WO2000052472A1 - Rapid assay for infection in small children - Google Patents

Abstract

A rapid assay for infection in small children allows excluding uninfected children from unnecessary treatment. The assay can be used to detect infections of the blood stream (sepsis), cerebrospinal fluid (CSF), or urinary tract. The level of CD11b (Mac-1, CR3) on the surface of polymorphonuclear leukocytes (PMN, neutrophil) is measured by flow cytometry (FACS) in whole blood samples. Neutrophils are identified using anti-CD15 antibodies. Spontaneous in vitro upregulation of CD11b is prevented by handling samples at 4 °C or adding a stabilizing compound such as adenosine or anti-CD14 antibody to the samples. A calibration composition defines an infection diagnosis threshold. The composition can be a CD11b-coated latex microparticle provided as part of a kit, or a solid-phase composition provided as part of a testing device or test strip. A kit further includes fluorescent anti-CD11b and anti-CD15 antibodies, as well as a stabilizing compound.

Description

RAPID ASSAY FOR INFECTION IN SMALL CHILDREN

U.S. GOVERNMENT RIGHTS This invention was made with U.S. Government support under grant Nos. M01 RR00070 and AI34762, awarded by the National Institutes of Health. The U.S. Government has certain rights in this invention.

BACKGROUND This invention relates to the diagnosis and treatment of infection, in particular sepsis, in young children.

Children with fever make up a substantial proportion of all ambulatory pediatric visits. Fever may signal a minor infection or a serious, life-threatening condition such as sepsis (bacteremia). Sepsis develops very rapidly, and if left untreated is often fatal. Because of the severe consequences of leaving sepsis untreated, treatment is commonly initiated empirically and many children who do not suffer from sepsis are unnecessarily treated. The treatment of small children suspected of sepsis often includes hospitalization and parenteral antimicrobial therapy for at least 48 hours.

For further information on conventional approaches to diagnosing and treating sepsis in young children see the articles by Baraff et al. "Management of the Young Febrile Child. Commentary on Practice Guidelines," Pediatrics 100:134-136 (1997); Baraff et al. "Practice Guideline for the Management of Infants and Children 0 to 36 Months of Age with Fever without Source," Pediatrics 92:1-12 (1993); Jaskiewicz et al. "Febrile Infants at Low Risk for Serious Bacterial Infection — An Appraisal of the Rochester Criteria and Implications for Management. Febrile Infant Collaborative Study Group," Pediatrics 94:390-396 (1994); Chiu et al. "Identification of Febrile Neonates Unlikely to Have Bacterial Infections," Pediatr. Infect. Pis. 16:59-63 (1997); and Teach et al. "Efficacy of an Observation Scale in Detecting Bacteremia in Febrile Children Three to Thirty-Six Months of Age, Treated as Outpatients. Occult Bacteremia Study Group," J. Pediatrics 126:877-881 (1995).

A rapid and reliable assay for infection would allow avoiding unnecessary antibiotic treatment and hospitalization for small children. Such a test would not only save money, but would also lessen the substantial social disruption and family separation resulting from hospital admissions.

SUMMARY The present invention provides a rapid and reliable method for testing a small child for infection, and determining whether to treat the child for infection. The test results are available within less than an hour, preferably within half an hour, of collecting a patient sample. The testing method comprises measuring the levels of CD11 b or a CD11 b complex in a blood sample of a small child, particularly the levels found on the cell surface of neutrophils. Prior to the measurement step, the sample is handled under conditions inhibiting the in vitro upregulation of neutrophil surface CD11 b. The CD11b or CD11b complex levels in the patient sample are then compared to a predetermined infection diagnosis threshold, where increased levels of CD11 b above the threshold are indicative of infection.

DETAILED DESCRIPTION In small children, i.e. children of from about 3 months of age to about 36 months of age, a rapid diagnosis of infection is made by measuring the level of CD11 b or CD11b complex present on the cell surface of blood cells. Of particular interest is measuring the levels on neutrophils present in the blood. Conveniently the testing is performed using flow cytometric analysis of a whole blood sample.

