Method for detecting Candida infection

Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Oxidoreductase

Reexamination Certificate

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C435S189000, C435S255400, C435S025000, C435S026000, C435S034000

Reexamination Certificate

active

06280988

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
At present the laboratory tools available to clinicians for the diagnosis of invasive candidiasis are limited. Surveillance cultures of peripheral sites have little predictive value, with the possible exception of
Candida tropicalis
infection in neutropenic patients. Blood cultures, even when they are tailored for optimal growth of fungi, are slow, insensitive, and nonspecific. Measurement of antibodies to Candida is not helpful in immunosuppressed patients who comprise the very group that is most vulnerable to invasive infections. Detection of circulating fungal products, particularly mannan, provides the desired level of specificity, but the clinical sensitivity of current assays is disappointing, and the technology for performing them is not readily portable to clinical laboratories. Commercially available latex agglutination kits detect uncharacterized antigen(s) and lack sensitivity and specificity.
Most medically important species of Candida produce micromolar amounts of the pentitol D-arabinitol in vitro and there is considerable evidence that patients with invasive candidiasis have higher serum D-arabinitol levels and higher serum D-arabinitol/creatinine ratios than uninfected patients. Therefore, D-arabinitol is potentially useful as a diagnostic marker for invasive candidiasis, a disease that often is difficult to diagnose antemortem by traditional methods.
Enantioselective measurements of D-arabinitol in human serum were originally made by combined microbiologic-gas chromatographic (GC) and enzymatic-GC techniques. These approaches were, however, time consuming and cumbersome. Recently, two GC methods have been developed that employ columns with a chiral stationary phase capable of separating enantiomers of arabinitol. Although these methods are highly specific for D-arabinitol and do not require serum pretreatment by enzymatic or microbiologic techniques, they are too cumbersome for routine use in the clinical laboratory. A more practical enzymatic fluorometric method has been developed that uses an
Enterobacter aerogenes
(
Klebsiella pneumoniae
) D-arabinitol dehydrogenase. Unfortunately, cross reactivity of the dehydrogenase with D-mannitol, a hexitol normally present in human serum, reduces the specificity of this assay.
2. Description of the Related Art
Soyama and Ono, in
Clinica Chimica Acta,
149 (1985) 149-154, describe an enzymatic fluorometric method for the determination of D-arabinitol in serum by initial rate analysis.
Soyama and Ono describe an improved procedure for determining serum D-arabinitol by a resazurin-coupled enzymatic method in
Clinica Chimica Acta,
168 (1987) 259-260.
The purification and properties of
Klebsiella aerogenes
D-arabinitol dehydrogenase are discussed by Neuberger, et al., in
Biochem. J.,
183 (1979) 31-42.
Kiehn, et al., in
Science,
206 (1979) 577-580, describe a gas-liquid chromatographic method for measuring D-arabinitol in human serum and assess the clinical usefulness of this method for detecting candidiasis. In actuality, total pentitol (D- and L-arabinitol, xylitol and ribitol) concentration is being measured by this method.
Bernard, et al., in
J. Infect. Dis.,
151(4)(1985) 711-715, describe a combined microbiologic-GC method for determining the stereoisomeric configuration of arabinitol in serum, urine, and tissues in invasive candidiasis. In this method, D-arabinitol concentrations are calculated as the difference between serum arabinitol levels determined by GC before and after sample incubation with a strain of
C. tropicalis,
which consumes D-arabinitol once preferred substances are exhausted. Unfortunately, the method requires a 24-hour incubation step, is susceptible to interference by anti-fungal drugs, and is insufficiently sensitive.
Wong and Brauer, in
J. Clin. Microbiol.
26 (1988) 1670-1674, describe a combined enzymatic-GC method for the enantioselective measurement of D-arabinitol in human serum. In this method, D-arabinitol dehydrogenase from
K. pneumoniae
is used instead of
C. tropicalis
cells for the removal of D-arabinitol from serum; and D-arabinitol levels are calculated as the difference between arabinitol levels determined by GC in the untreated and enzyme-treated serum. Although this combined enzymatic-GC method is unaffected by antifungal drugs, sufficiently sensitive to quantify D-arabinitol in most serum specimens, and can be completed within a few hours, the technique requires that each specimen be analyzed twice by GC to determine the concentration of D-arabinitol.
Enantioselective measurement of the Candida metabolite D-arabinitol in human serum using multidimensional gas chromatography and a new chiral stationary phase is disclosed by Wong and Castellanos, in
J. Chromatography,
495 (1989) 21-30. This technique is sensitive and highly specific for D-arabinitol, but requires that each specimen be fractioned successively over GC columns containing a conventional and chiral stationary phase.
The separation and quantification by gas chromatography-mass spectrometry of arabinitol enantiomers to aid the differential diagnosis of disseminated candidiasis is disclosed by Roboz, et al., in
J. Chromatog.,
500 (1990) 413-426. The columns used in this approach have a limited useful lifetime and the procedure is laborious and time-consuming.
A review of techniques for the diagnosis of invasive candidiasis is described by Jones in
Clin. Microbiol. Rev.,
3 (1990) 32-45.
Ness, et al., in
The Journal of Infectious Diseases,
159 (1989) 495-502 describe the Candida antigen latex test for detection of invasive candidiasis in immunocompromised patients.
Cabezudo, et al., discuss the value of the Cand-Tec Candida antigen assay in the diagnosis and therapy of systemic candidiasis in high-risk patients (
Eur. J. Clin. Microbiol. Infect. Dis.,
8 (1989) 770-777).
Walsh, et al., in
N. Engl. J. Med.,
324(15) (1991) 1026-1031, assess the clinical utility of the Candida enolase antigen test for detecting candidiasis in cancer patients.
SUMMARY OF THE INVENTION
One embodiment of the present invention is directed to an enzyme in purified form specific for D-arabinitol.
Another embodiment of the present invention is directed to a D-arabinitol dehydrogenase enzyme capable of catalyzing the oxidation of D-arabinitol and substantially incapable of catalyzing the oxidation of D-mannitol and that is substantially free of other enzymes capable of oxidizing D-mannitol.
Another embodiment of the present invention is a method for determining D-arabinitol. The method comprises the steps of providing in combination (1) a medium suspected of containing D-arabinitol and (2) a D-arabinitol dehydrogenase enzyme and examining the medium for the product produced as a result of the oxidation of the D-arabinitol. The enzyme utilized is capable of catalyzing the oxidation of D-arabinitol and substantially incapable of catalyzing the oxidation of D-mannitol.
Another aspect of the invention is a method for detecting the presence of a Candida organism in a host. The method comprises the step of examining a sample from the host for the presence of D-arabinitol utilizing a D-arabinitol dehydrogenase enzyme. The enzyme is capable of utilizing D-arabinitol as a substrate and substantially incapable of utilizing D-mannitol as a substrate.
Another embodiment of the invention concerns a method of detecting a Candida infection in a patient. The method comprises the steps of providing in combination a sample from the patient, a D-arabinitol dehydrogenase enzyme capable of catalyzing the oxidation of D-arabinitol by nicotinamide adenine dinucleotide (NAD
+
) and substantially incapable of catalyzing the oxidation of D-mannitol, and examining the combination for the amount of NADH formed in a given time. The term “NAD
+
” shall mean the natural coenzyme and NAD
+
mimetics including NAD
+
attached to a support and thio analogs, which contain one or more sulfur atoms in place of one or more oxygen atoms of the NAD
+
. The amount of NAD
+
redu

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