WO2013088396A1

WO2013088396A1 - Specimen collection apparatus

Info

Publication number: WO2013088396A1
Application number: PCT/IB2012/057316
Authority: WO
Inventors: Lizl CRONJE; Lubertus Klumperman
Original assignee: Stellenbosch University
Priority date: 2011-12-14
Filing date: 2012-12-14
Publication date: 2013-06-20

Abstract

A specimen collection apparatus which reduces the chances of contamination while collecting a specimen is provided. The apparatus has an elongate flexible member (3) with a material (9) at or near the leading end (7) which is selected to permit the capture of a predetermined target substance thereon. The material and leading end of the flexible material are protected by a protective covering (11) during insertion of the flexible member into a region from where the specimen is to be collected, and after insertion the covering is removed to expose the material. Thereafter, a cable (5) can be operated to withdraw the material into the flexible member to prevent contamination of the specimen. The material is typically a polymer functionalised with a moiety to which the target substance will bind, such as an aliphatic quaternary ammonium moiety when the target substance is M. tuberculosis.

Description

SPECIMEN COLLECTION APPARATUS

FIELD OF THE INVENTION

This invention relates to an apparatus and method for collecting a specimen through surface-capture of a pathogen, such as mycobacteria, from the gastrointestinal tract of a patient.

BACKGROUND TO THE INVENTION

Pathogenic mycobacteria are known to cause serious diseases in humans and animals. Mycobacteria are known for their characteristic cell wall, which is hydrophobic and waxy in nature and rich in mycolic acids. These mycolic acids are complex long chain a-alkyl, β- hydroxylated fatty acids with a chain length usually in the range of C₇₇.8o, although it can be shorter. The mycolic acids function as a very effective lipid barrier between the cell and its environment and are the primary reason for the extreme hydrophobic character of mycobacteria and their tendency to aggregate. Mycolic acids are found in the cell walls of the mycolata taxon, a group of bacteria that includes Mycobacteria tuberculosis, as well as the Rhodococcus sp. Other pathogenic mycobacteria include M. leprae, the causative agent for leprosy, mycobacteria of the MAC complex (including M. avium and M. intracellular), opportunistic pathogens found in AIDS patients, M. paratuberculosis, causing systemic infection and chronic inflammation of the intestines, M. kansasii, M. marinum and M. simiae, to name but a few. There are also a variety of non-pathogenic mycobacteria, such as M. smegmatis.

Diseases caused by pathogenic mycobacteria can be diagnosed by the presence of the causative mycobacteria in a specimen from a patient. For example, tuberculosis can be diagnosed by the presence of Mycobacterium tuberculosis in a biological specimen. M. tuberculosis can be detected using visual techniques such as microscopy or culture, or using a molecular technique such as polymerase chain reaction (PCR). PCR, a molecular detection method, is relatively fast to perform but has shown inconsistent results, probably due to variations in methodology, the high risk of contamination and the presence of PCR inhibitors, giving false negative PCR results. It is also a very expensive test, making it essentially unavailable to most patients in resource-limited settings.

For microscopy, the mycobacteria are stained using the Ziehl-Neelsen protocol and show up under the microscope as acid-fast bacilli. Although it is a relatively fast method, a substantial number of mycobacteria have to be present in the specimen in order to be detected under the microscope, i.e. the mycobacteria are only visible if there are 5 000- 10 000 bacilli per milliliter of specimen. Culture is more sensitive than microscopy as it is able to detect as little as 10-100 bacilli per milliliter of specimen. In spite of this sensitivity, culture is still only positive in less than 50% of children diagnosed with active tuberculosis. The main disadvantage of the culture method is the lengthy incubation period needed for mycobacterial growth. Due to the bacteria's slow doubling time of 18-24 hours, it takes 2-8 weeks for mycobacterial growth to be detected with conventional media and 1 -3 weeks with the liquid broth system. Culture plates must therefore be kept for 12 weeks before a test result may be indicated as negative. This is particularly problematic when dealing with children younger than 6 years of age, who can develop pulmonary, disseminated and central nervous system tuberculosis within 3 months of infection due to the short incubation period. This group of children also has the highest risk for disease development and death following primary infection. Another disadvantage of culture is that the volume of sputum required to provide optimum results for the growth detection of M. tuberculosis must exceed 5 ml_. Studies have also indicated that the best results are achieved if the high volume of sputum is collected overnight or over 24 hours, thus increasing the risk of contamination. Sputum specimens must therefore be decontaminated before culture to eliminate normal fast growing contaminants, such as normal flora, without killing off the mycobacteria. Unfortunately, even the mildest decontamination method, such as the frequently used N- acetyl-L-cysteine (NALC)-NaOH method, can kill up to a third of the mycobacteria in a clinical specimen.

It is therefore important that good quality specimens are obtained with the highest possible concentration of mycobacteria to enable the accurate diagnosis of tuberculosis. However, it is particularly difficult to collect adequate samples in young children, especially those who are unable to generate the necessary coughing force to aerolize M. tuberculosis into droplets and therefore cannot produce a spontaneous sputum specimen when required.

Sputum, induced sputum and gastric aspirates are the most commonly used specimen collection methods for children. Older children suspected of having pulmonary tuberculosis can usually produce sputum when required for use in culture or microscopy diagnostic methods. This is more likely to be positive than samples obtained by other methods. Children younger than 4 years of age, however, are unable to produce sputum spontaneously. Gastric aspiration using a nasogastric feeding tube remains a widely used modality in young children who cannot produce sputum on demand. Samples are collected on three consecutive mornings after an overnight fast period of 8-10 hours. This sample collection process usually requires hospitalization as it is vital that the correct technique is used and that the timing and processing of the samples are consistent. However, even under optimal conditions, less than 50% of the samples give a positive result in those with tuberculosis infection. Specimens obtained using the gastric aspirates method must be processed and decontaminated to kill or inhibit bacterial contaminants, but this procedure also results in the death of most of the mycobacteria in the specimen (as little as 10-20% of the mycobacteria may remain viable after optimal processing). Other disadvantages of this method are that gastric aspirates are uncomfortable, involve fasting and require repeated admission to a hospital or clinic and are therefore not really an option in resource-limited settings. Despite the low sensitivity of this method and the disadvantages described above, gastric aspirates are considered amongst the better methods for collecting specimens from children suspected of having tuberculosis, as the procedure is easy to perform, requires no special apparatus and is cost effective.

