WO1999014309A2

WO1999014309A2 - Rapid filter diagnostic device and method for the direct detection of micro-organisms in body fluids

Info

Publication number: WO1999014309A2
Application number: PCT/EP1998/005483
Authority: WO
Inventors: Roland Maes
Original assignee: Anda Biologicals S.A.
Priority date: 1997-08-27
Filing date: 1998-08-21
Publication date: 1999-03-25
Also published as: AU1752799A; WO1999014309A3

Abstract

The present invention is related to a diagnostic device for the detection of micro-organisms comprising a drum having one or more adsorbing pads with a specific pores size adapted for the micro-organisms to be detected. The present invention is also related to a diagnostic method for the diagnostic of micro-organisms present in a sample.

Description

RAPID FILTER DIAGNOSTIC DEVICE AND METHOD FOR THE DIRECT DETECTION OF MICRO-ORGANISMS IN BODY FLUIDS

Field of the invention

The present invention relates to a new and rapid detection method of micro-organisms and to the means and media used for performing said method.

Background of the invention

Many bacteria and parasites are known to acquire drug resistance and their detection is regularly increasing in importance for Public Health agencies. In particular, micro-organisms causing diseases of epidemic proportions in low income countries are of diagnostic importance because expensive detection means are often inapplicable in those countries where an early and accurate diagnostic is most useful. However, said parasites and bacteria are intrinsically difficult to characterise. Among these diseases, one may cite leishmaniasis, gonorrhoea, syphilis, tuberculosis, malaria, tick-borne Lyme disease, meningitis due to a variety of vectors such as Nei sseria , Haemophil s, Streptococcus, Lis teria and Mycobacteriu , unicellular and multicellular protozoa (amebae, fungi, worms) whose life cycle, mode of reproduction, characteristics of growth or other feature render then difficult either to isolate and grow in vi tro or to be easily recognised by direct observation. Diagnosis means for pathogens are available but are either too cumbersome, too inaccurate or too expensive to be used routinely. The smear microscopy done with sputum, applied for the detection of the tuberculosis bacille is based on bacilloscopy (visualisation of stained bacteria in one hundred microscopic fields on a microscopy glass slide) . The technique ill -applied and costly presents a sensitivity often well below 50% and cumbersome (seven hours of a technician's time is needed _..for each case diagnosed) . The limit of sensitivity reached by the bacilloscopy is around 10^", 000 bacilles per ml of sputum. For Sexually Transmitted Diseases (gonorrhoea and syphilis) , the available diagnostic methods are no better. The immunoenzymatic detection of Chlamydia is characterised by a high yield of false positive results. PCR, culture and immunofluorescence tests exist for said diagnosis, but are either too expensive or unpractical (lack of microscopes, lack of electricity) to be used on a routine basis.

Aims of the invention

The present invention aims to provide a new diagnostic device and method which do not present the drawbacks of the state of the art .

Another aim of the present invention is to provide said diagnostic method and device which is simple and not expensive, and could be applied and used easily without needing expensive apparatus and media like microscopes .

Summary of the invention

The Inventor has discovered that it is possible to retain large micro-organisms such as bacteria, protozoa and worms on the surface of a filtering membrane and thereafter visualise them with a simple immunodetection system based on enzyme, fluorophore, latex beads or gold micellae coupled to immunological reagents.

The invention consists of a drum able to receive a certain number of adsorbing pads. The diameter of these adsorbing pads is standard, and the drum is usually 5 cm in diameter and 1 to 5 cm deep. The depth of the drum is variable, depending on the number of adsorbing pads, and the degree of sensitivity one wishes to obtain (i.e. the volume of sample fluid processed) since the more fluid is processed, the more sensitive will be the method.

The drum is- filled with absorbing pads and topped by a filtering membrane (preferably made of nitrocellulose, nylon or any other adequate material) able to retain micro-organisms. On top of the filtering membrane, one may insert a funnel containing a prefilter (preferably made of glass fiber or paper) in order to hold and discard insoluble materials that may clog or obscure the retaining membrane (when charged with fluids such as sputum or whole blood) . The drum forms for the absorbing material for the fluid tested is very convenient but expensive. Simpler adsorbing devices are also suitable, composed of a flat sheet laminate of the retaining membrane and an underlying take-up pad enclosed within a cardboard holder. The sample to be analysed (sputum, blood, serum, plasma, cephalo-rachidian fluid, urine) is deposited within the pre- filter funnel and the sample fluid is directed into the centre of the retaining membrane, therewith creating de facto a "positive" area surrounded by a "negative" area. The sample fluid deposited in the funnel inserted on top of the retaining membrane is sucked within the drum by the absorbing pads .