Prior to the measurement step, the sample is handled under conditions inhibiting the in vitro upregulation of neutrophil surface CD11 b, for example by handling the sample at 4° C or by adding a stabilizing compound such as adenosine or anti-CD14 antibody to the sample. The CD11b or CD11b complex levels in the patient sample are then compared to a predetermined infection diagnosis threshold, where increased levels of CD11b above the threshold are indicative of infection.

CD11b is a beta-integrin adhesion molecule involved in neutrophil adhesion, diapedesis, and phagocytosis. CD11b is the β-subunit of the Mac-1 receptor, where the α-subunit of Mac-1 is CD18. For general information on CD11b and its structure see Corbi et al. (1988) J. Biol. Chem. 263:12403-12411 ; the Mac-1 sequence is described by Springer et al. (1985) Nature 314(6011):540-2. CD11b is stored in primary and secondary intraceliular granules within unstimulated neutrophils and certain other leukocytes. Mac-1 is the receptor for C3bi, a component of the complement cascade which mediates adhesion of monocytes and granulocytes to C3bi-sensitized particles. The Mac-1 complex is well-known in the art (for reviews see Gahmberg et al. (1998) Cell Mol Life Sci 54(6):549-55; Ross and Vetvicka (1993) Clin Exp Immunol 92(2):181-4).

The term "CD11 b complex" is intended to refer to stable molecular complexes that comprise CD11 b, and that are upregulated in the same manner as CD11 b. For example, in order to monitor the level of CD11 b on the surface of a neutrophil, one may assay for the presence of CD11b itself, or may monitor the levels of complexes that comprise CD11 b, such as Mac-1 , e.g. using binding agents that bind to epitopes found on CD18, on CD11 b, or present only when the two subunits are associated.

CD11b is found on the surface of a variety of cells, including monocytes and neutrophils. It is normally present at very low levels on the neutrophil cell surface and becomes elevated rapidly upon neutrophil encounter with pathogens or their products. CD11 b increases on the neutrophil surface within five minutes of exposure to bacterial products such as endotoxin (lipopolysaccharide, LPS), and surface expression peaks within 30 minutes. Neutrophil upregulation of CD11 b upon stimulation with LPS has been shown to depend on a complex by rapid cascade of events, initiated by the binding of LPS to CD14 on the neutrophil surface. In response to antigen, CD11b is transported from intracellular granules to the neutrophil surface. The upregulation of neutrophil surface CD11 b is not dependent on antigen-induced protein synthesis.

Neutrophils are also known to increase expression of CD11 b in vitro and in vivo upon stimulation with other factors such as the anaphylotoxin C5a, the bacterial product F-met-leu-phe, and the inflammatory cytokine tumor necrosis factor-α (TNF-α), presumably through a mechanism unrelated to CD14. Increase of neutrophil surface CD11 b during viral and yeast infections has been reported in several in vivo and in vitro studies.

The present invention relies on the surprising observation that immune system immaturity or dysfunction does not necessarily preclude the use of neutrophil surface CD11b as a diagnostic tool.

In a typical assay, a blood sample is assayed for the presence of CD11 b or CD11b complex by combining the sample with a CD11b or CD11b complex specific binding member, and detecting directly or indirectly the presence of the complex formed between the two members. The term "specific binding member" as used herein refers to a member of a specific binding pair, i.e. two molecules where one of the molecules through chemical or physical means specifically binds to the other molecule. In this particular case one of the molecules is CD11 b or CD11 b complex, where CD11b or CD11b complex is any protein substantially similar to the amino acid sequence of naturally occurring CD11 b, or a molecular complex comprising CD11 b, or a fragment thereof. The complementary members of a specific binding pair are sometimes referred to as a ligand and receptor. In addition to antigen and antibody specific binding pairs, peptide-MHC antigen and T cell receptor pairs; peptide ligands and receptor; autologous monoclonal antibodies, and the like. Antibodies and T cell receptors may be monoclonal or polyclonal, and may be produced by transgenic animals, immunized animals, immortalized human or animal B-cells, cells transfected with DNA vectors encoding the antibody or T cell receptor, etc. The details of the preparation of antibodies and their suitability for use as specific binding members are well-known to those skilled in the art.

