Comparison of the kidney fungal burden in experimental disseminated candidiasis by species of the Candida parapsilosis complex treated with fluconazole, amphotericin B and caspofungin in a temporarily neutropenic murine model.

BACKGROUND: The efficacy of fluconazole (FLU), amphotericin B (AMB) and caspofungin (CAS) was tested against three Candida orthopsilosis, three C. metapsilosis and two C. parapsilosis sensu stricto isolates in neutropenic mice. METHODS: Mice were immunosuppressed by 200 mg/kg cyclophosphamide. Five-day intraperitoneal treatment was started 24 h after infection. Kidney burden was analyzed using the Kruskal-Wallis test. RESULTS: FLU 10 and 20 mg/kg as well as AMB 1 mg/kg significantly decreased the fungal burden (p < 0.05) for all eight isolates of the three species. CAS 2 and 5 mg/kg were efficacious against all C. orthopsilosis and C. metapsilosis isolates (p < 0.05), but only 5 mg/kg CAS was effective against C. parapsilosis isolates (p < 0.05). CONCLUSIONS: The efficacy of FLU and AMB against the three species was comparable. Though the activity of CAS was higher against C. orthopsilosis and C. metapsilosis, the current treatment guidelines for C. parapsilosis sensu stricto seem to be applicable to other 'psilosis' species.

From farm to fork follow-up of thermotolerant campylobacters throughout the broiler production chain and in human cases in a Hungarian county during a ten-months period.

A study tracking thermotolerant campylobacters from the setting of the broilers throughout the whole rearing period, slaughter and sale of chicken products in five consecutive broiler rotations of the same henhouse as well as in two different other farms was conducted in a well-defined geographic area (Hajdú-Bihar county, Hungary) between March 2006 and Feb 2007. All notified cases of human campylobacteriosis in this area during the study period were also included. One hundred and one, 44, 23 and 282 Campylobacter jejuni and 13, 15, 20 and 60C. coli were isolated from broiler houses, slaughterhouses, retail shops and human samples, respectively. Sixty-two isolates collected from broilers or their environment selected from different flocks (57C. jejuni, 5C. coli), 92 isolates collected from abattoirs and retail shops (72C. jejuni, 20C. coli), as well as 85 randomly selected human isolates (74C. jejuni, 11C. coli) were subjected to PFGE analysis using restriction enzymes KpnI and SmaI. Sixty-six of the isolates produced unique Sma-Kpn profiles; the majority (46) of these were of human origin. The remaining isolates formed PFGE clusters of between 2-25 isolates with 14 (12C. jejuni and 2C. coli) main clusters comprised of five or more isolates with identical KpnI-SmaI patterns. Two genetic clones of C. jejuni (clone A, n=25; clone B, n=20) included 18% of isolates from different sources. Generally, isolates from one cluster were found in 1-3 different flocks, notably, clone B was present in three rotations including those from the two independent farms. Six of the seven investigated flocks had one or two characteristic prevalent clones. Transmission of clones between consecutive flocks was frequently seen. Spread of both C. jejuni and C. coli was traced multiple times along the food chain; eight C. jejuni, but no C. coli clones were detected both in broilers and humans. These data suggest that broilers were the major source for C. jejuni but not for C. coli in the studied area and period. For C. jejuni the carryover of strains between consecutive flocks may be a common event, but the strain is eventually replaced by another and consecutive carryover events seem to be infrequent. The majority of the human disease was due to nonepidemic strains; some clones were transmitted from more than one broiler flocks (including epidemiologically unrelated flocks) to humans multiple times.

Efficacy of a single 6 mg/kg versus two 3 mg/kg caspofungin doses for treatment of disseminated candidiasis caused by Candida albicans in a neutropenic mouse model.

We compared the efficacy of single six mg/kg and 2x3 mg/kg caspofungin doses to the traditional one mg/kg daily against C. albicans in a neutropenic murine model. In lethality experiments, all regimens improved survival (p<0.0014 for all three isolates); differences among the treated groups were not statistically significant. We calculated kidney fungal burdens on each study day for six days postinfection using two isolates in two experiments. In the first three days, only the six mg/kg dose produced significant decrease on all study days (p<0.05-0.001), but differences between the three treatment arms disappeared by 4-6 days postinfection (p<0.05 for all isolates on all days).

In vitro efficacy of amphotericin B, 5-fluorocytosine, fluconazole, voriconazole and posaconazole against Candida dubliniensis isolates using time-kill methodology.

