Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2001-01-12
2002-10-22
Horlick, Kenneth R. (Department: 1656)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100, C536S024100, C536S024300, C536S024330, C435S006120, C435S091100, C435S091200
Reexamination Certificate
active
06469156
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to detecting a pathogenic fungus,
Histoplasma capsulatum
, using oligonucleotide probes specific for
H. capsulatum
to amplify
H. capsulatum
DNA by means of the polymerase chain reaction. Test samples may originate from the environment, where
H. capsulatum
spores are found, or from clinical samples obtained from patients.
BACKGROUND OF THE INVENTION
Histoplasma capsulatum
is a dimorphic fungus that is pathogenic in humans. In one form, the fungus has the mycelia characteristic of molds, and produces spores as an aspect of its reproduction. This form is commonly found in the ambient environment, and is highly prevalent in soils containing guano from bats or birds. In general it proliferates in soils rich in nutrients such as those containing rotting organic residues. Its morphology includes multicellular filaments containing cylindrical structures (i.e., the hyphae or conidia). This form may further contain macroconidia and microconidia which may grow in soil or on media designed for fungal growth at 25° C.
In its second form,
H. capsulatum
adopts a single celled state characteristic of yeast, and reproduces by budding. This form is prevalent on suitable nutrient media in the laboratory, and is the form generally found after it infects a human. Infection may occur upon breathing dust containing fragments of
H. capsulatum
hyphae or conidia; as noted above, such fragments may be prevalent if dust is stirred up from soil containing the droppings from birds or bats or soil that is otherwise rich in organic nutrients, especially nitrogenous nutrients.
The infection produces a systemic fungal disease, histoplasmosis, which can initially result in a variety of symptoms often dependent on the type of infection, the general health of the host, and the exposure level. In acute self-limiting infections, especially those occurring in healthy individuals, the symptoms may be very mild, such as those of a flu-like respiratory illness, with symptoms such as general malaise, fever, chest pain, a dry cough, headache, loss of appetite, shortness of breath, joint and muscle pains and chills. The severity of the symptoms may depend on how many spores have been inhaled, and the general health of the subject.
The symptoms of respiratory infection are often suppressed by the immune response. This response includes endocytosis by pulmonary macrophages.
H. capsulatum
may then proliferate within macrophages and be transported systemically as a result. As a result, such suppression appears to result in long term latency rather than in elimination of the organism.
H. capsulatum
infection can also result in calcified deposits within the lungs, produced when growth of the yeast is inhibited by host immune responses. Both the infected macrophages and the calcified deposits serve as a reservoir of latent infection. As a consequence, if the individual subsequently becomes immune compromised or immune deficient, histoplasmosis can reappear. Thus individuals such as those receiving immune-suppressing therapy, the very young or the elderly, those suffering from cancer, or expressing the symptoms of acquired immune deficiency syndrome are susceptible to recrudescence of histoplasmosis as a chronic and/or a disseminated infection, and may include ocular infection. Resurgence of such latent histoplasmosis produces potentially lethal symptoms.
Assay for
H. capsulatum
infection or its presence in a sample is carried out by methods such as culturing, immunoassay for surface antigens, and a bioassay. Currently, histoplasmosis may be diagnosed by culture, or by the demonstration of a rise in complement-fixing antibody titers in serum. A definitive diagnosis of an
H. capsulatum
infection requires the isolation and propagation of the fungus, which is time-consuming and lacks sensitivity. It is also dangerous for laboratory personnel, who must take extreme caution to prevent inhalation of the pathogenic fungus, so as not to become ill with a pulmonary infection. Further, only small quantities of antigens of
H. capsulatum
for use as biological reagents may be prepared in this manner.
Conventional laboratory identification methods used to isolate and identify
H. capsulatum
include the culture of a clinical specimen at room temperature on specialized fungal media. This procedure isolates the slower growing
H. capsulatum
colonies from possible contaminants, such as bacteria, and from faster growing saprobic fungi. This method, however, has several disadvantages. Because the growth of
H. capsulatum
to a visible colony normally takes from about two to four weeks, and sometimes as long as 12 weeks, this identification procedure is very slow. (See, for example, Rippon,
Histoplasmosis
. In
Medical Mycology, The Pathogenic Fungi and the Pathogenic Actinomyestes
, 3rd Ed., Saunders, Ch. 15 (1988); Koneman at al.,
Laboratory Identification of Molds
, in
Practical Laboratory Mycology
, (3rd ed. Williams & Wilkins (1985)); and McGinnis,
Histoplasma capsulatum
, in
Laboratory Handbook of Medical Mycology
, Academic Press (1986).) Further, additional growth is required before the characteristic colony morphology and microscopic sporulation pattern with tuberculate macroconidia may be observed. In addition, approximately 10% of cultures produce only smooth-walled macroconidia, and some cultures fail to sporulate. Moreover, many species of fungi other than
H. capsulatum
, such as
Blastomyces dermatitides
, Chryacsporium sp., and Sepedonium sp., produce similar colony and sporulation characteristics. Thus, additional testing is usually necessary to definitively identify the organism.
One method of converting the mycelial colony of
H. capsulatum
to the yeast phase is performed by subculturing the organism onto highly enriched cysteine-containing media, and incubating it at 35-37° C. However, conversion to the yeast phase is often difficult, and may require several additional subcultures at three-day intervals.
Histoplasmin, an unpurified culture supernatant obtained from the mycelial phase of
H. capsulatum
contains
H. capsulatum
M antigens. Histoplasmin is currently used to probe both humoral and cell-mediated responses in patients with histoplasmosis. It is used for the serologic diagnosis of histoplasmosis, and as a skin test antigen to demonstrate delayed hypersensitivity to infection in skin tests for histoplasmosis. The purification of histoplasmin is described by Bradley et al., Infect. Immun. 9:870-880 (1974). The preparation of H and M antigens of
H. capsulatum
free of heterologous antigens is described by Green et al., Curr. Microbiol. 12:209-216 (1985). (See also, Pine, Contrib. Microbiol. Immunol. 3:138-168 (1977).) The preparation of antisera to the M antigen is described by Green et al., Infect. Immun. 14:826-831 (1976). General information concerning the serodiagnosis of fungal diseases is present in Kaufman et al., Serodiagnosis of Fungal Diseases, in
Manual of Clinical Laboratory Immunology
(3rd ed., American Society for Microbiology, Washington, D.C.(1988)).
Serologic evidence is a principal diagnostic indicator of histoplasmosis. Several serologic tests, such as the immunodiffusion test, detect precipitants against the species-specific H and M antigens found in histoplasmin. (See, for example, Kaufman, Clin. Infect. Dis. 14:23-29 (1992), and Wheat, Eur. J. Clin. Microbiol. Infect. Dis. 8:480 (1989).)
Although the M antigen of
H. capsulatum
is useful in immunoassays for the diagnosis of histoplasmosis, purification of the M antigen from a batch culture is a laborious and low-yield process. The use of a recombinantly-produced M antigen of
H. capsulatum
in such immunoassays would significantly diminish the labor necessary to obtain M antigens which are pure enough to be useful in the immunoassays, and would result in high yields of the M antigen.
Assay for
H. capsulatum
infection historically has been via immunoassay for surface antigens. Additionally, a widely practiced assay is the mouse inoculum/agar plate culture method. This me
Reid Thomas M.
Schafer Millie P.
Horlick Kenneth R.
Needle & Rosenberg P.C.
Spiegler Alexander H.
The United States of America as represented by the Department of
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