Monoclonal antibodies specific for CD11b and CD18 are known in the art and commercially available. Alternatively, monoclonal or polyclonal antibodies are raised to the proteins. The antibodies may be produced in accordance with conventional ways, immunization of a mammalian host, e.g. mouse, rat, guinea pig, cat, dog, etc., fusion of resulting splenocytes with a fusion partner for immortalization and screening for antibodies having the desired affinity to provide monoclonal antibodies having a particular specificity. The antibodies may be labeled with radioisotopes, enzymes, fluorescers, chemiluminescers, or other label which will allow for detection of complex formation between the labeled antibody and its complementary epitope.

In a preferred embodiment, antibody specific for CD11b or CD11b complex are fluorescently labeled, directly or indirectly, and measurement is performed using flow cytometry. A preferred sample is whole blood, although cellular fractions of whole blood, e.g. aphoresis samples, elutriated samples, and the like, may also find use. The volume of the blood sample can be less than 1 ml, preferably less than 0.1 ml. The whole blood sample is handled at all times under conditions inhibiting the spontaneous upregulation of neutrophil surface CD11b. In one embodiment, the sample is handled at 4° C. In another embodiment, a stabilizing compound such as adenosine or anti-CD 14 antibody is added to the sample. Particular care should be taken in handling samples under analysis, since neutrophil surface CD11b assays are extremely sensitive to sample handling conditions. The consequences of inadequate control over assay conditions are illustrated by Davis et al. (1995) Laboratory Hematoloαv 1:3-12, "CD64 Expression: Potential Diagnostic Indicator of Acute Inflammation and Therapeutic Monitor of Interferon- gamma Therapy". Davis et al. reports no correlation between leukocyte levels of many markers, including CD11 b, and the presence of infection in adults. Analysis of the methods in the study reveals that spontaneous upregulation of CD11b was not prevented. Samples were analyzed up to 24 hours after collection, and were not stabilized by temperature control or addition of a stabilizing compound.

Neutrophils in the whole blood sample or fraction thereof may be identified according to a neutrophil identification marker. A neutrophil identification marker of interest is a CD15 marker, such as a fluorescent anti-CD15 antibody. Preferably the antibody against CD15 is labeled with fluorochrome that is spectrally distinguishable from the label used to quantitate CD11b or CD11b complex; such that the two markers can be analyzed simultaneously on a two-laser flow cytometer. Fluorescent anti-CD15 antibodies are well known in the art. The measured CD11b or CD11 b complex level is then compared to a predetermined diagnosis threshold for the category of patient under evaluation. A measured level exceeding the threshold is indicative of infection. Factors other than the assay results may be used in the patient evaluation. The assay allows excluding uninfected children from unnecessary treatment. Because of the severe potential health consequences of not treating truly infected children, it is relatively more important to minimize the number of false negatives rather than the number of false positives. The diagnosis threshold can be chosen to yield desired negative and positive predictive values.

The diagnosis threshold preferably defines a sharp cutoff between infected and uninfected patients. The cutoff is chosen such that it separates infected and uninfected patients with a high (e.g. 99%) negative predictive value. Alternatively, multiple (e.g. two) diagnosis thresholds may be used for identifying infected and uninfected subpopulations of patients, as well as a subpopulation requiring further evaluation. A first diagnosis threshold then separates uninfected patients from patients requiring further evaluation, and a second diagnosis threshold separates patients requiring evaluation from infected patients. Also, a specific diagnosis threshold may be used for each one of a plurality of age groups. The diagnosis threshold is defined by a standardized calibration composition provided to all testing sites. In a preferred embodiment, the calibration composition comprises CD11b-coated latex microparticles on the order of 1 μm in diameter. Techniques for producing microparticles coated with a particular molecule are known in the art. In one such technique, microparticles are coated with mouse anti-latex antibodies conjugated to mouse anti-human CD11b antibodies.

