Pythium insidiosum is a pathogen that causes disease in both animals and humans. Human infection is rare; however, when it does occur, most patients, especially those having underlying hemoglobinopathy syndromes, such as thalassemia, exhibit a severe form. We identified four isolates of P. insidiosum. Two were recovered from tissue biopsy specimens from thalassemic and leukemic patients, one was derived from brain tissue from a thalassemic patient, and another was isolated from a corneal ulcer from a fourth patient. Western blotting and an enzyme-linked immunosorbent assay (ELISA) were performed with a serum sample derived from one thalassemic patient. The methods used to identify the P. insidiosum isolates were based on morphology, nucleic acid sequencing, and a PCR assay. To confirm the identification, portions of the 18S rRNA genes of these four isolates were sequenced. The sequences were shown to be homologous to previously described P. insidiosum DNA sequences. In addition, PCR amplification of the internal transcribed spacer region specific for P. insidiosum was positive for all four isolates. The ELISA with the serum sample from the thalassemic patient gave a positive result from a serum dilution of 1:800. Finally, Western immunoblotting with this serum sample showed positive immunoglobulin G recognition for proteins of 110, 73, 56, 42 to 35, 30 to 28, 26, and 23 kDa. The results of this study show that both molecularly based diagnostic and serodiagnostic techniques are useful for the rapid identification of human pythiosis. The predominant antigens recognized by Western blotting may be useful in the development of a more sensitive and specific diagnostic tool for this disease.

Superoxide dismutase (SOD) is an enzyme that converts superoxide radicals into hydrogen peroxide and oxygen molecules. SOD has been shown to contribute to the virulence of many human-pathogenic fungi through its ability to neutralize toxic levels of reactive oxygen species generated by the host. SOD has also been speculated to be important in the pathogenesis of fungal infections, but the role of this enzyme has not been rigorously investigated. In this report, we isolated and characterized the copper, zinc superoxide dismutase gene, designated sodA, from the important human pathogenic fungus, Penicillium marneffei. The putative SodA polypeptide consisted of 154 amino acids and exhibited a significant level of similarity to other fungal Cu, Zn SODs. Differential expression of the sodA gene in P. marneffei was demonstrated by semi-quantitative RT-PCR. Apparently, the sodA transcript accumulated in conidia, but expression was downregulated in the mycelia phase. In contrast, transcript expression was upregulated in the yeast phase as well as during macrophage infection. The significantly higher expression of the sodA transcript during macrophage infection suggests that this gene might play an important role in stress responses and in the adaptation of P. marneffei to the internal macrophage environment. The latter may serve as a putative virulence factor of this fungus allowing for survival in the host cell.

Penicillium marneffei is an intracellular dimorphic fungus that can cause a fatal disseminated disease in human immunodeficiency virus-infected patients. The factors that affect the pathogenicity of this fungus remain unclear. Here, we report the isolation and characterization of the gpdA cDNA and genomic clones encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in P. marneffei. Phylogenetic analysis of GAPDH amino acid sequences demonstrated the evolutionary relationship of P. marneffei to other fungi, including the intracellular pathogen Ajellomyces capsulatus. To assess the central importance of phagocytic cells in defence against P. marneffei infection, we used Northern blotting to investigate the response of the isocitrate lyase-encoding gene (acuD) and gpdA to nutrient deprivation inside macrophages. The results revealed that after macrophage internalization, the gene involved in the glyoxylate cycle, acuD, showed higher expression levels as early as 2 h from the start of co-incubation, and the differential expression could be observed again at 8 h after infection. In contrast, the expression of gpdA was downregulated in the yeast phase, as well as during macrophage infection after 2, 4 and 8 h of infection. The induction of P. marneffei acuD was shown to be coordinated with the downregulation of the glycolytic gpdA gene, implying that the cytoplasmic environment of macrophages is deficient in glucose and the glyoxylate pathway could be used by this pathogen to allow subsistence on two-carbon compounds within the host cell following its intracellular persistence.

The ethanol extracts of clove (Eugenia caryophyllus Bullock & Harrison) and sweet flag (Acorus calamus Linn.) were investigated for their antifungal activity in comparison with eugenol and amphotericin B (AmB) by using the National Committee for Clinical Laboratory Standards (NCCLs) M27-P broth microdilution method. Two medicinal plant extracts, eugenol and amphotericin B were used to determine their minimum inhibitory concentrations (MICs) and minimum fungicidal concentrations (MFCs) against 28 clinical isolates of Candida albicans and 25 clinical isolates of Cryptococcus neoformans. The MICs of clove, sweet flag, eugenol and AmB against C. albicans were 17.41+8.64 mg/ml, 28.8+16.32 mg/ml, 12.16+4.53 mg/ml and 0.23+0.1 µg/ml respectively. The MFCs were 67.5+15.39 mg/ml, >75 mg/ml, 15.4+6.47 mg/ml and 0.47+0.21 µg/ml respectively. The same extracts and antifungal drugs which were tested against C. albicans were also tested against C. neoformans. The MICs were 2.43+0.95 mg/ml, 3.02+1.97 mg/ml, 6.28+3.4 mg/ml and 0.28+0.15 µg/ml, respectively. The MFCs were 22.22+12.71 mg/ml, 30.82+27.11 mg/ml, 10.06+4.9 mg/ml and 0.51+0.25 µg/ml respectively. The results showed that C. albicans was significantly (p<0.01) more susceptible to the extract of clove than sweet flag, whereas C. neoformans was significantly susceptible to the clove extract (p>0.05). Moreover, the extract of clove showed significantly (p<0.01) more potent inhibitory activity against C. neoformans than eugenol, while it showed significantly (p<0.01) less inhibitory activity against C. albicans than eugenol. AmB, the drug of choice for invasive infection treatment, remains as one of the most effective antifungal drugs. These data indicate that the extracts of clove and sweet flag were potential fungistatic agents against yeasts, whereas AmB and eugenol showed fungicidal effects.

Penicillium marneffei is an opportunistic fungal pathogen that exhibits thermally regulated dimorphism. At 25°C, this fungus grows vegetatively as mycelia, but at 37°C or upon invasion of a host, a fission yeast form is established. Yet, despite increased numbers of molecular studies involving this fungus, the role of P. marneffei stress response-related proteins is not well characterized. Actin is one of the proteins that have been proposed to play a role not only in cell transition, but also in thermo-adaptation. Here, we report the isolation and characterization of the actin encoding gene, actA, from P. marneffei. Examination of the deduced amino acid sequence of the ActA protein revealed that it is closely related to Aspergillus nidulans and Aspergillus clavatus. Northern blot analysis of actin expression during the mycelium to yeast phase transition of P. marneffei showed that the actA transcripts were initially upregulated soon after shifting the incubation temperature from 25°C to 37°C, but subsequently decreased slightly and did not change during further growth or under stress conditions. When cultures were started with conidia, upregulation of actA gene was found to correlate with germ tube production at either 25°C or 37°C. However, the relative expression level of actA transcripts again showed no significant differences in different cell types (conidia, mycelium, and yeast cells) or during macrophage infection. These results suggest that actin may play an important role in the early stages of cellular development, but not in environmental stress responses.