WO1996013615A1 - Identification of infection with flow cytometry - Google Patents

Abstract

The invention encompasses flow cytometric methods for detecting intracellular pathogens in cells at extremely low levels of infection. In particular, the method provides for the early and accurate detection of Leishmania-infected monocytes in peripheral blood and other body fluids and liquid suspensions of tissue-derived cells. This specific and highly sensitive flow cytometric method is useful as a technique for assessment of parasites in blood, and may also identify persons and animals who are undergoing active infections with Leishmania sp. or other intracellular pathogens.

Description

IDENTIFICATION OF INFECTION WITH FLOW

CYTOMETRY

Field of the Invention

The invention relates to diagnosis of infection by intracellular pathogens, and more particularly to the detection and quantification of various intracellular pathogens using flow cytometric techniques.

Background of the Invention

Intracellular pathogens are parasitic microbes that cross cell barriers and dwell within cells, particularly body fluid cells, for extended periods of time without destroying the host cells. Parasitic microbes include the following; bacteria, such as Salmonella sp., Shigella sp., Bordatella sp., Rickettsia sp., Chlamydia sp., Rochelamaia sp., Coxiella sp., Yersinia sp., Mycobacteria sp., Listeria sp., Brucella sp., Francisella sp., Legionella sp., Ehrlichia sp.; viruses such as HIV, influenza, measles, mumps, CMV, arenaviridae, paramyxoviridae, coronaviridae, togaviridea, and retroviruses; parasites, including protozoan parasites, such as Plasmodium sp. (in red blood cells), Leishmania sp. (in monocytes and macrophages), certain trypanosomes (Trypanosoma cruz i in monocytes/macrophages and myocardial cells), Theileria (in T lymphocytes), Babesia (in red blood cells), Erwinia; and fungi, such as Candida albicans and other species Pneumocystis sp., Blastomyces sp., and Histoplasma sp.

The detection of infection by parasitic microbes is difficult because they reside within host cells. Consequently, they cannot easily be detected or measured directly in circulating body fluids, but rather can only be detected by indirect methods such as serological screens. However, serological testing fails to detect those microorganisms that do not elicit measurable changes in serology. Additionally, serological testing fails to detect parasitic infection during the early stages and at extremely low levels of infection.

Serological testing relies on the presence and increase in titer of antibodies (serum proteins) of a certain immunoglobulin class (either IgG or IgM) that specifically interact with a pathogen or its toxic secreted factors. Humoral immune responses, those that lead to the development of pathogen-specific antibodies, are generally stimulated in the first few days of host exposure to the infectious pathogen, and these specific antibody titers in vivo increase over time. Many intracellular pathogens, however, fail to induce detectable levels of antibodies in infected animals and man until disease is well established and clinical symptoms are apparent. Delayed recognition by the humoral immune system likely reflects the intracellular niche of such organisms and a resulting lack of antigens for stimulation of immune cells in the extracellular milieu. Diagnosis of these infections by standard antibody- based techniques (complement fixation, hemagglutination, ELISA, RIA) is difficult, if not impossible, during the earliest phases of disease. Yet successful eradication of these sequestered pathogens depends upon a rapid and correct diagnosis, and prompt treatment. There are a number of infections (Hanta virus infections, HIV infection, malaria, leishmaniasis) where antibodies appear well after infection, or death precedes immune recognition and antibody production.

Several approaches for direct detection of pathogens are attempted as an alternative to serology: culture and quantitation of the pathogen from blood and tissues, PCR detection of specific DNA or mRNA in infected tissues, in situ hybridization for specific DNA or mRNA in cell smears or tissue sections. Each of these methods has its strengths and weaknesses.

Culture of the organism from potentially-infected tissues is the least satisfactory, since intracellular pathogens are inherently fragile in extracellular fluids, and methods for disruption of cells to release the pathogen are generally quite drastic. In addition, some intracellular pathogens cannot be cultured in the absence of cells, which makes isolation and quantitation difficult. The advantage of this technique, when it works, is that one can actually see the pathogen, and its phenotype can be confirmed by a variety of techniques (isoenzyme analysis, indirect fluorescence, or others). Culture of organisms, then, can result in a remarkably specific diagnosis, but it is neither easy nor particularly sensitive. For many pathogens, it is also not quick: Leishmania amastigotes released from infected cells and cultured in special media take days to weeks to convert to promastigotes and replicate enough to be detectable.

PCR, on the other hand, has the potential to be very sensitive. Isolation of DNA or RNA is directly from infected tissues; if the probe is monospecific for a parasite antigen not present in host tissue, PCR can amplify the less abundant pathogen DNA or RNA to levels that can assuredly pinpoint the presence of the pathogen. This technique, however, has serious specificity problems. It could be used as a screening technique for classes of organisms, but speciation is problematic. Moreover, each stage of the technique, from the isolation of the nucleic acids to the amplification steps, is complicated, requires skilled technicians, and introduces a potential for false positives or false negatives. Unlike culture, PCR results cannot be verified by direct observation of the organisms: both the tissue and the pathogen are solubilized for isolation of nucleic acids. PCR, then, is sensitive and fairly specific (at least within classes of microorganisms), but is difficult to perform and has reproducibility problems from laboratory to laboratory.

In situ hybridization for specific nucleic acids is similar to PCR, but substantially less sensitive. If the frequency of infected cells in a tissue or cell smear is less than 0.1% (1 in a thousand), infected cells are difficult to detect. Most in situ protocols use radioactivity to enhance sensitivity, but use of radioactive reagents is problematic for a number of environmental and personal safety issues. Thus in situ hybridization is relatively specific (within classes of microorganism), but is not sensitive or easy to perform.

