MXPA00007636A - Methods and compositions for detection and diagnosis of infectious diseases - Google Patents

Abstract

Methods and compositions for the detection and diagnosis of infectious diseases are provided. In particular, efficient and sensitive methods and compositions for the detection of active mycobacterial disease are provided for distinguishing between individuals having active disease, and individuals who have been immunologically exposed, such as those infected with a mycobacterium but who are without active disease, or those who have been vaccinated with BCG. The methods comprise topical application of antigen compositions for transdermal delivery.

Description

METHODS AND COMPOSITIONS FOR THE DETECTION AND DIAGNOSIS OF INFECTIOUS DISEASES CROSS REFERENCE TO RELATED APPLICATIONS This application claims priority to US Provisional Application Serial No. 60 / 073,911, filed on February 6, 1998, and to United States Provisional Application Serial No. 60 / 096,140, filed on November 11, 1998. August 1998 FIELD OF THE INVENTION The present invention relates to methods and compositions for detecting and diagnosing infectious diseases. In particular, the invention relates to transdermal delivery devices or systems comprising microbacterial antigens wherein the application of such devices or systems stimulates a sufficient immune response for the detection and diagnosis of active microbacterial infection.

BACKGROUND OF THE INVENTION The detection of infectious diseases is often carried out by the use of tests that monitor immunological responses. Many times, however, such tests are uncomfortable and often produce inconsistent results. In addition, the absence of sophisticated laboratory equipment often reduces the availability of testing in individuals living in underdeveloped areas, where the occurrence of infectious disease can be disproportionately elevated. The detection and accurate diagnosis of the infectious disease is important not only for treatment purposes, but also for the prevention of the occurrence and spread of the disease. The need for accurate and sensitive detection methods has become particularly pronounced recently, especially in view of the increase in infections such as those caused by mycobacteria. Microbacterial infections often manifest as diseases such as tuberculosis Human infections caused by mycobacteria have spread since ancient times, and tuberculosis remains a leading cause of death today. Although the incidence of the disease declined in parallel with advancing standards of life since at least the mid-1990s, microbacterial diseases still constitute a major cause of pathology and mortality in countries with limited medical resources and can cause widespread disease. , overwhelming in immunocompromised patients. Despite the efforts of numerous health organizations around the world, the eradication of microbial diseases has never been achieved, nor is it imminent eradication. Almost one third of the world's population is infected with the M. t uberculi complex, commonly referred to as tuberculosis (TB), with approximately 8 million new cases and 3 million deaths attributed to TB every year. After decades of decline, TB is booming. In the United States it is believed that up to 10 million individuals are infected. Almost 28,000 new cases were reported in 1990, an increase of 9.4 percent above 1989. A • 5 sixteen percent increase from 1985 to 1990. Overcrowded living conditions and shared air spaces are especially conducive to the spread of TB, contributing to the increase in cases that have been observed in the US. in prisoners in prisons and among the homeless in larger cities. Approximately half of all patients with acquired immunodeficiency syndrome (AIDS) will acquire an infection microbacterial, with TB being a particularly devastating complication. Patients with AIDS are at high risk of • develop clinical TB and anti-TB treatment seems to be less effective than in patients without AIDS. As a result, the infection frequently progresses to a fatal disseminated disease. The different microbacteria M. t ubercul osi s are increasingly found in opportunistic infections that plague AIDS patients. Organisms of the complex M. a vi um-int ra cel l ul a re (MAC), especially serotypes four and eight, are considered for 68% of the microbacterial isolates of patients with AIDS, MAC numbers up to 10 are found 10 acid-resistant tablets per gram of tissue) and, consequently, the prognosis for the patient infected with AIDS is poor. World Health Organization (WHO) continues to promote the battle against TB, recommending prevention initiatives such as "Expanded Program on Immunization" (EPI) ("Expanded Program in Immunization") and therapeutic compliance initiatives such as "Directly Observed Tretament Short- Course "(DOTS) (" Directly Observed Short-Term Treatment "). For example, TB eradication, diagnosis, treatment and prevention are equally important. The rapid detection of patients with active TB will lead to early treatment, whereby approximately 90% cure is expected. Therefore, early diagnosis is critical to the battle against TB. In addition, the therapeutic compliance would ensure not only the elimination of the infection, but also the reduction in the emersion of drug-resistant chains. The emersion of M. tubers is resistant to the drug is an extremely disturbing phenomenon. The number of new TB cases tested resistance to at least one standard drug increased from 10 percent in the early 80's to 23 percent in 1991. Compliance with therapeutic regimens is therefore also a crucial component in efforts to eliminate TB and avoid emersion of drug-resistant chains. Although more than 37 species of microbacteria have been identified, more than 95% of all human infections are caused by six species of microbacteria: M. tuberculosis, M. avium-intracellulare, M. kansasii, M. fortuitum, M. chelonae and M. leprae. The most prevalent microbacterial disease in humans is tuberculosis (TB) which is caused by microbacterial species comprising M. tubers is, M. bovis or M. africanum (Merck Manual 1992). The infection is typically initiated by the inhalation of infectious particles that are capable of reaching the terminal trajectories in the lungs. Following absorption by alveolar macrophages, the bacilli are able to replicate freely with eventual destruction of the phagocytic cells. It results in a cascade effect, where the destruction of the phagocytic cells causes the lymphocytes and additional macrophages to migrate to the site of infection, where they are also finally eliminated. The disease spreads more during the initial stages by the infected macrophages that pass to the local lymph nodes, as well as to the bloodstream and other tissues such as the bone marrow, spleen, kidneys, central nervous system and bone. (See Murray et al I. Medi ca l Mi crobi olgy, The C.V. Mosby Company 219-230 (1990)). There is still no clear understanding of the factors that contribute to the virulence of mycobacteria. Many researchers have implicated the lipids of the cell wall and the bacterial surface as contributors to the virulence and morphology of colony. Evidence suggests that C-mycosides, on the surface of certain microbacterial cells, are important for facilitating the survival of the organism within macrophages. Trehalose 6,6 'dimicolate, a cord factor, has been implicated for other microbacteria. The interrelation of colony morphology and virulence is pronounced particularly in M. Avi um. The bacilli of M. a vi um occur in several different forms of colony. Bacilli that grow as clear or rough colonies in conventional laboratory media are capable of multiplying within macrophages in tissue culture, are virulent when injected into susceptible mice, and are resistant to antibiotics.