An alternative method for specimen collection in children is the induced sputum method, which uses a single hypertonic saline solution. This method provides the same yield as gastric aspirates, but there is a risk of nosocomial transmission if infection control measures are not adequate.

A recent study used a string test to retrieve M. tuberculosis from the stomach of sputum smear-negative adults infected with HIV with tuberculosis symptoms. A nylon string coiled up inside a conventional gelatin capsule is used in this type of test. A piece of the nylon string protrudes through a hole at the one end of the capsule and is taped to the cheek of the patient. The patient then swallows the rest of the weighted capsule and the string unravels through the same hole as the capsule descends into the stomach. When the gelatin capsule comes into contact with the gastric juices, it dissolves and releases the string into the stomach. After a period of about 4 hours, the string is retrieved by pulling it out via the esophagus and mouth, and is tested for the presence of certain pathogens. The yield of the string test in this particular study was higher than in the case of induced sputum, and the procedure was safer to health workers. No microbiological diagnosis, however, could be made during the study, possibly due to the decontamination procedure using NaOH-NALC, after which only 5-10% of the mycobacteria remained viable. Although M. tuberculosis is also found in the stomach of children (the result of swallowing sputum containing the TB bacilli), a string test as described above for children would not be feasible as the effect of the decontamination step would be even greater in children than in adults, due to the lower bacillary load in children. There is thus a need for a method for obtaining a specimen from a patient which is suitable for use in a mycobacterial diagnostic method and which overcomes at least some of the problems described above.

SUMMARY OF THE INVENTION

According to a first embodiment of the invention, there is provided a specimen collection apparatus comprising:

an elongate flexible member, a leading end of which is insertable through the oesophagus into the gastrointestinal tract of a patient;

a material at or near the leading end which is selected to permit the capture of a target substance thereon; and

a protective covering over the leading end of the flexible member and the material. The target substance may be a microorganism, pathogen, bacterium or mycobacterium, and more preferably is a mycobacterium such as M. tuberculosis.

The material may be a polymer functionalised with a functional moiety that binds the target substance. The polymer may be poly(styrene-a//-maleic anhydride) (SMA) and the functional moiety may include a positively charged hydrophobic group, and more preferably is a quaternary ammonium group with a hydrophobic aliphatic chain, such as a Ci₂ aliphatic quaternary ammonium group. The functionalised polymer may be styrene-[N- 3-(N',N'-dimethylamino)propyl maleimide] copolymer. Alternatively, the functional moiety on the polymer may inlcude a phosphonium group or a polypeptide or protein.

The material may be spun into fibers, and more preferably into nanofibers.

The flexible member may be a tube, such as a nasogastric feeding tube. The material may be housed inside the leading end of the flexible member or may be positioned outside the leading end of the flexible member.

The material may be attached near to or at one end of a cable which extends through the flexible member, and the other end of the cable may be operatable by a user of the specimen collection apparatus.

The cable may be operated to retract the material into the flexible member or to push the material out of the flexible member. The protective covering may be soluble in gastric fluid, such as a gelatine covering.

The protective covering may be a plug which is connected to the end of the cable at the leading end of the flexible member and the material may be attached to the cable adjacent to the protective member, so that in a closed configuration the material is housed inside the flexible member and the protective covering is seated against the leading end of the flexible member and in an open configuration the material and plug are positioned outside of the leading end of the flexible member, the position of the material and plug being controlled by operation of the cable. According to a second embodiment of the invention, there is provided a method of obtaining a specimen from the gastro-intestinal tract of a patient for use in a method of diagnosing a disease or infection, the method comprising the steps of:

inserting a specimen collection apparatus as described above through the oesophagus of the patient into the gastrointestinal tract, the specimen collection apparatus having a material at or near a leading end which is selected to permit the capture of a target substance thereon and a protective covering over the leading end of the flexible member and the material;

exposing the material to the gastrointestinal tract for a time which is sufficient for the target substance, if present, to be captured onto the material;

retracting the material into the specimen collection apparatus; and

removing the specimen collection apparatus from the patient.

The material preferably does not require a decontamination step prior to being used in the method of diagnosis.

According to a third embodiment of the invention, there is provided a method of diagnosing a disease or infection in a patient, the method comprising the steps of:

obtaining a specimen from the gastro-intestinal tract of the patient according to the method described above; and

identifying whether a target substance which causes the infection or disease is present in the sample;

wherein the method does not comprise a step of decontaminating the sample to remove contaminants which are not from the gastro-intestinal tract of the patient. The identification step may be performed using light microscopy, fluorescence microscopy or PCR.

According to a further embodiment of the invention, there is provided a polymer functionalised with a positively charged moiety with a hydrophobic aliphatic chain for use in capturing a target substance for use as a specimen in a method of diagnosing a disease or infection in a patient caused by the target substance, wherein the polymer is attached to a specimen collection apparatus as described above.

The target substance may be selected from microorganisms, pathogens, bacteria and mycobacteria, and more preferably is a mycobacterium such as M. tuberculosis.

The polymer may be poly(styrene-a//-maleic anhydride) (SMA) functionalised with the functional moiety. The functional moiety may include a quaternary ammonium group with a hydrophobic aliphatic chain, such as a Ci₂ aliphatic quaternary ammonium group.

The functionalised polymer may be styrene-[N-3-(N', N'-dimethylamino)propyl maleimide] copolymer.

