# Patent

## Title
Method for diagnosing invasive Candida infections

## Abstract

The present invention concerns an in vitro method for diagnosing invasive candidiasis (IC) in a subject which comprises detecting the presence of a Candida glycan and detecting the presence of antibody directed against a protein selected from the group consisting of fructose bisphosphate aldolase (Fba1), enolase 1 (Eno1), heat shock protein 90 (Hsp90), hyphal wall protein (Hwp1), and mannoprotein 65 (Mp65) in a blood, plasma or serum sample of the subject. The invention also relates to a method of determining a suitable treatment regimen for a patient and to a kit for implanting the methods described herein.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
This application claims priority to European Patent Application 12305778.8 filed Jun. 29, 2012. The entirety of this application is incorporated by reference herein.
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jul. 15, 2013, is named 243521.000002_SL.txt and is 42,027 bytes in size.
The present invention concerns an in vitro method for predicting or diagnosing invasive candidiasis (IC) in a subject which comprises detecting the presence of a Candida glycan and detecting the presence of antibody directed against a protein selected from the group consisting of fructose biphosphate aldolase (Fba1), enolase 1 (Eno1), heat shock protein 90 (Hsp90), hyphal wall protein (Hwp1), and mannoprotein 65 (Mp65) in a blood, plasma or serum sample of the subject. The invention also relates to a method of determining a suitable treatment regimen for a patient and to a kit for implanting the methods described herein.
Candidiasis is a fungal infection with yeast(s) of any Candida species, of which Candida albicans is the most common. Candidiasis encompasses infections that range from superficial infections of skin and mucosal membranes, which cause local inflammation and discomfort, to systemic or invasive candidiasis (IC). Candida infections of the latter category are also referred to as deep-seated tissue infection affecting one or multiple organ(s) revealed or not by blood culture (candidemia). This severe forms of the disease are usually confined to severely immunocompromised persons, such as cancer, transplant, and AIDS patients, but may occur also in medical and surgical intensive care units and intravenous catheter bearing patients.
Invasive candidiasis remains a problem of public health persisting in medical and surgical intensive care units, onco-haematology, bone marrow and hematopoietic stem cell transplantation and premature newborns wards. According to recent reports Candida spp. rank as the fourth most common cause of nosocomial bloodstream infections and are characterized by an important medical and economic impact linked to difficulties of the clinical and biological diagnosis. Systemic candidiasis is associated with long hospital stays and mortality rates of 18 to 70% and a shift in the spectrum of infecting species has also occurred. Non-Candida albicans species, although they may have a lower intrinsic pathogenic potential than C. albicans, are now identified in more than 45-60% of episodes of hematogenously disseminated candidiasis. Moreover, the increased morbidity as a consequence of candidemia generates important extra costs related to longer hospital stays and prophylactic, empirical, or curative antifungal treatment. While the availability of new antifungal drugs such as echinocandins and new azoles is a promising step toward improving outcomes for patients with invasive candidiasis, a definitive and early diagnostic approach is imperative to avoid delay in the initiation of treatment for infected patients and to prevent unnecessary therapy in high-risk individuals who are only colonized. This delay in the initiation of appropriate antifungal treatment is a cause for the high morbi-mortality rates of IC and is related to a lack of rapid and reliable tests discriminating patients with IC from patients with superficial colonization.
Histological examination of biopsies and blood culture are still considered as the “gold standard” for the diagnosis of IC; however these methods are rarely performed or poorly sensitive, respectively.
Several rapid, non culture based methods have been developed including the detection of anti-Candida antibody against immunogenic targets and the detection of circulating Candida antigen (Yeo and Wong. Clin Microbiol Rev 2002; 15:465-84). However, only a few assays have been standardized and are commercially available. The antigen assays target the detection of mannan (Weiner and Yount. J Clin Invest 1976; 58:1045-53; Herent et al. J Clin Microbiol 1992; 30:2158-64), D/L arabinitol (Switchenko et al. J Clin Microbiol 1994; 32:92-7), heat labile antigen (Fung et al. J Clin Microbiol 1986; 24:542-7) and (1→3)-β-D-glucans (Ostrosky-Zeichner et al. Clin Infect Dis 2005; 41:654-9; Senn et al. Clin Infect Dis 2008; 46:878-85). Another assay targeting Enolase has been abandoned (Walsh T J et al. N Engl J Med. 1991 Apr. 11; 324(15):1026-31). Antibody assays target the mannan, the major antigenic component of the yeast cell wall. However, these non-culture methods, even helpful, exhibit numerous drawbacks in their sensitivity and specificity, and sometimes require the combination of two or more assay results for accurate diagnosis of IC.
Improvement of specificity has been obtained by combining the detection of serum mannan and anti-mannan antibodies (Sendid et al. J Clin Microbiol 1999; 37:1510-7). Such an improvement can be explained by the fact that when a specific antigen and antibodies to this antigen are detected in a same patient, the antibodies may facilitate the clearance of the antigen. A recent meta-analysis study of diagnostic values of these two assays has been recently published (Mikulska et al. Crit Care. 2010; 14(6):R222). The use of mannan antigen and anti-mannan antibodies in the diagnosis of invasive candidiasis: recommendations from the Third European Conference on Infections in Leukemia. Detection of mannan appears to be very specific: 93% on 767 patients (11 studies) with a thin 95% IC (91%-94%). However the sensitivity is weak: 58% on 453 patients from 14 studies (95% IC: 53%-62%). Addition of the detection of anti-mannan antibodies allowed to increase the sensitivity up to 83% (95% IC: 79%-87%) but lowered the specificity to 72% due to the lower specificity of anti mannan antibodies detection especially in heavily colonized patients (Ellis et al. J Med Microbiol 2009; 58:606-15).
Many additional proteins that could help in the diagnosis of IC have been pointed out during the past 10 years especially with the development of proteomics analysis. Some of them seem to be specific to pathogenic process. Indeed, more than twenty Candida proteins have been reported to be over expressed during the switch saprophytic/pathogenic phases leading to investigate their values for the diagnosis of IC (Clancy et al. J Clin Microbiol 2008; 46:1647-54). The diagnostic value has not been confirmed for the majority of these identified proteins and only primary results on non standardized or sophisticated assay have been obtained for the others. Hyphal Wall Protein (Hwp1) [Staab et al. J Biol Chem 1996; 271:6298-305; Lain et al. BMC Microbiol 2007; 7:35; Martin et al. Int J Med Microbiol; 301:417-22], Agglutinin like sequence family (Als3) [Martin et al. Int J Med Microbiol; 301:417-22; Hoyer et al. Curr Genet 1998; 33:451-9; Green et al. Infect Immun 2005; 73:1852-5], Superoxide dismutase (Sod) [Martin et al. Int J Med Microbiol; 301:417-22], Methionine synthetase—Met6 [Pitarch et al. Proteomics 2001; 1:550-9; Pitarch et al. Proteomics 2004; 4:3084-106; Pitarch et al. Proteomics Clin Appl 2007; 1:1221-42], Malate deshydrogenase [Hernando et al. Int Microbiol 2007; 10:103-8], Fructose biphosphate aldolase [Pitarch et al. Proteomics 2001; 1:550-9; Hernando et al. Int Microbiol 2007; 10:103-8], Hsp70 family [La Valle et al. Infect Immun 2000; 68:6777-84; Pitarch et al. Proteomics 2001; 1:550-9], Hsp90 [Matthews R C. J Med Microbiol 1992; 36:367-70; Pitarch et al. Proteomics 2001; 1:550-9], Phosphoglycerate kinase (PGK) [Pitarch et al. Proteomics 2001; 1:550-9; Pitarch et al. Proteomics 2004; 4:3084-106; Hernando et al. Int Microbiol 2007; 10:103-8; Clancy et al. J Clin Microbiol 2008; 46:1647-54], Diacylglycerol kinase catalytic domain [Hernando et al. Int Microbiol 2007; 10:103-8], Glyceraldehyde 3-phosphate dehydrogenase (G3P) [Pitarch et al. Proteomics 2001; 1:550-9], Alcohol Dehydrogenase (ADH1) [Pitarch et al. Proteomics 2001; 1:550-9], Diacylglycerol kinase [Hernando et al. Int Microbiol 2007; 10:103-8], 65 kDa mannoprotein (CamP65) [Berzaghi et al. Clin Vaccine Immunol 2009; 16:1538-45] make part of all these proteins identified by proteomic approaches.
Limited results are available to conclude on the possibility to use these proteins for improving the diagnosis of IC and further prospective studies are required to confirm the usefulness in clinical practice.
The inventors evaluated the diagnostic performances of a panel of ELISA tests detecting total immunoglobulins against six Candida albicans recombinant proteins obtained from E. coli transfected by specific plasmids containing fructose biphosphate aldolase (Fba1), enolase 1 (Eno1), heat shock protein 90 (Hsp90), hyphal wall protein (Hwp1), mannoprotein 65 (Mp65), and superoxide dismutase 5 (SOD5) Candida albicans genes. These tests were compared to the already marketed mannanemia and antimannan antibody tests. The cohort consisted of patients with IC determined by Candida albicans (53 patients and 157 sera) or Candida non albicans species (40 patients and 142 sera). Control group consisted of 80 blood donors (80 sera) and 90 Candida colonized patients (90 sera) without evidence of IC. Preliminary investigations allowed selecting Hsp90, Hwp1, Fba1, Eno1 and Mp65 as the best candidates for further clinical evaluation of their usefulness for the diagnostic of IC.
To improve the diagnostic potential of these biomarkers, a combination with mannan antigenemia was performed. Indeed, with a specificity arbitrarily fixed at 80.0%, mannanemia/anti-recombinant protein antibody (RP-Ab) association, specific sensitivities of Hsp90 Ab, Fba1 Ab, Hwp1 Ab, Eno1 Ab and Mp65 Ab were 80.9%, 83.8%, 83.8%, 79.1% and 75.5%, while combination of mannanemia/anti-mannan antibodies lead to a sensitivity of 61.7% for IC patients versus controls (blood donors+hospitalized and colonized patients). When the date of positivity of RP-Ab/mannanemia was compared to the date of positive blood culture, the mean delay of 5 days before isolation of Candida species from blood was observed for all RP-Ab biomarkers.
Altogether, these results indicate that anti-Hsp90, anti-Fba1, anti-Hwp1, anti-Eno1 or anti-Mp65 Ab/mannanemia association can advantageously substitute anti-mannan antibodies/mannanemia for the prediction or early diagnosis of IC. Such a result was unexpected because whereas detection of mannan and anti-mannan antibodies is complementary in patients with invasive candidiasis, most probably due to the implication of anti-mannan antibodies in the clearance of soluble mannan, such a complementarity could not be expected for mannan and anti-Hsp90, anti-Fba1, anti-Hwp1, anti-Eno1 or anti-Mp65 antibody, based on a mechanism of antigen clearance. Therefore, it was entirely surprising that specificity and sensitivity of diagnostic of candidemia could be improved by replacing detection of anti-mannan antibodies by detection antibodies against Hwp1, Eno1, Fba1, Hsp90 or Mp65.
Method for Diagnosing Invasive Candidiasis
The invention relates to an in vitro method for diagnosing invasive candidiasis (IC) in a subject, said method comprising, or consisting of, the steps consisting of:
a) detecting the presence of a Candida glycan in a blood, plasma or serum sample of the subject;
b) detecting the presence of antibody directed against a Candida protein selected from the group consisting of fructose bisphosphate aldolase (Fba1), enolase 1 (Eno1), heat shock protein 90 (Hsp90), hyphal wall protein (Hwp1), and mannoprotein 65 (Mp65) in a blood or plasma sample of the subject; and
c) wherein the presence of said Candida glycan and/or of said antibody directed against a protein selected from the group consisting of Fba1, Eno1, Hsp90, Hwp1, and Mp65, is indicative of IC.
“Invasive candidiasis” or “IC” is also called candidemia and denotes systemic infection with yeast(s) of any Candida species, e.g. Candida albicans, Candida parapsilosis, Candida kruseï, Candida tropicalis, Candida glabrata, Candida lusitaniae, Geotrichum capitatum, Candida norvegiensis, and Candida guillermondii. Candida albicans is the most common Candida species. IC may be ultimately diagnosed by histological examination of biopsies and detection of Candida species in blood cultures.
Preferably, invasive candidiasis is due to infection with a Candida species selected from the group consisting of Candida albicans, Candida parapsilosis, Candida kruseï, Candida tropicalis, Candida glabrata, Candida lusitaniae, Geotrichum capitatum, and Candida norvegiensis. 
Therefore, a Candida glycan and/or a Candida protein may be preferably a glycan and/or protein which is found in one or more Candida species selected from the group consisting of Candida albicans, Candida parapsilosis, Candida kruseï, Candida tropicalis, Candida glabrata, Candida lusitaniae, Geotrichum capitatum, and Candida norvegiensis 