Candida dubliniensis is a recently described yeast that causes infections in mucosal surfaces as well as sterile body sites. Candida dubliniensis develops resistance to fluconazole (FLC) more rapidly than the closely related species C. albicans. The killing activity of amphotericin B (AMB), 5-fluorocytosine (5FC), FLC, voriconazole (VRC) and posaconazole (POS) was determined against six C. dubliniensis clinical isolates, identified using molecular biological methods and C. dubliniensis CD36 reference strain. Minimum inhibitory concentrations (MICs) were determined using the Clinical and Laboratory Standards Institute standard procedure. Time-kill assays were performed using RPMI-1640 as test media over a 48-h period. AMB proved to be fungicidal at >or=0.5 microg ml(-1) against all clinical isolates after 48 h. 5FC was only fungicidal at 32-64x MIC (4-8 microg ml(-1)) against all C. dubliniensis isolates. FLC, VRC and POS were fungistatic; decrease in colony number was observed only at the highest concentrations tested (8, 4 and 4 microg ml(-1), respectively). Triazoles invariably showed fungistatic effect at concentrations attainable in the serum. In clinical situations when a fungicidal antifungal is desirable, AMB may be used.

Harmonisation of ethics committees' practice in 10 European countries.

BACKGROUND: The Directive 2001/20/EC was an important first step towards consistency in the requirements and processes for clinical trials across Europe. However, by applying the same rules to all types of drug trials and transposing the Directive's principles into pre-existing national legislations, the Directive somewhat failed to meet its facilitation and harmonization targets. In the field of ethics, the Directive 2001/20/EC conditioned the way of understanding and transposing the "single opinion" process in each country. This led to a situation in which two models of research ethics committees organisation systems exist, being the model in which the "single opinion" is considered to be the decision made by a single ethics committee more effective and simpler in terms of administrative and logistic workload. METHOD: A survey was conducted in 10 European countries. Members of the European Clinical Research Infrastructures Network working party number 1, with expertise in the field of ethics, responded. RESULTS: There is a major heterogeneity in the composition of ethics committees among the surveyed countries based on the number of members, proportion of experts versus lay members and expertise of the scientific members. A harmonized education of the ethics committees' membership based in common curricula is recommended by the majority of countries. CONCLUSIONS: Despite the efforts for harmonization of the European Clinical Trial Directive, from an ethical point of view, there remains a plurality of ethics committees' systems in Europe. It is important to comprehend the individual national systems to understand the problems they are facing.

Difference in killing activity of caspofungin and paradoxical growth between Candida albicans and C. krusei clinical isolates in different media.

Minimum fungicidal concentration (mFC) of caspofungin was determined against 16 Candida albicans and 16 C. krusei in Rpmi-1640 and antibiotic medium 3 (Am3). time-kill tests were performed on six C. albicans and four C. krusei strains at 0.06-16 mg/l caspofungin. mFC ranges after 48 h were 0.5-1 and 1-2 mg/l for C. albicans and C. krusei, respectively; one C. albicans and the C. krusei reference strain showed paradoxical growth (pG) in Rpmi-1640, respectively. in killing experiments, after 48 h caspofugin was fungicidal against two and four C. albicans in Rpmi-1640 (at 16 mg/l) and in Am3 (at >0.5 mg/l), respectively; pG was noted in three and two cases, respectively. Caspofungin at >2 and 0.5 mg/l was fungicidal against all tested C. krusei strains even after 24 h in Rpmi-1640 and Am3, respectively. Killing activity of caspofungin against C. albicans and C. krusei could be exactly measured only by killing curves.

Prevalence and characterization of Salmonella infantis isolates originating from different points of the broiler chicken-human food chain in Hungary.

During the 10-month study period Salmonella contamination of broiler houses and the flocks reared in three farms (A, B and C), the slaughter houses where the flocks were slaughtered, as well as the carcass and retail raw meat products originating from them was investigated. In the broiler farm A five consecutive flocks, in the B and C farms one flock was sampled. Environmental samples were taken prior to the introductions. Environmental, drinking water, feed and faecal samples were collected regularly using standard methods. Before and during processing of the flocks, environmental and carcass samples were taken at the abattoirs. Salmonella contamination of the carcass, retail meat, as well as stool samples of farm and abattoir workers and from human illnesses registered in the same period and region were also examined. Isolation, sero-, phage- and antibiotic resistance typing, class 1 integron and plasmid profiling of the strains were performed; their genetic relationship was assessed by PFGE. Although the broiler house and the faecal samples of the 5 flocks of the farm A were negative for Salmonella, S. infantis was isolated from 20-100% of the abattoir carcass samples. The retail raw meat samples were 0-100% S. infantis positive. The environmental samples of farm B were Salmonella negative, but the examined flock was contaminated: S. infantis was identified from 43% of the faecal samples. This serotype was identified in 100% of the carcass and retail raw meat samples. From environmental samples taken before the arrival of the 1-day-old chicks in the broiler house C, S. infantis was cultured. S. infantis prevalence in the faecal samples was 35% and all the carcass and retail raw meat samples were S. infantis contaminated. Altogether 164 S. infantis strains were isolated out of which 145 were further characterized. The vast majority (142/145) of the strains belonged to phage types 217 and 213. All but one were characterized by the nalidixic acid-streptomycin-sulphonamide-tetracycline resistances, had an 885 bp class 1 integron and a large plasmid of > 168 kb in size. The strains showed > or = 88.7% genetic similarity. The results obtained shows that the same multi-drug resistant S. infantis clone was spread from the examined broiler farms contaminating the slaughter and the retail meat and appeared in the human illnesses of the examined region that was earlier detected as the dominant clone characteristic of the broiler and human population of the whole country.