Because of the limitations inherent in serological testing, tissue biopsies are often performed to detect the presence of infection by parasitic microbes. This method of testing also has several drawbacks. Tissue biopsies can be painful, and create a risk of infection. Assaying the tissue may require staining and microscopic observation, which is expensive and time consuming, and relies on subjective analysis by the person observing the specimen. Further, quantification of the degree of infection is difficult and is based on a very limited sample, thus degrading the statistical accuracy of the measurement. As an alternative to microscopic analysis, tissue biopsies may be cultured to determine the presence and amount of parasitic infection. The biopsy material may also be used as a source for the isolation of the parasitic microbe. These techniques are time consuming, complicated and expensive.

Still further, if there is sufficient material, tissue biopsies may be homogenized to create a cell lysate and used directly in immunological assays with detection antibodies of a known pathogen specificity. The production of cell lysate, however, creates handling problems and releases potentially interfering cellular substances that may adversely affect the performance of the subsequent immunological assay. Further, this method fails to discriminate between internally and externally located microbes, and does not provide information about cell cycle or stage of infection.

Leishmania are parasitic microbes that are difficult to detect and quantify. For example, military personnel who participated in Operations Desert Storm/Shield

(ODS) were screened for a range of infectious diseases endemic to the Middle East. Twenty eight reported cases of leishmaniasis were confirmed by a combination of serology and/or identification of parasites in tissue biopsies (Magill et al., 1992; Magill et al., 1993). All of the confirmed infections were caused by Leishmania tropica and L. major, both of these strains of Leishmania sp. are endemic to the Middle East, and are generally thought to cause simple self-curing cutaneous disease with no systemic complications. However, physicians identified 11 individuals with visceral manifestations of leishmaniasis (Magill et al., 1993); in most instances parasites were isolated from bone marrow aspirates or biopsies of lymph nodes. Based on isoenzyme characterization of those infections from which sufficient parasites were recovered, the etiologic agent was determined to be L. tropica.

The unexpected diagnosis of visceral leishmaniasis as a result of infection with a normally cutaneous form of the parasite is somewhat perplexing. Indeed, estimates of the extent of Operation Desert Storm military personnel potentially infected with viscerotropic L. tropica were complicated further by clinical symptoms which do not conform to those classically associated with visceral leishmaniasis or kala-azar (i.e., hepatosplenomegaly, pancytopenia, hyperglobulinemia).

The presence of . -ropt^'cα-infected monocytes in the bone marrow raised concerns regarding the potential transmission of leishmaniasis via transfusion of contaminated blood. In fact, transmission of visceral leishmaniasis via blood transfusion has been reported previously (Cohen et al., 1991).

Because of these concerns, all individuals who served in Operation Desert Storm were deferred from blood donation effective November, 1991. Deferral of blood donation was problematic, however, since a sensitive test to screen blood for Leishmania-mfect ά monocytes did not, and still does not, exist. A sensitive and specific test to screen blood for Leishman -infection remains a critical need for ensuring a safe blood supply under similar circumstances in the future. Such a test is also critical for the rapid detection and early diagnosis of e--./ιmα ^'α-infection through fairly non-invasive means.

The terms "flow cytometry" and "flow cytometric" and "cell sorter" refer to the instruments and procedures whereby a population of cells in a liquid suspension is directed through a fine liquid stream passing by the cytometer's laser into a device capable of measuring the physical and/or chemical characteristics of cells based on the light quanta emitted. The light passing through each cell is measured individually and can represent physical characteristics of cells that are unlabeled and also cells that have various types of labels attached. One type of label commonly used is fluorescent and may be attached to molecules on the outside or inside of cells. The labels generally are light emitting fluorescent tags such as phycoerythrin, fluorescein (green light) or rhodamine (red light). The fluorescent label often is conjugated to a binding agent, such as an antibody, capable of binding to a component of the cell surface. Cell fluorescence is indicative of the presence of the binding partner, such as an antigen, of the binding agent on the cell surface. The intensity of the fluorescence is a function of the number of fluorescent labels bound per cell, and is thus related to the number of binding partners available on the surface of the cell.

The cell sorter exposes the cells moving through the liquid stream to light, usually a specific wavelength, known as the excitation wavelength, that corresponds to the fluorescent label used. In response to excitation wavelengths, the fluorescent label fluoresces and emits light. The cell sorter detects and records the emission of light, both the number of discrete occurrences and the intensity of the light emitted. Optionally, the cell sorter can momentarily divert the cell stream so as to separate fluorescing cells from non-fluorescing cells, or separate cells having different wavelength fluorescence, or those having fluorescence exceeding a certain predetermined minimum intensity from the rest of the cells in the population. The light quanta emitted is measured for each cell individually and presented as such in data representations.

Thus, a population of cells can be characterized by parameters such as number of fluorescing cells, number of non- fluorescing cells, and population profiles of cells having various fluorescence intensities (See Figures 1 - 9).

There are a number of ways to enhance the sensitivity and specificity of the flow cytometry detection technique. These include, but are not limited to, labeling the cell preparations for both characteristic cell surface antigens and the intracellular pathogen to improve the frequency of incidences (especially good for pathogens with limited host cell range, such as the Leishmania), directly labeling the specific anti-pathogen antibody to improve fluorescence intensity, and identification of monoclonal antibodies that recognize intraspecies and interspecies variations to improve specificity.

The Leishmania replicate primarily in monocytes and tissue macrophages. One can gate the cell cytometry machine to specifically select only monocytes and macrophages in a cell population, thereby reducing the number of cells examined in the assay. Leishmania sp. upregulate several cell surface receptors on macrophages which can also be used for diagnostic purposes. Thus, the sensitivity and specificity of this assay may be greatly enhanced by using a cocktail of antibodies directed at the infected cell type, up-regulated surface receptors, and the parasite itself. Other pathogens are found specifically in monocytes in peripheral circulation, including Mycobacteria, Rickettsia, Francisella, Trypanosoma cruzi. Still other pathogens are found specifically in lymphocytes, including HIV and Theileria. Lymphocytes can be selected for by both size and specific surface markers.

The specificity of the assay depends upon the use of monoclonal antibodies to identify infected cells. In addition, cells that do not have antibodies or fluorescent labels attached have characteristic light diffraction patterns based on their physical properties. The diffraction patterns may be indicative of changes in cellular physiology and morphology within a particular cell population.