A “subject” or “patient” may be a human or a non human mammal, such as monkeys, dogs, cats, guinea pigs, hamsters, rabbits, cows, horses, goats and sheep. Preferably, the subject is a human, in particular a man, a woman or a child (0-18 year old).
By “detecting the presence” of an antigen or antibody, it is meant that in the largest extent it is determined in the antigen or antibody is present or absent in the sample to be analysed. According to an embodiment, the level of said Candida glycan and/or antibody directed against said Candida protein is detected.
Accordingly, the method may comprise or consists of the steps consisting of:
a) detecting the level of a Candida glycan in a blood, plasma or serum sample of the subject;
b) detecting the level of antibody directed against a Candida protein selected from the group consisting of fructose biphosphate aldolase (Fba1), enolase 1 (Eno1), heat shock protein 90 (Hsp90), hyphal wall protein (Hwp1), and mannoprotein 65 (Mp65) in a blood, plasma or serum sample of the subject; and
c) wherein an elevated level of said Candida glycan, and/or an elevated level of said antibody directed against said Candida protein selected from the group consisting of Fba1, Eno1, Hsp90, Hwp1, and Mp65, relative to a reference level is indicated of invasive candidiasis.
As used herein, a “Candida glycan” denotes an oligosaccharide or polysaccharide component of the cell wall of a Candida species. Carbohydrates account for 80 to 90% (wt/wt) of the cell wall of Candida albicans. The major carbohydrates are
(i) mannan, i.e. polymers of mannose in a variety of α and β linkage arrangements which are covalently associated with proteins to form glycoproteins also known as mannoproteins. The term “mannan” is also used to refer to the main soluble immunodominant component present in the outer cell wall layer of Candida species. A representative structure of Candida albicans mannan has been described in Martinez et al. Clinical Microbiology Reviews, 1998, 11(1), 121-141. Mannan is made up of three major sugar components: the longer outer chain, the N-linked inner core attached to the polypeptide, and the shorter O-glycosidically linked or base labile oligosaccharides attached to the polypeptide. The mannose polymers are linked to proteins by N-glycosidic bonds (through two GlcNAc [di-N-acetylchitobiose] units) to asparagine residues and by O-glycosidic, alkali-labile linkages to threonine or serine residues. The N-glycosidically linked carbohydrate has a backbone chain of α-1,6-linked mannopyranosyl residues with oligosaccharide branches containing mannopyranosyl residues with α-1,2, α-1,3, β-1,2, β-1,4 and single α-1,6-linked mannose units and phosphodiester bonds. Single mannose residues and short, unbranched mannose oligosaccharides constitute the O-glycosidically linked sugar component;
(ii) β-glucans, i.e. polymers of glucose, in particular branched polymers, containing β-1,3 and β-1,6 linkages. The β-glucans include in particular (1,3)-β-D-glucan; and (1,6)-β-D-glucan;
(iii) chitin, which is an unbranched homopolymer of N-acetyl-D-glucosamine (GlcNAc).
By “detecting the presence or level of a Candida glycan”, it is meant that the presence or level of at least one Candida glycan is detected. According to an embodiment, a presence or level of a Candida glycan which is determined may be the presence or level of mannan, β-glucan (in particular (1,3)-β-D-glucan), or chitin. In particular, the presence or level of a Candida glycan which is determined may be the presence or level of mannan, only. Although characterization of the assay has shown that combinations of more than two of the markers defined herein (Candida glycan and antibody directed against a Candida protein as recited above) did not significantly improved discriminatory potency of the method, the presence or level of more than one Candida glycan could nevertheless be determined, for instance the presence or level of mannan and the presence or level of at least one β-glucan (in particular (1,3)-β-D-glucan), or the presence or level of mannan and the presence or level of chitin, or the presence or level of at least one β-glucan (in particular (1,3)-β-D-glucan) and the presence or level chitin.
The “presence or level of a Candida glycan in a blood, plasma or serum sample of the subject” is representative of the presence or level of the Candida glycan in circulating blood of the subject. The presence or level of Candida glycan may denote in particular the presence or level of soluble Candida glycan, as for instance mannan is released from mannoproteins by proteolytic cleavage. Thus, the “presence or level of mannan in a blood, plasma or serum sample of the subject” may denote in particular the presence or level of the soluble immunodominant component of Candida mannoproteins, mannan. The detection or level of mannan antigen is also called mannanemia.
The presence or level of a Candida glycan may be determined by any suitable chemical or biochemical method known from the one skilled in the art, such as chromatography, enzyme-based chemoluminescent methods, Biacore or mass-spectrometry.
For instance the chromogenic enzymatic test Fungitell® (which is marketed by Associates of Cape Cod Inc.) could be used for detecting (1,3)-β-D-glucan.
Additionally, lectins which bind specifically to chitin, or to (1,3)-β-D-glucan or (1,6)-β-D-glucan, could be used for the detection of chitin, or (1,3)-β-D-glucan or (1,6)-β-D-glucan.
However, as antibody-based methods, which are described in more details below, are preferably used. In this context, immunoassays are particularly suitable
The method further includes detecting the presence or level of antibody directed against a Candida protein selected from the group consisting of fructose bisphosphate aldolase (Fba1), enolase 1 (Eno1), heat shock protein 90 (Hsp90), hyphal wall protein (Hwp1), and mannoprotein 65 (Mp65) in a blood, plasma or serum sample of the subject.
The presence or level of a Candida glycan and of an antibody directed against the Candida protein may be detected (i) in one and the same blood, plasma or serum sample of the patient or (ii) in several blood, plasma or serum samples sequentially obtained from the same patient, (iia) either at the same time (i.e. essentially simultaneously or no more than 3 minutes apart) or at least no more than 72 hours, no more than 48 hours, no more than 24 hours, preferably no more than 12 hours, preferably no more than 6 hours, still preferably no more than 3 hours apart, or (iib) in the course of patient monitoring, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days apart.
The method of the invention is preferably carried out at least twice a week. The method of the invention is also preferably carried out as long as the subject is at risk of developing invasive candidiasis, in particular as long as the patient is hospitalised.
“Fructose bisphosphate aldolase” or “Fba1” is an enzyme involved in glycolysis and gluconeogenesis and which is regulated by yeast-hyphal switch. A representative sequence for Fba1 is Fba1 from Candida albicans SC5314 which is encoded for instance by a polynucleotide comprising sequence SEQ ID NO:1, and which consist of the amino acid sequence SEQ ID NO:2. These sequences were published in Jones et al. Proc. Natl. Acad. Sci. U.S.A. 101 (19), 7329-7334 (2004). The diploid genome sequence of Candida albicans is available from National Center for Biotechnology Information database under accession number XM_717597 (as available on 14 Feb. 2012).
“Enolase 1” or “Eno1” denotes a glycolytic enzyme (EC 4.2.1.11) present in the cytoplasm and, in minor amounts, in the inner layer of the cell wall of C. albicans. A representative sequence for Eno1 is Eno1 from Candida SC5314 which is encoded for instance by a polynucleotide comprising sequence SEQ ID NO:3, and which consist of the amino acid sequence SEQ ID NO:4. These sequences were published in Jones et al.; Proc. Natl. Acad. Sci. U.S.A. 101 (19), 7329-7334 (2004). The diploid genome sequence of Candida albicans is available from National Center for Biotechnology Information database under accession number XM_706819 (as available on 14 Feb. 2012).
“Heat shock protein 90” or “Hsp90” is a molecular chaperone which is involved, in Candida albicans, in the morphogenetic transition from yeast to filamentous growth. Hsp90 has been reported to be present in cell wall of Candida albicans and it circulates in body fluids of patients with invasive candidiasis. A representative sequence for Hsp90 is Hsp90 from Candida SC5314 which is encoded for instance by a polynucleotide comprising sequence SEQ ID NO:5, and which consist of the amino acid sequence SEQ ID NO:6. These sequences were published in Chibana et al. Genetics 170 (4), 1525-1537 (2005). Sequence finishing and gene mapping for Candida albicans are available from National Center for Biotechnology Information database under accession number XM_883730 (as available on 14 Feb. 2012).
“Hyphal wall protein” or “Hwp1” denotes a mannoprotein specifically expressed in the cell wall surface of the hyphae of C. albicans. A representative sequence for Hwp1 is Hwp1 from Candida SC5314 which is encoded for instance by a polynucleotide comprising sequence SEQ ID NO:7, and which consist of the amino acid sequence SEQ ID NO:8. These sequences were published in Jones et al. Proc. Natl. Acad. Sci. U.S.A. 101 (19), 7329-7334 (2004). The diploid genome sequence of Candida albicans is available from National Center for Biotechnology Information database under accession number XM_707905 (as available on 14 Feb. 2012).
“Mannoprotein 65” or “Mp65” denotes a 65-kDa mannoprotein which is present in the cell wall and also in Candida culture supernatants and which is thought to have adhesive properties. A representative sequence for Mp65 is Mp65 from Candida SC5314 which is encoded for instance by a polynucleotide comprising sequence SEQ ID NO:9, and which consist of the amino acid sequence SEQ ID NO:10. These sequences were published in Jones et al. Proc. Natl. Acad. Sci. U.S.A. 101 (19), 7329-7334 (2004). The diploid genome sequence of Candida albicans is available from National Center for Biotechnology Information database under accession number XM_709288 (as available on 14 Feb. 2012).
It is to be understood that Candida Fba1, Eno1, Hsp90, Hwp1, and Mp65 proteins are intended to encompass any naturally existing variant or isoform protein differing from the respective representative amino acid sequences identified above by modification of the amino acid sequence and/or of the glycosylation pattern of the protein. By modification of the amino acid sequence it is meant deletion, addition or substitution, of at least one amino acid, for instance by 1 to 15 amino acids, such as by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids. Preferably the variant or isoform protein has at least 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with the representative amino acid sequence, as can be determined by global pairwise alignment using the Needleman-Wunsch algorithm. The percentage of sequence identity can be readily determined for instance using the program Needle, with the BLOSUM62 matrix, and the following parameters gap-open=10, gap-extend=0.5.
The Candida Fba1, Eno1, Hsp90, Hwp1, and Mp65 proteins can elicit antibodies in subjects infected with Candida species and developing IC.
The antibodies to be detected may be an IgM, IgD, IgG (in particular IgG1, IgG2, IgG3 or IgG4), IgA and IgE, or any combination thereof.
As used herein, detecting “the presence or level of antibody directed against a Candida protein selected from the group consisting of Fba1, Eno1, Hsp90, Hwp1, and Mp65” means that the presence or level of antibodies against at least one of Hsp90, Fba1, Hwp1, Eno1 and Mp65 is detected. According to an embodiment, the presence or level of antibodies against Fba1, or antibodies against Eno1, or antibodies against Hsp90, or antibodies against Hwp1, or antibodies against Mp65, only, is detected. Although combinations of more than two of the markers defined herein (Candida glycan and antibody directed against a Candida protein) may not significantly improve discriminatory potency of the method, the presence or level of antibodies against more than one Candida protein, in particular 2 or 3 Candida proteins, as recited herein may be detected. For instance the presence or level of antibody against Fba1 and the presence or level of antibody against Eno1, or the presence or level of antibody against Fba1 and the presence or level of antibody against Hsp90, or the presence or level of antibody against Fba1 and the presence or level of antibody against Hwp1, or the presence or level of antibody against Fba1 and the presence or level of antibody against Mp65, or the presence or level of antibody against Eno1 and the presence or level of antibody against Hsp90, or the presence or level of antibody against Eno1 and the presence or level of antibody against Hwp1, or the presence or level of antibody against Eno1 and the presence or level of antibody against Mp65, or the presence or level of antibody against Hwp1 and the presence or level of antibody against Mp65.
When the Candida protein is a glycoprotein, such as Hwp1, Hsp90 and Mp65, the antibodies directed against a Candida protein which are detected or measured may the antibodies binding to a deglycosylated form of said Candida protein.
The presence or level of antibody may be determined by any suitable immunoassay method known from the one skilled in the art, as described below.
