Coccidioides is a fungal pathogen of humans which can cause a life-threatening respiratory disease in immunocompetent individuals. Recurrent epidemics of coccidioidal infections in Southwestern United States has raised the specter of awareness of this soil-borne microbe, particularly among residents of Arizona and Southern California, and has galvanized research efforts to develop a human vaccine against coccidioidomycosis. In this review, we discuss the rationale for such a vaccine, examine the features of host innate and acquired immune response to Coccidioides infection, describe strategies used to identify and evaluate vaccine candidates, and provide an update on progress toward development of a vaccine against this endemic pathogen.

We designed two primer systems that amplify a fragment of the gene coding for the small ribosomal subunit (18S rRNA). A broadly reactive, yet fungus-specific, primer cocktail comprises two previously published primers, TR1 and TR2, which specifically amplify dermatophytes, and two newly designed primers, CA1 and AF2, which specifically amplify Candida and Aspergillus respectively. This primer cocktail amplifies a DNA fragment of approximately 578 basepairs (bp) in length (from position 838 to 1415), which contains variable, possibly species-specific regions (V5, partly V7). Another newly designed primer, UF1 (universal fungal primer 1), along with the eukaryotic primer S3 amplifies a 926-bp fragment (from position 263 to 1188) that includes the variable regions V3, V4 and V5. Both primer systems amplified DNA from Saccharomyces cerevisiae, Candida albicans, Cryptococcus neoformans, Aspergillus fumigatus, Penicillium marneffei, Fusarium oxysporum and Trichophyton mentagrophytes, but not the DNA from Prototheca zopfii, Escherichia coli or humans. The previously published oligonucleotides TR and HC, which are specific for dermatophytes and Histoplasma respectively, and the newly designed group-specific oligonucleotides, CA and AF, hybridized with T. mentagrophytes, Histoplasma capsulatum, C. albicans and A. fumigatus respectively, but not with the other six fungi or with the three controls.

Four primer systems, amplifying fragments of the gene coding for the small ribosomal subunit (18s rRNA) were characterised with pure cultures of 65 medically relevant fungal species plus two mushrooms. A primer cocktail (TR1 /CAl-TR2/AF2) amplified 59 of 67 fungal species; the universal fungal primer 1 (UF1) in combination with the eukaryotic primers S3 or EUl amplified 64 and 65 of 67 fungal species, respectively. The design of an additional primer (RZY1) enabled the amplification of the missing members of the zygomycetes. The primer systems amplified all the medically relevant fungi tested. These included eight Candida spp. and seven other yeast species, 13 dermatophytes, 32 moulds (including six zygomycetes and five dimorphic fungi) and two mushrooms. Eleven controls including DNA from Schistosoma mansoni, Escherichia coli, Mycobacterium tuberculosis and man were not amplified. The oligonucleotide CA hybridised with C. albicans, C. tropicalis and C. parapsilosis; the oligonucleotide TR hybridised with the 13 dermatophytes; the oligonucleotide AF hybridised with Aspergillus fumigatus, A. Jlavus, A. terreus, A. nidulans, A. versicolor, A. tamarii, A. clavatus, A. Jischeri, but not with A. niger or A. versicolor; and the oligonucleotide HC hybridised with three varieties of Histoplasma capsulatum. These oligonucleotides did not hybridise with the other fungi nor the controls. The specificity of the newly designed primer systems was confirmed by selective amplification of fungal DNA from human lung tissue spiked with fungal biomass and from vitrectomy fluid of a patient with candida endop h t halmitis.

Thermostable alkaline proteinase was produced by a strain of Chrysosporium keratinophilum when cultured in lactose/mineral salt medium incorporating keratin solubilized with DMSO. The proteinase, partially purified by cold-acetone precipitation followed by gel-filtration on Sephadex G-75, was optimally active at pH 9 and stable from pH 7 to 10 with over 90% relative residual activity after incubation at 25°C for 24 h. The optimum temperature for enzyme activity was 90°C at which the activity half-life was 30 min. Enzyme activity was stimulated by Fe(2+) and inhibited by 1,10 o-phenanthroline. Gel-filtration indicated an M r of 69 kDa.

The effects of the methanol extract of the stem bark of Prosopis africana (Guill., Perrott. and Rich.) Taubert (Fabaceae) on bleeding/clotting and coagulation time, excision and dead space wounds were studied in rats. Also, the extract was subjected to antibacterial, and acute toxicity and lethality (LD50) tests. The extract significantly (P<0.05) reduced bleeding/clotting and coagulation time in rats. It also reduced epithelialization period of excision wounds in rats and inhibited the growth of laboratory strains of Staphylococcus aureus, Bacillus subtilis, Salmonella typhi, Pseudomonas aeruginosa and Klebsiella pneumoniae to varying extents. Acute toxicity and lethality (LD50) test on the extract established an LD50 of 774 mg/kg (i.p) in mice while phytochemical analysis gave positive reactions for alkaloids, saponins, tannins, flavonoids, steroids, terpenoids and carbohydrates. The results of this study demonstrate the beneficial effects of the stem bark of P. africana in wound care.

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The LightCycler system (two-step reverse transcription-PCR-fluorescent hybridization [LC RT-PCR-FH]) was used to quantifyCandida species are frequently found as saprophytes that colonize the surfaces of certain mucous membranes in human. The superficial and disseminated infections caused by them are largely opportunistic, and Candida albicans accounts for most of the etiologic species isolated from cutaneous candidiasis. Microscopic demonstration of yeast elements in potassium hydroxide preparations of infected tissue and cultural isolation of yeast from skin scrapings are used to confirm clinical diagnosis of cutaneous candidiasis. Rapid and highly sensitive diagnostic assays, based on PCR of DNA, have also been used to identify candidal pathogens in tissue (3,4,5,7,8).The amplification of a targeted DNA sequence by PCR does not, however, indicate the viability of the source organism, important information for evaluating the efficacy of a particular therapy and thus for avoiding an unnecessary prolongation of therapy. For this reason, reverse transcription (RT)-PCR of mRNA molecules has been used to assess the viability of microorganisms (9,11,14). However, RT-PCR is a qualitative assay, and this places a limitation on the inferences that can be drawn from the result; differences in the quantities of target mRNAs cannot easily be ascertained from the intensities of amplification bands. A determination of quantitative differences is needed in an assessment of the potencies of different concentrations of an agent or the progressive efficacy of a particular treatment. Furthermore, with the template at low levels, bands are seldom visualized in gel, resulting in falsenegative interpretations (14).In this study, we estimated the viability of C. albicans in a reconstituted-skin model of cutaneous candidiasis by detection of C. albicans ACT mRNA using the LightCycler (LC) system (a two-step RT-PCR-fluorescent hybridization [FH] assay) (10). LC DNA-based identification of fungi has been shown to be highly sensitive and rapid (10), and we applied of this system to rapidly quantify C. albicans ACT mRNA. The ACT mRNA was specifically targeted because of the constitutive nature of the gene and the major role of actin in cell division (6,15).