi-synthetic factors however still contain mixtures of different homologue.
Therefore particular suitable mycolic acid derivatives for use in the context of the present invention are synthetic cord factors, for example the synthetic cord factors described in WO 2010/08667, i.e. compounds of formula (M) x (S)y (M')z, wherein x is from 1 to 6, y is from 1 to 12, z is from 0 to 10, each M and each M' is independently a mycolic acid residue including a β-hydroxy acid moiety and each S is a monosaccharide unit.
The mycolic acid antigen may be in a form selected from homogenous and heterogenous compound mixtures. The mycolic acid derived antigen may for instance be used in combination with a phospholipid such as phosphatidylcholine .
The mycolic acid antigen may be immobilised on the substrates in various ways that are known to the skilled person. Synthetic mycolic acid derived antigens may be synthesised with particular active groups that enable immobilisation to a substrate material.
In case of a nitrocellulose substrate the mycolic acid derived antigen may be suitably immobilised as follows. Mycolic acid derived antigens may be obtained in lyophilized form and be reconstituted in a solvent mixture, for instance a chloroform: methanol: water mixture, and diluted to a concentration in the order of several nanomolars, for instance 1 nM. This dilution can then be spotted on a nitrocellulose membrane, wherein each spot is separated at a predetermined distance, e.g. 1 cm. After drying of the spots the mycolic acid derived antigens are immobilized on the membrane. Alternative immobilisation methods may for instance involve dissolving the antigens in hexane or hot PBS to form an antigen coating solution before spotting the solution on a membrane .
Claims (26)
Priority Applications (13)
Application Number | Priority Date | Filing Date | Title |
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NL2015553A NL2015553B1 (en) | 2015-10-02 | 2015-10-02 | A method for detecting a marker for active tuberculosis. |
RU2017127905A RU2712270C2 (en) | 2015-01-05 | 2016-01-04 | Methods for detecting marker for active tuberculosis |
ES16720577T ES2755679T3 (en) | 2015-01-05 | 2016-01-04 | Procedures for detecting an active tuberculosis marker |
PL16720577T PL3243076T3 (en) | 2015-01-05 | 2016-01-04 | Methods for detecting a marker for active tuberculosis |
KR1020177021903A KR20170102344A (en) | 2015-01-05 | 2016-01-04 | How to Detect Active Tuberculosis Markers |
BR112017014491A BR112017014491A2 (en) | 2015-01-05 | 2016-01-04 | Methods for detecting a marker for active tuberculosis |
US15/541,683 US10921322B2 (en) | 2015-01-05 | 2016-01-04 | Methods for detecting a marker for active tuberculosis |
PCT/NL2016/050002 WO2016111619A1 (en) | 2015-01-05 | 2016-01-04 | Methods for detecting a marker for active tuberculosis |
EP16720577.2A EP3243076B1 (en) | 2015-01-05 | 2016-01-04 | Methods for detecting a marker for active tuberculosis |
CN201680012300.9A CN107438767B (en) | 2015-01-05 | 2016-01-04 | Method for detecting markers of active tuberculosis |
PH12017501240A PH12017501240B1 (en) | 2015-01-05 | 2017-07-04 | Methods for detecting a marker for active tuberculosis |
ZA2017/05206A ZA201705206B (en) | 2015-01-05 | 2017-08-01 | Methods for detecting a marker for active tuberculosis |
HK18105520.8A HK1246393A1 (en) | 2015-01-05 | 2018-04-27 | Methods for detecting a marker for active tuberculosis |
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NL2015553A NL2015553B1 (en) | 2015-10-02 | 2015-10-02 | A method for detecting a marker for active tuberculosis. |
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