Time-kill studies investigating the killing activity of caspofungin against Candida dubliniensis: comparing RPMI-1640 and antibiotic medium 3.

OBJECTIVES: We evaluated the in vitro activity of caspofungin against Candida dubliniensis strains using MIC and minimum fungicidal concentration (MFC) measurements and time-kill methodology. METHODS: We used six C. dubliniensis clinical isolates and the CD 36 type strain. MICs and MFCs of caspofungin were determined using the standard broth microdilution method with normal (10(3) cells/mL) and elevated (10(5) cells/mL) starting inocula in RPMI-1640 and antibiotic medium 3 (AM3). MIC was determined after 24 h, and plating for MFC determination was performed after 48 h. In time-kill tests, all strains were tested at 0.06-16 mg/L caspofungin concentrations in RPMI-1640 and AM3. RESULTS: In RPMI-1640, the MIC range was 0.06-8 mg/L. Trailing growth was observed regardless of the starting inoculum after 48 h, but not after 24 h. In AM3 regardless of starting inoculum, MICs were 0.03 mg/L. After 48 h, trailing was not detected; two isolates grew at a concentration of 8 mg/L using 10(5) cells/mL as the starting inoculum [paradoxical growth (PG)]. All MFCs in RPMI-1640 and AM3 were >8 and < or =0.12 mg/L, respectively. In AM3, all but a single isolate showed PG in the MFC tests. Time-kill tests confirmed the results obtained by MFC tests both in RPMI-1640 and AM3. CONCLUSIONS: In vitro activity of caspofungin against C. dubliniensis depended on the starting inoculum and medium used. Using AM3 eliminated trailing from MIC determinations but not PG in MIC, MFC and time-kill tests.

Evaluation of the new Micronaut-Candida system compared to the API ID32C method for yeast identification.

A new system, Micronaut-Candida, was compared to API ID32C to identify 264 yeast (Candida albicans, C. parapsilosis, C. tropicalis, C. krusei, C. inconspicua, C. norvegensis, C. lusitaniae, C. guilliermondii, C. dubliniensis, C. pulcherrima, C. famata, C. rugosa, C. glabrata, C. kefyr, C. lipolytica, C. catenulata, C. neoformans, Geotrichum and Trichosporon species, Rhodotorula glutinis, and Saccharomyces cerevisiae) clinical isolates. Results were in concordance in 244 cases. Eighteen out of the 20 of discordant results were correctly identified by Micronaut-Candida but not by API ID32C, as confirmed by PCR ribotyping.

DNA fingerprinting analysis of breakthrough outbreaks in vaccine-protected poultry stocks.

We report recurrent outbreaks of Yersinia pseudotuberculosis conjunctivitis in ducks and of fowl cholera in geese, occurring in stocks previously vaccinated with inactivated autogenous vaccines. Enterobacterial repetitive intergenic consensus sequence-based PCR and pulsed-field gel electrophoresis indicated reinfection with a new Y. pseudotuberculosis strain and vaccine evasion by the same Pasteurella multocida strain.

In vitro study of Candida tropicalis isolates exhibiting paradoxical growth in the presence of high concentrations of caspofungin.

Paradoxical growth was noted in RPMI 1640 and antibiotic medium 3 in the case of 14 and 1 of 15 Candida tropicalis strains, respectively, at a caspofungin concentration of 12.5 microg/ml using minimum fungicidal concentration tests. Time-kill assays showed that against isolates killed at lower concentrations, caspofungin at a concentration of 12.5 microg/ml was only fungistatic.

Correlation of posaconazole minimum fungicidal concentration and time kill test against nine Candida species.

