Impact of iron chelators on growth and expression of iron-related genes of Cryptococcus species.

INTRODUCTION: Iron chelator has previously demonstrated fungicidal effects. This study aimed to investigate the antifungal activity of the iron chelators deferoxamine (DFO) and deferasirox (DSX) against Cryptococcus. MATERIALS AND METHODS: Cryptococcus neoformans and Cryptococcus gattii were used to determine the minimal inhibitory concentrations (MICs) of DFO and DSX, and the fractional inhibitory concentration index (FICI) of DFO and DSX when combined with amphotericin B (AMB). Expression of cryptococcal CFT1, CFT2, and CIR1 genes was determined using real-time polymerase chain reaction (PCR). RESULTS: Neither DFO nor DSX alone showed antifungal activity against Cryptococcus strains. When combined with AMB, the MICs of DFO and DSX decreased from>200µg/mL to 6.25 or 12.5µg/mL. The MIC of AMB decreased one-fold dilution in most strains when combined with iron chelators. The FICI of DFO+AMB and DSX+AMB was 0.5 and 1, respectively. C. neoformans showed significant growth retardation when incubated with a combination of sub-MIC concentrations of AMB and DFO; whereas, C. gattii demonstrated lesser growth retardation in DFO+AMB. No cryptococcal growth retardation was observed when DSX was combined with AMB. When C. neoformans was grown in DFO, the CFT1, CFT2, and CIR1 proteins were expressed 1.7, 2.0, and 0.9 times, respectively. When C. neoformans was grown in DSX, the CFT1, CFT2, and CIR1 genes were expressed 0.5, 0.6, and 0.3 times, respectively. CONCLUSION: Synergistic antifungal activity of combination DFO and AMB was observed in Cryptococcus. Relatively increased CFT1 and CFT2 expression may be associated with the effect of DFO that inhibits the growth of fungi.

A novel structure of Tn4001-truncated element, type V, in clinical enterococcal isolates and multiplex PCR for detecting aminoglycoside resistance genes.

A multiplex polymerase chain reaction (PCR) was established for detecting aacA-aphD, aph(2'')-Ib, aph(2'')-Ic and aph(2'')-Id, encoding high-level gentamicin resistance (HLGR), and aadA and aadE, encoding high-level streptomycin resistance (HLSR), in enterococci. The assay was implemented for 419 enterococcal blood and urine isolates recovered from patients at a university hospital in Thailand. Among the isolates tested, 56.1% (235 isolates) and 58.9% (247 isolates) contained aacA-aphD and aadE, respectively. The aph(2'')-Ib, aph(2'')-Ic, aph(2'')-Id and aadA genes were not found in any isolate. Among the isolates carrying the aacA-aphD gene, 99.1% exhibited a HLGR phenotype. All 235 enterococcal isolates containing aacA-aphD were further studied by PCR to characterise the structure of the resistance determinants carrying the aacA-aphD gene. The result revealed that only 22.6% carried Tn4001-related element, whereas the remaining isolates contained Tn4001-truncated element. No Tn4001-IS257 hybrid structure was detected. The majority of isolates carrying Tn4001-related element were Enterococcus faecalis (77.4%). Among Tn4001-truncated elements detected, all previously reported types (types I-IV) were found. Furthermore, a novel Tn4001-truncated type, designated type V, was also identified.

Protozoan enteric infection in AIDS related diarrhea in Thailand.

The aim of this study was to determine the prevalence of enteric protozoa and other pathogens in AIDS patients with diarrhea in Bangkok, Thailand. Of 288 consecutive patients screened in the 10 month period between November 1999-August 2000 inclusive, 55 (19.2%) had Cryptosporidium spp, 13 (4.5%) had Isospora oocyst, 11 (3.8%) had Giardia lamblia, 3 (0.9%) had Entamoeba histolytica, and 1 (0.3%) had Iodamoeba butschlii infection. The prevalence of microsporidia was 11% in this study. Of 251 patients for whom stool culture for bacteria was performed, enteric bacterial pathogens isolated were Campylobacter spp in 18 (7.1%), Salmonella spp in 11 (4.3%), and Shigella spp in 1 (0.5%). Other pathogens found in these patients were Clostridium difficile in 16/102 (15.6%). Mycobacterium spp in 18/287 (6.2%), and Strongyloides stercoralis in 23/288 (8.0%). Overall, parasitic and bacterial pathogens were identified in 140 (48.6%) patients. These pathogens were identified by the routine simple wet smear technique in 32, formalin-ether concentration method in 46, culture for S. stercoralis in 5, and culture for bacteria in 30. Additional test, using modified Ziehl-Neelsen staining, identified cryptosporidial oocyst, isospora oocyst, and Mycobacterium spp in 72. The microsporidia, initially identified by modified trichrome blue staining, all were then determined to be Enterocytozoon bieneusi by thin sectioning electron microscopy. Protozoan and bacterial pathogens were confirmed to be important etiologic agents in diarrhea in AIDS in Thailand. They were all associated with increased mortality. Routine stool examination by simple wet smear detected only one-fourth of these pathogens. Therefore all diagnostic techniques for these organisms should be made more widely available in Thailand.

Zoonotic species of Cryptosporidium are as prevalent as the anthroponotic in HIV-infected patients in Thailand.

The epidemiology of chronic diarrhoea in adults with late-stage HIV infection was investigated in a prospective study in Bangkok, Thailand. During this investigation, 34 Cryptosporidium isolates were obtained from the faeces of 36 patients, with mean CD4(+) counts of only 14 x 10(6) CD4(+) cells/litre (range = 2 x 10(6) - 53 x 10(6)/litre), who had symptomatic cryptosporidiosis. Genotyping of these isolates, by RFLP analysis and DNA sequencing of the hypervariable region of the 18S rRNA gene, indicated that only 17 (50%) were of the C. parvum human genotype. The rest were of C. meleagridis (seven), the C. parvum 'bovine' genotype (five), C. felis (three) and C. canis (two). Extensive genotypic heterogeneity was observed among the C. parvum isolates, and two other isolates, one of C. meleagridis and the other of C. felis, produced atypical restriction patterns and were only identified by sequencing. This appears to represent the first report of C. canis and the 'bovine' genotype of C. parvum in HIV-infected Thai patients.