Rough or clear bacilli that are kept in laboratory culture medium with > Frequently spontaneously assume an opaque colony morphology at the time they fail to grow in macrophages, are avirulent in mice, and are highly susceptible to antiobiotics. The differences in colony morphology between the transparent, rough and opaque chains of M. a vi um are almost certainly due to the presence of a glycolipid coating on the surface of harsh and transparent organisms that acts as a protective capsule. This capsule, or coating, is • 5 consists mainly of C-mycosides that apparently protect the virulent M. a vi um organisms from lysosomal enzymes and antibiotics. In contrast, non-virulent opaque forms of M. a vi um have very little C-10 mycoside on their surface. Both antibiotic resistance and resistance to death by macrophages have been attributed to the glycolipid barrier on the surface of M. a vi um. The diagnosis of infection microbacteria is confirmed by the isolation and identification of the pathogen, although the conventional diagnosis is based on • Sputum, X-ray examination of the chest (CXR) and clinical symptoms. The isolation of the Mycobacterium in a medium takes a period of four to eight weeks. The identification of the species takes two more weeks. There are several other techniques to detect mycobacteria such as the reaction in polymerase chain (PCR), direct test of microbacterial tuberculosis, or amplified microbacterial tuberculosis test (BAT), and detection tests that use radioactive labels. A diagnostic test that is widely used to detect infections caused by M. t uberc u l os i s is the tuberculin skin test. Although numerous versions of the skin test are available, typically one of the two preparations of tuberculin antigens is used: old tuberculin (OT) or purified protein derivative (PPD). The preparation of the antigen is either injected into the skin int radiically or applied topically and then transported invasively into the skin with the use of a multiprong inoculator (Tine test). There are several problems with the diagnostic method of the skin test. For example, the Tine test is generally not recommended because the amount of antigen injected into the intradermal layer can not be controlled accurately (See Murray et al., Medi ca l Mi crobi olgy, The CV Mosby Company 219-230 ( 1990)) . Although tuberculin skin tests are widely used, they typically require 2-3 days to generate results, and often, the results are inaccurate as false, the positives are sometimes observed in subjects who have undergone microbiota but have they are healthy In addition, cases of false diagnosis are frequent because a positive result is not observed only in patients with active TB, but also in people vaccinated with BCG and those who have been infected with mycobacteria but who have not developed the disease. Therefore, it is hard to distinguish patients with active TB from the others, such as domestic TB contacts, by the tuberculin skin test. Additionally, the tuberculin test often produces a cross reaction in those individuals who were infected with microbacteria other than M. t ub rcu l os i s (MOTT). The diagnosis using the skin tests currently available, is often subject to error and inaccuracies. What is needed are effective tests to detect the presence of microbacterial infection. In particular, a test that does not require invasion of the skin surface of the tested person would minimize the exposure of the physician administering the test to the bodily fluids of the tested person and decrease the risk of transmission of other infectious agents that may be present in the tested person. the person tested. further, a test that is easily administered and has a positive or negative result determined easily is essential when monitored according to a therapeutic regimen for highly infectious diseases such as tuberculosis, particularly in individuals such as homeless people, inmates in prisons, schoolchildren and retirees . Also what is needed are inexpensive and accurate methods to distinguish between people who have active disease states and those who have only been immunologically exposed to infectious agents (such as those previously infected with a microbacteria) but without active disease, or those people who have been vaccinated with BCG. Additionally, there is no known method for monitoring the effects of drug therapy in persons infected with a microbacterium, such as tests that can distinguish between active tuberculosis and other stages of cure or prior exposure. In addition, what is also needed is a test that can be easily administered to children, who are especially afraid of currently used skin tests that include needles or puncture the skin. Such tests are particularly desirable for monitoring patients particularly patients with AIDS who are highly susceptible to microbacterial infection. In addition, tests that are easily administered and that have a positive or negative result determined easily are essential when monitoring a disease such as tuberculosis in homeless people or inmates in prisons.