The prior art is further characterized by the following references:

Bertho AL, Cysne L, Coutinho SG: Flow cytometry in the study of the interaction between murine macrophages and the protozoan parasite Le ishmania amazonensis. J Parasitol 78: 666, 1992 Bjerknes R, Bassoe,C-F: Phagocyte C3-mediated attachment and intemalization: flow cytometric studies using a fluorescence quenching technique. Blut 49: 315, 1984

Cohen C, Corazza F, Mol PD, Brasseur D: Leishmaniasis acquired in Belgium. Lancet 338: 128, 1991

Eperon S, McMahon-Pratt D.: Extracellular amastigote-like forms of Leishmania panamensis and L. braziliensis. II. Stage- and species-specific monoclonal antibodies. J Protozool 36: 510, 1989

Hallden G, Andersson U, Hed J, Johansson S: A new membrane permeabilization method for the detection of intracellular antigens by flow cytometry. J Immunological Meth 124: 103, 1989

Jackson PR, Pappas MG, Hansen BD: Fluorogenic substrate detection of viable intracellular and extracellular pathogenic protozoa. Science 227: 435, 1985

Jaffe JL, McMahon-Pratt D: Monoclonal antibodies specific for Leishmania tropica. I. Characterization of antigens associated with stage- and species-specific determinants. J Immunol 131: 1987, 1983

Handman E: Study of Leishmania major-infected macrophages by use of lipophosphoglycan-specific monoclonal antibodies. Infect Imm 58: 2297, 1990

Magill AJ, Gasser RA, Oster CN, Grogl M, Sun W: Viscerotropic leishmaniasis in persons returning from

Operation Desert Storm - 1990-1991. MMWR 41: 131, 1992

Magill AJ, Grogl M, Gasser RA, Sun W, Oster CN: Visceral infection caused by Leishmania tropica in veterans of Operation Desert Storm. New Eng J Med 328: 1383, 1993 Pattanapanyasat K, Webster HK, Udomsangpetch

R, Wanachiwanawin W, Yongvanitchit K: Flow Cytometric two-color staining technique for simultaneous determination of human erythrocyte membrane antigen and intracellular malarial DNA. Cytometry 13: 182, 1992

Sander B, Anderson J, Andersson U. Assessment of cytokines by immunofluorescence and the paraformaldehyde-saponin procedure. Immunol Rev 119: 65, 1991.

Pattanapanyasat et al. (1992) detected malarial parasites within red blood cells by staining the DNA chemically with propidium iodide. This indirect approach would be not be applicable to nucleated cells, such as monocytes, because the staining of monocytic DNA with propidium iodide would interfere with detection of viral DNA.

Furthermore, this approach does not directly identify the organism based on its inherent distinguishing characteristics, for example a surface antigen or membrane amino acid sequence found only in a particular organism such as Leishmania tropica. What is needed is a specific and highly sensitive flow cytometric method for the detection of intracellular pathogens in body fluids. Such a flow cytometric method would be useful for rapidly and accurately screening the large numbers of samples handled by blood banks. This method would aid in the prevention of contamination of blood banks, thereby decreasing the spread of infectious disease to individuals receiving transfusions. Another benefit of this method is the rapid identification of infected individuals so that treatment can be initiated at an earlier stage. Summary of the Invention

Accordingly, the present invention encompasses flow cytometric methods capable of detecting intracellular pathogens in cells at extremely low levels of infection. The invention provides methods for accurate detection of pathogen- infected cells in blood (red blood cells, monocytes, lymphocytes, polymorphonuclear leukocytes), bone marrow, and other tissues and body fluids. In particular, the method provides for the early and accurate detection of parasite infected monocytes in peripheral blood and other body fluids and liquid suspensions of tissue-derived cells. This specific and highly sensitive flow cytometric method is useful as a technique for assessment of parasites in blood, and may also identify persons and animals who are undergoing active infections with Leishmania sp. or other intracellular pathogens such as HIV, Brucella and others.

It is an object of the present invention to provide a method for detecting intracellular pathogens in body fluids and tissues.

It is yet another object of the present invention to provide a method to detect intracellular pathogens specifically in cells isolated from bodily fluids including, but not limited to, blood, lymph, bile, cerebrospinal fluid, saliva, lacrimal secretions, gastrointestinal secretions, peritoneal fluid, pleural fluid, and urine.

Still another object of the present invention is to provide a method to detect intracellular pathogens in cells derived from various tissues of the body including, but not limited to, epithelium, muscle, connective tissue, nervous tissue, and glandular tissue. Cells may be derived from scrapings, biopsies, lavages, crude homogenates, and dispersions. Cells may also be obtained from cultures of cells derived from organisms suspected of infection. Yet another object of the present invention is to provide a method to detect infections of the cells of the female reproductive system, obtained through scrapings, biopsies, lavages or organ removal.

Still another object of the present invention is to provide a method to screen for infections of the respiratory system, gastrointestinal system, nervous system, muscular system, urinary system, and lymphatic system.

Another object of the present invention is to provide a sensitive method to detect intracellular pathogens, such pathogens including, but not limited to, parasites, bacteria, viruses, and fungi.

Brief Description of the Figures

Figure 1 graphically depicts monoclonal antibody- specific indirect single color fluorescence in human monocytes infected with L. major, or left uninfected. In panels A-D a monoclonal antibody to vimentin was used. In panels E-F a monoclonal antibody to L. major was used. The fluorescence of control monocytes is shown for comparative purposes.

Figure 2 graphically depicts monoclonal antibody- specific indirect single color fluorescence in L. major infected human monocytes as a function of serial dilution of the monocytes in uninfected monocytes.