Selective retention of micro-organisms on the retaining membrane is achieved by the use of membranes with different pore sizes. Micro-organisms like mycobacteria, whose size is comprised between 0.3-0.6 μm in width and 1-4 μm in length, are retained on a membrane with a pore-size of 0.8 μm to 0.45 μm, or less. The spirochete Trepone a pallidum, that causes syphilis, is 5 to 20 μm in length and 0.09 to 0.5 μm in diameter. Borrelia, that causes relapsing fever, and Borrelia burgdorferi , that causes Lyme disease, are 0.2 to 0.5 μm wide and 3 to 20 μm in length, similar in size to Treponema . . These spirochetes are expected to be retained by membranes of -a pore size of 0.8 μm or less. Neisseria meningi tidis and Neisseria gonorrhoea are gram- negative cocci 0.6 to 1.0 μm in diameter. These organisms are retained by membranes whose pore size is 0.45 μm or less. The trypanosomes causing sleeping sickness, leishmaniasis and Chagas disease are at least 1 to 3 μm. Plasmodium, causing malaria, and Toxoplasma gondii , causing toxoplasmosis, are sporozoa 4 to 7 μm in length that are retained on membranes with a pore diameter of 1.2 μm or less. Worms are retained by membranes that have a pore-size of 3.0 to 5.0 microns or less. Haemophilus (0.2 to 0.3 μm by 0.5 to 0.8 μm) , Streptococcus (2 μm in diameter) and Listeria (chains of 3 to 5 organisms, each 0.4 to 0.5 μm by

0.5 to 2 μm) are etiological agents for meningitis and are also retained on membranes of the appropriate pore-size ranging from 0.2 to 1.2 μm.

The spontaneous retaining power of the device according to the invention does not need to be sensitised with a coupling agent to specifically retain the organisms on its surface (i.e. by protein A that captures antibodies or by antibodies specific for the micro-organisms under investigation) . As micro-organisms are retained on the surface of the membrane, the device according to the invention considerably simplifies and cheapens the elaboration of rapid diagnostic tests for these microorganisms and allows their combination with means and media used in common rapid immunochromatographic tests useful to detect antigens or antibodies (i.e. the pregnancy hormone HCG, the ovulation hormone LH, the cancer marker CEA, the antibodies against HIV, the antibodies against rubella etc . ) . In the diagnostic device of the invention, the whole surface of the retaining membrane may be used to trap the micro-organisms under investigation but an improvement consists in a funnel with a prefilter of glass fiber or paper fitted on top of the membrane, that cleans up and concentrates the sample fluid in a small central spot on the retaining membrane that composes the top surface of a drum containing absorbing pads . Once the micro-organisms under diagnostic examination are collected on the retaining membrane (0.2 to 5.0 μm pore-size, depending on the size of the analysed micro-organism) , the primary clearing prefilter made of glass fiber (to eliminate the solid particles present in sputum and in urine) or paper (for the elimination of red and white blood cells) , is discarded and the retaining membrane is washed with a small amount of washing fluid. This retaining membrane, that is used in conventional tests as a solid carrier for the immobilisation of specific reaction partners whose role is to isolate the reagents under analysis, is used in this described method solely as a filter, retaining spontaneously on its surface the microorganisms under analysis. After the wash, the microorganisms retained on the surface are thereafter labelled with a primary antibody specific for the surface components of the micro-organisms under investigation. This primary antibody is conveniently used under the form of antiserum appropriately diluted but antibodies may be purified and may be coupled directly or indirectly to a marker (such as gold micellaes, enzymes, chromophores, stained latex beads, radioactive labels or fluorophores) . After a second washing step, the primary antibodies attached immunologically to the surface of the micro-organisms retained on the membrane are recognised and visualised by a marker to..which they are coupled or visualised by the coupling with a secondary specific binding partner that may recognise the primary antibody. The secondary binding partner reactive with the primary antibody may be an antibody or any other reagent such as protein A or protein G, coupled with a marker that allows its recognition. Said device and method allow a direct recognition of the retained micro-organisms, either with the naked eye or after enzymatic or radioactive development, or under a light or a fluorescent microscope.

