High resolution genotyping of clinical Aspergillus flavus isolates from India using microsatellites.

BACKGROUND: Worldwide, Aspergillus flavus is the second leading cause of allergic, invasive and colonizing fungal diseases in humans. However, it is the most common species causing fungal rhinosinusitis and eye infections in tropical countries. Despite the growing challenges due to A. flavus, the molecular epidemiology of this fungus has not been well studied. We evaluated the use of microsatellites for high resolution genotyping of A. flavus from India and a possible connection between clinical presentation and genotype of the involved isolate. METHODOLOGY/PRINCIPAL FINDINGS: A panel of nine microsatellite markers were selected from the genome of A. flavus NRRL 3357. These markers were used to type 162 clinical isolates of A. flavus. All nine markers proved to be polymorphic displaying up to 33 alleles per marker. Thirteen isolates proved to be a mixture of different genotypes. Among the 149 pure isolates, 124 different genotypes could be recognized. The discriminatory power (D) for the individual markers ranged from 0.657 to 0.954. The D value of the panel of nine markers combined was 0.997. The multiplex multicolor approach was instrumental in rapid typing of a large number of isolates. There was no correlation between genotype and the clinical presentation of the infection. CONCLUSIONS/SIGNIFICANCE: There is a large genotypic diversity in clinical A. flavus isolates from India. The presence of more than one genotype in clinical samples illustrates the possibility that persons may be colonized by multiple genotypes and that any isolate from a clinical specimen is not necessarily the one actually causing infection. Microsatellites are excellent typing targets for discriminating between A. flavus isolates from various origins.

[Melioidosis: the importance of a detailed medical history, including recent travels]. / Melioidosis: het belang van een goede reisanamnese.

A 70-year-old woman was admitted to hospital with fever and signs of bronchopneumonia following a recent visit to Southeast Asia. She was diagnosed with melioidosis and treated with ceftazidime i.v. for two weeks, followed by oral co-trimoxazol and folinic acid. She recovered and had no recurring disease in the first year following recovery. Melioidosis is caused by an infection with Burkholderia pseudomallei. Clinical presentation can vary, but pneumonia is present in most patients. The diagnosis should be considered in patients with reduced immunological resistance who have been in endemic areas such as Southeast Asia, especially during the rainy season. It is important to determine which countries have been visited by patients who have recently returned from tropical areas. In addition, the time of onset, the duration of symptoms and a detailed physical examination are essential in the assessment of patients presenting with exotic diseases.

Utility of CSP typing to sub-type clinical Aspergillus fumigatus isolates and proposal for a new CSP type nomenclature.

CSP typing is a newly developed sub-typing strategy that employs comparative DNA sequence analysis of the 12-mer tandem repeat region of the AFUA_3G08890 gene. In order to allow standardization of analysis and exchange of results between laboratories, we propose a new nomenclature for individual CSP repeats as well as for CSP types. A collection of 209 clinical isolates of Aspergillus fumigatus recovered from various hospitals throughout The Netherlands was analyzed by using CSP typing and this newly proposed nomenclature. Eighteen different CSP types were recognized, positioning the CSP gene as a typing target between the relatively low discriminatory MLST loci and the highly discriminatory microsatellite markers. CSP typing may be a welcome addition to the existing molecular methods to study the diversity of A. fumigatus at the sub-population level. The results also show the presence of lineages of closely related CSP types within the A. fumigatus population, adding unique and valuable information about the population structure of A. fumigatus.

Molecular typing and colonization patterns of Aspergillus fumigatus in patients with cystic fibrosis.

Aspergillus fumigatus is a chronic colonizer of the respiratory tract of patients with cystic fibrosis (CF). A total of 204 A. fumigatus isolates from 36 CF patients from three different medical centers, collected over a period of four months till 9.5 years, were genotyped using the short tandem repeat panel for A. fumigatus (STRAf assay). Four different colonization patterns were observed. Colonization patterns with only unique genotypes were found in 36% of the patients. In contrast 17% of the patients were chronically colonized with a single genotype. The remaining patients showed a predominant genotype or genotypes that succeed each other. In this collection no relation was found between colonization patterns and allergic bronchopulmonary aspergillosis.

In vitro activities at pH 5.0 and pH 7.0 and in vivo efficacy of flucytosine against Aspergillus fumigatus.

The antifungal agent flucytosine was found to be active in vitro against Aspergillus fumigatus isolates when the MIC was determined at pH 5.0 instead of pH 7.0. The in vitro MIC at pH 5.0 corresponded to the in vivo efficacy of flucytosine monotherapy in a murine model of invasive aspergillosis.