The functionalised polymer may be spun into fibers, and more preferably, into nanofibers.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a perspective view of a first embodiment of a specimen collection apparatus;

Figure 2 is a perspective view of a second embodiment of a specimen collection apparatus;

Figure 3 shows fluorescent microscopy (FM) images of washed SMI-qCi₂ nanofibers after incubation with decreasing concentrations of BCG at 37 <C and pH 2 for one hour;

Figures 4A and 4B show FM images (left) and light microscopy (LM) images (right) of washed SMI-qCi₂ nanofibers after incubation with decreasing concentrations of M. tuberculosis at 37 <C and pH 2 for one hour;

Figure 5 shows PCR products in a 2.5% agarose gel for BCG concentration studies.

Lanes A and A¹ contain DNA 1 00 bp size marker, lane B contains lysed BCG as positive control and lane C contains H₂0 as negative control. Numbered lanes contain the following PCR products of decreasing concentration of BCG incubated with SMI-qC^: lanes 1 and 10, 10⁸ cells; lanes 2 and 1 1 , 10⁷ BCG/mL; lanes 3 and 12, 1 0^s BCG/mL; lanes 4 and 1 3, 1 0⁵ BCG/mL; lanes 5 and 14, 10⁴ BCG/mL; lanes 6 and 15, 10³ BCG/mL; lanes 7 and 16, 1 0² BCG/mL; lanes 8 and 17, 1 0 BCG/mL; lanes 9 and 18, 0 cells;

Figure 6 shows 14 PCR products in a 2.5% agarose gel for M. tuberculosis concentration studies. Lanes A and A¹ contain DNA 100 bp size markers, lane B contains lysed M. tuberculosis as positive control and lane C contains H₂0 as negative control. Numbered lanes contain the following PCR products of increasing concentration of M. tuberculosis incubated with SMI-qd₂: lanes 1 and 10, 0 cells; lanes 2 and 1 1 , 10 Mtb/mL; lanes 3 and 12, 10² Mtb/mL; lanes 4 and 13, 10³ Mtb/mL; lanes 5 and 14, 10⁴ Mtb/mL; lanes 6 and 15, 10⁵ Mtb/mL; lanes 7 and 16, 10^s Mtb/mL; lanes 8 and 17, 10⁷ Mtb/mL; lanes 9 and

18, 10^s Mtb/mL; and

Figure 7 shows FM images of washed SMI-qCi₂ nanofibers after incubation with BCG at 37 °C and pH 2 for (a) 15 min., (b) 30 min., (c) 45 min. and (d) 60 min.

DETAILED DESCRIPTION OF THE INVENTION

A specimen collection apparatus and method of collecting a specimen are described herein. The specimen collection apparatus comprises an elongate flexible member the leading end of which is insertable through the oesophagus into the gastrointestinal tract of a patient and with a material at or near the leading end which is selected to permit the capture of a predetermined target substance thereon. The material and leading end of the flexible material are protected by a protective covering during insertion of the flexible member, and after insertion the covering is removed to expose the material. Thereafter the material can be withdrawn into the flexible member, which can then be removed from the patient.

The specimen collection apparatus enables a specimen to be collected and retrieved from the gastric region of a patient in a protected manner which reduces the chances of contamination of the specimen from other regions of the body. A decontamination step, which would significantly reduce the yield of mycobacteria, is therefore not required, thus providing an overall yield of mycobacteria which is sufficiently high to allow for the sensitivity of existing tests to be improved and/or for a faster and more accurate diagnosis to be made, especially in young children.

On embodiment of a specimen collection apparatus (1 ) according to the invention is shown in Figure 1 and includes an elongate flexible member (3), in this embodiment a conventional 3-4mm nasogastric feeding tube open at both ends, with a thin cable (5) extending there through. A small wad of a nanofiber or nonwoven material (9) is secured to the cable (5) at the leading end (7) of the tube (3). A membrane (1 1 ), in this embodiment a hard gelatine capsule providing a friction fit over the end (7) of the tube (3), covers the material (9). The free end (13) of the cable (5) extends from the end (15) tube (3) and has a ring (17) secured thereto.

The material (9) is selected to permit the capture thereon of a predetermined target substance, in this embodiment the pathogen M. tuberculosis. This is achieved by using a functionalised polymer. A synthesized aliphatic (long chain hydrocarbon) quaternary ammonium group, in particular a C₁₂ aliphatic quaternary ammonium moiety, is attached to the polymer to functionalize it for M. tuberculosis attachment. In this embodiment the functionalization is not specific for M. tuberculosis binding. Any functional group consisting of a quaternary ammonium (to provide a (+) charge in any pH environment) with a long aliphatic chain (to provide a hydrophobic character) will suffice (a hydrophobic moiety or a positive charge moiety on its own is not sufficient for mycobacteria capture). The current functional group targets the hydrophobic cell wall of M. tuberculosis that is (-) charged under normal physiological conditions. The functionalized polymer thus interacts with M. tuberculosis in two-ways: ionic interaction due to +/- charge attraction, and hydrophobic interaction between the mycolic acids of the M. tuberculosis cell wall and the Ci₂ hydrocarbon chain of the modified polymer.

The functionalized polymer is electrospun to produce nanofibers so as to increase the available surface area for binding, and hence capture, of the pathogen. The nanofibers can be twisted into a yarn for easier handling. A yarn of approximately 10 cm is used in the present embodiment.

In use, the leading end (7) of the tube (3) is inserted through the oesophagus of the patient (not shown), either nasally or orally, into the stomach. The gelatine capsule (1 1 ) covering the leading end (7) protects the material (9) against contamination and facilitates easy insertion of the tube into the stomach. Once in the stomach the gelatine dissolves in the gastric fluid within a few minutes and the material is then exposed to the gastric fluid. The material is left exposed to the gastric fluid for a period of time sufficient to permit binding of any M. tuberculosis which may be present thereto. Hereafter the ring (17) is used to pull on free end (13) of the cable (5) to withdraw the material (9) into the tube (3) and the tube is withdrawn from the patient. Withdrawing the material (9) into the tube limits contamination of the material (9) during removal of the apparatus (1 ) from the oesophagus.

Hereafter the material (9) is removed from the apparatus, washed with PBS and analysed for the presence of M. tuberculosis using conventional light microscopy, fluorescence microscopy, PCR or any other micro-organism appropriate detection method. An increased yield of M. tuberculosis over conventional methods can be obtained using the apparatus and without the need for gastric aspiration.