Figure 3 graphically depicts monoclonal antibody- specific indirect single color fluorescence in L. major infected human monocytes as a function of serial dilution of the monocytes in uninfected monocytes, wherein lymphocytes have been removed from the data analysis and only dilutions up to 1/16,386 have been included, wherein (A) is an uninfected control; (B) is a 1/4 dilution; (C) is a 1/32 dilution; (D) is a

1/256 dilution; (E) is a 1/2,048 dilution; and (F) is a 1/16,384 dilution. The area indicated by the rectangle to the right of Rl includes the area where infected cells are detected.

Figure 4 graphically depicts monoclonal antibody- specific indirect single color fluorescence in L. major infected human monocytes as a function of serial dilution of the monocytes in uninfected monocytes and shows a theoretical lower level limit of detection of approximately 75 infected cells per 10,000 uninfected cells.

Figure 5 shows the relative fluorescence intensity of monoclonal antibodies (MoAbs) specific for vimentin (A-F) and Leishmania major (G-L), in the presence of PBS or saponin, when used to stain uninfected human monocytes (column 1, A,D,G,J), and those infected with promastigotes (column 2, B,E,H,K) or amastigotes (column 3, C,F,I,L) of L. major. Monocytes reactive for these MoAbs were identified by indirect, single-color immunofluorescence with phycoerythrin conjugated goat anti-mouse IgG (Fab')2 MoAb. In each instance, staining for vimentin or L. major (gray open histogram) is presented in comparison to staining with control

MoAb (solid red or blue histogram).

Figure 6 displays the relative fluorescence intensity of MoAb specific for Leishmania major with infected monocytes (~65% infected) serially diluted (1/2-1/16) in uninfected monocytes. For clarity, lymphocytes have been gated-out from these analyses. Monocytes considered to be infected appear as red dots in the region 1 (Rl), which was established based on MoAb treated, uninfected monocytes. Monocytes reactive for the L. major-MoAb were identified by indirect single-color immunofluorescence with phycoerythrin conjugated goat anti-mouse IgG (Fab')2 MoAb.

Figure 7 graphically depicts the low dilutions of Le ishmania-m^' f ecteά monocytes represented in Figure 6. Monocytes are represented as histograms based on relative fluorescent intensity with infected monocytes demonstrating increased fluorescence compared to uninfected cells. For clarity of presentation, lymphocytes have been gated out from data analyses. Monocytes reactive for these MoAbs were identified by indirect single-color immunofluorescence with phycoerythrin conjugated goat anti-mouse IgG (Fab')2 MoAb.

Figure 8 shows the detection of Leishmania- infected monocytes in peripheral blood seeded with monocytes previously infected in vitro. Monocytes were infected with L. major in vitro, serially diluted in uninfected monocytes, and 1 x 10*5 monocytes from each dilution were introduced into peripheral blood collected in LeucoPREP tubes (E-I). Based on control MoAb treated cells (D), region Rl (the area enclosed by the rectangle to the left) was established to represent where infected monocytes (represented as red dots) were expected to be observed. Based on calculations of infected monocytes in culture, it was predicted that tubes E-I would contain 20, 10, 5, 2.5, and 1.25 infected cells per 2,000 events, respectively. Contained within Rl is the actual number of infected monocytes determined by flow cytometric analysis. Note the high degree of correspondence between expected and observed results. Monocytes reactive for these MoAbs were identified by indirect single-color immunofluorescence with phycoerythrin conjugated goat anti-mouse IgG (Fab')2 MoAb.

Figure 9 presents the identification of Leishmania- infected monocytes in peripheral blood of human patients suspected of being infected with L. chagasi. Patient #1 was reported to be culture negative (Figure 9A), while patient #2 (Figure 9B) was culture positive for infection with L. chagasi. Cells from each patient were stained with vimentin, control MoAb, and anύ-Leishmania MoAb. Determinations of infection were based on a comparison of staining between control MoAb and MoAb specific for Leishmania spp.

Figure 10 shows a photomicrograph of Wright stained slides of a Leishmania infected population of human monocytes.

Figure 1 1 represents flow cytometric identification of CD45, CD13 and Leishmania. Panels A and B represent populations of uninfected control cells screened for

CD45 (panel A) or CD 13 (panel B) respectively. Panels C and D represent populations of infected cells screened for CD45 (panel C) or CD 13 (panel D) respectively.

Detailed Description

The invention encompasses a flow cytometric method for the detection of intracellular pathogens. An advantage of the method is the rapid, early, and accurate detection and quantification of infection, even at extremely low levels (figures 3, 4, and 7). The novel method is useful for rapidly testing large numbers of samples, and provides a high degree of reliability and assurance. Thus, the method is particularly suited to screening of blood banks and is an invaluable aid to establishing and maintaining the integrity of the blood supply.

Intracellular pathogens detectable using the disclosed method include: bacteria such as; Salmonella sp., Shigella sp., Bordatella sp., Rickettsia sp., Chlamydia sp., Rochelamaia sp., Coxiella sp., Yersinia sp., Mycobacteria sp., Listeria sp., Brucella sp., Francisella sp., Legionella sp., and E IPhhrrlliirchhiina s snp * viruses such as; HIV, influenza, measles, mumps, CMV, etc. [arenaviridae, paramyxoviridae, coronaviridae, togaviridea, and retroviruses];

parasites such as; many protozoan parasites, including Plasmodium sp. (in red blood cells), Leishmania sp.

(in monocytes and macrophages), certain trypanosomes (Trypanosoma cruzi in monocytes/macrophages and myocardial cells), Theileria (in T lymphocytes), Babesia (in red blood cells), and Erwinia; and

fungi such as; Candida albicans and other species

Pneumocystis sp., Blastomyces sp., and Histoplasma sp.

Cells used in the flow cytometric method can be derived from any source. Body fluids such as blood, lymph, cerebrospinal fluid, peritoneal fluid, pleural fluid, urine, and saliva are suitable sources of cells. Additionally, tissue samples, such as bone marrow or liver, may be treated so as to dissociate the tissue into a suspension of individual cells. Body fluids are particularly desirable sources of cells to be screened for intracellular pathogens because they are easily obtained and do not require extensive handling such as tissue disruption and filtering to remove undissociated cellular debris. Further, blood, lymph and bone marrow contain cells such as monocytes that commonly serve as the host cell for intracellular pathogens. Studies in our laboratory confirmed that Leishmania sp. could survive in whole blood maintained under blood bank conditions for > 35 days (Leiby et al., unpublished observations).