The method preferably does not include detection of additional biomarkers, other than Candida glycan(s) and antibody(ies) directed against a Candida protein selected from the group consisting of Fba1, Eno1, Hsp90, Hwp1, and Mp65. In particular, the method preferably does not comprise detecting antibody(ies) directed against Candida glycan(s), especially against mannan.
Immunoassay Methods
Immunoassays are antibody-based methods which enable for measuring antigens or antibodies. They typically include indirect, sandwich, or competition immunoassays, for instance in a radioimmunoassays (RIA) or EIA (Enzyme ImmunoAssay) format. A sandwich immunoassay is a method using two antibodies specific for the antigen to be detected, which bind to different sites on the antigen or ligand. The primary antibody (or capture antibody), which recognizes the antigen, is attached to a solid surface. The antigen is then added followed by addition of a second antibody referred to as the detection antibody. The detection antibody binds the antigen to a different or a repeated epitope(s). As the antigen concentration increases the amount of detection antibody increases leading to a higher measured response.
To quantify the extent of binding different reporters can be used: a radioactive tracer in RIA, or a fluorophore or enzyme in EIA (in particular in ELISA). In ELISA, an enzyme is typically attached to the detection antibody or to a third antibody which binds the detection antibody and which must be generated in a different species than detection antibodies (i.e. if the detection antibody is a rabbit antibody than the third antibody would be an anti-rabbit from goat, chicken, etc., but not rabbit). The substrate for the enzyme is added to the reaction that forms a colorimetric readout as the detection signal. The enzyme step of the assay can be replaced with a fluorophore-tagged detection and the fluorescent signal is measured in a fluorescent plate reader. The signal generated is proportional to the amount of target antigen present in the sample.
A competitive binding assay is based upon the competition of labelled and unlabeled antigen for a limited number of antibody binding sites. A fixed amount of labelled antigen and a variable amount of unlabeled antigen are incubated with an antibody bound to a solid phase. The amount of labelled antigen is a function of the total concentration of labelled and unlabeled antigen. As the concentration of unlabeled antigen is increased, less labelled antigen can bind to the antibody and the measured response decreases. Thus the lower the signal, the more unlabeled antigen there is in the sample.
In indirect immunoassay, an antigen is coated onto a solid surface. The coated solid surface is then incubated with a sample to analyse if it contains antibodies specific for the antigen. Detection of antigen-bound antibodies is then performed using a detectably labelled secondary antibody which binds to the antibody specific for the antigen. The secondary antibody may be labelled with a fluorophore which generates a fluorescent signal measurable in a fluorescent plate reader, or with an enzyme which can produce a colorimetric signal upon addition of the enzyme substrate.
The immunoassay may be performed in a lateral flow immunochromatographic assay format, high throughout and/or multiplex immunoassay format such as Luminex®, platform or any microfluidic derived format such as microchip immunoassays, etc. . . .
As used herein, “antibody” or “immunoglobulin” have the same meaning, and are used equally. In natural conventional antibodies, two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chain, lambda (λ) and kappa κ. There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG (such as IgG1, IgG2, IgG3 or IgG4), IgA and IgE. The light chain includes two domains, a variable domain (VL) and a constant domain (CL). The heavy chain includes four domains, a variable domain (VH) and three constant domains (CHI, CH2 and CH3, collectively referred to as CH). The variable regions of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen. The specificity of the antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant (epitope). Antibody combining sites are made up of residues that are primarily from the hypervariable or complementarity determining regions (CDRs). Occasionally, residues from non hypervariable or framework regions (FR) influence the overall domain structure and hence the combining site. The CDR refers to amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site. The light and heavy chains of an immunoglobulin each have three CDRs, designated L-CDR1, L-CDR2, L-CDR3 and H-CDR1, H-CDR2, H-CDR3, respectively. An antigen-binding site, therefore, includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region. FR refers to amino acid sequences interposed between CDRs.
Antibodies may be monoclonal antibodies (mAb), polyclonal antibodies, or fragment thereof. Monoclonal antibodies are monospecific antibodies, produced by clones of a hybridoma cell line, which bind to the same epitope on an antigen. Polyclonal antibodies are a combination of immunoglobulins directed against a specific antigen, each immunoglobulin possibly binding to a different epitope on the antigen. Polyclonal antibodies are generally produced by immunisation of a suitable mammal, such as a mouse, rabbit or goat. Antibody fragments include for instance Fab, Fab′, F(ab′)2, Fv, scFv, and nanobodies (single chain antibodies).
For the detection of a Candida glycan, antibodies binding preferentially or specifically to said Candida glycan may be used. The antibodies which may be used for the detection of a Candida glycan may be monoclonal antibodies (mAb), polyclonal antibodies, or fragment thereof.
As used herein, an antibody binding “preferentially” to said Candida glycan, is an antibody showing less that 15% cross reactivity, preferably less than 10% or 5% cross reactivity, with an antigen different from said Candida glycan. An antibody binding “specifically” to said Candida glycan is an antibody showing no cross-reactivity with any other antigen.
For the detection of mannan, antibodies binding to α-1,2-linked oligomannose sequences of more than four residues, or to β-1,2-linked oligomannosides could be used for instance. The monoclonal antibody EBCA1, which is available in the Platelia Candida antigen and Platelia Candida antigen Plus (Ag) tests (Bio-Rad Laboratories, Marnes La-Coquette, France), binds to a minimal epitope consisting of α-1,2-linked oligomannose sequences of more than four (or at least five) residues which are present in the acid-stable fraction of mannan. Other monoclonal antibodies could be used for the detection of β-1,2-linked oligomannosides, such as antibody 5B2 which preferentially binds with mannobiosides (Hopwood et al. Infect. Immun. 1986, 54, 222-227; Collot M et al J. Med. Chem. 2008; 51:6201-6210), antibody B6.1 which specifically binds with mannotriose (Han, Y et al. Infect. Immun. 1997; 65:4100.) and antibody 26G7 which recognizes an heteropolymer 2 beta and 2 alpha mannosides (Elguezabal et al., Oral Dis, 2004, 10, 81-86).
In particular, mannan may be conveniently detected by a sandwich enzyme immunoassay which uses, as capture and detection antibodies, an antibody recognizing sequences of α-linked oligomannoses constituted of more than four (or at least five) residues, such as the monoclonal antibody EBCA1.
β-glucans and chitin are immunogenic as circulating anti-β-D-glucans and anti-chitin antibodies have been found in humans (Sendid et al. Clin. Vaccine Immunol. 2008, 15, 1868-1877). Accordingly antibodies can be elicited against (1,6)-β-D-glucan, (1,3)-β-D-glucan or chitin in order to be used in an immunoassay for detecting a (1,6)-β-D-glucan or chitin.
For the detection of (1,3)-β-D-glucan and/or (1,6)-β-D-glucan, an antibody such as antibody 2G8 which was described in the international patent application WO2006/030318, or an anti-(1,3)-β-D-glucan antibody such as available from Biosupplies Australia Pty. Ltd. may be used.
For the detection of antibody(ies) directed against a Candida protein, purified Candida protein, or epitopic fragments thereof, or a polypeptide comprising full length protein, epitopic fragments or variants thereof which have been recombinantly produced or chemical synthesized may be used. When the Candida protein is a glycoprotein, the purified Candida protein, or epitopic fragments thereof, may have been deglycosylated. Purified and recombinant full length proteins or epitopic fragments thereof may or may not include glycosylation(s). Preferably the polypeptide used for detection of an antibody directed against a Candida protein is devoid of glycosylation.
As used herein, the term “epitopic fragment” refers to a polypeptide fragment of a protein which consists of a stretch of contiguous amino acids of the protein and comprises one or more epitopes of said protein. An epitopic fragment retains the capacity to be bound by at least part, preferably all, of the antibodies that bind to the full length protein from which the fragment is derived. An epitope may be linear when made of contiguous amino acids, or conformational when an antibody binds to discontinuous amino acids that come together in three dimensional conformation. It is generally admitted that a linear epitope is constituted by at least 4 or 5 amino acid contiguous residues. Accordingly, an “epitopic fragment” according to the invention preferably comprises at least 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 40, 50 or 100 contiguous amino acids of the protein from which the epitopic fragment derives. An “epitopic fragment” may comprise less than 300, 250, 200, 150, 100, 50, 40, 30, 20 or 15 amino acids.
The fragments can be generated for instance by processing the full length protein, or a longer fragment of said protein, with a proteolytic enzyme (classically trypsin, chymotrypsin, or collagenase), or by chemical treatment with cyanogen bromide (CNBr), for instance.
Variant polypeptides of the full length Candida protein or of an epitopic fragment thereof may be obtained by modification of its amino acid sequence by deletion, addition or substitution, of at least one amino acid, for instance by 1 to 15 amino acids, such as by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids. Preferably the variant polypeptide has at least 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with the full length Candida protein or epitopic fragment thereof, as can be determined by global pairwise alignment using the Needleman-Wunsch algorithm, in particular using the program Needle, with the BLOSUM62 matrix, and the following parameters gap-open=10, gap-extend=0.5. A variant polypeptide retains the capacity to be bound by at least part, preferably all, of the antibodies that bind to the full length protein or the fragment thereof from which it derived. A variant polypeptide may typically comprise a heterologous polypeptide sequence, such as a tag (His-tag, GFP, etc. . . . ) to facilitate purification.
Deglycosylation of Candida (glycol) protein, of fragments thereof, may be performed by any chemical or enzymatic method compatible with the preservation of the antigenicity of the protein (i.e maintaining epitope structures). Reference may be made for instance to trifluoromethanesulfonic acid (TFMS) hydrolysis for complete glycan removal without altering the protein component, or to processing with the enzymes PNGase, or F Endoglycosidases F1, F2, and F3 for removal of N-linked glycans.
Recombinant Candida full length protein, epitopic fragments or variants thereof may be produced by any method known to the one skilled in the art. Typically, a nucleic acid sequence comprising a sequence encoding the desired polypeptide, under the control of or operatively associated with transcriptional and translational control sequences (in particular a promoter sequence) is inserted in an expression vector. A vector is the vehicle by which a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence. Vectors include plasmids, phages, viroses, etc. An “expression system”, i.e. the host cell and a compatible vector for the expression of Candida protein, or epitopic fragment thereof, may include E. coli host cells and plasmid vectors, yeast host cells (such as Pichia pastoris) and vectors and plasmid vectors, insect host cells and Baculovirus vectors, and mammalian host cells (such as COS-1 or CHO cells) and vectors. Preferably, recombinant Candida protein or epitopic fragments thereof are produced in E. coli. The recombinant proteins may be produced with tags, such as His-tags, in order to facilitate their purification, which may be eliminated afterwards.
For epitopic fragments preferably containing no more than 20 amino acids, chemical synthesis such as solid phase synthesis may be employed for preparing the epitopic fragments.
It has been reported that a 47-kDa antigen which is a heat-stable breakdown product of Hsp90 may be found in patients with systemic candidiasis. Cloning of this 47-kDa fragment (consisting of amino acids at positions 313 to 707 of Hsp90 sequence SEQ ID NO:6) has been described in Matthews and Burnie, 1989, FEMS Microbiol. Lett. 60:25-30 and in U.S. Pat. No. 5,686,248. It has been further shown that antibodies to the 47-kDa antigen of Hsp90 cross react with peptides of sequences STDEPAGESA (SEQ ID NO:13), LSREM (SEQ ID NO:14), LKVIRK (SEQ ID NO:15) and LKVIRKNIVKKMIE (SEQ ID NO:16). Accordingly, a polypeptide which may be used for detecting anti-Hsp90 antibodies may be selected in particular from the group consisting of a polypeptide comprising, or consisting of:

    
    
        a) SEQ ID NO:6 or a naturally existing variant or isoform thereof;
        b) an epitopic fragment of a polypeptide defined in a), in particular a fragment consisting of amino acids at positions 313 to 707 of SEQ ID NO:6, or a variant thereof; and
        c) SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:16 or a variant thereof.
    
    


While full length Hwp1 protein can be used for detecting anti-Hwp1 antibodies, it has been reported that a 161 amino acid long N-terminal fragment thereof (amino acids at positions 41 to 200) increased antibody detection by ELISA in sera of patients with invasive candidiasis (Lain et al., 2007, BMC Microbiology, 7:35). Furthermore, a fragment of Hwp1 consisting of amino acids at positions 27 to 203 of Hwp1 was shown to be an epitopic fragment of Hwp1 (Fradin et al., 2008 Infect. Immun. 76, 4509-4517).
Accordingly, a polypeptide which may be used for detecting anti-Hwp1 antibodies may be selected in particular from the group consisting of a polypeptide comprising, or consisting of:

    
    
        a) SEQ ID NO:8 or a naturally existing variant or isoform thereof;
        b) an epitopic fragment of a polypeptide defined in a), in particular a fragment consisting of amino acids at positions 41 to 200, or 27 to 203 of SEQ ID NO:8, or a variant thereof.
    
    


A polypeptide which may be used for detecting anti-Fba1 antibodies may be selected in particular from the group consisting of a polypeptide comprising, or consisting of:

    
    
        a) SEQ ID NO:2 or a naturally existing variant or isoform thereof;
        b) an epitopic fragment of a polypeptide defined in a), or a variant thereof.
    
    


A polypeptide which may be used for detecting anti-Eno1 antibodies may be selected in particular from the group consisting of a polypeptide comprising, or consisting of:

    
    
        a) SEQ ID NO:4 or a naturally existing variant or isoform thereof;
        b) an epitopic fragment of a polypeptide defined in a), or a variant thereof.
    
    


For the detection of antibodies against Mp65, the Scw1 protein, i.e. the protein homologous to Mp65 in Saccharomyces cerevisiae, may also be used. Scw1 coding sequence and amino acid sequence are shown in SEQ ID NO:11 and 12, respectively. Accordingly, a polypeptide which may be used for detecting anti-Mp65 antibodies may be selected in particular from the group consisting of a polypeptide comprising, or consisting of:

    
    
        a) SEQ ID NO:10 or a naturally existing variant or isoform thereof, in particular a polypeptide comprising, or consisting of SEQ ID NO:12;
        b) an epitopic fragment of a polypeptide defined in a), or a variant thereof.
    
    


Diagnosis of Invasive Candidiasis
According to an embodiment, diagnosing invasive candidiasis may be performed by comparing the level of said Candida glycan with a reference level, and comparing the level of antibody directed against said Candida protein selected from the group consisting of Fba1, Eno1, Hsp90, Hwp1, and Mp65, with another reference level. If an elevated level of said Candida glycan, and/or an elevated level of antibody directed against said Candida protein selected from the group consisting of Fba1, Eno1, Hsp90, Hwp1, and Mp65, relative to their respective reference level is detected, then the subject is developing or has developed invasive candidiasis. In this embodiment, a combined interpretation of the separated assays is thus performed.
The reference level(s) may be determined as a single value or a range of values which is determined based on the level of said Candida glycan or the level of antibody directed against said Candida protein, as appropriate, measured in a population of healthy subjects, in a population of subjects superficially infected with a Candida strain, or in a population of subjects suffering from invasive candidiasis.
The reference level can also be determined by analysing a sample from the same subject for instance at an earlier time point prior to onset of invasive candidiasis or prior to suspicion of invasive candidiasis.
Typically, the analysed population could be divided into quantiles based on the measured level of antigen or antibody. The reference level could be defined as the median, or the second tertile, or the second or third quartile, or the third or fourth quintile etc. . . .
Comparison with a reference level may also be performed by comparing the level of said Candida glycan or the level of antibody directed against said Candida protein with the level of Candida glycan or antibody, as appropriate, measured in a standard sample constituted by a pool of sera obtained from patients having invasive candidiasis.
The reference level for a given marker may vary depending on the method used for measuring.
According to another embodiment, a combined analysis of the levels of said Candida glycan and of said antibody directed against said Candida protein is performed in order to determine if the subject has or has not invasive candidiasis. In this embodiment, a combined analysis of the biomarkers is thus performed.
According to this embodiment, the level of a Candida glycan and the level of an antibody directed against the Candida protein are detected in one and the same blood, plasma or serum sample of the patient or in several blood, plasma or serum samples sequentially obtained from the same patient essentially simultaneously.
Based on the analysis of a reference set of blood, plasma or serum samples from subjects with invasive candidiasis and subjects without invasive candidiasis, a Relative Operating Characteristic (ROC) curve can be generated for each marker analysed. A ROC curve is a graphical representation of the sensitivity (or true positive rate) against the false positive rate (i.e. [1-specificity], specificity being the true negative rate) of a marker-based test. A ROC space is defined by sensitivity and (1-specificity) as x and y axes respectively. The best possible prediction method would yield a point in the upper left corner or coordinate (0,1) of the ROC space, representing 100% sensitivity (no false negatives) and 100% specificity (no false positives). A completely random guess would give a point along a diagonal line (the so-called line of no-discrimination) from the left bottom to the top right corners. The diagonal divides the ROC space. Points above the diagonal represent good classification results (better than random), points below the line poor results (worse than random). The Area Under the Curve (AUC) of a ROC curve may be calculated. The higher the AUC, the higher the diagnostic accuracy of the diagnostic marker.
For combined analysis of markers, such as the level of said Candida glycan and the level of said antibody directed against said Candida protein, a new virtual marker Z may be calculated based on a linear combination of the levels of the individual markers, i.e. the level of said Candida glycan and the level of antibody directed against said Candida protein. Z is calculated as follows: Z=Σai×[Markeri] where ai are calculated coefficients and [Markeri] are individual levels of marker (optionally in normalised units). The values of the ai coefficients are determined in order to maximize the Area Under the Curve (AUC) of the ROC curve for the selected marker combination.
Determination of the coefficient values may be readily achieved using for instance mROC program or any other program implementing an algorithm for maximising the AUC of ROC which may be used for multivariate ROC (Wang, Computational Statistics and Data Analysis 2007; 51:2803-2812; Xiong et al. Med Decis Making. 2004 November-December; 24(6):659-69; Ma and Huang, Bioinformatics. 2005 Dec. 15; 21 (24):4356-62; Pepe et al., Biometrics. 2006 March; 62(1):221-9; Wang et al.; Wang et al., J Proteomics Bioinform. 2012, 5:3; Liu A et al., Stat Med. 2005 Jan. 15; 24 (1):37-47. PubMed PMID: 15515132).
Examples of values for ai for two-marker combinations, depending on the marker combination, and based on the analysis of the serum samples obtained from the patients identified in Tables 3 and 4 of the instant application, are shown in Table 1. These ai values were those used to generate the ROC curves shown in FIGS. 6-10.







TABLE 1







Exemples of a1 and a2 coefficients maximizing


AUC of ROC curve for two-marker combination










Marker combination
Z = a1 × Marker1 + a2 × Marker2







Ag_Mannan + Ab_Fba1
Z = 0.499 × [Ag_Mannan*] +




2.324 × [Ab_Fba1*]



Ag_Mannan + Ab_Hwp1
Z = 0.481 × [Ag_Mannan*] +




2.416 × [Ab_Hwp1*]



Ag_Mannan + Ab_Hsp90
Z = 0.481 × [Ag_Mannan*] +




2.416 × [Ab_Hsp90*]



Ag_Mannan + Ab_Eno1
Z = 0.453 × [Ag_Mannan*] +




1.916 × [Ab_Eno1*]



Ag_Mannan + Ab_Mp65
Z = 0.486 × [Ag_Mannan*] +




2.261 × [Ab_Mp65*]



Ag_Mannan + Ab_Mannan
Z = 0.599 × [Ag_Mannan*] +




0.357 × [Ab_Mannan*]







*levels of markers normalised by a log10 transformation.






The above values of a1 and a2 coefficients for two-marker combinations given in Table 1 are purely indicative as these values are liable to be modified with standardization of the methods. Furthermore, the a1 and a2 coefficients may depend on the measure units used for mannan antigen and antibody against Candida protein.
Once the values of the ai coefficients have been determined for a given marker combination, by standardisation on subject and control samples, a reference level of the virtual marker Z may be determined as a single value or a range of values, for instance as quantiles.
When the level of a Candida glycan and the level of an antibody directed against the Candida protein have been measured in a sample, or in at least two samples obtained at the same time from the same subject, a value of the virtual marker Z may be calculated for the test sample. If the calculated value for the virtual marker Z is higher than the reference level of the virtual marker Z, then the subject is developing or has developed invasive candidiasis.
Therefore, the invention also relates to a method which comprises the steps consisting of:

    
    
        a) detecting the level of a Candida glycan in a blood, plasma or serum sample of the subject;
        b) detecting the level of antibody directed against a Candida protein selected from the group consisting of fructose biphosphate aldolase (Fba1), enolase 1 (Eno1), heat shock protein 90 (Hsp90), hyphal wall protein (Hwp1), and mannoprotein 65 (Mp65) in the same blood, plasma or serum sample of the subject or in another blood, plasma or serum sample sequentially obtained from the same patient, essentially simultaneously; and
        c) wherein a combined analysis of the level of said Candida glycan and the level of antibody directed against said Candida protein selected from the group consisting of Fba1, Eno1, Hsp90, Hwp1, and Mp65, is performed by calculating the level of a virtual marker Z=Σai×[Markeri] where [Markeri] are individual levels of Candida glycan marker and of antibody directed against said Candida protein selected from the group consisting of Fba1, Eno1, Hsp90, Hwp1, and Mp65, and ai are coefficients which values are determined in order to maximize the Area Under the Curve (AUC) of the Relative Operating Characteristic (ROC) curve for the combination of Markeri; and
        d) wherein if the level of the virtual marker Z calculated in step c) is higher than a reference level of the virtual marker Z, then the subject is developing or has developed invasive candidiasis.
    