BRIEF DESCRIPTION OF THE INVENTION The present invention comprises methods and compositions for the detection of infectious diseases. According to a preferred embodiment of the present invention, transdermal delivery devices or systems are provided, such as patches containing microbacterial antigenic compositions. Such perches are worn on the skin and removed after a predetermined period of time. The skin is then examined for an immunogenic response to the presence of the antigen in the patch. The various methods of the prior art, the diagnostic methods and the compositions provided therein are highly sensitive and specific. More importantly, the diagnostic methods and compositions of the present invention are especially effective in detecting M. tubercules infection in patients with active tuberculosis, thus eliminating the possibility of individuals with a false diagnosis receiving vaccines or vaccines. otherwise they have been exposed to the organism without manifestation of the disease. Diagnostic methods described herein include the topical application of compositions comprising microbacterial antigens including, but not limited to MPB44, MPB45, MPB51, MPB59, MPB64, MPB70, MPB80 or MPB83, for transdermal delivery to the skin and for the subsequent detection of an immunogenic response. Antigens can be applied in an individual way • 5 or in combination. Particularly preferred is the topical application of an antigenic composition comprising MPB64. The present invention contemplates any antigen having the characteristics of MPB64, in which there is a delayed-type hypersensitivity reaction to the antigen in the presence of active TB disease, and no reaction where there has been no exposure to the microbacteria, or on exposure through the vaccine and other state of non-active tuberculosis. According to the above, it is an object of the present invention to provide • methods and compositions for the detection of infectious diseases. Another object of the present invention is to provide methods and compositions for the detection of active tuberculosis. It is another object of the present invention to provide methods and compositions for the Detection of active tuberculosis using the topical application of antigenic compositions for transdermal delivery to the skin. Another object of the present invention is to provide methods and compositions for the detection of microbacterial infections. Still another object of the present invention is to provide methods and compositions for the detection of active disease caused by microbacterial species comprising M. tuberculosis complex, M. avium-intracellulare, M. kansasii, M. fortuitum, M. chelonae, M. leprae, M. africanum and M. microti. Another object of the present invention is to provide methods and compositions for the detection of active disease by M. tubers ulos. It is still another object of the present invention to provide methods and compositions for the detection of active disease caused by M. bovis. Another object of the present invention is to provide methods and compositions for the immunological detection of microbacterial infection, which use topical application without requiring invasive procedures.

Still another object of the present invention is to provide compositions and sensitive diagnostic methods for the detection of active disease caused by microbacteria, wherein the antigenic compositions are applied topically and delivered transdermally and the skin is subsequently screened for an immunogenic response. Another object of the present invention is to provide methods and compositions for the detection of active disease caused by microbacteria wherein the topically applied microbacterial antigenic composition comprises MPB44. MPB45, MPB51, MPB59, MPB64, MPB70, MPB80 or MPB83. Still another object of the present invention is to provide methods and compositions for the detection of active disease caused by microbacteria wherein the topically applied antibacterial microbacterial composition comprises MPB44, MPB45, MPB51, MPB59, MPB64, MPB70, MPB80 or MPB83, wherein the Antigen is applied either individually or in combination with another - 1! microbacterial antigen. It is another object of the present invention to provide methods and compositions for the detection of active disease caused by microbacteria wherein the topically applied microbacterial antigenic composition comprises MPB64. Still another object of the present invention is to provide methods and compositions for the diagnosis of the infectious disease that is easy to administer. A further object of the invention is to provide methods and compositions for the detection of active tuberculosis to monitor the efficacy of therapeutic treatments. Another object of the present invention is to provide equipment for the diagnosis and detection of active disease caused by microbacteria. Still another object of the present invention is to provide methods and compositions for the detection of active disease caused by microbacteria in micronebacterial disease contacts or domestic TB It is another object of the present invention to provide methods and compositions for monitoring the clinical condition of a patient• 5 infected with mycobacteria after chemotherapy. Another object of the present invention is to provide sensitive diagnostic compositions and methods for children with active tuberculosis. It is a further object of the present invention to provide sensitive compositions and methods for the detection of active disease caused by microbacteria wherein the The method includes the use of a skin patch. These and other objects, features and advantages of the present invention will be • apparent after a review of the following detailed description of * the described embodiments and the appended claims BRIEF DESCRIPTION OF THE FIGURES. "Figure 1 shows photographs of the dose response of varying amounts of MPB64 antigen by cutaneous reactions in guinea pigs of India, Figure 2 is a graph showing the chronological course of the response of • Delayed-type hypersensitivity to a patch with the MPB64 antigen after inoculation of guinea pigs with BCG.

DETAILED DESCRIPTION 10 The present invention can be understood • more easily by reference to the following detailed description of the specific modalities included herein. Although the present invention has been described with reference to the specific details of certain modalities of the same, it is not intended that such details should be considered as • limitations on the scope of the invention. The full text of the references mentioned in is hereby incorporated in its entirety for reference, including US Provisional Application Serial No. 60 / 073,911, filed on February 6, 1998 and the United States Provisional Application Number of series 60 / 096,140 filed on August 11, 1991 Microbacterial infections such as those that cause tuberculosis, once thought to be diminished in occurrence, have impacted and once again constitute a serious health threat. Areas where humans are housed together or living in substandard housing are increasingly found to have people infected with mycobacteria. People who are immunocompromised are at a higher risk of becoming infected with mycobacteria and dying from such an infection. In addition, the emersion of chains resistant to the microbacterial drug has added to the treatment problems of such infected persons. Many people who are infected with mycobacteria are poor or live in areas with inadequate health care services. Such people are not easily tested for microbacterial infection and need non-invasive and inexpensive methods for detecting infections. In addition, people who are in prison or are homeless, generally have inadequate health care, poor physical condition, and adequate or successful health care intervention is typically not available. The present invention provides methods and compositions comprising topical applications of antigenic composition for transdermal delivery of antigens, particularly microbacterial antigens. More particularly, the present invention provides methods and compositions for detecting diseases such as active tuberculosis, and distinguishing persons with the active disease from persons who have only been immunologically exposed to infectious agents such as microbacteria. The methods and compositions of the present invention can be used to test for the presence of infection by microbacteria in humans as well as other animals. For example, the present invention may be particularly useful for the detection of diseases in cows infected with M. bovi s. "Medically active tuberculosis" is diagnosed by well-known medical procedures such as chest x-rays (CXR), sputum tests, or other symptoms. Because the precise identification of the presence of infectious microbacterial agents is expensive and takes a long time, the diagnosis of active disease does not necessarily include the identification of the presence of the microbacteria. According to the foregoing, the diagnosis of active disease such as tuberculosis may depend on the detection of other aspects of microbacterial infection such as the generation of particular immune responses or the manifestation of certain symptoms. As used herein, the term "tuberculosis" encompasses the disease states usually associated with infections caused by species of microbacteria comprising Comple. Or M. tuberculosis. Microbacterial infections caused by microbes other than M. tuberculosis (MOTT) are usually caused by microbacterial species comprising, M. avium-intracellulare, M. kansasii, M. fortuitum, M. chelonae and M. leprae, M. africanum and M. microti The present invention includes methods and compositions for topical application that allows transdermal delivery of antigens that elicit an immune response, such as a delayed-type hypersensitivity response in persons having active disease such as tuberculosis caused by microbacterial infections. Such antigens are derived from microbacteria, or are cross-reactive with microbacterial proteins or carbohydrate residues. Preferred antigens comprise those that are derived from microbacteria including, but not limited to MPB44, MPB45, MPB51, MPB59, MPB64, MPB70, MPB80 or MPB83. A particularly preferred antigen is MPB64. (See Ka waj i ri e t a l Japanese Patent Application No. Pub. 09206092 which is incorporated herein by reference in its entirety). Another preferred combination of antigens comprises the combined use of MPB64 and MPB59. For example, the combination of MPB59 and MPB64 may indicate infection of atypical microbacteria since a subject infected with other classes of microbacteria that do not secrete MPB64 will show a positive response to MPB59.