Flow cytometric analysis of cells for the presence and quantity of intracellular pathogens generally requires the steps of: (i) obtaining a sample of cells suspected of being infected with an intracellular pathogen comprising a suspension of cells;

(ii) adding to the suspension of cells a fixing agent in amount sufficient to fix the cells;

(iii) washing the fixed cells to remove the fixing agent;

(iv) adding to the suspension of fixed cells a permeabilizing agent in an amount sufficient to permit the entry into the cell of a binding agent-label conjugate capable of selectively binding to an intracellular pathogen antigen;

(v) adding the binding agent-label conjugate to the suspension of fixed and permeabilized cells under conditions sufficient to permit the binding of the binding agent-label to the intracellular pathogen antigen;

(vi) washing the cells to remove unbound binding agent-label conjugate; and

(vii) detecting bound binding agent-label conjugate using flow cytometric techniques.

A sample comprising a suspension of isolated cells is treated with a fixing agent to stabilize the cellular structure such that the cellular structure does not fall apart when exposed to permeabilizing agent and other solutions. Examples of suitable fixing agents include glutaraldehyde and paraformaldehyde. The range of concentrations of these fixatives is generally 0.01 to 8% for paraformaldehyde, and 0.001 to 2% for glutaraldehyde. Combinations of these two reagents may also be employed. Preferred concentrations of paraformaldehyde are 0.1 to 6% with the most preferred concentrations of 1 to 4%. Preferred concentrations of glutaraldehyde are 0.01 to 1% with the most preferred concentrations of 0.05 to 0.5%. The particular concentrations of fixatives are adjusted to optimally fix a particular cell type.

Concurrently or subsequently the cells are exposed to a permeabilizing agent that disrupts the cellular membrane sufficiently to permit a binding agent conjugated to a detectable label to enter the cell. Permeabilization agents generally are detergent or detergent-like compounds and may include, but are not limited to the following reagents; Saponin, Triton X-100, lysolecithin, n-Octyl-B-D-glucopyranoside, and Tween 20. A desirable permeabilizing agent is saponin (Figure

1 and 5). When permeabilization is performed after fixation, the cells are rinsed several times to remove the fixative. Optionally, a blocking step may be included at some point prior to the addition to the suspension of the intracellular pathogen-specific binding agent. For example, excess human

IgG can be added after fixing to reduce non-specific binding of the binding agent to the cell surface. Verification of adequate permeabilization is obtained by immunocytochemically labeling a normal cellular component such as cytoskeletal elements. Antisera to vimentin or other cytoskeletal proteins may be used (Figures 1, 5, 6, 8, and 9). In order to analyze the optimal concentration of permeabilizing agent that does not deleteriously affect cellular morphology, cells may be examined in the light microscope.

Either concurrent with, or subsequent to, permeabilization of the cellular membrane, but in the absence of the fixing agent, a binding agent conjugated with a detectable label is contacted with the cells. The mixture of permeabilized cells and binding reagent-label conjugate is incubated under conditions sufficient to permit the binding agent-label conjugate to penetrate the cellular membrane, enter the cells and bind to intracellular pathogens. Next the unbound binding agent-label conjugate is washed away from the cells. This step can be conducted either in the presence or absence of permeabilizing agent. Usually, all steps involving exposure of the cells to the binding agent-label conjugate or other immunological reagents are performed in the presence of permeabilizing agent, although in some circumstances, permeabilization may be conducted in a single step.

Alternatively, the permeabilizing agent can be removed from the cell suspension in subsequent wash steps. Optionally, the permeabilizing agent may be maintained in the cell suspension. When saponin is used as a permeabilizing agent, it is usually present in all steps. Concentrations of saponin which are employed are between 0.001 and 5%, with preferred concentrations of 0.05 to 1%, with the most preferred concentration of 0.01 to 0.1 %. The optimal concentration is determined for each cell type employed.

The cell suspension is then analyzed by flow cytometry to detect and quantify the number of cells that fluoresce or the amount of fluorescence in response to exposure to the excitation wavelength of the fluorescent label. Forward and side scatter patterns are produced by the flow cytometer.

Binding agents are characterized by their ability to selectively bind to intracellular pathogens while not binding to non-intracellular pathogen material. Binding agents must also be capable of being conjugated to labels detectable by flow cytometry. Examples of suitable binding agents include monoclonal and polyclonal antibodies that bind specifically to intracellular pathogen antigens, ligands that bind specifically to intracellular pathogen receptors, receptors that bind specifically to intracellular pathogen ligands, and lectins, selectins and the like that bind to intracellular pathogen components. Labels include any label detectable by flow cytometry that can be conjugated to binding agents. Suitable labels may be fluorescent, including but not limited to fluorescein, rhodamine, Texas red and phycoerythrin. The binding reagent-label conjugate is made according to any of the many conjugation methods known to one skilled in the art. The conjugate may be formed as the result of direct conjugation of the binding agent to the label, or by crosslinking through a hetero- or homobifunctional crosslinker. For example, there are a variety of amine-based and sulfhydryl-based conjugation methods well known to those skilled in the art. Other conjugation methods include glutaraldehyde. Binding agents such as antibodies may also be directly coupled to labels such as fluorescein isothiocyanate.

Other indirect methods of visualizing binding agents include avidin-biotin methods coupled with fluorescent label or enzymes capabale of generating a color product in the presence of the appropriate substrate to amplify the signal to noise ratio.

We initially identified infected cells by an indirect method: a fluorescence labeled secondary antibody is used to identify the primary antibody specific for the pathogen. Direct staining would greatly increase sensitivity and reduce background fluorescence. One can directly conjugate monoclonal antibodies with fluorescent or avidin-biotin labels to enhance sensitivity of the detection system.