    


Method for Determining a Suitable Treatment Regimen
The high rates of mortality associated with invasive candidiasis (IC) are mainly due to the delay in identifying that a subject is affected with IC and initiating the appropriate antifungal treatment.
The invention further relates to a method for determining a treatment regimen in a subject, said method comprising:
a) diagnosing if the subject has invasive candidiasis by carrying out the method as described above, and
b) if the subject is diagnosed as having invasive candidiasis, determining an antifungal treatment for said subject.
The subject may be a subject suspected of having invasive candidiasis or at risk of invasive candidiasis. The subject may be in particular a subject having superficial infection of skin and/or mucosal membranes. The subject may also be for instance an immunocompromised subject bearing intravenous catheter.
An antifungal treatment may be for instance treatment with echinocandins which notably include Caspofungin (1-[(4R,5S)-5[(2-aminoethyl)amino]-N2-(10,12-dimethyl-1-oxotetradecyl)-4-hydroxy-L-ornithine]-5-[(3R)-3-hydroxy-L-ornithine]pneumocandin B0), Micafungin and/or Anidulafungin (see for instance Patterson T F. Curr Infect Dis Rep 2006; 8:442-8). Echinocandins are synthetically modified lipopeptides which inhibit the synthesis of (1,3)-β-D-glucan. Another antifungal treatment may be for instance treatment with Enfumafungin and Enfumafungin derivatives such as described in the patent application published as US 20110224228. Amphotericin B and its lipidic formulations, or antifungal azole derivatives such as fluconazole, itraconazole, and Voriconazole (see for instance Pfaller et al. J Clin Microbiol 2006; 44:819-26; Mean et al. Crit Care 2008; 12:204) may also be used.
Combined administration of theses compounds, or combination with other active principles is possible.
The treatment is preferably administered as long as circulating mannan can be detected in the serum of the subject.
Kit for Diagnosing Invasive Candidiasis
The invention further relates to a kit comprising, or consisting of:

    
    
        a) means for determining the presence or level of a Candida glycan as described above; and
        b) means for determining the presence or level of antibody directed against a Candida protein selected from the group consisting of fructose bisphosphate aldolase (Fba1), enolase 1 (Eno1), heat shock protein 90 (Hsp90), hyphal wall protein (Hwp1), and mannoprotein 65 (Mp65), described above.
    
    


Means for determining the presence or level of a Candida glycan may be in particular means for determining the presence or level of Candida mannan, (1,3)-β-D-glucan or chitine. Such means may be in particular antibodies such as those described above (e.g. EBCA1 for mannan). Such means may also comprise a chromogenic substrate and enzyme(s) as are available in the Fungitell test (Associates of Cape Cod, Mass., USA).
Means for determining the presence or level of antibody directed against a Candida protein selected from the group consisting of Fba1, Eno1, Hsp90, hwp1 or Mp65 may comprise purified Candida protein, or epitopic fragments thereof, or a polypeptide comprising full length protein, epitopic fragments or variants thereof which have been recombinantly produced or chemical synthesized, as disclosed above.
The invention will be further illustrated in view of the following figures and examples.



FIGURES
FIGS. 1-5. Boxplot representation of diagnosis potential of anti-recombinant protein-antibody [RP-Ab; respectively anti-Fba1 Ab (FIG. 1); anti-Hwp1 Ab (FIG. 2); anti-Hsp90 Ab (FIG. 3); anti-Eno1 Ab (FIG. 4); anti-Mp65 Ab (FIG. 5)] or anti-mannan antibodies associated with mannanemia. Boxplot representation is a convenient way of graphically depicting groups of numerical data through their five-number summaries (the smallest observation, lower quartile (Q1), median (Q2), upper quartile (Q3), and largest observation). Boxplots can be useful to display differences between populations without making any assumptions of the underlying statistical distribution. CTRL represents controls and IC represents invasive candidiasis patients.
FIGS. 6-10. ROC curves of combinations of RP-Ab [respectively anti-Fba1 Ab (FIG. 6); anti-Hwp1 Ab (FIG. 7); anti-Hsp90 Ab (FIG. 8); anti-Eno1 Ab (FIG. 9); anti-Mp65 Ab (FIG. 10)] with mannanemia. The actual diagnosis (mannan Antibody and mannan Antigen) test was represented with dotted lines and association of RP-Ab and mannanemia was represented with black line.



EXAMPLES
Example 1
Recombinant Protein Production in E. coli 
1) Strains and Plasmids
Candida albicans SC5314 was used as fungal DNA source to generate the different recombinant proteins. Escherichia coli strain DE3 was transformed to produce the recombinant proteins while the strain DH5α was used to amplify the plasmids.
2) Cloning
Six recombinant proteins were expressed in E. coli as described previously (Fradin et al., Infect Immun (2008), 76: 4509-4517): N-terminal fragment of Hwp1 (amino acids 27 to 203), Eno1 (full length), Mp65 without its peptide signal (amino acids 1 to 22), Fba1 (full length), Sod5 without its peptide signal (amino acids 1 to 22) and its C-terminal GPI consensus sequence (last 24 terminal amino acids) and Hsp90 (full length).
Six sets of primers (Table 2 below) were designed to clone the different genes or truncated genes. PCR amplified fragments with high fidelity Expand Taq polymerase (Roche) were directly cloned in pEXP5-NT/TOPO plasmid.







TABLE 2







Set of primers used for gene cloning








Recombinant



proteins
Primers set





N-term Hwp1
5′CAAGGTGAAACAGAGGAAGCT3′ (SEQ ID NO: 17) and



5′TCAAGCAGGAATGTTTGGAGTAGT3′ (SEQ ID NO: 18)


 


Eno1
5′ATGTCTTACGCCACTAAAATCCACGC3′ (SEQ ID NO: 19) and



5′TTACAATTGAGAAGCCTTTTGGAAATCTTTAC3′ (SEQ ID NO: 20)


 


Mp65
5′GCTCATCAACATCATCAACAT3′ (SEQ ID NO: 21) and



5′TTAGTTAGAGTAAATACCCCAGTA3′ (SEQ ID NO :22)


 


Fba1
5′ATGGCTCCTCCAGCAGTTTTA3′ (SEQ ID NO: 23) and



5′TTACAATTGTCCTTTGGTGTG3′ (SEQ ID NO: 24)


 


Sod5
5′GATGCACCAATCTCAACTGAC3′ (SEQ ID NO: 25) and



3′TTAACCTTGAGGAGCAGTAGAAGC3′ (SEQ ID NO: 26)


 


Hsp90
5′ATGGCTGACGCAAAAGTTGAA3′ (SEQ ID NO: 27) and



5′TTAATCAACTTCTTCCATAGC3′ (SEQ ID NO: 28)









Example 2
Evaluation of Diagnosis Potential of Anti-Recombinant Protein-Antibodies or Anti-Mannan Antibodies Associated with Mannanemia
1) Materials and Methods
Patients
Between January 2005 and December 2007, 157 serum samples were retrospectively collected in different clinical departments of Lille University Hospital (LUH), from 53 patients (24 females and 29 males [mean age, 56.78+/−23.71 years]) with proven Candida albicans candidiasis. The average number of samples per patient in this group was 2.68+/−2.13 (Table 3).







TABLE 3







Clinical features of patients with systemic Candida albicans


infection. Patients were classified according to clinical wards

















No. Of
Date of serum sampling in



Patient
Sexa
Age (yr)
Hospital ward
sera
relation to blood culture (days)
Candida species
















1
F
34
Burn unit
7
−10, −3, 0, 4, 13, 18, 22
Candida albicans


2
M
43
Burn unit
4
−16, −9, 12, 27
Candida albicans


3
M
54
Burn unit
3
−6, −4, 10
Candida albicans


4
F
54
Burn unit
4
−10, −3, 4, 11
Candida albicans


5
F
59
Burn unit
1
17
Candida albicans


6
M
68
Burn unit
4
−10, −3, 4, 18
Candida albicans


7
F
75
Burn unit
2
7, 14
Candida albicans


8
M
81
Cardiology
1
6
Candida albicans


9
F
31
Gastroenterology
1
0
Candida albicans


10
M
53
Gastroenterology
1
2
Candida albicans


11
M
59
Gastroenterology
3
−6, −2, 6
Candida albicans


12
M
78
Heat surgery
1
1
Candida albicans


13
F
46
Clinical hematology
8
−8, −5, 1, 5, 5, 16, 22, 27
Candida albicans


14
F
62
Clinical hematology
7
−3, 4, 12, 14, 20, 27, 29
Candida albicans


15
M
70
Clinical hematology
6
3, 10, 13, 17, 19, 24
Candida albicans


16
F
86
Infectious diseases
3
1, 7, 14
Candida albicans


17
M
10
Intensive care unit
2
3, 23
Candida albicans


18
F
14
Intensive care unit
1
4
Candida albicans


19
M
32
Intensive care unit
2
14, 30
Candida albicans


20
M
35
Intensive care unit
1
−1
Candida albicans


21
F
36
Intensive care unit
2
3, 10
Candida albicans


22
M
46
Intensive care unit
2
−2, 5
Candida albicans


23
M
47
Intensive care unit
6
−3, 4, 13, 20, 27, 28
Candida albicans


24
M
49
Intensive care unit
2
−6, −2
Candida albicans


25
F
50
Intensive care unit
1
0
Candida albicans


26
M
57
Intensive care unit
6
−8, −2, 5, 12, 20, 27
Candida albicans


27
F
59
Intensive care unit
2
3, 4
Candida albicans


28
M
60
Intensive care unit
5
−10, −3, 4, 11, 25
Candida albicans


29
F
62
Intensive care unit
4
−12, −5, 2, 9
Candida albicans


30
M
64
Intensive care unit
2
2, 9
Candida albicans


31
M
66
Intensive care unit
5
2, 6, 8, 12, 13
Candida albicans


32
M
72
Intensive care unit
4
−5, 2, 6, 9
Candida albicans


33
F
75
Intensive care unit
1
3
Candida albicans


34
F
76
Intensive care unit
9
4, 7, 11, 14, 17, 22, 25, 27, 28
Candida albicans


35
F
79
Intensive care unit
9
−1, 3, 4, 5, 6, 13, 14, 20, 27
Candida albicans


36
F
82
Intensive care unit
1
5
Candida albicans


37
F
87
Intensive care unit
4
2, 7, 22, 29
Candida albicans


38
M
89
Intensive care unit
2
−7, −1
Candida albicans


39
M
48
intensive surgical care unit
1
2
Candida albicans


40
M
72
intensive surgical care unit
1
0
Candida albicans


41
M
72
intensive surgical care unit
3
−3, 4, 16
Candida albicans


42
F
73
intensive surgical care unit
2
−5, 25
Candida albicans


43
M
73
intensive surgical care unit
1
11
Candida albicans


44
M
78
intensive surgical care unit
1
2
Candida albicans


45
F
83
intensive surgical care unit
2
−9, 19
Candida albicans


46
M
15
Oncology
2
−5, 1
Candida albicans


47
M
6
Paediatrics
6
15, 17, 18, 19, 24, 26
Candida albicans


48
F
14
Paediatrics
1
−1
Candida albicans


49
F
17
Paediatrics
3
−15, −8, 6
Candida albicans


50
M
53
Pneumology
2
−11, −10
Candida albicans


51
F
80
Pneumology
1
4
Candida albicans


52
M
64
Transplantation
1
−1
Candida albicans


53
F
49
Traumatology
1
0
Candida albicans





aM, male; F, female






A second group of 142 serum samples was also collected in different departments of LUH from 40 patients (10 females and 30 males [mean age, 58.00+/−21.97]) with proven invasive candidiasis determined by non-albicans yeast species (Table 4). The average number of samples per patient in this group was 3.59+/−2.66. This group contains 7 different yeast species: Candida parapsilosis (17 patients; 49 sera), Candida kruseï (3 patients; 10 sera), Candida tropicalis (5 patients; 20 sera), Candida glabrata (12 patients; 40 sera), Geotrichum capitatum (1 patient; 12 sera), Candida norvegiensis (1 patient; 3 sera) and Candida lusitaniae (1 patient; 8 sera).