MPB64 is a frequent microbial antigen associated with the M. tubercules complex. It was first described as MPT64 by Harboe et al. (Infect Immun 1986; 52: 293-902, which is incorporated herein in its entirety), and has been well characterized and used in several laboratories since then. (See for example Yamaguchi et al Infect and I mu 1989, 57: 283-288, which is also incorporated herein in its entirety). "MPB64" and "MPT64" refer to the same antigen: MPT64 was isolated from the cultured filtrate of M. t ubercu l os isy therefore it was named as a microbacterial protein of tuberculosis and MPB64 was finally isolated from the culture filtrate of M. bovi s (or BCG) and therefore was named bovis microbacterial protein. Subsequently it was discovered that both proteins are the same.

MPB64 and MPT64 refer to antigens segregated from species of microbacteria including, but not limited to M. t uberc ul os i s, M. bovi s and some chains of BCG M. bovi s. The antigen secretes during bacterial growth and is immunogenic, producing delayed-type hypersensitivity (DTH) in guinea pigs and humans. Recombinant antigens can also be used in the compositions and diagnostic methods contemplated by the present invention. See for example Do et a l. Journ of Leukocyte 1995; 57: 221-225; Roche et al., Clin. Exp. Immunol 1996; 103 (2) 226-232; and Roche et al., J. Infect. Dis. 1994; 107 (5): 1326-30, each of which is incorporated herein in its entirety. The antigens of the present invention are applied topically for transdermal delivery in the skin of the person to be tested. The antigen is applied by maintaining a composition comprising the antigen in close contact with the skin. The concentration of the antigen in the composition is in a range of about 1 to 150 micrograms / two is applied, more particularly 10 to 100 micrograms / dose applied, more particularly 30 to 75 micrograms / two is applied. The antigenic composition may comprise a physiologically active solution comprising surfactants, regulators and solvents that allow the transdermal delivery of the antigenic composition. Preferably the surfactants, regulators and solvents that improve permeabilization are used. and antigen transport, and that do not initiate a reaction by themselves or interfere with the immunogenicity of the antigen. The surfactants for the antigenic composition comprise Tween 20, Tween 40, Tween 60 and Tween 80; each of which can be used in concentrations ranging from 0.001-10%, 0.001-1% and preferably 0.005% in regulated phosphate salt. A preferred embodiment for the application comprises from 30 to 70 micrograms of antigen in 100 microliters of phosphate buffered salt which further comprises the preferred surfactant Tween 80. A most preferred embodiment for the application is 75 micrograms of antigen in 100 microliters of regulated salt of phosphate (PBS) with 0.005% Tween 80. The prepared antigenic compositions can be stored in suitable plastic or glass aseptic containers, in groups or prorated according to desired quantities. The present invention is particularly directed to methods of transdermal delivery of antigenic compositions to skin cells for the detection of active disease. According to the above, they are included with the same antigenic and solvent combinations all contemplated that allow the supply of antigens of the infectious agent to the cutaneous cells, and result in the detection of the active disease. As used herein, the thermal "transdermal delivery" refers to the delivery of a composition to all layers of the skin, including but not limited to, the epidermis (stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum, stratum). basale), the dermis and the subcutaneous layer. As used in this, the term "topical application" refers to the application or placement of a composition on the skin without puncturing or otherwise invasively introducing the skin by the use of needles and the like. A preferred embodiment of the present invention comprises a transdermal delivery device or system for holding the compositions described above in close contact with a person's skin. A highly preferred embodiment comprises a patch band, such as a cutaneous patch band, to hold. the composition in close contact with the skin. Materials that are suitable for use in the patch for delivering the antigenic composition of the present invention include the TORII "Toriiba ™" patch band (obtained from Torii and Co., Ltd, Tokyo), Finn chamber and Perme-aid S . (Nitto-Denkou Co. Japan). In addition, materials such as medical adhesive tape or plaster can also be used where a portion of the tape or plaster includes a portion of a material impregnated with the antigenic composition, and wherein the material is located so that it is in contact direct with the skin. Suitable medical adhesive fabrics, tapes and plasters are made by numerous manufacturers such as Nichiban (Japan), Kimberly-Clark (Neenah, Wisconsin) and 3M (St. Paul, Minnesota).

The device can be held in place by various clamping methods well known to those skilled in the art.