The signal delivered by the secondary label may be further amplified. If gold was used as a marker, a silver amplifier can be applied, that results in the deposit of black silver grains around each gold micelle. If an enzyme as peroxidase is used, an enhancer commercially available (Dako Envision™ kit) may be used to amplify and visualise the enzymatic products of reaction. Advantageously, a third binding partner coupled to a marker and able to recognise the secondary partner of reaction may be used to amplify a third time the positive signal present on the membrane . Various aspects of the present invention will be described in the following non limiting examples, which are presented as illustrations of the present invention. Examples Exampl e 1

Rabbit antibodies against mycobacteria were purchased (Dako, Denmark) . Mycobacterium bovis, strain BCG, was obtained from the Pasteur Institute (Paris, France) and was diluted in physiologic water containing 2% bovine albumin and 0.2% tween 20 to a concentration of 10,000 organisms per ml. The determination of the concentration was made by staining (Ziehl-Nielsen stain) mycobacteria smeared on a microscopic slide and counting the stained microscopic bodies with a microscope. This method of diagnostic is standard. One stained bacterium visible in a single microscopic field is roughly indicative of the presence of 10,000 mycobacteria per ml of solution. Two hundred microliters of the mycobacterial suspension was passed through a nitrocellulose membrane whose mean diameter pore-size was 0.45 μm. After the fluid was passed through the membrane, the membrane was washed with 2 times 100 μl of physiological saline containing 0.2% tween 20. One hundred microliters of rabbit antibodies against mycobacteria, appropriately diluted 1:80 in saline containing 0.2% tween 20, were passed through the membrane, that was thereafter again washed to eliminate non-adsorbed antibodies. One hundred microliters of an appropriately diluted (1:160) solution of a peroxidase-labelled goat antibody against rabbit IgG was then placed on the membrane and subsequently washed twice with 100 μl of saline containing 0.2% tween 20. The PODase retained on the membrane was then visualised with an enzymatic substrate for peroxidase, DAB (3 , 3 ' -diaminobenzidine tetrahydrochloride) , currently used in histochemistry because it precipitates as a black stain after reaction with PODase and H2O2. After the passage of 100 μl of a DAB solution containing H2O2 , the membrane was washed twice with saline. After the enzymatic development and removal of the unreacted DAB, the membrane was dotted with black spots, indicating the presence of mycobacteria on the membrane .

Example 2

A nitro-cellulose membrane with a pore-size of 0.45 μm was used to collect the mycobacteria in an experiment where the experimental conditions were similar to those applied in example 1. After labelling the mycobacteria present on the membrane with specific rabbit antibodies, a secondary partner of reaction consisting of a dextran backbone to which are covalently linked about 100 PODase molecules and up to 20 antibody molecules recognising rabbit IgG per backbone, was applied (Dako

Envision™) . After a wash, the presence of PODase was revealed with DAB. This system considerably increases the sensitivity of the method : solutions containing about 1,000 mycobacteria per ml could be classified as positive.

Exampl e 3

A membrane with a pore-size of 0.8 μm was used to collect the mycobacteria in an experiment were the experimental conditions were similar to those applied in example 1. After labelling the mycobacteria present on the membrane with specific rabbit antibodies, a secondary reaction partner consisting in gold-sensitised goat anti- rabbit IgG was applied (Jackson ImmunoResearch, PO Box 9, West Grove PA 19390) . This method is considerably simpler than one based on PODase, but the sensitivity achieved is 10,000 mycobacterial bodies /ml, roughly identical to that of bacilloscopy. Exampl e 4

A membrane with a pore-size of 0.8 μm was used to collect the mycobacteria. To simplify the revelation step, the gamma-globulin fraction of the rabbit antiserum against mycobacteria was isolated and sensitised with gold micellae.

The isolation of the gamma-globulin fraction proceeded according to a standard protein A-sepharose method : 10 ml of serum were brought to pH 8 with 1/10 volume of 1 M Tris buffer at pH 8.0 and passed through a 10 ml column of Protein A-sepharose at pH 8. Elution of the gamma-globulins occurs at low pH (100 mM glycine, pH 3.0). Gold micelles were prepared according to a standard procedure (Bioconjugate techniques, Greg T. Hermanson Academic Press 1996, Chapter 14) : 1 ml of a 4% HAUCI4 ^was diluted in 400 ml water and brought to boiling. Six ml of a 1% sodium citrate solution is added and boiling pursued during 30 minutes. This method of obtention yields micellae whose average size is 30 nm. Cool at room temperature. The antibody-gold complex was made according to the recipe given in the same textbook (page 600 and following) .