It will be appreciated, however, that many other embodiments of apparatus exist which fall within the scope of the invention, particularly regarding the configuration of the apparatus. For example, as shown in Figure 2, the elongate flexible member (20) could be a sleeve with a stiff cable running therein (22) and a tubular enclosure (24) at its leading end (26). A plug (28) which provides a sealing fit within the open end of the enclosure (24) is secured to the end of the cable (22). The pathogen selective material (30) is secured to the cable (22) adjacent to the plug (28).

After the enclosure (24) is inserted into the stomach, the cable (22) is operated to push the plug (28) away from the enclosure (24), exposing the material (30) to the gastric fluid. A flange (32) can be provided over the cable (22) to assist in pushing the material (30) out of the enclosure (24).

After a period of time sufficient to permit binding of a pathogen to the material (30) the cable is again operated to withdraw the material (30) into the enclosure (24) and seat the plug (28) over the end thereof. The apparatus is then removed from the patient and the material is analysed.

Many other apparatus configurations exist. Also, many different substances can be targeted and many suitable materials can be used to capture the target substance which can be selected from microorganisms, pathogens, bacteria, mycobacteria, organic and inorganic molecules or compounds, including minerals and toxins.

It will be appreciated that many mechanisms exist whereby a functional group may interact with a pathogen to cause it to be captured. Consequently, "capture" has its widest meaning in this specification and includes binding through specific or non-specific interaction of compounds or organisms, for example, but not limited to, metal-ligand interaction, van der Waals forces, hydrogen bonding, ion pair interaction and the like. It is also preferable to use a polymer that can be chemically modified with the functional group and used as is or processed into fibers, nonwovens or related textiles, such as SMA, but any suitable high surface area material coated with the functional group can be used.

Other functional groups that may be considered as capture agents include any combination of aliphatic hydrocarbon chains that will provide the necessary hydrophobic character to facilitate the capture of mycobacteria when coupled with any positively charged center, such as phosphonium, etc. The capture agent must, however, not be too hydrophobic in character as this will result in poor wetting of the functionalized nanofibers, thus preventing close contact with the mycobacteria and a reduction in the capture effectivity of the polymer nanofibers. Another option would be a host protein that can capture mycobacteria through interaction with its lipoarabinomannan or other cell surface structures that can act as ligands; as well as host ligands that can bind to mycobacteria proteins, such as fibronectin-binding proteins, heparin-binding haemagglutinin adhesion (HBHA) and laminin-binding proteins.

It will be appreciated that the cable can be a string, wire, cord or the like, can be woven or non-woven and can be stiff or flaccid.

It is also possible for the sample or specimen to be a liquid, such as sputum, gastric juices, bronchial lavage, blood, urine, and so forth, or a solid, such as a tissue biopsy or stool, which has been treated to extract the micro-organism into a liquid. The pH for capture of the biological sample can range from 0 to 10. The quaternary ammonium moiety is positively charged under all pH conditions and will thus not lose its effectivity as the pH of the biological sample fluctuates, and the hydrophobic character of the capture agent is also not influenced by the pH of the biological sample.

The processing of the micro-organisms captured onto the surface of the modified polymer does not include decontamination, due to the capture surface being removed from the biological specimen in a protected manner. Processing of the captured micro-organism will include washing the polymer coated with captured microorganisms with PBS to remove any loosely adhered micro-organisms not properly captured onto the surface of the polymer. The method for detection of the micro-organisms may be any appropriate method for detecting the relevant micro-organism. For mycobacteria in general and M. tuberculosis in particular, these detection methods can include microscopy detection (either convention light or fluorescence after staining), PCR or other amplification and detection methods based on the nucleic acids of the relevant organism in question. Synthesis of functionalized polymers according to an embodiment of the invention will be described in more detail below. The invention, however, is not intended to be limited to these specific functionalized polymers or the described methods of making them.

Synthesis of functionalized polymer nanofibers

Poly(styrene-a//-maleic anhydride) (SMA), synthesized using conventional radical copolymerization to yield SMA (M_n = 128 000 g/mol, D = 2.7, MAnh content: 50%) was modified via the nucleophilic addition reaction of a primary N-alkylamine to the reactive maleic anhydride unit of SMA. The cyclic maleic anhydride underwent ring opening to form a secondary amide and carboxylic acid group, also known as maleamic acid. Ring closure was achieved with the application of heat and corresponding loss of water. The ring-closed maleimide is more stable than the ring-opened compound and less susceptible to hydrolysis.

Repeating units of SMA

Synthesis of polymer (SMA)

Conventional radical copolymerization was used to synthesize an alternating copolymer of styrene and maleic anhydride in a 1 :1 molar ratio styrene:maleic anhydride.

Maleic anhydride (MAnh) (14 g, 0.14 mol), styrene monomer (15 g, 0.14 mol) and 2,2' azobis(isobutyronitrile) (AIBN, 0.1 182 g, 7.20^*10^"4 mol) were dissolved in 200 mL methyl ethyl ketone (MEK). The reaction mixture was degassed with N₂ for 30 minutes and stirred overnight (16 hours) at 60 °C. The reaction mixture was cooled to room temperature, precipitated in 500 mL iso-propanol and washed with n-hexane. The polymer was dried under vacuum at room temperature to remove any unreacted monomer and residual solvent and then analyzed using SEC. M_n = 128 000 g/mol, D =2.7 Synthesis of functionalized polymer (SMI-qC₁₂)

SMI-qCi₂ was prepared via a precursor, namely styrene-[A/-3-(A/',A/-dimethylamino)propyl maleimide] copolymer (SMI-tC). This precursor was formed upon reaction of the maleic anhydride unit of SMA with 3-(/V,A/-dimethylamino)-1 -propylamine at room temperature and without a catalyst to form the ring-opened secondary amide and carboxylic acid, followed by heat-induced cyclization (Scheme 1 ). SMI-tC was subsequently reacted with suitable bromoalkane compounds in excess which resulted in the quaternization of the tertiary amine moiety of SMI-tC to yield the relevant modified styrene-maleimide copolymer, ready for electrospinning into polymer nanofibers.