The quantity of CD11b on the surface of each microparticle defines the diagnosis threshold. For example, the level of CD11b on the surface of the microparticle is chosen to be substantially equal to the surface CD11b level on a neutrophil corresponding to the relevant diagnosis threshold. For calibration, the CD11 b-coated microparticle is stained with the quantity of anti-CD11b fluorescent antibody used for staining neutrophils in the assay. The stained microparticle is analyzed on the device used for performing the assay, and the device's settings are calibrated according to the microparticle readings. For example, for a conventional flow cytometer, photomultipiier settings are adjusted such that the fluorescence of anti-CD11 b antibodies on the microparticle surface has a predetermined value.

The calibration composition is preferably provided as part of a kit for testing children for infection. The kit comprises the calibration composition, a marker for CD11b, a neutrophil identification marker, and a stabilizing compound. Preferably, the kit comprises a CD11b-coated microparticle, a fluorescent anti-CD11 b antibody, a fluorescent anti-CD15 antibody spectrally distinguishable from the anti-CD11b antibody, and adenosine or an anti-CD14 antibody. The kit concentrations and protocols are chosen so as to yield a suitable (mid-range) signal on the device used for analysis.

As is apparent to the skilled artisan, there are many well-known methods other than flow cytometry for measuring levels of a given molecule. In particular, methods and devices (test strips, test cartridges) for performing desired solid-phase assays are well known in the art and will not be described here in detail. Test devices can include a composition defining a diagnosis threshold. The diagnosis threshold can also be defined by multiple device components.

An assay of the present invention can involve obtaining measurement values other than CD11b levels. Measuring a CD11b level can be thought of as probing a larger physiological response of the patient to infection. The physiological response to infection involves a number of molecules other than CD11b. Other molecules may exhibit changes that are temporally associated with neutrophil CD11 b upregulation. For example, monocyte surface levels of CD14 can decrease, or serum TNF-α levels increase, at the same time as CD11b is upregulated.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the subject invention, and are not intended to limit the scope of what is regarded as the invention. It will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. Efforts have been made to ensure accuracy with respect to the numbers used (e.g. amounts, temperature, concentrations, etc.) but some experimental errors and deviations should be allowed for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees centigrade; and pressure is at or near atmospheric.

As used herein the singular forms "a", "and", and "the" include plural referents unless the context clearly dictates otherwise. All technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs unless clearly indicated otherwise.

All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

EXAMPLE

To test whether small child is infected, a whole blood sample is obtained from the child and tested by flow cytometry. 0.1 ml of whole blood is obtained from the child and is diluted with 2.5 ml cold staining medium. A suitable staining medium is composed of deficient RPMI 1640 Medium (Applied Scientific, Irvine, CA), 1 % fetal calf serum (Gemini Bioproducts, Calabasas, CA), with 1M HEPES, pH 7.2 (Sigma, St. Louis, MO). 10% sodium azide stock solution is added to a concentration of 0.02%. If stored, the sample is maintained at 4° C. The sample is washed once with staining medium, resuspended in 0.5 ml staining medium and stained for 15 minutes with FITC-conjugated CD15 (to identify neutrophils) and PE-conjugated CD11b mouse anti-human monoclonal antibodies (Becton Dickinson, Milpitas, CA). The sample is then washed three times and resuspended in 1 % para- formaldehyde/staining medium. Prior to staining, the FITC-conjugated anti-CD 15 is diluted with unconjugated anti-CD15 antibody in order to bring the neutrophil CD15 staining into mid-range on the FACS sorter (FACScan, Becton Dickinson). Before a run, the instrument is adjusted so that the fluorescence of calibration beads in each channel has a predetermined value.