Using a single gold-labelled antibody, specific for the microbial antigen retained on the membrane, the sensitivity achieved is 10,000 entities /ml.

Exampl e 5

A membrane with a pore-size of 0.8 μm was used to collect the mycobacteria as described in example 1. The labelling of the mycobacteria proceeded with gold- sensitised rabbit antibodies against mycobacteria, as per example 4. The secondary gold label was made of gold- sensitised goat antibodies against rabbit IgG. The sensitivity of the system improved, to reach about 3,000 mycobacteria / ml .

Example 6 A membrane with a pore-size of 0.8 μm was used to collect the mycobacteria in an experiment where the experimental conditions were similar to those applied in example 1. After labelling the mycobacteria present on the membrane with specific rabbit antibodies-, a secondary reaction partner consisting in gold-sensitised goat anti- rabbit IgG was applied (Jackson ImmunoResearch, PO Box 9, West Grove PA 19390) . After washing the unattached gold away, the gold signal remaining on the membrane was intensified by reaction with silver. A silver enhancing kit (Sigma chemical Co, St Louis, Mo) consists of a silver salt, an initiator and a fixer (sodium thiosulfate) . This method of enhancement of the signal improved the sensitivity of the method to about 3,000 particles per ml.

Example 7

A membrane with a pore- size of 0.45 μm was used to collect the mycobacteria in an experiment were the experimental conditions were similar to those applied in example 1. After labelling the mycobacteria present on the membrane with specific rabbit antibodies, a secondary binding partner consisting of Protein A-20 nm colloidal gold, was applied (Sigma Chemical Co, St Louis, MO) . The results were similar to those obtained in example 6, when the secondary antibody was gold-sensitised goat anti-rabbit IgG, i.e. 3,000 particles/ml. Exampl e 8

These experiments showed us that, when no sputum is used, a membrane with a pore size 0.45 μm has a retention power superior to membranes whose pore size is 0.8 μm. However, when sputum is used, the 0.45 μm pore sized membrane tends to clog up easier. To further favour flow, a glass fiber pre-filter is needed, that retains unsolubilised matter and viscous matter present in sputum. Sputum from a TB patient was collected at the Pneumology department of t e University Hospital of Strasbourg (France) . The sputum underwent regular analysis and was found to contain TB bacilli by the bacilloscopy technique based on the Ziehl-Nielsen Stain. The sputum was fluidised by addition of 0.25% N-acetyl-cysteine (final concentration) and 1% NaOH (final concentration) . This fluidising method is standard in mycobacteriology for the treatment of sputum before its use in cultures. The NaOH is added as a fluidisation medium and also in order to decontaminate the sample and kill all other bacteria present, before culture for tubercle bacilli is initiated. The NaOH is not indispensable for the liquefaction and N- acetyl-cysteine, at concentrations between 0.5% and 2% and alkaline pH, is able to liquefy sputum in two minutes. Other methods of fluidisation, based on hypochlorite (bleach, also called Javel water) or sodium dodecyl sulphate, described in the literature can also be used. Bleach may be appropriate for low income countries as it is cheap available locally and because it better respects the integrity of the retaining membrane than NaOH. An alteration of the retaining membrane, made of nitrocellulose, was observed when the sample fluid was treated with NaOH at 1%. A neutralisation of the fluid with HCl was needed before passing it on a nitro-cellulose retaining membrane. Other means of fluidisation (bleach, Dithiothreitol) proved compatible with the retaining membranes used. An additional problem (not observed in the present case) was the possibility that the fluidising agent would affect the detection of the mycobacterial antigens recognised by the primary rabbit antibodies. However, any fluidising agent that respects the antigenicity of the bacteria and does not alter the retaining membrane could be used. A fiber-glass prefilter pad was placed on top of the nitrocellulose retaining membrane, so as to retain the unsolubilised material remaining in the fluidised sputum. Two hundred μl of fluidised sputum were passed through the prefilter and the 0.8 μm nitrocellulose membrane, before the fiberglass prefilter was removed. The retaining membrane was washed with 2 times 100 μl of 0.2M phosphate pH 7.2 containing 0.2% tween 20. After the wash, a rabbit antiserum against M. tuberculosis appropriately diluted 1:160 in phosphate-buffered saline pH 7.2 containing 0.2% tween 20, was placed on the membrane. This primary antiserum was obtained by inoculation of rabbits with heat -killed mycobacteria (M. tuberculosis H37 RA, DIFCO, Detroit, MI) . The secondary partner of reaction was 30 nm gold-sensitised goat anti-rabbit IgG. After a washing step, the gold signal present on the membrane was amplified with a reagent consisting in 30 nm gold-sensitised rabbit anti-goat IgG. The sensitivity of the system was good, detecting about 3,000 mycobacterial entities/ ml.