Synthesis of styrene-[A/-3-(A/',A/'-dimethylamino)propyl maleimide] copolymer (SMI-tC) 3-(/V,A/-dimethylamino)-1 -propylamine (3.3 g, 33 mmol) was added dropwise to a solution of SMA (5 g, 25 mmol) in 25 mL DMF at room temperature. The reaction was heated and refluxed at 170 °C for 1 hour whereafter the reaction was cooled, the polymer was precipitated in diethyl ether and filtered. The product was dissolved in methanol/THF, precipitated into diethyl ether, filtered and dried under vacuum at 60 'C for 24 hours to remove any residual solvent.

Scheme 1: Synthesis of styrene-[N-3-(N',N'-dimethylamino)propyl maleimide] copolymer Synthesis of styrene-[A/-3-(A/'-dodecyl-A/',A/'-dimethylammonium)propyl maleimidel copolymer (SMI-gCi?)

1 -Bromododecane (1 .15 g, 4.6 mmol) was added dropwise to a solution of styrene-[/V-3- (A/',A/-dimethylamino)propyl maleimide] copolymer (1 .0 g, 3.5 mmol) in 18 mL DMF at room temperature. The reaction was heated to 1 10 °C for 48 hours, whereafter the reaction was cooled, the polymer was precipitated in diethyl ether, filtered and washed thoroughly three times with pentane (Scheme 2). The polymer was dried under vacuum at 60 °C for 24 hours to remove an residual solvent.

Scheme 2: Synthesis of styrene-[N-3-(N'-dodecyl-N',N'-dimethylammonium)propyl maleimide] copolymer

Electrospinning of SMI-qCi₂

SMI-qCi₂ was dissolved in 1 :1 DMF:methanol (25 wt. %). The prepared solution was placed in a 1 mL plastic syringe connected to a syringe pump (Harvard, Model 33 Twin Syringe Pump). An electrode lead of a high voltage power supply capable of generating positive DC voltages from 0 to 25 kV was connected to the blunt metal needle of the syringe. The positive charge was set at 7.5 kV. The flow rate was set at 0.0025 mL/min and the needle diameter was 21 gauge. A stationary foil covered collector was placed 15 cm from the needle tip and connected to a negative electrode. The negative charge was set at 7.5 kV. The collected electrospun fibers were placed under vacuum at 60 'C to remove any residual solvents. Diagnosis of captured M. tuberculosis

Concentration studies with mycobacteria at low pH

BCG culture

One mL BCG (containing pJV 75 Amber) freezer stock was inoculated in 10 mL Middlebrook 7H9 medium containing 0.2% glycerol, 0.05% Tween-80, 10% ADC and 25 μg/ml kanamycin and grown to an optical density of 0.6-0.8, when measured at 600 nm in a spectrophotometer (OD₆₀o)- The cells were pelleted by centrifugation at 3000 x g for 10 minutes at 4 °C and resuspended in 10 mL Middlebrook 7H9 medium containing 0.2% glycerol and 10% ADC (no Tween). The centrifugation and resuspension steps were repeated. The cells were inoculated to approximate OD₆₀o of 0.05 in 7H9 medium containing 0.2% glycerol, 10% ADC and 25 μg mL kanamycin (no Tween) (50-100 mL cultures) and grown to approximate OD₆₀o of 0.6-0.8 (as the cells clumped in the media without Tween it was difficult to take an accurate OD measurement).

M. tuberculosis culture

One mL H37Rv M. tuberculosis isolate (harbouring an rpoB531 mutation - confers rifampicin resistance) freezer stock was inoculated in 10 mL Middlebrook 7H9 medium containing 0.2% glycerol, 0.05% Tween-80, 10% ADC and grown to OD₆₀₀ of 0.6-0.8. The cells were pelleted by centrifugation at 3000 x g for 10 minutes at 4 ^<C and resuspended in 10 mL Middlebrook 7H9 medium containing 0.2% glycerol and 10% ADC (no Tween). The centrifugation and resuspension steps were repeated. The cells were inoculated to approximate OD₆₀₀ of 0.05 in Middlebrook 7H9 medium containing 0.2% glycerol, 10% ADC and 2 μg mL rifampicin (no Tween) (2 x 50 mL cultures) and grown to approximate OD₆oo of 0.6-0.8 (as the cells clumped in the media without Tween it was difficult to take an accurate OD measurement, but 10-14 days of growth were sufficient to reach the specified OD).

Mycobacteria (M. tuberculosis or BCG) culture dispersion was decanted from the tissue culture flask into a tube and centrifuged at 3000 rpm for 15 minutes. The supernatant was discarded and the pellet was resuspended in pH 2-adjusted PBS (pH adjustment done using concentrated HCI) to a total volume of 50 mL. The resulting pH-adjusted culture dispersion was diluted serially 1 :10 in pH 2-adjusted PBS, from approximately 10⁸ mycobacteria/mL to 10 mycobacteria/mL. Aliquots of 10 mL of the final dispersion were pipetted into tubes with screw lids. A tube with PBS, instead of culture, was included as negative control. A 10 mg piece of polymer nanofiber mat was added to each of the tubes, taking care that the polymer did not stick to the side of the tube and was moving freely in the culture. The tubes were closed and incubated at 37 °C for one hour, whilst shaking. The polymer piece was subsequently removed and washed twice in PBS for 5 minutes and returned to a clean eppendorf tube. Mycobacteria-polymer interaction was determined using FM, LM and PCR (after DNA extraction) as detection methods.