The sample is analyzed on the FACScan with a threshold set such that only cells with the correct range of CD15 fluorescence for neutrophils are accepted as events. This threshold excludes erythrocytes and all other leukocytes. The median fluorescence of neutrophil CD11b is determined for each sample based on the distribution of CD11b fluorescence for the counted cells.

It will be clear to one skilled in the art that the above embodiments may be altered in many ways without departing from the scope of the invention. For example, a CD11b test may be used to diagnose young patients one to three months of age. A solid-phase composition defining an infection diagnosis threshold may be provided as part of a test strip or testing device. Markers other than fluorescent antibodies are suitable for use in a method of the present invention. Such markers include, among others, sugars, polynucleotides, enzymes, and enzyme substrates. While at least one marker is preferably capable of specific and direct binding to CD11 b, markers capable of indirectly coupling to CD11b through other parts of the molecular complex which includes CD11 b may also be used. Scanning microscopy and light refraction techniques can be used instead of flow cytometry. Assay techniques not involving analysis of specific blood parts or cell types (e.g. neutrophils) can be used. Such techniques include whole blood staining only with a marker for CD11 b, followed by flow cytometric or laser scanning microscopy analysis of the whole blood sample. CD11 b levels may be measured in specific parts of a whole blood sample, for example in serum, on the surface of particular cell types (leukocytes, monocytes, neutrophils), and in the cytoplasm of particular cell types. Various clinical techniques may be used in conjunction with the assay. Moreover, test parameters, including diagnosis thresholds, can be chosen according to particular clinical applications and desired positive and negative predictive values. Accordingly, the scope of the invention should be determined by the following claims and their legal equivalents.

Claims

WHAT IS CLAIMED IS:

1. A method of testing a small child for infection, comprising: a) measuring a CD11b or CD11b complex level in a blood sample of said child; and b) determining whether to treat said child for infection using said CD11b or CD11 b complex level.

2. The method of claim 1 , wherein said measuring said CD11b or CD11b complex level is performed by flow cytometry.

3. The method of claim 2, wherein said sample is a whole blood sample.

4. The method of claim 1 , wherein said CD11 b or CD11 b complex level is a neutrophil surface CD11 b level.

5. The method of claim 4, further comprising exposing said blood sample to a condition inhibiting an in vitro upregulation of said CD11b or CD11 b complex level, prior to measuring said CD11 b level.

6. The method of claim 1 , wherein said determining whether to treat said child for infection comprises comparing said CD11b or CD11 b complex level to an infection diagnosis threshold.

7. The method of claim 1 , wherein said measuring said CD11b or CD11b complex level comprises exposing said sample to a fluorescent anti-CD11 b antibody, and measuring an fluorescence signal from said anti-CD11 b antibody in said sample.

8. The method of claim 1 wherein said measuring said CD11b or CD11b complex level comprises coupling a marker to CD11b in said sample, and measuring a level of said marker in said sample.

9. The method of Claim 1 , wherein said small child is from 3 to 36 months of age.

10. A composition defining a CD11b an infection diagnosis threshold for a child three to thirty-six months of age.

11. The composition of claim 10, comprising microparticles with a predetermined surface level of CD11b defining said diagnosis threshold.

12. A kit comprising the composition of claim 11 , and a marker for CD11 b.

13. The kit of claim 11 further comprising a neutrophil identification marker.

14. The kit of claim 13 wherein: a) said marker for CD11b comprises a fluorescent anti-CD11b antibody; and b) said neutrophil identification marker comprises a fluorescent anti-CD15 antibody spectrally distinguishable from said anti-CD11b antibody.

15. The kit of claim 13 further comprising a stabilizing compound capable of inhibiting an in vitro upregulation of neutrophil surface CD11b.

16. A method of testing for infection comprising: a) obtaining a measurement value indicative of an upregulation of neutrophil surface CD11b in a blood sample of a child three to thirty-six months of age; and b) determining whether to treat said child for infection using said measurement value.