OBJECTIVES: We evaluated the in vitro activity of posaconazole against nine Candida species using minimum fungicidal concentration (MFC) measurements and time-kill methods. METHODS: MFCs of posaconazole were determined for 209 clinical isolates (32 Candida albicans, 30 Candida glabrata, 21 Candida tropicalis, 29 Candida krusei, 28 Candida parapsilosis sensu stricto, 50 Candida inconspicua, 13 Candida kefyr, 3 Candida lusitaniae and 3 Candida guilliermondii) and 7 ATCC Candida strains. The following strains were tested in time-kill studies: 3 strains each of C. glabrata, C. kefyr, C. guilliermondii and C. lusitaniae; 2 C. tropicalis; 4 C. albicans; 4 C. inconspicua; 9 C. krusei; 12 C. parapsilosis; and 7 ATCC strains. RESULTS: Posaconazole was fungicidal in both MFC and time-kill experiments (at 2 mg/L within 48 h in time-kill assays) against each C. krusei, C. inconspicua and C. lusitaniae strain and was fungistatic against each C. albicans, C. glabrata, C. tropicalis and C. guilliermondii strain. For the C. parapsilosis strains, posaconazole MFCs were

Novel PCR assay for identification of Salmonella enterica serovar Infantis.

AIMS: We developed, optimized and tested two novel PCR assays specific for Salmonella enterica subspecies enterica serovar Infantis. METHODS AND RESULTS: The fljB gene was chosen as the target sequence. Primers were designed on a consensus sequence built by sequencing the fljB gene of five genetically unrelated Hungarian S. Infantis strains and using sequence data from the GenBank (http://www.ncbi.nih.gov). Two alternative assays were designed, which share the reverse primer. Both proved to be highly specific to S. Infantis, neither reacted with 42 other nontyphoidal serovariants tested. The detection limit of the assays was determined to be 10(5) CFU ml(-1) from pure culture, and 10(6) CFU g(-1) from artificially spiked chicken faeces samples. CONCLUSIONS: Although the detection limit is rather high to allow for using them for direct detection, the assays may be useful in identification of S. Infantis both for diagnostic and for research purposes. SIGNIFICANCE AND IMPACT OF THE STUDY: The described PCR assays allow for the correct identification of S. Infantis even when traditional serotyping methods fail because lack of expression of flagellar antigens.

Characterization of Staphylococcus aureus strains isolated from bovine milk in Hungary.

Staphylococcus aureus is a major foodborne pathogen due to its capability to produce a wide range of heat-stable enterotoxins. The primary purpose of this research was to characterize S. aureus isolates recovered from mammary quarter milk of mastitic cows and from bulk tank milk produced on Hungarian dairy farms of different sizes. Macrorestriction analysis of chromosomal DNA from S. aureus isolates was performed using the restriction enzyme SmaI followed by pulsed-field gel electrophoresis (PFGE). The prevalence rates of nine S. aureus enterotoxin genes (sea, seb, sec, sed, see, seg, seh, sei, and sej) and of the toxic shock syndrome toxin 1 gene (tst) were determined by multiplex polymerase chain reaction (PCR). The bulk tank milks of 14 out of 20 farms were contaminated with S. aureus at levels of up to 6.0x10(3 )CFU/ml. Farm size had no significant effect (P>0.05) on the S. aureus counts in bulk milk. The prevalence rates of penicillin resistance were 88.9% and 20.0% among the S. aureus recovered from mastitic quarter milk and bulk tank milk, respectively. After phenotypic characterization, a total of 59 S. aureus isolates were selected for genotyping. PFGE analysis revealed 22 distinct pulsotypes, including 14 main types and 8 subtypes, at a similarity level of 86%. Only one or two main types were observed on each of the farms tested, indicating a lack of genetic diversity among S. aureus isolates within farms, and there were only two pulsotypes which occurred on more than one farm. The PFGE patterns showed genetic relatedness between the S. aureus strains recovered from quarter milk and bulk milk on two large farms, implying that on farms having a high number of mastitic cows, S. aureus from infected udders may contaminate bulk milk and, subsequently, raw milk products. Sixteen (27.1%) of the S. aureus isolates tested by multiplex PCR were found to be positive for enterotoxin genes, with 15 of them carrying just one gene and one strain carrying two genes (seg and sei). The most commonly detected toxin genes were seb, sea, and sec, whereas none of our isolates possessed the see, seh, sej, or tst genes. On 75% of the dairy farms surveyed, no enterotoxigenic staphylococci were recovered from either mastitic quarter milk or bulk tank milk.

Pacheco's disease in a Hungarian zoo bird population: a case report.