TABLE 4







Clinical features of patients with systemic Candida infection. Patients were


classified according to yeast species involved in IC and to clinical wards

















No. of
Date of serum sampling in



Patient
Sex a
Age (yr)
Hospital ward
sera
relation to blood culture (days)
Candida species
















54
M
21
Burn unit
2
−1, 12
Candida parapsilosis


55
M
43
Burn unit
4
−6, 0, 7, 21
Candida parapsilosis


56
F
82
Clinical hematology
2
2, 7
Candida parapsilosis


57
M
24
Intensive care unit
4
−11, −6, 0, 1
Candida parapsilosis


58
M
51
Intensive care unit
1
25
Candida parapsilosis


59
M
54
Intensive care unit
5
−10, −3, 4, 10, 18
Candida parapsilosis


60
M
65
Intensive care unit
1
−11
Candida parapsilosis


61
M
67
Intensive care unit
4
−11, −4, 3, 17
Candida parapsilosis


62
F
75
Intensive care unit
1
23
Candida parapsilosis


63
M
76
Intensive care unit
5
−15, −12, 2, 6, 9
Candida parapsilosis


64
M
78
Intensive care unit
3
−8, −3, 4
Candida parapsilosis


65
M
87
Intensive care unit
7
−14, −7, 0, 7, 14, 21, 22
Candida parapsilosis


66
F
87
Intensive care unit
2
−5, 8
Candida parapsilosis


67
M
65
Intensive surgical care unit
2
0, 1
Candida parapsilosis


68
M
57
Intensive surgical care unit
4
−5, 1, 9, 23
Candida parapsilosis


69
M
90
Intensive surgical care unit
1
3
Candida parapsilosis


70
F
10
Paediatrics
1
3
Candida parapsilosis


71
M
47
Clinical hematology
3
−11, −4, 3
Candida kruseï


72
M
68
Clinical hematology
6
−9, −7, −1, 4, 12, 19
Candida kruseï


73
M
75
Oncology
1
4
Candida kruseï


74
M
39
Burn unit
5
−15, −8, 10, 13, 28
Candida tropicalis


75
M
51
Clinical hematology
1
3
Candida tropicalis


76
F
62
Clinical hematology
10
−15, −6, −2, −1, 0, 1, 3, 5, 7, 12
Candida tropicalis


77
M
8
Intensive care unit
3
1, 8, 18
Candida tropicalis


78
M
67
Oncology
1
−5
Candida tropicalis


79
M
24
Hyperbare
2
−2, 5
Candida glabrata


80
M
31
Intensive care unit
1
3
Candida glabrata


81
M
57
Intensive care unit
5
0, 6, 13, 20, 27
Candida glabrata


82
M
63
Intensive care unit
4
−10, 2, 10, 11
Candida glabrata


83
M
63
Intensive care unit
8
−5, −4, −1, 0, 1, 3, 7, 12
Candida glabrata


84
F
69
Intensive care unit
1
14
Candida glabrata


85
F
76
Intensive care unit
5
−9, −5, 2, 9, 16
Candida glabrata


86
F
87
Intensive care unit
3
−7, 7, 12
Candida glabrata


87
F
24
Intensive surgical care unit
5
−11, −7, −5, 0, 10
Candida glabrata


88
M
71
Intense surgical care unit
2
1, 15
Candida glabrata


89
M
85
intensive surgical care unit
3
2, 4, 11
Candida glabrata


90
F
60
Oncology
1
8
Candida glabrata


91
M
64
Clinical hematology
12
−15, −11, −9, −6, −3, −1, 1, 4, 6, 11, 13, 15
Geotrichum capitatum


92
M
45
Clinical hematology
3
1, 7, 12
Candida norvegensis


93
M
76
Clinical hematology
8
−10, −3, −1, 1, 4, 6, 8, 11
Candida lusitaniae





a M, male; F, female






The following criteria were applied as retrospective selection rules when the laboratory and clinical files were examined: (i) positive blood culture from Candida species; (ii) availability of serum samples obtained within a range of 3 weeks before and 1 month after positive cultures, (iii) the presence of risk factors (cancer and chemotherapy, abdominal surgery, AIDS, major health problems requiring hospitalization in intensive care units -ICUs-, and use of broad-spectrum antibiotics, indwelling intravascular catheters, and hyperalimentation; and (iv) the presence of an infectious syndrome (namely, fever) that did not respond to antibacterial therapy but that did respond to antifungal therapy. In order to evaluate performances of biomarkers for the early diagnosis of IC, selection of serum samples was restricted to the period ranging from 2 weeks before to 1 month after the date of isolation of yeast species from blood culture. After blood sampling blood samples were centrifuged and serum aliquots were stored at −80° C. until required.
Control Group.
Two groups of control sera were included in this study:

    
    
        (i) Group 1 comprised 90 serum specimens from 90 hospitalized patients (32 females and 58 males; mean age, 64+/−15.5 years) without evidence of invasive candidiasis. This group of patients was enrolled in a prospective study conducted in an ICU of LUH for 6 months. The study was designed for the assessment of risk factors for nosocomial candidiasis. These patients were under clinical and mycological survey for periods ranging from 1 to 74 days (mean, 12 days). Samples of blood, oral swabs, urine, and stools were collected biweekly. Among them 90 patients, 71 were colonized by yeast species with evaluation of number of colonized body sites: 1 site (16.9%), 2 sites (26.8%), 3 sites (26.8%), 4 sites (25.3%) and 5 sites (4.2%).
        (ii) Group 2 consisted of 80 serum samples from healthy blood donors.
    
    


EIA Detection of Anti-C. albicans Mannan Antibodies in Human Sera.
Antibodies to Candida albicans mannan were detected using the Platelia Candida antibody (Ab) Plus test (Bio-Rad Laboratories, Marnes La-Coquette, France) according to manufacturer's instructions. Briefly, Enzyme ImmunoAssay (EIA) was performed with BEP III automate (Behring Laboratories, Paris, France). For individual sera, 100 μl of serum diluted 1/400 was applied to each well, and the plate was incubated for 1 h at 37° C. After washing, 100 μl of horseradish peroxidase-conjugated anti-human immunoglobulins was then added, and the plates were incubated for 1 h at 37° C. After intensive washing, the reaction was revealed by 30 min of incubation in darkness with 200 μl of tetramethylbenzidine solution. The absorbance at a λ of 450/620 nm was measured. The results were reported in arbitrary units (AU) in relation to the results on the standard curve.
Detection of Mannanemia.
Circulating mannan was detected using the Platelia Candida antigen Plus (Ag) test (Bio-Rad Laboratories, Marnes-La-Coquette, France) as described previously (Sendid et al., J Med Microbiol. 2002 May; 51(5):433-42). Briefly, microtiter plates were sensitized in an industrial setting with monoclonal antibody (monoclonal antibody EBCA1 of Platelia Candida antigen Plus (Ag) test). 300 μl of patient sera was denatured with 100 μl of EDTA treatment solution, and the mixture was boiled for 3 min and centrifuged at 10,000 g for 10 min. 50 μl of supernatant, obtained from patient serum and treated as described above, was mixed in a plate well with 50 μl of horseradish peroxidase-conjugated EBCA1. After incubation for 90 min at 37° C., the plates were washed intensively and the reaction was revealed by 30 min of incubation in darkness with 200 μl of tetramethylbenzidine solution. The optical density was read at a λ of 450/620 nm on a PR2100 reader (Sanofi Pasteur Diagnostics). Reactions were performed in duplicate. Each experiment included a calibration curve for a pool of normal human sera supplemented with concentrations of mannan of 0.1 to 27 ng/ml.
EIA Detection of Anti-C. albicans Recombinant Proteins Antibodies in Human Sera
Recombinant proteins were coated on ELISA plates (NUNC IMMUNO-MODULE 468680) at a concentration of 2 μg/ml for Mp65 and Eno1, and at a concentration of 4 μg/ml for Fba1, Hwp1 and Hsp90 produced in Escherichia coli, with carbonate solution pH 9.5+/−0.2 filtered 0.22 μm for all antigens. These preparations were incubated at room temperature overnight.
After coating, the wells were blocked by 100 μl of Bovine Serum Albumin/Saccharose solution pH 7.2+/−0.2 filtered 0.22 μm, emptied and filled with 200 μl of the same solution. Plates were then frozen at −20° C. Protocols for antigens coating, preparation of diluent solutions, dilution of patient sera, and conjugate solutions were based on preliminary experiments performed with a pool of sera from IC patients known to display high titers of anti-Candida antibodies.
Patient sera were diluted 1/100 in Tris saline buffer and BSA pH 7.6 and were incubated for 1 h at 37° C. on coated plate in dry incubator, 3 times washed with 800 μl of Tris saline buffer and Tween 20 (0.1%) and proclin (0.07%), incubated 1 h at 37° C. with a secondary anti-total immunoglobulin antibody conjugated to peroxidase (Bio-Rad, Marnes La Coquette, France), washed 3 times as previously described and incubated 30 minutes with 200 μl of tetramethyl benzidine (TMB) and stored at room temperature in dark. The reaction was stopped by the addition of 100 μl of stopping solution containing 2M H2SO4 and ODs were measured at 450/620 nm. In parallel, all sera were tested at the same time with the home-made EIA tests involving recombinant proteins, the PlateliaTM Candida Ab Plus and the PlateliaTM Candida Ag.
Statistical Analysis
Statistical analysis was performed in collaboration with SysDIAG: Systèmes Complexes pour le Diagnostic (UMR3145 CNRS/Bio-Rad, Montpellier, France). All statistics and figures were computed with the “R/Bioconductor” statistical open source software (Ge et al. Test 2003; 12:1-77; Gentleman et al. Genome Biol 2004; 5:R80) or SAS software v9.2 (SAS institute Inc). A differential analysis was carried out with the non-parametric Wilcoxon rank sum test and the Welch test. With the multiple testing methodologies, it is important to adjust the p-value of each marker to control the False Discovery Rate (FDR). The Benjamini and Hochberg (BH) procedure (Benjamini et al. Behav Brain Res 2001; 125:279-84) was applied on all statistical tests with the “multitest package” and an adjusted p-value below 0.05 was considered as statistically significant. A logarithmic transformation (log 10) was applied on the biomarker expression levels to ensure the data normality. All data distributions are illustrated as medians and boxplots for each biomarker. A Pearson test correlation was applied to identify biomarker correlation for all patient groups.
The marker diagnostic performance could be characterised by sensitivity, which represents its ability to detect the IC population, and specificity which represents its ability to detect the control population.
The results of the evaluation of a diagnostic test can be summarised in a 2×2 contingency table comparing these two well-defined populations. By fixing a cut-off the two populations could be classified into categories according to the results of the test, categorised as either positive or negative. Given a particular marker, we can identify a number of subjects with a positive test result among the “cases” population (the “True Positive”: TP) and b subjects with a positive test result among the “controls” population (the “True Negative”: TN). In the same fashion, c subjects with a negative test result among the cases (the “False Positive”: FP) and d subjects with a negative test result among the controls (the “False Negative”: FN) are observed. Sensitivity is defined as TP/(TP+FN); which is herein referred to as the “true positive rate”. Specificity is defined as TN/(TN+FP); which is herein referred to as the “true negative rate”.
The accuracy of each marker and its discriminatory power was evaluated using a Receiving Operating Characteristics (ROC) analysis. ROC curves are the graphical visualization of the reciprocal relation between the sensitivity (Se) and the specificity (Sp) of a test for various values.
In addition, all markers were combined with Mannan antigen (Ag) to evaluate the potential increase in sensibility and specificity using several approaches as mROC program (Kramar et al. Computer Methods and Programs in Biomedicine 2001; 66:199-207), logistic regression (Kleinbaum, D. G., Kupper, L. L., Muller, K. E. (1988) Applied Regression Analysis and other Multivariate Methods. Duxbury Press, Belmont, Calif.) and with two supervised learning algorithms, CART (Breiman L. Classification and regression trees. Wadsworth International Group, 1984.) and wKNN (Hechenbichler K, Schliep K. Weighted k-Nearest-Neighbor Techniques and Ordinal Classification. Volume 399, 2004).
mROC is a program developed by Kramar et al. (Comput Methods Programs Biomed, 2001, 66:199-207) which is dedicated to identify the linear combination which maximizes the AUC (Area Under the Curve) of ROC curves. The use of this program was described for instance in Staack et al. BMC Urol 2006; 6:19. This program implements an algorithm for maximising rank correlation estimation which is also an estimate for the area under the ROC curve (Su and Liu. Journal of the American Statistical Association 1993; 88:1350-1355; Wang, Computational Statistics and Data Analysis 2007; 51:2803-2812). The equation for the respective combination is provided and can be used as a new virtual marker Z, as follows:

Z=a×Marker1+b×Marker2+c×Marker3,

where a, b, c are calculated coefficients and Marker1,2,3 are individual level of markers.
A logistic regression model was also applied for univariate and multivariate analysis to estimate the relative risk of IC at different biomarkers values. We analyzed biomarkers as both continuous (data not shown) and categorical (using the quartile values as cutpoints) variables. In the last cases, the odds ratio (OR) and their 95% confidence interval are computed.
A CART (Classification And Regression Trees) approach was also applied to assess (biomarker+Mannan Ag) combinations. This decision tree approach allows to produce a set of classification rules, represented by a hierarchical graph easily understandable for the user. At each node of the tree, a decision is made. By convention, the left branch corresponds to a positive response to the question of interest and the right branch corresponds to a negative response to the question of interest. The classification procedure can then be translated as a set of rules ‘IF-THEN’.
A wKNN (weighted k-nearest neighbours) approach was applied as previously to assess (biomarker+Mannan Ag) combinations. The wKNN algorithm is one of the variations of KNN method which uses the K nearest neighbours, regardless of their classes, but then uses weighted votes from each sample rather than a simple majority or plurality voting rule. Given a patient x, each of the K samples provides a weighted vote that is usually equal to some decreasing function of its distance from the unknown sample x. These weighted votes are then summed for each neighbour, and the class with the largest total vote is attributed to x.
CART and wKNN are supervised learning methods. These methods require the use of a training set used to construct the model and a test set to validate it. So, we have shared our data set: 2⁄3 of the dataset are used for the learning phase and 1⁄3 are used for the validation phase. This sharing has been randomized and respect the initial proportion of the various statutes in each sample. To estimate the errors prediction of these two classifiers, we used the 10-fold cross-validation method, repeated 10 times in order to avoid overfitting problems. For these approaches, we used the “ipred package”, the “rpart package” and the “kknn package” of the R software.
Hierarchical Ascendant Clustering Analysis (HAC) is a method of cluster analysis, based on a pairwise distance matrix, which builds a hierarchy of clusters with sequentially agglomerative and divisive approaches. We have used this method to organize the map and to group the sample according to the nearest level of biomarker intensity. For this analysis, raw data were mean-centred and Pearson correlation matrix and average linkage were chosen as parameters.
2) Results
Standardization of Tests.
For each experiment, 3 serum controls were used. Negative control collected from healthy subject and 2 positive sera consisted of 1 pool of sera with known reactivity against Candida albicans mannan and one serum collected from patient belonging to IC group selected from previous series of experiments. All these controls allowed us to reduce inter-experiments variations.
A study of diagnosis potential of different recombinant proteins was performed by comparison of medians. When serological data were analyzed, antibodies against Fba1 (Fba1 Ab), Hwp1 (Hwp1 Ab), Hsp90 (Hsp90 Ab), Eno1 (Eno1 Ab) and Mp65 (Mp65 Ab) are the best biomarkers to discriminate IC patients from controls, however humoral response against Sod5 was less discriminant for both groups.
Comparison of Serological Reactivity Against a Panel of Antigens within IC and Control Groups
Using all group of patients (IC versus controls), boxplots were performed for each combination of mannanemia test and EIA tests involving recombinant proteins (FIGS. 1-5). When antibody response against each recombinant protein associated to mannanemia test was compared to mannanemia and anti-mannan antibody tests, significant differences were observed for Fba1 (p<0.0001; FIG. 1), Hwp1 (p<0.0001; FIG. 2), Hsp90 (p<0.0001; FIG. 3), Eno1 (p<0.0001; FIG. 4) and Mp65 (p<0.0001; FIG. 5).
Analysis of Discriminatory Potential of Each RP-Ab/Mannanemia Combination in Compared with the Mannanemia and Anti-Mannan Antibody Association
Combinations of more than 2 markers were tested however none have significantly improved the results obtained combining mannanemia and one of anti-protein recombinant antibodies.
ROC curves show the improvement of IC diagnostic with combination of RP-Ab and mannan antigen compared with combination of mannan antigen and anti-mannan antibody (FIGS. 6-10).
Comparison of Sensitivity and Specificity of Mannanemia and RP-Ab Combined Analysis.
Retrospective analysis of the cohort allowed sensitivity and a specificity of 26.6% and 99.4% respectively for mannanemia alone when combination of PlateliaTM Candida Ag and Ab showed an improvement of sensitivity (80.0%) and decrease of specificity (61.7%).
The ROC curves obtained from combination of RP-Ab/mannanemia (combined marker analysis by sera) showed a significant improvement of the diagnostic performances as reveled by AUCs: 0.902, 0.886, 0.884, 0.872, and 0.853 for Fba1, Hwp1, Hsp90, Eno1, and Mp65 respectively versus 0.769 for mannanemia and anti-mannan antibody combination (Table 5).
Furthermore, the association of RP-Ab and mannanemia increases significantly sensitivity and specificity. With a specificity arbitrarily fixed at 80.0% for the combination of mannan Ag+mannan Ab, sensitivities of mannan Ag+Hsp90, mannan Ag+Fba1 Ab, mannan Ag+Hwp1 Ab, mannan Ag+Eno1 Ab, and mannan Ag+Mp65 Ab were 80.9%, 83.8%, 83.8%, 79.1%, and 75.5%, respectively while the current serological diagnosis tests that combined mannan Ag+mannan Ab has a sensitivity of 61.7% (Table 5).







TABLE 5







Diagnosis potential (mROC approach) of RP-Ab associated with mannanemia for IC diagnosis.














Combination of 2

Cut-







biomarkers
AUC
off*
Se(%)
Sp(%)
PPV(%)
NPV(%)
CI95%





Mannan Ag* + Fba1 Ab*
0.902
−1.975
83.8
80.0
86.9
75.0
[0.871; 0.927]


Mannan Ag* + Hwp1 Ab*
0.886
−2.629
83.8
80.0
87.2
75.2
[0.851; 0.915]


Mannan Ag* + Hsp90 Ab*
0.884
−2.031
80.9
80.0
86.8
72.0
[0.848; 0.911]


Mannan Ag* + Eno1 Ab*
0.872
−2.209
79.1
80.0
86.6
70.1
[0.835; 0.901]


Mannan Ag* + Mp65 Ab*
0.853
−1.483
75.5
80.0
86.4
66.8
[0.815; 0.885]


Mannan Ag* + Mannan Ab*
0.769
−0.869
61.7
80.0
83.8
56.3
[0.723; 0.809]





*Markers normalized by a log10 transform.


AUC: area under the curve; Se: sensibility; Sp: specificity; PPV: positive predictive value (measures the proportion of subjects with positive test results who are correctly diagnosed); NPV: negative predictive value (measures the proportion of subjects with negative test results who are correctly diagnosed); CI 95%: 95% confidence interval. The cut-off value was set in order to specificity to 80%.






Noteworthy, the contribution of mannanemia and RP-Ab association was significantly higher for Candida parapsilosis infected patients where RP-Ab reached a sensitivity of 67.2% versus 21.3% for anti-mannan antibodies. Such an improvement was also observed for episodes determined by Candida albicans, Candida kruseï, Candida glabrata, Candida tropicalis and Candida lusitaniae. 
As compared with the combination of mannanemia with anti-mannan antibodies, mannanemia and RP-Ab combination improved specificity of detection of IC associated with Geotrichum capitatum or Candida norvegiensis. 
Analysis of RP-Ab/Mannanemia for the Precocity of IC Diagnosis
The contribution of RP-Ab/mannanemia association to early diagnosis of IC was performed by considering only serum samples collected during the period day −15 and the day of isolation of yeasts species from blood culture (day of positivation of blood culture).
All combinations were able to significantly differentiate IC from Controls (p<0.0001) with higher values of AUC than mannan Ag/Ab mannan (Table 6).







TABLE 6







Analysis of biomarkers association performance in two


weeks before isolation of yeasts from blood samples.


















Nfold



Marker combinations
pWILCOX
pWILCOX_FDR
pWELCH
pWELCH_FDR
Median
AUC





Mannan Ag + Fba1 Ab
0.0001
0.00011
0.00010
0.00012
2.06
0.892


Mannan Ag + Hwp1 Ab
0.0001
0.00011
0.00010
0.00012
1.63
0.872


Mannan Ag + Hsp90 Ab
0.0001
0.00011
0.00010
0.00012
1.64
0.871


Mannan Ag + Eno1 Ab
0.0001
0.00011
0.00010
0.00012
1.48
0.863


Mannan Ag + Mp65 Ab
0.0001
0.00011
0.00010
0.00012
1.63
0.826


Mannan Ag + Mannan Ab
0.0001
0.00011
0.00010
0.00012
1.19
0.719









Determination of mean day of positivity of different biomarkers was performed between day −15 and the day of isolation of yeast from blood samples. For all of these markers, the mean of positivity is between −5 and −6 days before the isolation of yeasts from blood samples.







TABLE 7







Determination of mean day of positivity of different RP-Ab


and mannanemia association in comparison with


the isolation day of yeasts in blood samples.










Marker combinations
Mean (positive Day)







Mannan Ag + Mannan Ab
−5.05



Mannan Ag + Fba1 Ab
−5.59



Mannan Ag + Hsp90 Ab
−5.28



Mannan Ag + Eno1 Ab
−5.48



Mannan Ag + Hwp1 Ab
−5.41



Mannan Ag + Mp65 Ab
−5.31










Accordingly, all RP-Ab/mannanemia remained discriminant for IC even if no significant difference with mannanemia/anti-mannan antibody in terms of mean delay of positivity before blood culture (5-6 days before positive blood culture).
Determination of Diagnostic Odds Ratio of Different Antibody and Antigen Combination For the Diagnosis of IC
Odds ratio reflect the scale of risk for developing IC according to the intensity of antibody response against RP and mannanemia levels. Knowing that the prognosis of IC is closely correlated with the delay of initiation of antifungal therapy, this ratio could help to identify patients that need an early antifungal treatment.
For all the associations of markers, 3 modalities of response intensity were determined in function of the repartition of values obtained in the cohort (using the quartile values as cutpoints). So, for each combination of markers the values of modality1 (mod1) are relative to the interval [Min, T1[(1st tertile), the values of modality2 (mod2) are relative to the interval [T1, T2[(2nd tertile) and the values of modality3 (mod3) are relative to the interval [T2, Max[(3rd tertile). The more the intensity of response is high, the more the risk of being IC is important.
The Anti-mannan antibody and mannanemia combination was associated with significant and adjusted Odds Ratios (OR) varying between 2,4 and 17.5. In comparison, the (Mp65 Ab and mannanemia) combination was associated with significant and adjusted Odds Ratios varying between 5.5 and 47.9. The (Eno1 Ab and mannanemia) combination was associated with significant and adjusted Odds Ratios varying between 7.7 and 59.4. The (Hwp1 Ab and mannanemia) combination was associated with significant and adjusted Odds Ratios varying between 7.1 and 65.5. The (Hsp90 Ab and mannanemia) combination was associated with significant and adjusted Odds Ratios varying between 6.8 and 77.5. The (Fba1 Ab and mannanemia) combination was associated with significant and adjusted Odds Ratios varying between 8.8 and 108.2 for (Tables 8-13).