The invention will be more fully understood in light of the following examples which are not to be construed in any way as imposing limitations upon the scope thereof. On the contrary, it is to be clearly understood that resort may be had to various other embodiments, modifications, and equivalents thereof which, after reading the description herein, may suggest themselves to those skilled in the art without departing from the spirit of the present invention and/or the scope of the appended claims. Example I

Isolation and culture of Leishmania.

All experimental Leishmania-infecύons were initiated with a cutaneous isolate of L. major (NIH strain 173) that is maintained by serial passage in footpads of BALB/cByJ

(Jackson Laboratory, Bar Harbor, ME) and/or CB-17/scid (Taconic Farms, Germantown, NY) mice. Monodispersed amastigotes (intracellular form of the parasite) were obtained by disruption of infected footpad tissue and passage through no. 50 stainless steel mesh screen into RPMI (GIBCO, Grand

Island, NY) containing 10% heat inactivated fetal bovine serum (ΔFBS; HyClone Laboratories Inc, Logan, UT) and 50 μg/ml gentamicin sulfate. Amastigotes were released from infected macrophages by homogenization in a tissue homogenizer; tissue debris was removed by centrifugation of the suspension at 200 x g. Parasites were stained by fluorescein diacetate/ethidium bromide, and were counted in a hemacytometer (Jackson et al., 1985).

Cultures of L. major promastigotes (extracellular

7 form) were initiated by seeding 1 x 10 amastigotes into 75 c m flasks containing 3 ml of SM media (GIBCO) supplemented with DMEM (GIBCO), ΔFBS, glutamine, and gentamicin (Cunningham, 1977). Promastigote cultures were maintained at 25°C for 8-10 days, or until promastigotes had reached stationary phase.

Isolation and culture of monocytes.

Monocytes were recovered from peripheral blood mononuclear cells (PBMC) of HIV and hepatitis B- seronegative donors after leukapheresis and purified by countercurrent centrifugation elutriation. Cell suspensions were generally >95% monocytes by criteria of cell morphology on Wright-stained slides prepared by cytocentrifugation (Diff-Quick, Dade Diagnostics, Aquado, PR), by granular oxidase, and by non-specific esterase. Monocytes were cultured as suspensions in RPMI 1640 containing 10% ΔFBS and gentamicin, at a concentration of 1 x 10 cells/ml in polypropylene tubes.

Isolation of mononuclear cells from peripheral blood.

Mononuclear cells were separated from peripheral blood using LeucoPREP tubes (available from Becton-Dickinson, Rutherford, NJ). Blood was drawn from a laboratory volunteer directly into the LeucoPREP tube, inverted several times and centrifuged at 1,500 x g for 20 min, at room temperature. Following centrifugation, mononuclear cells were contained within the top plasma layer, separated from the remainder of the cells by a gel. The tube was inverted several times to resuspend the mononuclear cells which were then decanted off. Cells were washed repeatedly in RPMI 1640 containing ΔFBS and resuspended in polypropylene tubes at a concentration of 1 x 10 monocytes/ml using the same media.

Leishmania-infection of monocyte/mononuclear cell targets.

Purified monocytes or mononuclear cells obtained from LeucoPREP tubes were infected at multiplicities of infection ranging from 5: 1 to 1 : 1 (i.e., amastigotes or promastigotes: cells). Amastigotes or promastigotes (1 x 10 - 5 x 10 ) were added to tubes containing mononuclear cells and incubated at 37°C for 1-5 days. Prior to flow cytometric analyses, the intensity of /zmα ^'α-infection was determined by examination of Wright-stained slides prepared by cytocentrifugation. Development of reactive monoclonal antibodies.

Selection of the monoclonal antibody with the greatest specificity for our isolate of L. major was determined by immunofluorescence experiments carried out using a panel of monoclonal antibodies specific for Leishmania spp.

Preparation and characterization of these antibodies were described by Jaffe and McMahon-Pratt (1983), which is hereby incorporated by reference in its entirety. Briefly, various strains of Leishmania were obtained from stock cultures. Leishmania membranes were prepared by suspending the parasites in 20 mM tris(hydroxymethyl aminomethane), pH 7.3, containing 40 mM sodium chloride, 10 mM ethylenediaminetetraacetic acid, and 2 mM phenylmethylsulfonyl fluoride. Leishmania promastigotes were disrupted by nitrogen cavitation (1500 psi for 10 rnin.) in a cell disruption bomb (available from Parr Instrument Co., Moline, IL.) and the homogenate was subfractionated by differential centrifugation. Monoclonal antibodies were raised in Balb/c female mice by injection of Leishmania membranes in incomplete Freund's adjuvant. Hybrid cells secreting monoclonal antibodies were prepared by fusing NS-1 (P3- X63/Ag8) mouse plasmacytoma cells (1 x 10 cells) with o spleen cells (1 x 10 cells) isolated from the immunized mice. Cloned secreting hybrid cells were injecting i.p. into pristane- primed BALB/c mice to obtain ascites fluid.

Immunocytochemistry

Immunofluorescence histological experiments were carried out on amastigotes and/or promastigotes of L. major as described previously (Eperon and McMahon-Pratt, 1989). Briefly, amastigotes and/or promastigotes were air dried on slides, stained with Leishmania spp. specific monoclonal antibodies, followed by a fluorescein conjugated goat anti-mouse IgG (Fab')2 (Boehringer Manheim, Indianapolis, IN), and examined by fluorescence microscopy.

Flow cytometric analysis.