For example, the device can be attached to the subject's arm by the use of a tape, or it can be attached by the use of an adhesive. Preferably, the material such as the adhesive used to hold the device in place should be gas permeable, and resistant to water so that it does not fall off as a result of wetting due to perspiration or bathing. A particularly advantageous aspect of the present invention relates to the ease with which the invention can be used and executed. For example, the integrity of the antigenic compositions can be preserved by transporting or preserving the compositions in aseptic containers under appropriate temperature conditions. The antigenic compositions may be stored in aliquots of desired amounts, for example, 100 or 200 μl, and then applied to gauze, plaster or tape or the like, as necessary for test patients. In addition, each of the components necessary for the compositions and methods of the present invention can be provided together in a kit for ease of use.

In addition to a patch type embodiment, the present invention also takes the form of other transdermal drug delivery vehicles known to those skilled in the art, including, but not limited to, gels, creams, liquid sprays and the like. A preferred method contemplated by the present invention comprises topical administration of a composition comprising the microbacterial antigen for transdermal delivery to the skin of a human. For example, a patch containing the antigenic composition is applied to a person's forearm and held in close contact with the skin. The patch is left in place for a predetermined period of time to allow sufficient transdermal administration of the antigen. Such period of time may vary from 1 to 5 days, preferably 2 to 5 days, more preferably 3 days. After the specified time has passed, the patch is removed and the skin examined for an immunogenic response. It is known in the art that a delayed-type hypersensitivity reaction is observed in the skin in response to the presence of some antigens. Typically such reactions are observed following the invasive introduction of an antigenic composition, and usually such reactions are characterized by redness, erythema, induration (increased thickness of the skin), presence of ulcers or red vesicles. The response sought in the present method is similar to the response observed with the transdermal injection of the microbacterial antigen. The inventors of the present invention have surprisingly found that transdermal delivery by topical application of the antigens of the present invention causes an immunogenic response (specifically a delayed type hypersensitivity reaction (DTH) in persons having active microbacterial disease such as tuberculosis.) As shown in the examples herein, guinea pigs infected with TB express a delayed-type hypersensitivity skin reaction. to the MPB64 antigen as long as the bacteria continue to grow, in contrast the guinea pigs in India immunized with BCG-Tokyo lost the delayed-type hypersensitivity to MPB64 at some time after vaccination. • 5 discovered by the inventors, the methods and compositions of the present invention, comprising microbacterial antigens, particularly MPB64, can be used for the diagnosis of microbacterial disease active such as active TB. Surprisingly, individuals who were infected with M. t ubercu l os but have not developed tuberculosis, and individuals who have previously received BCG vaccinations, do not show a positive skin reaction in response MPB64. The new discovery of the transdermal antigen supply by topical application, • particularly MPB64, for the diagnosis of The active microbacterial disease such as tuberculosis is especially desirable because no invasive procedures are required. In fact, the intradermal delivery of MPB64 has recently shown that it is not successful for distinguish between patients with TB and healthy controls (Wilcke et al., Tubercle and Lung Disease (1996) 77: 250-256). Effective transdermal delivery of microbacterial antigens by topical application until now was unknown, and • 5 using the methods and compositions of the present invention, the inventors have successfully developed diagnostic procedures to distinguish between patients with active TB and healthy controls of healthy PPD. Although it is not desired to be limited by the following theory, it is thought that one reason why the intradermal injection may be less effective than the transdermal application is related to the solubility aspects of the antigen and / or inappropriate opportunity for antigen presentation. For example, it is possible that with intradermal injection the • antigen dissolves rapidly in body fluids with insufficient time for the immune system recognize and / or respond to it. In contrast, the novel methods of the present invention allow the antigen to be gradually introduced into the subject's immune system, and as the antigen slowly infiltrates the Through the pores of the skin and sweat glands in the intradermal local portion, there is sufficient time for the immune system to mount a response, typically in the form of a delayed hypersensitivity reaction. • 5 It is also possible that the intradermal or transdermal introduction of the antigen produces different immune responses. Although you do not want to be limited by the following theory, it is thought that the reactions Hypersensitivity (also known as cell-mediated or Type IV immunity reactions) are extensively mediated by T cells with consequent inclusion of monocytes. Such reactions typically have responses that are observe after a passage of time, that is, 18-24 hours, and therefore they are referred to as delayed-type hypersensitivity. HE • thinks that a cell that presents the antigen presents the antigen to a T cell and after When activated, the T cells release lymphokines that cause the accumulation and activation of non-immune macrophages, monocytes and lymphocytes. The present invention can also be used to detect active tuberculosis in household contacts of previously infected TB patients. This allows the monitoring of the spread of the disease to others in close contact with the people originally infected. Because the • 5 members of the public may have tuberculosis and spread it when coughing, the present invention can be used to monitor the exposure of people working with the public such as stewards or doctors. The people that work with the populations of inmates in • Prisons or homeless are also easily monitored for the presence of active tuberculosis. An especially desirable use of The present invention is the monitoring of the effectiveness of the treatment of people with microbacterial infections. For example, the ability to detect when the patient with active tuberculosis no longer has active TB, this way, the treatment is effective, it is highly desirable with the increase in drug resistant microbacteria. A preferred method includes topically applying the antigens of the present invention to the person with active TB before treatment, and after a sufficient time, observe the skin reaction. At a later point in the treatment regimen, the antigens are applied again, the cutaneous reaction is observed. The lack of response through the skin indicates that the treatment has been effective in changing the state of active tuberculosis. A continuous response through the skin indicates that the treatment either is not effective or that there has not been enough time for the treatment to be effective, and the person still has active TB. At this time, the treatment could be changed, the drug's sensitivity of the infection microbial could be determined, or the same treatment could be continued for a longer period of time. One use of the present invention could be the rapid selection of a population such as when investigating a neighborhood or poor neighborhood for the amounts of infection, for testing the incoming prisons, or a group of homeless people in a shelter or on the street . For example, incoming prisoners could have patches containing the antigens of the present invention delivered to them at the entrance to prison. After the specified time of use of the patch, the skin reactions of the prisoners are examined. Those with positive reactions could • 5 stay separately. Because the present invention detects active TB, those prisoners could be isolated from other prisoners and immediately begin treatment. Other tests for TB could allow the detection of prisoners who have been exposed to TB at some point in their lives and who may not be able to transmit TB to others. There is no need to isolate and treat people who are not able to transmit TB to others. In many parts of the world, people are vaccinated against TB. It is extremely • difficult to detect active TB in these people because with TB skin tests, all people exposed to the microbacterium exit positive, whether there is active TB or the effects of vaccination. The present invention is used in such areas to detect the presence of active TB in individuals and distinguish such individuals from those who were previously vaccinated. The ease of administration is a particularly beneficial aspect of the present invention. For example, children do not know • 5 scare by the application of a topical device, such as a patch, and are not hesitant to use such a device for a sufficient time to create a cutaneous response. Such topical devices as patches are easily stored and transported to • isolated places that may lack refrigeration and clean water. The present invention can be made of inexpensive materials that can be produced at low cost and used by health care organizations around the world. This invention is further illustrated by the following examples, which are not for • be constructed in no way as limitations that are imposed on the scope of it. On the contrary, it is to be clearly understood that recourse can be had to several other modalities, modifications and equivalents thereof, which, after reading the description in the Presently, they can suggest themselves to those skilled in the art without departing from the spirit of the present invention.