TABLE 8







Determination of Odds Ratios on Mannan Ag + Mannan Ab


according to the intensity of signals


Odds Ratio Estimates













95% Wald




Point
Confidence


Effect
Modality Interval
Estimate
Limits














Mannan Ag + Mannan
[0.05; 0.64[ vs
2.414
1.519
3.838


Ab mod2 vs mod1
[−2.19; 0.05[


Mannan Ag + Mannan
[0.64; 8.65[ vs
17.485
9.041
33.817


Ab mod3 vs mod1
[−2.19; 0.05[


Mannan Ag + Mannan
[0.64; 8.65[ vs
7.242
3.763
13.936


Ab mod3 vs mod2
[0.05; 0.64[
















TABLE 9







Determination of Odds Ratios on Mannan Ag + Mp65 Ab


according to the intensity of signals


Odds Ratio Estimates












Point
95% Wald


Effect
Modality Interval
Estimate
Confidence Limits














Mannan Ag + Mp65
[−0.09; 1.43[ vs
5.456
3.324
8.956


Ab mod2 vs mod1
[−6.89; −0.09[


Mannan Ag + Mp65
[1.43; 10.37[ vs
47.887
21.578
106.276


Ab mod3 vs mod1
[−6.89; −0.09[


Mannan Ag + Mp65
[1.43; 10.37[ vs
8.777
3.979
19.360


Ab mod3 vs mod2
[−0.09; 1.43[
















TABLE 10







Determination of Odds Ratios on Mannan Ag + Eno1 Ab


according to the ntensity of signals


Odds Ratio Estimates












Point
95% Wald


Effect
Modality Interval
Estimate
Confidence Limits














Mannan Ag + Eno1
[−0.30; 1.61[ vs 
7.739
4.613
12.983


Ab mod2 vs mod1
[−3.75; −0.30[


Mannan Ag + Eno1
[1.61; 20.74[ vs 
59.421
26.538
133.049


Ab mod3 vs mod1
[−3.75; −0.30[


Mannan Ag + Eno1
[1.61; 20.74[ vs
7.678
3.466
17.011


Ab mod3 vs mod2
[−0.30; 1.61[
















TABLE 11







Determination of Odds Ratios on Mannan Ag + Hwp1 Ab


according to the intensity of signals.


Odds Ratio Estimates












Point
95% Wald


Effect
Modality Interval
Estimate
Confidence Limits














Mannan Ag + Hwp1
[−0.34; 1.90[ vs
7.065
4.237
11.781


Ab mod2 vs mod1
[−4.45; −0.34[


Mannan Ag + Hwp1
[1.90; 11.66[ vs
65.464
28.095
152.538


Ab mod3 vs mod1
[−4.45; −0.34[


Mannan Ag + Hwp1
[1.90; 11.66[ vs
9.266
4.018
21.365


Ab mod3 vs mod2
[−0.34; 1.90[
















TABLE 12







Determination of Odds Ratios on Mannan Ag + Hsp90


according to the intensity of signals.


Odds Ratio Estimates













95% Wald




Point
Confidence


Effect
Modality Interval
Estimate
Limits














Mannan Ag + Hsp90
[−0.35; 1.89[ vs
6.777
4.068
11.289


Ab mod2 vs mod1
[−7.45; −0.35[


Mannan Ag + Hsp90
[1.89; 12.40[ vs
77.458
31.551
190.161


Ab mod3 vs mod1
[−7.45; −0.35[


Mannan Ag + Hsp90
[1.89; 12.40[ vs
11.430
4.705
27.771


Ab mod3 vs mod2
[−0.35; 1.89[
















TABLE 13







Determination of Odds Ratios on Mannan Ag + Fba1 Ab


according to the intensity of signals.


Odds Ratio Estimates












Point
95% Wald


Effect
Modality Interval
Estimate
Confidence Limits














Mannan Ag + Fba1
[−0.39; 2.17[vs
8.762
5.180
14.822


Ab mod2 vs mod1
[−8.08; −0.39[


Mannan Ag + Fba1
[2.17; 11.96[vs
108.250
40.808
287.149


Ab mod3 vs mod1
[−8.08; −0.39[


Mannan Ag + Fba1
[2.17; 11.96[vs
12.354
4.745
32.165


Ab mod3 vs mod2
[−0.39; 2.17[









Thus, according to this scale, association of anti-mannan antibody and mannanemia allows a risk at a maximum of 17.5 while the risk obtained with the association with mannanemia and RP-Ab reaches 47.9, 59.4, 65.5, 77.5 and 108.2 for respectively Mp65, Eno1, Hwp1, Hsp90 and Fba1.
Performance of the Combined Interpretation of the Separated Biomarker Assays
The performance of the diagnosis method based on the combined interpretation of the separated biomarker assays, i.e. based on determining if an elevated level of said Candida glycan, and/or an elevated level of antibody directed against said Candida protein selected from the group consisting of Fba1, Eno1, Hsp90, Hwp1, and Mp65, relative to their respective reference level is detected, was evaluated both on sera and patients. In the evaluation on sera, it is evaluated if the biomarker levels measured in each serum sample of any patient correctly led to the identification of the patient as having or not having invasive candidiasis. In the evaluation on patients, it is evaluated if altogether the biomarker levels measured in sera samples of a given patient correctly led to the identification of the patient as having or not having invasive candidiasis.
The performances on sera using combined interpretation of separated assays, as detailed in Table 14, show that for a specificity set at 79.9% (about 80%) the levels of sensitivity obtained for the different biomarker combinations Mannan Ag+protein Ab, although not identical to those obtained on sera using the combined analysis of the biomarkers (results shown in Table 5), are all improved as compared with the reference test Mannan Ag+Mannan Ab.







TABLE 14







Performances on sera using combined interpretation of separated assays











Markers
Sensitivity (%)
Specificity (%)







Mannan Ag + Hsp90 Ab
74.8
79.9



Mannan Ag + Mp65 Ab
75.6
79.9



Mannan Ag + Fba1 Ab
83.5
79.9



Mannan Ag + Eno1 Ab
78.3
79.9



Mannan Ag + Hwp1 Ab
81.5
79.9



Mannan Ag + Mannan Ab*
62.6
75.1







*sensitivity and specificity values of the current Platelia Candida antigen (Ag) and Platelia Candida Ab Plus tests (Bio-Rad Laboratories, Marnes-La-Coquette, France).






The comparison of Table 14 and Table 15 shows that the performances on sera or on patients using combined interpretation of separated assays are similar.







TABLE 15







Performances on patients using combined


interpretation of separated assays











Markers
Sensitivity (%)
Specificity (%)







Mannan Ag + Hsp90 Ab
80.6
79.9



Mannan Ag + Mp65 Ab
83.9
79.9



Mannan Ag + Fba1 Ab
84.9
79.9



Mannan Ag + Eno1 Ab
82.8
79.9



Mannan Ag + Hwp1 Ab
84.9
79.9



Mannan Ag + Mannan Ab*
61.3
75.1







*sensitivity and specificity values of the current Platelia Candida antigen (Ag) and Platelia Candida Ab Plus tests (Bio-Rad Laboratories, Marnes-La-Coquette, France).

## Claims

1. An in vitro method for diagnosing invasive candidiasis (IC) in a subject, said method comprising the steps:
a) detecting a level of a Candida mannan in a blood, plasma or serum sample of the subject using a method selected from the group consisting of an immunoassay, a chromatography, an enzyme-based chemoluminescent method, surface plasmon resonance, mass-spectrometry, and a lectin-based detection;
b) detecting by an immunoassay a level of antibodies directed against a Candida protein selected from the group consisting of fructose bisphosphate aldolase (Fba1), enolase 1 (Eno1), heat shock protein 90 (Hsp90), hyphal wall protein (Hwp1), and mannoprotein 65 (Mp65) in the same sample of the subject or in another sample sequentially obtained from the same subject, essentially simultaneously or no more than 3 hours apart;
c) performing a combined analysis of the level of said Candida mannan detected in step a) and the level of antibodies directed against said Candida protein detected in step b) by calculating a level of a marker Z =Σ ai ×[Marker i], wherein [Marker i] are individual levels of said Candida mannan and of the antibodies directed against said Candida protein, and ai are coefficients which values are determined in order to maximize an Area Under the Curve (AUC) of a Relative Operating Characteristic (ROC) curve for the combination of [Marker i]; and
d) deducing that the subject is developing or has developed invasive candidiasis if the level of the marker Z calculated in step c) is higher than a reference level of marker Z.

2. The method according to claim 1, wherein at step b) the level of antibodies against Fba1, or antibodies against Eno1, or antibodies against Hsp90, or antibodies against Hwp1, or antibodies against Mp65, only, is detected.

3. The method according to claim 1, wherein said method does not comprise detecting an antibody directed against mannan.

4. The method according to claim 1, wherein said Candida mannan is detected by a sandwich enzyme immunoassay which uses, as capture and detection antibodies, an antibody recognizing sequences of α-linked oligomannoses comprising more than four residues.

5. The method according to claim 1, wherein said reference level of the marker Z is a single value or a range of values determined based on the level of said Candida mannan or the level of antibodies directed against said Candida protein, as appropriate, measured in a population of healthy subjects, in a population of subjects superficially infected with a Candida strain, in a population of subjects suffering from invasive candidiasis, or in a sample from the same subject obtained at an earlier time point.

6. The method according to claim 1, wherein invasive candidiasis is due to an infection with a Candida species selected from the group consisting of Candida albicans, Candida parapsilosis, Candida kruseï, Candida tropicalis, Candida glabrata, Candida lusitaniae, Geotrichum capitatum, and Candida norvegiensis.

7. The method according to claim 1, wherein in step b) the antibodies are directed against a Candida Fba1.

8. The method according to claim 7, wherein the level of the antibodies directed against the Candida Fba1 is detected using a polypeptide comprising:
a) SEQ ID NO:2; or
b) a variant polypeptide of the polypeptide defined in a), wherein said variant polypeptide has at least 80% sequence identity with the polypeptide defined in a), over the whole length of the polypeptide.

9. The method according to claim 1, wherein in step b) the antibodies are directed against a Candida Eno1.

10. The method according to claim 9, wherein the level of the antibodies directed against the Candida Eno1 is detected using a polypeptide comprising:
a) SEQ ID NO:4; or
b) a variant polypeptide of the polypeptide defined in a), wherein said variant polypeptide has at least 80% sequence identity with the polypeptide defined in a), over the whole length of the polypeptide.

11. The method according to claim 1, wherein in step b) the antibodies are directed against a Candida Hsp90.

12. The method according to claim 11, wherein the level of the antibodies directed against the Candida Hsp90 is detected using a polypeptide comprising:
a) SEQ ID NO:6;
b) amino acids at positions 313 to 707 of SEQ ID NO:6;
c) a sequence SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:16; or
d) a variant polypeptide of the polypeptide defined in a), b), or c), wherein said variant polypeptide has at least 80% sequence identity with the polypeptide defined in a), b), or
c) over the whole length of the polypeptide.

13. The method according to claim 1, wherein in step b) the antibodies are directed against a Candida Hwp1.

14. The method according to claim 13, wherein the level of the antibodies directed against the Candida Hwp1 is detected using a polypeptide comprising:
a) SEQ ID NO:8;
b) amino acids at positions 41 to 200, or at positions 27 to 203 of SEQ ID NO:8; or
c) a variant polypeptide of the polypeptide defined in a) or b), wherein said variant polypeptide has at least 80% sequence identity with the polypeptide defined in a) or b), over the whole length of the polypeptide.

15. The method according to claim 1, wherein in step b) the antibodies are directed against a Candida Mp65.

16. The method according to claim 15, wherein the level of the antibodies directed against the Candida Mp65 is detected using a polypeptide comprising:
a) SEQ ID NO:10 or SEQ ID NO:12; or
b) a variant polypeptide of the polypeptide defined in a), wherein said variant polypeptide has at least 80% sequence identity with the polypeptide defined in a), over the whole length of the polypeptide.