Detection methods Fluorescence microscopy (FM)

Images of the functionalized SMI fibers were obtained using an Olympus IX-81 microscope, coupled to an MT-20 Xenon burner. The samples were placed on a microscope slide and incubated with SYTO-9 nucleic acid and propidium iodide for 15 minutes. The stained samples were returned to an eppendorf and heated in a heating block at 85 °C for 20 minutes. The stained samples were returned to a microscope slide and were excited using a Xenon-Arc burner (Olympus Biosystems GMBH) as light source, with the 472 nm or 572 nm excitation filter. Emission was collected using a UBG triple- bandpass emission filter cube. Light microscopy (LM)

The polymer samples were fixed to a microscope slide with 2 drops of albumin fixative and heat set at 85 'Ό for 2 hours on a heating block. The polymers were covered with carbol fuchsin solution, heated from below using a lit cotton swap until steam started to rise from the slide, and left for 5 minutes. The stained polymers were carefully rinsed with water, taking care that the polymer remained on the slide. The stained polymers were subsequently destained with dilute hydrochloric acid for one minute and counter stained with methylene blue for two minutes. The stained polymers were carefully rinsed with water, taking care that the polymer remained on the slide. The stained slides were air- dried and the samples were viewed using a 100x oil immersion objective.

DNA extraction and polymerase chain reaction (PCR)

DNA extraction:

Proteinase K (10 of 10 mg/mL) was added to each tube and incubated overnight at 42 °C. The tubes with polymer were removed from the oven and equal volumes of buffer 2 containing guanidine hydrochloride were added to each tube containing a washed polymer sample ~ 200 μΙ_. NucliSENSE lysis buffer (1 .6 ml_) was added to each tube. After vortexing and incubating the tubes for 30 minutes, they were centrifuged and the polymer was removed. The silica suspension was subsequently vortexed and a 50 μΙ_ aliquot was added to each of the lysed samples. Thereafter, the tubes were briefly vortexed immediately and left for 10 minutes without mixing. The tubes were centrifuged for 2 minutes at 1500g and the supernatant was carefully decanted. The silica was washed as follows:

- 400 μΙ_ wash buffer 1 for 30 seconds;

- 500 μΙ_ wash buffer 2 to each of the test tubes and wash for 30 seconds;

- 500 μΙ_ wash buffer 3 to each of the test tubes and wash for 15 seconds;

- 50 μΙ_ elution buffer onto the silica pellet in the micro tubes and close the caps. The tubes were placed in the Thermoshaker and incubated for 5 minutes at 60 °C at 1400 rpm to elute any nucleic acid from the silica. Thereafter the tubes were placed in the miniMAG and the magnet was placed close to the silica to compact it. The supernatant containing the extracted nucleic acid was transferred to a fresh clean tube, properly marked. Care was taken not to transfer any silica particles.

The extracted nucleic acid was amplified using PCR. The primers used were as follows: BCG: 5 -AAGCGGTTGCCGCCGACCGACC-3' (SEQ ID NO: 1 ),

5 -CTGGCTATATTCCTGGGCCCGG-3' (SEQ ID NO: 2) and

5 -GAGGCGATCTGGCGGTTTGGGG-3' (SEQ ID NO: 3).

M. tuberculosis: 5 -CAAGTTGCCGTTTCGAGCC-3' (SEQ ID NO: 4),

5'-CAATGTTTGTTGCGCTGC-3' (SEQ ID NO: 5) and

5'-GCTACCCTCGACCAAGTGTT-3' (SEQ ID NO: 6).

The PCR reactions were carried out in a total volume of 25 μΙ_, containing 1 μΙ_ DNA template, 1 x enzyme buffer, 3.5 mM MgCI₂, 4.0 mM dNTP's, 25 pmol of each primer and 0.5 U HotStarTaq DNA polymerase (Qiagen Germany). Two additional tubes were added, one with 2 μΙ_ of lysed mycobacteria as positive control and one with RNA-nuclease free water as negative control. Amplification was initiated by incubation at 95 °C for 15 min., followed by 45 cycles at 94 ^<C for 0.5 min., 62 ^<C for 0.5 min., and 72 °C for 0.5 min. After the last cycle, the samples were incubated at 72 °C for 10 min. The presence of the PCR products was determined by electrophoresing 10 μΙ_ of the reaction product on a 2.5% agarose gel in 1 XTAE buffer (pH 8-3) at 5 V/cm for 4 hours. 5 μΙ_ of a 100 bp DNA size marker was co-electrophoresed. The gel was stained with ethidium bromide (50 μΙ_ (10mg/ml_ stock solution) added to 1700 ml_ TAE buffer) and photographed under UV to visualize. To minimise the risk of laboratory cross-contamination during the PCR amplification, each procedure (preparation of the PCR reaction mixes, the addition of the DNA, the PCR amplification and the electrophoretic fractionation) was conducted in physically separated rooms. Negative controls (water) were included to control for reagent contamination.

Results of concentration studies

Concentration studies were carried out with BCG and M. tuberculosis as test cultures, and SMI-qCi₂ nanofibers to determine whether the interaction between the mycobacteria and this polymer was dependent on the concentration of the mycobacteria. Interaction between SMI-qCi₂ and decreasing concentrations of BCG at pH 2 were visualized using FM. Figure 3 shows the representative FM images of the washed SMI-qCi₂ nanofibers, after incubation with decreasing concentrations of BCG at 37 °C and pH 2 for one hour. Analysis of the FM images of Figure 3 indicated that the number of BCG captured onto the SMI-qCi₂ nanofibrous surface appeared to decrease with a decreasing concentration of BCG from approximately 10⁸ BCG/mL to 10⁵ BCG/mL.

Interaction between SMI-qCi₂ and decreasing concentrations of M. tuberculosis after incubation at 37 ^<C and pH 2 was visualized using FM and conventional light microscopy with ZN staining (LM). Figure 4 shows the representative FM images (left) and LM images (right) of the washed SMI-qCi₂ nanofibers after incubation with decreasing concentrations of M. tuberculosis at 37 ^<C and pH 2 for one hour. Analysis of the FM images indicated that the number of M. tuberculosis captured onto the SMI-qCi₂ nanofibrous surface appeared to decrease with a decreasing concentration of M. tuberculosis from approximately 10⁸ Mtb/mL to 10² Mtb/mL. According to the FM images, no M. tuberculosis was captured onto the surface of the SMI-qCi₂ nanofibers at a concentration below 10² Mtb/mL. These results differ from literature where it is reported that 5000-10 000 acid-fast bacilli/mL are required to give a positive microscopy result.^52"54 A possible explanation for this result may be that the inclination of M. tuberculosis to clump together in clusters on the hydrophobic surface of the SMI-qCi₂ nanofibers made it easier for the microscope operator to see them. The microbes are not spread out and do not need to be identified individually. Analysis of the LM images indicated that the number of M. tuberculosis captured onto the SMI-qCi₂ nanofibrous surface appeared to decrease with a decreasing concentration of M. tuberculosis from approximately 1 0⁸ Mtb/mL to 10² Mtb/mL. According to the LM images, no M. tuberculosis was captured onto the SMI-qCi₂ nanofibrous surface at a concentration below 10² Mtb/mL.