The contents of culture tubes containing monocytes/mononuclear cells were transferred to 1.5 ml eppendorf tubes and washed in several changes of PBS by centrifugation. This step and each successive step was done at 4°C. Cells were then fixed and permeabilized using paraformaldehyde/saponin treatment (Sander et al., 1991). Briefly, cells were first fixed in paraformaldehyde (final concentration 2%) for 10 min followed immediately by a blocking step with human IgG for 10 min to reduce non¬ specific binding. Cells were then permeabilized by addition of 1 ml of 0.1% saponin-PBS, centrifuged immediately, and the resulting supernate discarded. The pellet was resuspended and stained with monoclonal antibodies (MoAbs) specific for Leishmania sp., vimentin, or control IgG for 30 min. Vimentin, a cytoskeletal element of cells, served as a marker/control for intracellular staining. Cells were then washed in 0.1% saponin in PBS, the supernate discarded, pellet re-suspended, and stained with phycoerythrin conjugated goat anti-mouse IgG (Fab')2 (Boehringer Mannheim) for 30 min. Cells were then washed in successive changes of 0.1% saponin- PBS (IX), PBS (3X), and analyzed on a FACScan and/or FACSort (Becton-Dickinson, San Jose, CA).

Example II

Selection of monoclonal antibody specific for L. major.

The selection of a highly specific MoAb for identification of Leishmania-mfected monocytes was important in the development of the invention. Equally important was a determination of the optimal dilution of this MoAb that produced the most intense staining with the least amount of background. A panel of MoAbs, specific for Leishmania spp., were tested at several dilutions for reactivity with our isolate of L. major and compared based on immunofluorescence. This panel included a series of MoAbs specific for L. major

(T-series), a cross-reactive MoAb (L-l), and several MoAbs specific for components of L. amazonensis and L. pifanoi (A- series) (Jaffe and McMahon-Pratt, 1987). The most intense staining reactions were produced with MoAbs from the T-l, T-2, and T-3 groups, with the T-3 MoAbs producing the most intense staining with both amastigotes and promastigotes, even at low dilutions (data not shown). Because the most intense staining, with the least amount of background, was observed at a 1/50 dilution of T-3, all subsequent staining with this MoAb was done at this dilution. Surprisingly, there was the general lack of staining with L-l, a MoAb previously identified as cross-reactive for many species of Leishmania. As expected, staining with MoAbs specific for L. amazonensis and L. pifanoi were negative.

Identification of Leishmania-infected monocytes.

Initially, monocytes were infected with amastigotes harvested from BALB/c mice. However, parasite preparations obtained in this manner contain murine IgG that was recognized by the secondary antibody (goat anti-mouse IgG (Fab')2). This resulted in large amounts of background staining even in controls (data not shown). To avoid this problem, promastigotes were used to infect monocytes because they are grown axenically, and thus, are free of contaminating murine IgG.

L. major is an obligate intracellular pathogen of monocytes/macrophages; therefore, optimal visualization of infected cells by flow cytometry requires permeabilization of cells prior to staining. To verify that the cells were indeed permeabilized following saponin treatment, we initially stained for vimentin using cells permeabilized with saponin or treated with only PBS. As shown in Figures 1 and 5, cells which were treated with only PBS, regardless of whether these cells were uninfected (Figure 1A and 5 A) or infected (Figures IB, 5B and 5C), showed no staining with anti-vimentin antibodies. However, treatment of these cells with saponin resulted in nearly 100% of cells with antibody-stained vimentin (Figures ID, IE, and 5D).

Sensitivity of detection using L. major-infected monocytes.

To determine the detection sensitivity for the system, infected monocytes (-90% monocytes, of which -65% were infected) which were serially diluted in a similar population of uninfected monocytes were used. At each dilution 1 x 10 cells were processed for flow cytometry and 5 x 10 cells were cultured in supplemented SM media (25°C) to determine if viable parasites were present by culture. Cells used for flow cytometry were stained using the indirect methods described previously. As demonstrated in Figure 6, infected cells could be identified clearly in dilutions down to

1/16 when compared to uninfected control antibody treated cells. For each serial dilution the representative population of infected cells decreased with a concomitant increase in the proportion of uninfected monocytes.

Additional dilutions of this same infected cell population are shown in Figure 7. In this instance, the presence of infected monocytes was detected in dilutions through 1/1024, a dilution at which the level of sensitivity approaches 1 infected cell in 10,000 uninfected cells (Figure 7). In vitro culture of cells confirmed that viable L. major were present at each dilution examined by flow cytometry. This series of experiments, however, was not designed to determine the limits of detection, but merely to demonstrate the sensitivity of this methodology when cultured monocytes were examined. Sensitivity could be substantially increased by using anύ-Leishmania MoAbs directly conjugated to fluorescent labels or by merely increasing the number of events collected by the cytometer (represented data is only

2,000 events).

Isolation and detection of Leishmania-infected monocytes in peripheral blood.

In order for flow cytometric techniques to be effective as a screening technique for Leishmania spp. in blood, it is necessary to isolate and detect infected monocytes in peripheral blood. For this reason, we isolated mononuclear cells from peripheral blood using citrated LeucoPREP tubes that had been seeded with several different concentrations of human monocytes previously infected in vitro with L. major.

Once isolated and washed several times, the mononuclear cell fraction was stained for . w-α/ør-infected monocytes as described previously. The resulting cytometric profiles for several low concentrations of infected cells are found in Figure 8. As expected for a population of peripheral blood cells, lymphocytes were the predominate subpopulation (-90%). Theoretical calculations based on the percentage of infected monocytes used to seed the LeucoPREP tubes and number of cells recovered from each LeucoPREP tubes suggests that the seeded tubes should contain 20, 10, 5, 2.5, and 1.25 infected cells per 2,000 events (Fig. 8E-I, respectively). As indicated in Figure 8E, 15 infected cells were observed compared to the background levels of 2 observed in the uninfected, antibody treated controls (Fig. 8D). Indeed, the observed number of infected cells at each dilution (Fig. 8E-I) was above background levels (Fig. 8D) in each instance. The numbers of cells actually detected were 15, 11, 8, 4, and 3, respectively. Subtracting the background level of 2 (Figure 8D) from these numbers results in values of 13, 9, 6, 2, and 1 demonstrating a close correspondence between the expected and observed values. This surprising result demonstrates the exquisite sensitivity and accuracy of this method.

Example III

Flow cytometric analysis of patient samples infected with L. chagasi.