Utility of a microsatellite assay for identifying clonally related outbreak isolates of Aspergillus fumigatus.

A microsatellite assay based on short tandem repeats (STRAf) has been recently described as a discriminatory, high throughput assay for fingerprinting Aspergillus fumigatus isolates. However, the STRAf assay has not been tested for its utility in outbreak settings where it is critical to distinguish clonal clusters from genetically unrelated genotypes. In the present study, employing a panel of epidemiologically linked A. fumigatus isolates obtained from 6 different outbreaks of invasive aspergillosis (IA), we demonstrate that the STRAf assay can be a valuable molecular tool to support epidemiological investigations. We also report for the first time the detection of microvariation events in the A. fumigatus population studied.

Retrotransposon insertion-site context (RISC) typing: a novel typing method for Aspergillus fumigatus and a convenient PCR alternative to restriction fragment length polymorphism analysis.

Retrotransposon(-like) sequences in Aspergillus fumigatus have been used as typing targets through restriction fragment length polymorphism (RFLP)/Southern blotting approaches. Differences in fingerprints between unrelated isolates are the result of variations in copy-number and differences in the regions flanking the retrotransposon elements. Here, we present retrotransposon insertion-site context (RISC) typing as a novel and convenient PCR-based typing alternative to the RFLP approach. RISC typing aims at amplifying the sequences flanking the retrotransposon-like sequences in A. fumigatus and allows large numbers of isolates to be analyzed in a timely fashion with excellent discriminatory power.

Activity and post antifungal effect of chlorpromazine and trifluopherazine against Aspergillus, Scedosporium and zygomycetes.

The phenothiazine compounds chlorpromazine and trifluopherazine are antipsychotic agents that exhibit antimicrobial activity against bacteria, some protozoa and yeasts. Data of activity against filamentous fungi are lacking. The in vitro activity and postantifungal effect (PAFE) of chlorpromazine and trifluopherazine was determined against Aspergillus species, zygomycetes and Scedosporium species. In vitro susceptibility testing was performed with CLSI M38A and the PAFE was determined with previously established methods. Both drugs inhibited the growth of all fungi tested at concentrations of 16 to 64 microg ml(-1). For Aspergillus species the mean PAFE was 3.7 and 4.7 h; for zygomycetes, 3.1 and 3.4 h; for Scedosporium, 4.3 and 5.3 h for chlorpromazine and trifluoroperazine respectively. These are the first drugs shown to induce PAFE against Scedosporium. We show that phenothiazine compounds have in vitro antifungal activity and exhibit PAFE against a broad range of filamentous fungal pathogens. Although the exact mechanism of action is unknown, further studies are needed to explore the clinical usefulness of phenothiazine compounds.

Microsatellite based typing of Aspergillus fumigatus: strengths, pitfalls and solutions.

Microsatellites, or short tandem repeats (STR's), are popular tools to discriminate between microbial isolates. Here, we report on the robustness of a microsatellite panel for discrimination of Aspergillus fumigatus isolates. Two major PCR artefacts (stutter peaks and minus-A peaks) can complicate correct interpretation of STR data. We investigated the effect of alterations to the various components of the PCR amplification mixtures on these PCR artefacts and on the reproducibility of this assay. Some extreme conditions led to a loss of signal, but, under all conditions where a signal was obtained, identical typing results were produced. Furthermore, pitfalls with the exchange of results between labs are discussed. These pitfalls are primarily associated with sizing of the obtained PCR fragments.

Comparison of two highly discriminatory molecular fingerprinting assays for analysis of multiple Aspergillus fumigatus isolates from patients with invasive aspergillosis.

Two highly discriminatory fingerprinting assays, short tandem repeat typing and amplified fragment length polymorphism (AFLP), were compared to determine the genetic relatedness between 55 isolates of Aspergillus fumigatus obtained from 15 different patients suffering from proven invasive aspergillosis. Both techniques showed that interpatient isolates belonged to different genotypes and that intrapatient isolates from deep sites were all of the same genotype. By contrast, multiple genotypes were found among isolates originating from respiratory samples. Both techniques have specific advantages and disadvantages. AFLP is more universally applicable, but short tandem repeat analysis offers better discriminatory power and should be the preferred method for standardizing typing of clinical isolates of Aspergillus fumigatus.

Clostridium cadaveris bacteraemia: two cases and review.