EXAMPLE 1 To test the development of a rapid, simple and novel diagnostic method for active tuberculosis, subjects are tested for the skin reaction to the MPB64 antigen by a transdermal delivery method after the Topical application of an antigenic composition • using removable patches. Preparation of the Cutaneous Screen Although antigens such as MPB59, MPB70, MPB44, MPB45 or MPB51 or MPB64 can used in the patch test of the present invention, in the following example the antigen used was MPB64 at a concentration of approximately 75 μg per patch. Is contemplated • that approximately 50 to 100 μg of antigen can be used successfully by patch. A preferred antigenic solution comprises approximately 750μg of antigen per ml of phosphate buffered solution (PBS), wherein the PBS consists of 0.005% Tween 80. 25 The patches were applied to the skin of the test subjects, were left for three days and the results were interpreted by observing the site after removal of the patch. The presence of an immune response such as a delayed-type hypersensitivity reaction (DTH) (redness, induration or small red vesicles indicated a positive result), ie the presence of active tuberculosis. No change in the site was concluded as a negative response. Trial Subjects 53 patients with active tuberculosis, and 43 positive controls of purified, healthy protein derivative (PPD) were tested to determine whether the reaction to MPB64 was positive or not only in patients with active tuberculosis. We examined patients with tuberculosis from four clinics, in the vicinity of Manila, Philippines, Our Lady of Grace Paris, Sto. Child from Tondo Parish, Canossa Health and Social Center and Health Care Development Center. Of the 53 patients with active tuberculosis, 52 showed positive reaction to MPB64, while none of the 43 positive PPD controls had a positive reaction to MPB64. The specificity of MPB64 for active tuberculosis was 100% and the sensitivity was 98.1%. The effectiveness of the test was 98.9%. The patch test with MPB64 is an effective and accurate method for the diagnosis of active tuberculosis, which distinguishes such patients from individuals vaccinated with BCG and those naturally infected, but who do not develop tuberculosis. The experimental design for this discovery is described more fully in Example 2.

EXAMPLE 2 To determine the integrity of MPB64 as a specific antigen to diagnose active TB using the skin patch method, comparative tests were conducted among three different classes of individuals: (1) patients with active TB (2) healthy tuberculin positive persons ( 3) Domestic TB contacts The correlation between the cutaneous reaction to MPB64 and the clinical status of TB in humans was observed. Because the purpose of this study was to determine the integrity of MPB64 as a diagnostic antigen specific for active TB, the selection of patients with active TB was not important. The clinical records of outpatients entering the clinics were verified. Those patients who were positive in saliva spots, had a normal CXR, and who had clinical symptoms indicating active TB were classified as active TB patients, Group 1. Culture results are not available in most cases. Patients who started chemotherapy shortly before were preferred because the long-term effect of chemotherapy on the cutaneous reaction of MPB64 was not known. However, there were some patients in Group I who had been treated for 6 months. They were considered as patients with active TB due to the positive symptoms in recent examinations and due to their symptoms. The patients lived near the clinics where the socioeconomic conditions were very poor. The geographical location of their residences is important because they were scheduled to return to the clinic 3 days later to be read the • 5 results. Only 12 of the 105 patients tested did not return. Among the patients who returned, 53 were available for the final analysis. The analysis is shown in Example 1. The rest of the subjects were excluded because their patches were removed or dropped before the reading day. The patients were selected according to their clinical records and only those who were positive in spots, had an abnormal CXR and had other symptoms such as cough, fever, or weight loss to indicate TB, were selected as patients with active TB. Positive culture patients were preferred, but crop results are available in only seven cases. The majority of patients with active TB have been on chemotherapy for 1-4 months. Some have been treated for 6 months at the time of the study.