Based on these FM and LM results it can be concluded that M. tuberculosis can be captured onto the SMI-qCi₂ nanofibrous surface due to ionic interaction between the negatively charged M. tuberculosis cell wall and the positively charged quaternary ammonium moiety of the functionalized polymer, as well as the hydrophobic-hydrophobic interaction between the mycolic acids of the M. tuberculosis cell wall and the aliphatic Ci₂ hydrocarbon chain of the functionalized polymer. This interaction is also a function of concentration, i.e. the higher the concentration of M. tuberculosis, the more M. tuberculosis is captured onto the nanofibrous surface.

The extent of interaction between SMI-qCi₂ and BCG was also analysed using PCR. Figure 5 shows a representative image of the agarose gel analysis of the PCR products of BCG after the affinity study between SMI-qCi₂ and decreasing concentrations of BCG. The PCR gel shows clear and distinct bands at 196 bp for the presence of BCG at concentration levels of 10⁸ BCG/mL to 1 0⁵ BCG/mL for both sets, as well as 1 0³ and 1 0 BCG/mL for the one set and 1 0² and 1 0 BCG/mL for the other set, indicating that SMI- qCi₂ was able to capture BCG at a concentration of 10 BCG/mL and higher. There are some bands in the duplicate sets that did not amplify properly. A possible explanation for this occurrence may be that the DNA of BCG was not properly released during the DNA purification procedure and was therefore not detected by PCR, or that the DNA was lost during the amplification process. Non-specific bands of primer dimers at approximately 50 bp were sometimes present on the gel that has no bearing on this study. These may be as a result of non-specific amplified products or the formation of primer dimers.

PCR was also used as diagnostic tool to determine the extent of interaction between M. tuberculosis and the surface of the SMI-qCi₂ nanofibers. Figure 6 shows a representative image of the agarose gel analysis of the PCR products of M. tuberculosis after the affinity study between SMI-qCi₂ and decreasing concentrations of M. tuberculosis. The PCR gel shows clear and distinct bands at 235 bp for the presence of M. tuberculosis at concentration levels of 10⁴ Mtb/mL to 10⁸ Mtb/mL and faint bands at 10 Mtb/mL and 10² Mtb/mL, indicating that SMI-qCi₂ was able to capture M. tuberculosis at a concentration of 10 Mtb/mL and higher. There are some bands in the duplicate sets that did not amplify properly. A possible explanation for this occurrence may be that the DNA of M. tuberculosis was not properly released during the DNA purification procedure and was therefore not detected by PCR, or that the DNA was lost during the amplification process. The faint bands in lanes 1 and 10 at 235 bp may indicate some cross contamination during the DNA extraction process. Non-specific bands of primer dimers at approximately 1 15 bp were sometimes present on the gel. These may be as a result of non-specific amplified products or primer dimers.The bands on the PCR gel, correlating to M. tuberculosis DNA, also showed an increase in intensity from 10⁴ Mtb/mL to 10⁸ Mtb/mL, indicating an increase in the amount of M. tuberculosis DNA detected through amplification. This result correlates well with the results from the FM and LM images that also showed an increase in the amount of M. tuberculosis captured by SMI-qCi₂ with an increase in the concentration level of M. tuberculosis.

In conclusion, the FM, LM and PCR results indicated that the SMI-qCi₂ nanofibrous surface was able to capture M. tuberculosis effectively at low pH conditions and that this interaction was concentration dependent.

Time studies with mycobacteria at low pH

The pH of the BCG culture dispersion was adjusted to 2 using concentrated HCI. A 10 mL aliquot of pH-adjusted BCG culture dispersion was pipetted into 4 tubes with screw lids. A tube with PBS, instead of BCG culture, was included as negative control. A 10 mg piece of functionalized SMI nanofibrous mat was added to each of the tubes, taking care that the polymer did not stick to the side of the tube. The tubes were closed and incubated at 37 °C, whilst shaking. After 15 minutes of incubation time, the polymer was removed from one tube and washed twice in PBS for 5 minutes. After 30 minutes of incubation time, another polymer was removed from a tube and washed twice with PBS and returned to a clean eppendorf tube. The same procedure was followed after 45 minutes and 60 minutes of incubation time. BCG-polymer interaction was determined using FM as the detection method. Figure 7 shows the representative FM images of the washed SMI-qCi₂ nanofibers after incubation with BCG at 37 °C and pH 2 for the specified time periods. Analysis of the FM images indicated an increase in the number of BCG captured onto the surface of the SMI-qCi₂ nanofibers with an increase in incubation time. Based on these FM images (Figure 7), it can be concluded that the extent of interaction between BCG and the surface of the SMI-qCi₂ nanofibers appeared to be a function of time. The longer the incubation time between BCG and the SMI-qCi₂ nanofibers, the more BCG was captured onto the surface of these polymer nanofibers.

Claims

1 . A specimen collection apparatus comprising:

a protective covering over the leading end of the flexible member and the material.

2. A specimen collection apparatus according to claim 1 , wherein the target substance is selected from the group consisting of microorganisms, pathogens, bacteria and mycobacteria.

3. A specimen collection apparatus according to either of claims 1 or 2, wherein the target substance is a mycobacterium.

4. A specimen collection apparatus according to any one of claims 1 to 3, wherein the target substance is Mycobacterium tuberculosis.