Clostridium cadaveris is a strict anaerobic Gram-positive rod that is the most prominent bacterium during the decay of dead bodies. We present 2 rare cases of bacteraemia with C. cadaveris. The source of both infectious episodes was most probably of gastrointestinal origin.

Use of a novel panel of nine short tandem repeats for exact and high-resolution fingerprinting of Aspergillus fumigatus isolates.

Here we describe a new panel of short tandem repeats (STRs) for a novel exact typing assay that can be used to discriminate between Aspergillus fumigatus isolates. A total of nine STR markers were selected from available genomic A. fumigatus sequences and were divided into three multicolor multiplex PCRs. Each multiplex reaction amplified three di-, tri-, or tetranucleotide repeats, respectively. All nine STR markers were used to analyze 100 presumably unrelated A. fumigatus isolates. For each marker, between 11 and 37 alleles were found in this population. One isolate proved to be a mixture of at least two different isolates. With the remaining 99 isolates, 96 different fingerprinting profiles were obtained. The Simpson's diversity index for the individual markers ranged from 0.77 to 0.97. The diversity index for the multiplex combination of di-, tri-, and tetranucleotide repeats ranged from 0.9784 to 0.9968. The combination of all nine markers yielded a Simpson's diversity index of 0.9994, indicative of the high discriminatory power of these new loci. In theory, this panel of markers is able to discriminate between no less than 27 x 10(9) different genotypes. The multicolor multiplex approach allows large numbers of markers to be tested in a short period of time. The exact nature of the assay combines high reproducibility with the easy exchange of results and makes it a very suitable tool for large-scale epidemiological studies.

Assessing in vitro combinations of antifungal drugs against yeasts and filamentous fungi: comparison of different drug interaction models.

Non-parametric and parametric approaches of two competing zero-interaction theories--the Loewe additivity and the Bliss independence - were evaluated for analyzing the in vitro interactions of various antifungal drugs. Fifty-one data sets, derived from three drug combinations, tested in triplicate against 17 clinical yeast and mold isolates with a two-dimensional checkerboard microdilution technique, were selected to span from strong synergy to strong antagonism. These were analyzed with the standard FIC index model and modern concentration-effect response surface models: the fully parametric model developed by Greco et al. and the 3-D analysis developed by Prichard et al. The FIC index model is subjective, sensitive to experimental errors and resulted in approximated results and variable conclusions depending on the MIC endpoints determined and interpretation endpoints used. By using the MIC-2 endpoint (lowest drug concentration showing 50% of growth) for calculating the FIC indices, problems due to trailing phenomena were reduced and weak interactions could be detected; higher levels of reproducibility and agreement with the other models were achieved using the MIC-0 and MIC-1 (lowest drug concentration showing 10 and 25% of growth, respectively). High reproducibility was achieved in interpreting the FIC indices when the cutoffs of 0.25 and 4 (for single experiments) and the cutoff of 1 (for replicates) were used for defining the limits of additivity/indifference. Although the fully parametric Greco model did not describe precisely the entire response surface of all antifungal drug interactions, it was able to differentiate synergistic from non-synergistic interactions with a non-unit, reproducible, concentration-independent interaction parameter, including its uncertainty, without requiring replication. The Bliss independence based models resulted in mosaics of synergistic and antagonistic combinations, raising questions about the concentration-dependent nature of antifungal drug interaction. The sum of all statistically significant interactions were used as a summary interaction parameter for the entire response surface, concluding synergy or antagonism when it was positive or negative, respectively. The cutoffs of 100% and 200% were used to distinguish weak and moderate interactions, respectively in 12-16 x 8-12 checkerboard formats. Semi-parametric approaches need particular care as experimental errors are not eliminated from the entire response surface.

Pericarditis as complication of appendicitis.

Pericarditis as a complication of appendicitis is a rare event. In a 25-year period we encountered two pediatric cases with this severe complication due to (a)typical presentation of appendicitis resulting in small bowel obstruction, intraabdominal abscesses, constrictive pericarditis, and purulent pericarditis.

Activity of posaconazole in treatment of experimental disseminated zygomycosis.

Three isolates of zygomycetes were used to produce a disseminated infection in nonimmunocompromised mice. Against all zygomycete strains, amphotericin B significantly prolonged survival. Itraconazole was inactive against Rhizopus microsporus and Rhizopus oryzae but was partially active against Absidia corymbifera. Posaconazole had no beneficial effects against R. oryzae but showed partial activity against A. corymbifera. Posaconazole had a clear dose-response effect against R. microsporus.