Exampl e 9

The detection of mycobacteria in sputum was done as in example 8, with a membrane whose pore size was 0.45 μm. This small pore size was found acceptable although it retarded the flow rate, but a slower flow of the reagents enhanced the sensitivity of the method by allowing a longer contact and prolonged reaction time of the reagents with the antigens. Rabbit anti-mycobacterial serum was used to label the antigen retained on the membrane. The secondary partner of reaction was composed of antibodies against rabbit IgG raised in donkeys. The enhancer was 30 nm-gold-labelled Protein G. , which reacts well with equine antibodies. The sensitivity reached was. about 3,000 entities/ml.

Exampl e 10

Fluidised sputum was analysed, as per example 8, on 0.45 μm pore-sized membranes, labelling first the mycobacteria with a rabbit antibody. The secondary partner of reaction was gold-labelled Protein A. The enhancer used was gold-sensitised guinea-pig IgG, because guinea pig gamma-globulins react better with protein A than goat gamma-globulins do. A sensitivity of 1,000 mycobacteria /ml, (which is about ten times more sensitive than the regular bacilloscopy) was riched.

The determination of the presence of pathogens on the retaining membrane was preferentially done by first labelling the trapped organism with an antibody against the pathogen, applying Protein A-gold as the secondary reaction partner and finally applying gold- sensitised guinea-pig gamma-globulins as an enhancer. Said method and device based on the use of protein A and nonspecific guinea pig gamma-globulins labelled with gold micellae is advantageously simple, cheap and results in a satisfactory sensitivity.

The enhancement of the gold signal is routinely done with a silver enhancer. The gold-labelled enhancer consisting in a gold-sensitised antibody or gold- sensitised protein A or protein G or other partner of reaction directed against the secondary gold-sensitised antibody or partner of reaction itself reacting with the primary antibody , that may itself also bear a gold signal, is stable, neither light nor heat sensitive (PODase is heat sensitive, the silver enhancer is light sensitive and must be prepared extemporaneously) , and gives per se a stable amplified signal that does not require further development (e.g. an enzymatic substrate of reaction) nor fixation (e.g. the silver enhancer ^"must be prepared extemporaneously and must be fixed by thiosulfate) .

The amplification of a gold signal by a secondary gold marker proceeds from the fortuitous observation that the gold micellae are of a size considerably superior to that of the antibodies or lectins

(e. g. Protein A, protein G) used to stabilise them. The sensitisation of the gold micellae results in a layer of antibodies or other partners of reaction covering the gold, of which only a fraction is engaged in an immunological or binding reaction. The free-remaining reaction partners saturating the gold micellae are free to react with a second partner of reaction, which may also be presented as a layer covering gold particles. Gold-sensitised antibodies and partners of reaction used as secondary and tertiary amplification means are much easier to use than enzyme labelled products or than the silver enhancer. They are suitable to visualise the presence of micro-organisms trapped on a retaining membrane composed of a flat sheet laminate and an underlying take-up pad, with a sensitivity superior to that obtained by conventional diagnostic means as bacilloscopy. As an alternative to antibodies, one may use a gold- labelled rabbit, human or guinea pig IgG to react with gold-Protein A, if gold-protein A was the secondary partner of reaction applied in the test. Another approach is to use gold-sensitised protein A as an amplifier of the gold signal given by a gold-antibody marker based on human or guinea pig antibodies against rabbit IgG. A further variation of the protocol involves the use of Protein G, which is more versatile than protein A in that it reacts well with human, equine, bovine, pig and rabbit gamma globulins .