Healthy tuberculin positive volunteers were Filipinos and Japanese who show no signs of TB. At the time of the test, some family members came to the clinic along with patients with TB. They were tested as "domestic TB contacts". All subjects were informed of the profile of the study and gave their consent for the test. The number of subjects in the three groups was as follows; Group 1, patients with active TB, 53; Group 2, healthy controls, 43; and Group 3, domestic TB contacts, 41. The MPB64 was isolated from an 8-day culture filtrate of M. Bovi s BCG Tokyo (obtained from Japan BCG Laboratory, Tokyo, Japan). The purified protein was suspended in PBS and stored at -20 ° C. The amount of protein was measured by the Lowry method. The complete precipitated protein of ammonium sulfate from the 8-day culture filtrate of M. Bovi s BCG was named PPD-eT to distinguish it from tuberculin-derived purified protein (PPD), and was used as the control for the patch test. . PPDs prepared from M. t uberculos is Aoyama B were obtained from Japan BCG Laboratory (Tokyo, Japan). Five tuberculin units (TU) of PPDs suspended in 0.1 ml of reconstitution buffer were used for the Mantoux test with an intradermal injection. Ma teria for the Test Patch The TORII patch band "Toriiban ™" (obtained from Toríi and Co., Ltd, Tokyo) gauze size of 15mm, was used. The antigenic solution (75 μg of the antigen in 100 μl of PBS containing 0.005% Tween 80) was applied to the gauze and the patch was attached to a forearm of a human subject after cleaning the area of the skin with alcohol. which is about to apply the patch. The patch was placed on the skin of the subject in such a way that the gauze was impregnated with the antigenic solution that makes contact with the skin directly. The patch was left for 72 hours. Time of the Pap Test Patients with active TB and positive tuberculin positive controls were tested for a skin reaction to the MPB64 patch on the left arm, and to the PPD-eT patch on the right arm. Each patch contained 75 μg of antigen.

A dose of PPD (5 TU / 100 μl) was injected intradermally into the right forearm at a site separate from the patch. A PPD-eT patch was used to confirm that the antigens of • 5 proteins did not enter the body transdermally. If the PPD test was positive and the PPD-eT patch test was negative, the transdermal administration was incomplete. Such cases were excluded from the test results. • The united patch was removed 3 days later (72 hr) and the reaction was read as positive or negative. No change in the skin on the site was "negative", while Erythema, induration or a few small red vesicles at the site were recorded as a "positive" response to the antigen. We have compared applying the patch in • the forearm and the upper arm. The patches are more easily separated from the upper arm (41.2%) than the forearm (17.6%) before the day of reading. Therefore, the forearm is recommended for testing in adults. It is tadí sti cas 25 The two-by-two contingency test was used to evaluate the results of the MPB64 patch tests in humans. Pa ci tes with TB Acti va y Con trol es Sa l udabl es T i m ector P roject Table 1 shows the current numbers in Groups 1 and 2 with positive or negative feedback to MPB64. All subjects were positive for the Mantoux test of PPDs and the PPD-eT patch test. From these results, the following values were calculated: Sensitivity, 98.1%, Specificity, 100%; False positive rate, 0%; False negative rate, 1.9%, Positive predicted value, 100%; Negative predicted value (97.7%; Effectiveness of the test, 98.9%.) The results indicated that the MPB64 patch test is an effective method to distinguish between people with active TB and healthy positive tuberculin.

TABLE 1 MPB64 Patch Test Two-by-Two Contingency Test Between Group 1 and Group 2 • TB ta tates domes ti cos • The number of household TB contacts (Group 3) was 41 which included 12 males and 29 females. The results of the patch test are shown in Table 2. There were 26 subjects who show a positive reaction to both the PPD-eT patch and the MPB64 patch (63.4%), and nine subjects positive to PPD-eT but negative to MPB64 (22.06%). Six people (14.6%) were • negative to PPD-eT and MPB64. Among these doubly negative people, three were negatives in the Mantoux test of PPDs. Subjects in Group 3 were not registered as TB patients in the clinic. The clinical status of each person was not known although some symptoms were observed that suggest TB.

TABLE 2 Patch Test with MPB64 and PPD-eT in Domestic TB Contacts ^ F From this study it is suggested strongly that the patch test of MPB64 is a promising tool for rapid diagnosis of active TB. You can distinguish between patients with active TB from individuals who were vaccinated with BCG or those who became infected with TB but has not developed the disease with 98.1% sensitivity and 100% specificity. The patch test also has advantages over an intradermal injection in the technical facility and the safety of its application. Although Although it is not desired to limit by any theory, it is thought that the patch test can continuously supply the antigen for patient response.

EXAMPLE 3 In order to determine the integrity of MPB64 as a specific antigen for active TB using the skin patch method, comparative tests were conducted in guinea pigs in India. Hartley India guinea pigs, albinos, females weighing 300 to 400 g at the beginning of the experiments, were purchased from Japan Laboratory Animáis, Inc., Tokyo. The animals were kept under conditions pathogen-free specific in Japan BCG • Laboratory. Antigens The antigens were prepared according to the methods and materials established in the Example 2. Immunization of the Rabbit of India Live BCG vaccine (Japan BCG • Laboratory, Tokyo, Japan) is reconstituted according to the manufacturer's instructions and injected subcutaneously without adjuvant in guinea pigs in a dose of 0.5 mg per animal. The animals were tested between 4 and 25 weeks after the BCG injection. Ma teria for Pa rache of Proof 25 The TORII patch band "Toriiban ™" (obtained from Torii and Co., Ltd, Tokyo) of gauze size of 7 mm was used. An antigenic solution (75 μg of the antigen in 15 μl of PBS containing 0.005% Tween 80) was applied to the gauze and the patch was attached to a shaved area of each guinea pig. MPB64 was applied to a patch in several doses as indicated in Figure 1 and the patches were attached to the left sides and • 10 right of a guinea pig immunized with BCG in India where the hair had to be removed. Pache Test Schedule Patches were removed in 24 hours and the reaction was read immediately. No change in skin on the site was "negative", while erythema, induration or a few • Small red vesicles on the site were recorded as a "positive" response to antigen. Dose Response to MPB 64 in the Patch Test in India Rabbits Immunized with BCG Guinea guinea pigs were used previously immunized 4 weeks with BCG Tokyo for the patch test of MPB64 with several doses of the antigen. The highest dose for the patch test was 75 μg / patch. The animals were tested with patches containing MPB64 in various doses between 2.3 and 7.5 μ / patch. The patches were removed 24 hours later and the reaction was read as positive or negative. To ensure that the animals were sensitized to BCG, 0.05 μg of PPDs in 0.1 ml of the regulates were injected radically • "the cutaneous reaction was measured in 24 hours.The response to MPB64 was positive in a dose of 4.7 μg / patch or in highest concentration. The positive reaction was not observed in a dose of 2.3 μg of MPB64 per patch. Negative control patches • that contained only PBS containing Tween 80 at 0.005% did not produce any reaction cutaneous in guinea pigs immunized with BCG. Unimmunized guinea pigs did not show any response to PPDs or MPB64. Chronological Course of the Rea ct io n C o m ent to MPB 64 in the Indians Immunized with BCG.

