SUMMARY Penicillium marneffei infection is an important emerging public health problem, especially among patients infected with human immunodeficiency virus in the areas of endemicity in southeast Asia, India, and China. Within these regions, P. marneffei infection is regarded as an AIDS-defining illness, and the severity of the disease depends on the immunological status of the infected individual. Early diagnosis by serologic and molecular assay-based methods have been developed and are proving to be important in diagnosing infection. The occurrence of natural reservoirs and the molecular epidemiology of P. marneffei have been studied; however, the natural history and mode of transmission of the organism remain unclear. Soil exposure, especially during the rainy season, has been suggested to be a critical risk factor. Using a highly discriminatory molecular technique, multilocus microsatellite typing, to characterize this fungus, several isolates from bamboo rats and humans were shown to share identical multilocus genotypes. These data suggest either that transmission of P. marneffei may occur from rodents to humans or that rodents and humans are coinfected from common environmental sources. These putative natural cycles of P. marneffei infection need further investigation. Studies on the fungal genetics of P. marneffei have been focused on the characterization of genetic determinants that may play important roles in asexual development, mycelial-to-yeast phase transition, and the expression of antigenic determinants. Molecular studies have identified several genes involved in germination, hyphal development, conidiogenesis, and yeast cell polarity. A number of functionally important genes, such as the malate synthase- and catalase-peroxidase protein-encoding genes, have been identified as being upregulated in the yeast phase. Future investigations pertaining to the roles of these genes in host-fungus interactions may provide the key knowledge to understanding the pathogenicity of P. marneffei.

Because of the limited ability of the National Committee for Clinical Laboratory Standards proposed M27P methodology to detect resistance to amphotericin B by Candida isolates, we sought to identify alternative media and pH conditions that could reliably identify resistant isolates. Antibiotic Medium 3 broth (also known as Penassay broth) buffered to pH 5 or pH 7 produced superior results and readily identified a series of resistant isolates.Antifungal susceptibility testing is rapidly evolving. The M27P methodology currently under development by the National Committee for Clinical Laboratory Standards (NCCLS) gives reproducible results for testing of Candida and Cryptococcus isolates (3, 4, 6), and establishment of interpretive breakpoints is now beginning. However, it is becoming clear that the M27P methodology may be inadequate for certain organisms and antifungal agents. For example, Cryptococcus neoformans grows poorly in the RPMI 1640 medium suggested as part of the M27P methodology, and other media may therefore be preferable (5). In addition, concern has been raised that determination of susceptibility of Candida isolates to amphotericin B may be a problem (9). In this case, the M27P methodology yields a range of MICs that spans only three to four twofold serial dilutions. While MICs for putatively resistant isolates tend be at the high end of the range, the inherent variability of MIC determination precludes reliable discrimination between susceptible and resistant isolates. In this study, we have sought to resolve this problem by examining alternative media and pH conditions. We find that Antibiotic Medium 3 broth (also known as Penassay broth) buffered to pH 5 or pH 7 identifies amphotericin B-resistant isolates of Candida and is a candidate for standardization studies. MATERIALS AND METHODSIsolates. A series of putatively amphotericin B-resistant Candida isolates were obtained from a variety of sources (Table 1). In particular, a set of three Candida isolates previously characterized as amphotericin B resistant or susceptible on the basis of in vivo testing (1) was included. In addition, a pair of previously described bloodstream isolates (8) from a recent therapy trial comparing fluconazole with amphotericin B as therapy of candidemia (7) was also studied. Isolates were stored at Ϫ70ЊC and were passaged at least twice on Sabouraud dextrose agar at 35ЊC prior to being tested. Isolates were identified to the species level by using the API 20C system (Analytab Products, Plainview, N.Y.).Susceptibility testing. Broth macrodilution MICs were determined by using the NCCLS M27P methodology. Briefly, yeasts at final concentrations of 0.5 ϫ 10 3 to 2.5 ϫ 10 3

A multicenter study was conducted to expand the generation and analysis of data that supports the proposal of a reference method for the antifungal susceptibility testing of filamentous fungi. Broth microdilution MICs of amphotericin B and itraconazole were determined in 11 centers against 30 coded duplicate pairs of Aspergillus spp., Fusarium spp., Pseudallescheria boydii, and Rhizopus arrhizus. The effect of inoculum density (approximately 10 3 and 10 4 CFU/ml), incubation time (24, 48, and 72 h), and procedure of MIC determination (conventional and colorimetric [Alamar Blue] evaluation of growth inhibition) on intra-and interlaboratory agreement was analyzed. Based on intra-(97 to 100%) and interlaboratory (94 to 95%) agreement for both drugs, the overall optimal testing conditions identified were determination of colorimetric MICs after 48 to 72 h of incubation with an inoculum density of approximately 10 4 CFU/ml. These testing conditions are proposed as guidelines for a reference broth microdilution method.