Exampl e 11

Fluidised sputum was analysed, as per example 8, on 0.8 μm pore-sized membranes, labelling first the mycobacteria with a rabbit antibody. The secondary partner of reaction was coloured latex-labelled Protein A. The red latex beads sensitised with protein A were purchased (Stock n° C00002992JR, diameter 0.29 μm) from Bangs laboratories, 9025 Technology Drive, Fishers IN 46038-2886. The system was improved by the additional enhancement of the signal with gold-sensitised guinea-pig IgG. Guinea pig gamma- globulins react better with protein A than goat gamma- globulins do. A sensitivity of 1,000 mycobacteria /ml, which is about ten times more sensitive than the regular bacilloscopy was obtained. The system worked satisfactorily provided abundant volumes of washing fluids were applied to eliminate the latex marker. The pore size of the membrane used (0.8μm) was too close from the diameter of the latex beads to allow an easy flow-through and elimination of the unbound marker. With big latex beads, a membrane with a large pore size is needed, that restricts the use of latex beads to large organisms (worms, eukaryotic and multiceilular parasites) retained by membranes of a large pore size. Example 12

Human infections caused by Chlamydia tracho a tis primarily involve the eyes and the genital tract. The morphology of the chlamydiae resolves into Elementary Bodies (0.2 to 0.4 μm) that can be detected by immunofluorescence or enzyme linked immunosorbent assays and Reticulate Bodies which is the intracellular, metabolically active form (0.6 to- 1..0 μm) , arely observed microscopically. The laboratory diagnosis is based on a variety of methods (complement fixation, microimmunofluorescence, enzyme linked immunosorbent assays, direct microscopic examination, isolation of the micro-organisms and nucleic acid techniques) . For genital tract infections, specimens for assay are urethral and cervical swabs, and urine, with the aim to detect the etiological agent.

Chlamydia trachoma tis Elementary bodies (Cat N° 1990-3104) were purchased from Quartett GmbH (12307 Berlin, Germany) and human urine was spiked with decreasing amounts of the pathogen. Antibodies raised in rabbits were purchased from Quartett (Cat N° 1990-1204) and used to label the pathogen. One and a half millilitres of the spiked urine were passed on a filter with mean more size 0.2μm, that was able to retain the pathogen on its surface. No prefilter was useful. Revelation of the presence of the pathogen was done as per example 10, with Gold-protein A, that reacts with rabbit gamma-globulins. In a second analysis, antibodies raised in goats (Quartett Cat N° : 1990-0404) were used. Gold-labelled protein G was used to reveal the presence of the goat antibodies and an enhancer consisting in Gold-labelled pig gamma-globulins was finally applied. The sensitivity of this detection system was compared to that obtained by the rapid test chlamydia, a commercial diagnostic kit made by Abbott. Whereas a single amplification system (rabbit antibodies and Gold-protein A) yielded similar results, the use of an enhancer (Gold- labelled pig antibodies) increased the sensitivity fivefold.

Exampl e 13

Toxoplasmosis is caused by Tox plasma gondii , a sporozoan whose individual cells are 4 to 7 μm long. Outbreaks are due to the consumption of raw meat, inhaling oocysts at a riding stable and drinking contaminated water. Immunosuppressive treatments and immunodeficiency permits exacerbation of infections. Diagnosis relies essentially on detection of specific antibodies in serum but the sites most commonly attacked are the lymph nodes, brain, eyes and lungs. Direct examination of sputum, vaginal exudates, spinal, pleural and peritoneal fluids are possible but rarely practised. No diagnostic kits exist for direct examination of sputum and body fluids.

Toxoplasma gondii organisms grow easily in the peritoneal cavity of mice. The organisms were purchased from the Institut Pasteur of Lille (France) , together with rabbit antiserum. The organisms were mixed with sputum, the sputum was liquefied and 1 ml passed on an 0.8 μm membrane fitted with a prefilter. The same procedure as in example 8 was followed (Gold-labelled goat-anti-rabbit IgG and, as enhancer, gold-labelled rabbit anti-goat IgG) . The results were excellent, with the detection of about 200 pathogenic entities in the sample. Example 14

Few diagnostic kits for the detection of Neisseria gonorrhoeae are available. The laboratory diagnosis of gonococcal infection is based primarily on the identification of the etiologic agent by microscopic examination and by culture. Urethral smears are positive in only 60 percent of men with asymptomatic urethritis. In women, smears from the endocervix are positive in 50 to 60 percent of the symptomatic cases. Blood- cultures are positive in- disseminated disease and synovial fluid from patients with pyarthrosis^"usually yield positive cultures. Cultures derived from sterile sites (cerebrospinal fluid, blood, synovial fluid) usually provide a definitive diagnostic but positive cultures from non-sterile sites are of uncertain value. No serologic test is commercially available yet .