It is known that guinea pigs immunized with BCG lost the cutaneous reaction to MPB64 15 weeks after BCG immunization when tested by intradermal injection of MPB64. To address the question of whether this was true in the patch test case, guinea pigs in India were immunized with BCG Tokyo and tested with MPB64 patches at various times after the BCG injection. Individual animals were tested only once to avoid the reinforcement injection effect. As the control, a PPD-et patch test was applied to each animal at the same time. The results are shown in Figure 2. DTH was expressed as 3+, 2+, etc., because the diameter of a reaction was regulated by the size of a patch, not by the dose of the antigen. The cutaneous reaction to the MPB64 patch test was positive in all animals up to 13 weeks after the BCG injection. It becomes hard to detect later, and was completely negative in 23 weeks. In contrast, the reaction to the PPD-eT patch test remained positive until the end of the experiment at 25 weeks after the BCG injection. Delayed-type hypersensitivity (DTH) to MPB64 and PPD-eT was examined at various times after the BCG injection. Each point in Figure 2 represents the delayed-type hypersensitivity of 3 guinea pigs from India expressed as follows: 3+, erythema and induration; 2+, erythema; 1 +, small vesicles; +/-; faint colored -, Without reaction. Patch tests on guinea pigs from India confirmed that 1/16 of the dose of antigen used for humans produced a positive reaction. It should be understood, of course, that the above refers only to preferred embodiments of the present invention and that numerous modifications or alterations may be made therein without departing from the spirit and scope of the invention.

Claims (20)

1. CLAIMS Having described the invention as an antecedent, the content of the following claims is claimed as property. A transdermal delivery device for diagnosing infectious diseases comprising the topical application of a composition comprising antigen and physiologically effective solution. The device according to claim 1, characterized in that the device comprises a material selected from the group consisting of medical tape, medical plaster, gauze, TORII band, Finn chamber and Perm-aide S. 3. The device according to claim 2, characterized in that the antigen is derived from microbacterial species comprising complex of M. tubers is, M. avium-int racellulare, M. kansas ii, M. ortuitum, M. chelonae, M. leprae, M. africanum and M. mi croti. 4. The device according to claim 3, characterized in that the antigen comprises MPB44, MPB45, MPB51, MPB59, MPB64, MPB70, MPB80 or MPB83. The device according to claim 1, characterized in that the physiologically effective solution comprises surfactants, regulators or solvents. The device according to claim 5, characterized in that the physiologically effective solution comprises phosphate buffered solution comprising Tween 20, Tween 40, Tween 60 and Tween 80. 7. The device according to claim 2, characterized in that the antigen comprises MPB64 and the physiologically effective solution comprises regulated solution of phosphate and Tween 80. 8. The device according to claim 7, characterized in that the infectious disease comprises tuberculosis. 9. A method for detecting active disease comprising: topically applying a transdermal delivery device to the skin, wherein said device comprises an antigenic composition; withdrawing said transdermal delivery device after a predetermined period of time; observe the skin for an immune response; and correlate the presence of the immune response with the active disease. The method according to claim 9, characterized in that the transdermal delivery device comprises a material selected from the group consisting of medical tape, medical plaster, gauze, TORII band, Finn chamber and Perm-aide S. 11. The method according to claim 9, characterized in that the antigenic composition comprises a physiologically effective solution; it also comprises an antigen derived from microbacterial species selected from the group consisting of M. tubers is complex, M. avium-intracellulare, M. kansasii, M. fortuitum, M. chelonae, M. leprae, M. a ricanum and M. microti. The method according to claim 11, characterized in that the antigen comprises MPB44, MPB45, MPB51, MPB59, MPB64, MPB70, MPB80 or MPB83. 13. The method according to claim 11, characterized in that the physiologically effective solution comprises surfactants, regulators or solvents. 5 14. The method according to the indication 13, characterized in that the physiologically effective solution comprises phosphate buffered solution further comprising Tween 20, Tween 40, Tween 60 or Tween 80. The method according to the claim 11, characterized in that the antigenic composition comprises MPB64 and phosphate buffered solution further comprising Tween 80. 16. The method according to claim 15, characterized in that the active disease comprises tuberculosis. 17. A composition for detecting infectious disease comprising: an antigen, and a physiologically effective solution for its transdermal delivery after topical application. 18. The composition according to claim 17, characterized in that the 25 antigen is derived from microbacteria. 19. The composition according to claim 18, characterized in that the antigen comprises MPB44, MPB45, MPB51, MPB59, MPB64, MPB70, MPB80 or MPB83. The composition according to claim 19, characterized in that the physiologically effective solution for its transdermal delivery comprises surfactants, regulators and solvents.