5. A specimen collection apparatus according to any one of claims 1 to 4, wherein the material is a polymer functionalised with a functional moiety that binds the target substance.

6. A specimen collection apparatus according to claim 5, wherein the polymer is poly(styrene-a//-maleic anhydride) (SMA).

7. A specimen collection apparatus according to either of claims 5 or 6, wherein the functional moiety on the polymer comprises a positively charged hydrophobic group.

8. A specimen collection apparatus according to any one of claims 5 to 7, wherein the functional moiety on the polymer comprises a quaternary ammonium group with a hydrophobic aliphatic chain.

9. A specimen collection apparatus according to any one of claims 5 to 8, wherein the functional moiety on the polymer comprises a C₁₂ aliphatic quaternary ammonium group.

10. A specimen collection apparatus according to any one of claims 5 to 9, wherein the functionalised polymer is styrene-[N-3-(N',N'-dimethylamino)propyl maleimide] copolymer.

1 1 . A specimen collection apparatus according to any one of claims 5 to 7, wherein the functional moiety on the polymer comprises a phosphonium group with a hydrophobic aliphatic chain.

12. A specimen collection apparatus according to any one of claims 5 to 7, wherein the functional moiety is a polypeptide or protein.

13. A specimen collection apparatus according to any one of claims 1 to 12, wherein the material is nonwoven.

14. A specimen collection apparatus according to any one of claims 1 to 12, wherein the material is spun into fibers.

15. A specimen collection apparatus according to claim 14, wherein the fibers are nanofibers.

16. A specimen collection apparatus according to any one of claims 1 to 15, wherein the flexible member is a tube.

17. A specimen collection apparatus according to claim 16, wherein the tube is a nasogastric feeding tube.

18. A specimen collection apparatus according to any one of claims 1 to 17, wherein the material is housed inside the leading end of the flexible member.

19. A specimen collection apparatus according to any one of claims 1 to 17, wherein the material is positioned outside the leading end of the flexible member.

20. A specimen collection apparatus according to any one of claims 1 to 19, wherein the material is attached near to or at one end of a cable which extends through the flexible member.

21 . A specimen collection apparatus according to claim 20, wherein the other end of the cable is operatable by a user of the specimen collection apparatus.

22. A specimen collection apparatus according to either of claims 20 or 21 , wherein the cable can be operated to retract the material into the flexible member.

23. A specimen collection apparatus according to any one of claims 20 to 22, wherein the cable can be operated to push the material out of the flexible member.

24. A specimen collection apparatus according to any one of claims 1 to 23, wherein the protective covering is soluble in gastric fluid.

25. A specimen collection apparatus according to any one of claims 1 to 24, wherein the protective covering is formed from gelatine.

26. An apparatus according to any one of claims 21 to 25, wherein the protective covering is a plug which is connected to the end of the cable at the leading end of the flexible member and the material is attached to the cable adjacent to the protective member, so that in a closed configuration the material is housed inside the flexible member and the protective covering is seated against the leading end of the flexible member and in an open configuration the material and plug are positioned outside of the leading end of the flexible member, the position of the material and plug being controlled by operation of the cable.

27. A method of obtaining a specimen from the gastro-intestinal tract of a patient for use in a method of diagnosing a disease or infection, the method comprising the steps of:

inserting a specimen collection apparatus according to any one of claims 1 to 26 through the oesophagus of the patient into the gastrointestinal tract, the specimen collection apparatus having a material at or near a leading end which is selected to permit the capture of a target substance thereon and a protective covering over the leading end of the flexible member and the material;

retracting the material into the specimen collection apparatus; and

removing the specimen collection apparatus from the patient.

28. A method according to claim 27, wherein the disease or infection to be diagnosed is caused by pathogens selected from the group consisting of microorganisms, bacteria and mycobacteria.

29. A method according to either of claims 27 or 28, wherein the disease or infection is caused by a mycobacterium.

30. A method according to any one of claims 27 to 29, wherein the disease is tuberculosis and the target substance is M. tuberculosis.

31 . A method according to any one of claims 27 to 30, wherein the material does not require a decontamination step prior to being used in the method of diagnosis.

32. A method of diagnosing a disease or infection in a patient, the method comprising the steps of:

obtaining a specimen from the gastro-intestinal tract of the patient according to the method of any one of claims 27 to 30; and

wherein the method does not comprise a step of decontaminating the sample to remove contaminants which are not from the gastro-intestinal tract of the patient.

33. A method according to claim 32, wherein the identification step is performed using light microscopy, fluorescence microscopy or PCR.

34. A polymer functionalised with a positively charged moiety with a hydrophobic aliphatic chain for use in capturing a target substance for use as a specimen in a method of diagnosing a disease or infection in a patient caused by the target substance, wherein the polymer is attached to a specimen collection apparatus according to any one of claims 1 to 26.

35. A polymer according to claim 34, wherein the target substance is selected from the group consisting of microorganisms, pathogens, bacteria and mycobacteria.

36. A polymer according to either of claims 34 or 35, wherein the target substance is a mycobacterium.

37. A polymer according to any one of claims 34 to 36, wherein the target substance is M. tuberculosis.

38. A polymer according to any one of claims 34 to 37, which is poly(styrene-a//-maleic anhydride) (SMA) functionalised with the functional moiety.

39. A polymer according to any one of claims 34 to 38, wherein the functional moiety comprises a quaternary ammonium group with a hydrophobic aliphatic chain.

40. A polymer according to any one of claims 34 to 39, wherein the functional moiety comprises a Ci₂ aliphatic quaternary ammonium group.

41 . A polymer according to any one of claims 34 to 40, which is styrene-[N-3-(N',N'- dimethylamino)propyl maleimide] copolymer.

42. A polymer according to any one of claims 34 to 41 , which is nonwoven.

43. A polymer according to any one of claims 34 to 41 , which is spun into fibers.

44. A polymer according to claim 43, wherein the fibers are nanofibers.

45. A specimen collection apparatus substantially as herein described with reference to either one of Figures 1 and 2.