An epizootic of Pacheco's disease is reported from a zoo bird population. The infection was introduced by wild-captured Patagonian conures (Cyanoliseus patagonus) despite 61 days of quarantine. The disease affected several parrot species and, interestingly, three out of seven bearded barbets (Lybius dubius). The mortality rate was 30.93%. Autopsy revealed abdominal hyperaemia with liver haemorrhages and, in less rapid cases, yellowish discoloration and fragility of the liver. Death was caused by the collapse of circulation. Histopathology demonstrated liver cell necrosis, disintegration of the lobular structure, and a few intranuclear inclusion bodies. Icosahedral virions were detected by electron microscopy. The virus was isolated in the allantoic cavity of embryonated chicken eggs as well as in chicken embryo fibroblast cell culture. A 281-bp-long fragment of psittacid herpesvirus DNA was detected by PCR in cell culture material and liver samples of the affected birds. To our knowledge this is the first report of Pacheco's disease in bearded barbets as well as the first occurrence of Pacheco's disease in Hungary.

Posaconazole susceptibility testing against Candida species: comparison of broth microdilution and E-test methods.

Posaconazole (POS) is a newer triazole with activity against yeasts and moulds. POS and fluconazole were tested in vitro against 32 Candida albicans, 30 C. glabrata, 21 C. tropicalis, 29 C. krusei, 28 C. parapsilosis, 50 C. inconspicua, 13 C. kefyr and 5 C. famata isolates using CLSI broth microdilution method (BMD). We compared E-test and a modified BMD using polyethylene-glycol (PEG) as solvent to the CLSI method. BMDs and E-test were performed according to CLSI and the manufacturer's instructions respectively. Geometric means of POS MICs using BMD were 0.71, 0.22 and 0.21 microg ml(-1) against C. glabrata, C. krusei and C. inconspicua, respectively, and remained below 0.1 microg ml(-1) against all other species tested. One of two C. albicans and two of three C. glabrata isolates resistant to fluconazole showed MICs above 8 microg ml(-1) to POS. The impact of using PEG instead of DMSO had only a minor effect (agreements above 95% with the exception of C. parapsilosis). E-tests read after 24 h showed good agreement with the BMD. POS exhibited excellent in vitro activity against Hungarian Candida strains. E-test showed good correlation with the CLSI method, but to facilitate the comparability of results we believe that DMSO should be used as solvent in the BMD.

Rapid and simultaneous detection of avian influenza and newcastle disease viruses by duplex polymerase chain reaction assay.

A duplex reverse transcription-polymerase chain reaction (dRT-PCR) assay has been developed for the simultaneous, rapid and specific detection/discrimination of avian influenza virus (AIV) and Newcastle disease virus (NDV). Primers targeting the matrix protein gene (M) of AIV and the fusion protein gene (F) of NDV were evaluated experimentally with 13 AIV and 19 NDV strains. PCR products of the expected size of 144 bp and 316 bp were amplified from AIV/NDV samples, respectively, while no cross-reaction was observed with negative controls or with 16 other avian pathogens. The endpoint of detection was defined as approximately 10(+0.5) 50% egg infectious dose (EID(50))/0.2 ml for AIV and 10(+2.2) EID(50)/0.2 ml for NDV. The assay was able to detect AIV/NDV with similar sensitivity in spiked stool samples and in specimens from vaccinated birds. The developed dRT-PCR assay is a rapid, cost-effective tool, which provides powerful novel means for the early diagnosis of avian influenza and Newcastle disease.

Development of a novel PCR assay specific for Riemerella anatipestifer.

AIMS: Riemerella anatipestifer is a significant pathogen of waterfowl and turkeys. Due to their similar ecology and morphological and cultural characteristics it is important to differentiate R. anatipestifer infections from those caused by Pasteurella multocida. Present study describes a novel PCR assay that is capable of rapid and species-specific identification of R. anatipestifer from bacterial cultures. METHODS AND RESULTS: An ERIC (enterobacterial repetitive intergenic consensus)-PCR fragment common to all tested isolates was used as a target for primer design. After optimization, the assay was tested on 72 R. anatipestifer strains isolated from clinical samples and identified using biochemical tests. All of these gave positive results, while heterologous pathogens, including different serotypes of P. multocida, proved to be negative. The assay was also capable of demonstrating R. anatipestifer directly from five clinical samples. CONCLUSIONS: The presented PCR is suitable for proper identification of R. anatipestifer from culture. Preliminary investigation showed that the test could be suitable for detection of the pathogen from clinical samples as well. SIGNIFICANCE AND IMPACT OF THE STUDY: The described PCR assay will improve the fast and proper identification of R. anatipestifer.