Neisseria gonorrhoeae antigen was obtained from Scripps laboratories (6838 Flanders Dr. San Diego, CA 92121-2904) and sputum originating from the University Hospital of Strasbourg (France) was spiked with known concentrations of the antigen, as in example 8. The pore- size of the retaining membrane was 0.45 μm, compatible with the size of the organisms (0.6 to 1. Oμm in diameter) . The solubilisation of the sputum was done with 0.2% Sodium dodecyl sulphate at pH 9.5, treatment that was found not to alter the immunoreactivity of the pathogen. For the purpose of the demonstration that the etiological agent of gonorrhoea can be detected by the method of this invention, rabbit antiserum was purchased (Biochemed Co, Huntington Beach, CA 92646) but monoclonal antibodies, specific in an absolute way for Neisseria gonorrhoeae, are also available (Quartett, Berlin, Germany). The presence of rabbit IgG on the surface of the membrane, indicative of the presence of the pathogen, was further pursued as per example 8, with similar results.

Exampl e 15 The etiological agents of malaria are sporozoan, of which Plasmodium falciparum is the most virulent species. In man, it first multiplies in exoerythrocytic foci, mainly the liver. The progeny enters the blood stream and initiates erythrocytic infection. Within the red blood cells, the parasite passes through three stages : the trophozoite, the schizont and finally the gametocyte that reinfects blood-sucking mosquitoes.

The microscopic observation of the adult schizonts in blood smears is used for the diagnostic of malaria.

Formalin-inactivated schizonts were obtained from Antibody systems, P.O Box 212022, Bedford TX 76095. Monoclonal IgG2 antibodies to adult schizonts were obtained from Valbiotech, 57, Blvd de la Villette, 75010, Paris, France. Human fresh whole blood was spiked with the pathogen. After the spiking, the blood was hemolysed with 1% Nonidet P 40 (final concentration) and 1 ml of the fluid was passed on a glass prefilter and collected on a retaining membrane with pore size 0.8 μm. The mouse antibodies were revealed with Gold-labelled Protein G. Gold-labelled horse IgG was used as an enhancer. Distinct red spots on the membrane signalled the presence of individual organisms.

Claims

1. Diagnostic device for the detection of micro-organisms comprising a drum covered by one or more adsoromg pads with specific pores size adapted for the micro-organisms to be detected.

2. Diagnostic device according to claim 1, wherein the adsorbing pads are covered by a filtering membrane .

3. Diagnostic device according to claim 2, wherein the filtering membrane is made of microcellulose or nylon.

4. Diagnostic device according to any one of the preceding claims, which comprises on top of the filtering membrane a funnel containing prefilter means, preferably made of glass fiber or paper, to hold or discard insoluble materials such as solid particles present in sputum or urine or red or white blood cells present in a blood sample.

5. Diagnostic device according to any one of the preceding claims, comprising reactants for the selective recovering and/or detection of micro-organisms upon the adsorbing pads of the drum.

6. Diagnostic device according to claim 5, wherein reactants are antibodies specific for the surface components of said micro-organisms, possibly coupled directly or indirectly to a marker.

7. Diagnostic device according to claim 6, wherein the marker is selected from the group consisting of gold micellaes, enzymes, cnromophores, stained latex beads, radioactive labels, fluorophores or a mixture thereof.

8. Diagnostic method for the diagnostic of a micro-organism present in a sample, comprising the steps of

- taking a sample of a biological fluid from a patient, - depositing said sample upon the diagnostic device according to any one of the preceding claims, and

- obtaining a detection of a micro-organism present in said biological fluid by adding reagents for the selective recovering and/or detection of said micro-organism.

9. Method according to claim 8, wherein said reagents are antibodies specific for the surface components of said micro-organisms, possibly coupled directly or indirectly to a marker, preferably selected ^: from the group consisting of gold micellaes, enzymes, chromophores, stained latex beads, radioactive labels, fluorophores or a mixture thereof .

10. Method according to claim 8 or 9, wherein the biological fluid is selected from the group consisting of sputum, blood, serum, plasma, cephalo-rachidian fluid or urine obtained from a patient.