Peripheral blood samples from two individuals suspected of being infected with a visceral form of leishmaniasis, L. chagasi, were kindly provided by Dr. Selma M.B. Jeronimo, Universidade Federal do Rio Grande do

Norte, Natal, Brazil. Samples were shipped in LeucoPREP tubes which had been centrifuged prior to shipping to isolate the mononuclear cells. Upon receipt, the mononuclear cells were removed, washed several times in PBS, and processed for flow cytometry as described above for Example 1.

Samples from these individuals were important for validation and verification of our techniques since L. chagasi presents as a visceral infection in the host, and therefore, these patients are likely to have infected monocytes circulating in their blood. Blood samples were drawn into

LeucoPREP tubes and processed for flow cytometry as described for peripheral blood drawn under laboratory conditions. Flow cytometric data were analyzed blind. Figure 9A is from an individual who originally had culture positive leishmaniasis, but after 30 days of therapy no longer presented as being infected. Indeed, bone marrow aspirates from this individual were negative for imαn-α-infection. The flow cytometric data support the clinical findings; a comparison of staining patterns for control- and Λmαm^'α-antibody treated cells from patient #1 appeared virtually identical (see bottom 4 panels of Figure 9A). Patient #2, however, had a markedly different staining profile when the imαmα-antibody was used compared to control antibody (see bottom 4 panels of Figure 9B). This individual had a positive bone marrow aspirate at the time peripheral blood was collected.

These results demonstrate that the method of the present invention detected infection in monocytes from a patient with confirmed infection in a bone marrow aspirate. Furthermore the results show that the method is sufficiently sensitive to demonstrate the absence of infection in monocytes from a patient who was culture negative for L. chagasi.

Example IV

Determination of Permabilizing Parameters

To determine the optimal permeabilizing agent and its concentration, various concentrations of several detergents were tested. Desirable detergents function to maintain cellular structure as measured by FACS, and allow for intracellular identification of pathogens such as L. major. Table 1 shows the results of various detergents used at various concentrations in a FACS assay for CD45 and CD 13 antigens. Optimal permeabilization was determined based on maintenance of CD45 and CD 13 recognition, ability to identify intracellular L. major, and visual appearance of treated cells. The data presentated are representative of three similar experiments.

Table 1

* NP40 and SDS data, not collected due to clogging of fluidics by cellular debris. Cells visibly destroyed when examined on slides.

** High values associated due to total disruption of the cells free parasites Example V

Leishmania Infected Cells are Concentrated and Isolated by Use of Cell Sorting Flow Cytometer.

Populations of human monocytes, wherein some monocytes were infected with Leishmania were cytologically stained using Wright's stain. Only 10% of the unsorted sample were found to be infected. An aliquote of the sample of human monocytes were sorted to select for infected cells. After cell sorting the percentage of infected monocytes was nearly 95%.

Figure 10 shows a photomicrograph of Wright stained slides of a Leishmania infected population of human monocytes selective cell sorting designated to enrich for the infected cell population. After sorting the infected population had been enriched to nearly 95%.

Example V

Identification of Cell Surface Antigens for Enhanced Identification of Leishmania

Staining of infected cells could be enhanced by identification of cell surface antigens. The CD45 antigen is a mojor cell-surface glycoprotein confined to lymphoid and myeloid lineages. Staining for CD45 provides a clear differentiation between an infected cell and free components of parasites. The CD45 antigen is a mojor cell-surface glycoprotein. CD 13 staining identifies the particular type of monocytic cells observed to be infected with L. major. The antibody designated aLeish + FITC is a Leishmania specific antibody conjugated to FITC. For any cell to be considered infected with Leishmania it must be CD45+, CD 13+ and Leishmania. Such multi-marker positives are shown in Figure 11, panels C and D.

This and other embodiments of the invention are encompassed within the scope of the appended claims.

Claims

ClaimsWe claim:

1. A method of detecting intracellular pathogens in body fluids or tissues comprising the steps of:

(i) adding to a suspension of cells suspected of being infected with an intracellular pathogen a fixing agent in an amount sufficient to fix the cells to form a suspension of fixed cells;

(ii) washing the suspension of fixed cells to remove the fixing agent;

(iii) adding to the suspension of fixed cells a permeabilizing agent in an amount sufficient to permit the entry of a binding agent-label conjugate capable of selectively binding to an intracellular pathogen into the fixed cells, under conditions sufficient to permit the binding of the binding agent-label to the intracellular pathogen;

(iv) washing the fixed and permeabilized cells to remove unbound binding agent-label conjugate; and

(v) detecting the bound binding agent-label conjugate using flow cytometric techniques.

2. The method of Claim 1 wherein the permeabilizing agent is selected from the group consisting of Triton X- 100, lysolecithin, n-octyl-b-D-glucopyranoside,

Tween 20, and saponin.

3. The method of Claim 1 wherein the permeabilizing agent is saponin.

4. The method of Claim 1 wherein the binding agent-label conjugate is an antibody-fluorescent label conjugate, antibody-enzyme label conjugate, or antibody-biotin label conjugate.

5. The method of Claim 1 wherein the intracellular pathogen is selected from the group consisting of

bacteria selected from the group consisting of Salmonella sp., Shigella sp., Bordatella sp., Rickettsia sp., Chlamydia sp., Rochelamaia sp., Coxiella sp., Yersinia sp., Mycobacteria s ^■Tp.*,f Listeria sp., Brucella sp., Francisella sp., LLeeggiioonneellllaa sspp..,, aanndd Ehrlichia sp.;

viruses selected from the group consisting of HIV, influenza, measles, mumps, CMV, arenaviridae, paramyxoviridae, coronaviridae, togaviridea, and retroviruses;

parasites selected from the group consisting of Plasmodium sp. , Leishmania sp., trypanosomes, Theileria ,

Babesia, and Erwinia;; and,

fungi selected from the group consisting of Candida albicans , Pneumocystis sp., Blastomyces sp., and Histoplasma sp.