Molecular epidemiology of Aspergillus fumigatus isolates recovered from water, air, and patients shows two clusters of genetically distinct strains.

There has been an increase in data suggesting that besides air, hospital water is a potential source of transmission of filamentous fungi, and in particular Aspergillus fumigatus. Molecular characterization of environmental and clinical A. fumigatus isolates, collected prospectively during an 18-month period, was performed to establish if waterborne fungi play a role in the pathogenesis of invasive aspergillosis. Isolates recovered from water (n = 54) and air (n = 21) at various locations inside and outside the hospital and from 15 patients (n = 21) with proven, probable, or possible invasive aspergillosis were genotyped by amplified fragment length polymorphism analysis. Based on genomic fingerprints, the environmental A. fumigatus isolates could be grouped into two major clusters primarily containing isolates recovered from either air or water. The genotypic relatedness between clinical and environmental isolates suggests that patients with invasive aspergillosis can be infected by strains originating from water or from air. In addition, 12 clusters with genetically indistinguishable or highly related strains were differentiated, each containing two to three isolates. In two clusters, clinical isolates recovered from patients matched those recovered from water sources, while in another cluster the clinical isolate was indistinguishable from one cultured from air. This observation might open new perspectives in the development of infection control measures to prevent invasive aspergillosis in high-risk patients. The genetic variability found between airborne and waterborne A. fumigatus strains might prove to be a powerful tool in understanding the transmission of invasive aspergillosis and in outbreak control.

Evaluation of the post-antifungal effect (PAFE) of amphotericin B and nystatin against 30 zygomycetes using two different media.

The post-antifungal effect (PAFE) of amphotericin B and nystatin against 30 clinical zygomycetes was evaluated using two different media. PAFE is a suppression of fungal growth after limited drug exposure. The MICs of both drugs were determined using NCCLS M38-P guidelines. A spectrophotometric method was used to determine PAFE in vitro. Spores were exposed to amphotericin B and nystatin in RPMI-1640 or AM3 at concentrations of 4 x and 1 x MIC for 4 h for Absidia sp. and at 1 x and 0.5 x MIC for 1 h for the other strains. Drugs were eliminated by washing. Exposed and control spores were cultured in microtitre wells and incubated for 48 h. PAFE was calculated as T - C (Delta t) between the control and the exposure fungi. The first increase in optical density (OD0) was used to calculate PAFE and was considered significant when the value of the lower 95%CI of the exposed strain was greater than the upper 95%CI of the control. MIC ranges in RPMI-1640 were: 0.06-4 mg/L for amphotericin B and 0.5-8 mg/L for nystatin; MIC ranges in AM3 were: 0.06-2 mg/L for amphotericin B and 0.5-4 mg/L for nystatin. Killing was not observed at the concentration and exposure time used. In RPMI-1640, for amphotericin B the rank order for PAFE was Absidia corymbifera (5.6 h) > Rhizopus oryzae (5.2 h) > Mucor spp. (3.5 h) > Rhizopus microsporus (3 h), and for nystatin the rank order was Mucor spp. (5.8 h) > R. oryzae (3.3 h) > A. corymbifera (2.9 h) > R. microsporus (1.7 h). PAFE was not induced in Rhizomucor spp. PAFE was dependent on drug concentration.

In vitro susceptibilities of zygomycetes to conventional and new antifungals.

In vitro susceptibilities of 36 zygomycete isolates, belonging to six genera, to itraconazole, posaconazole, voriconazole, terbinafine, amphotericin B and 5-fluorocytosine were determined by using a broth microdilution adaptation of the National Committee for Clinical Laboratory Standards M-38P reference method. The influence of incubation time on MIC values, and the performance of a spectrophotometric method for MIC determination in comparison with the visual reference method, were also evaluated. Amphotericin B was active against most of the isolates. All the isolates were highly resistant to 5-fluorocytosine (MICs > 256 mg/L). Voriconazole was significantly less active than the other drugs with an overall MIC(90) (MIC at which 90% of the isolates were inhibited) of 32 mg/L. In contrast, posaconazole showed good activity (MIC(90) 1 mg/L). A wide range of MICs, from 0.03 to > or =32 mg/L, was obtained for itraconazole and terbinafine. Differences in susceptibility between and within genera were noted. Rhizopus spp. were significantly less susceptible to itraconazole, posaconazole, terbinafine and amphotericin B than Absidia spp., and less susceptible than Mucor spp. to amphotericin B. Terbinafine appeared to be more active against Rhizopus microsporus than against Rhizopus oryzae (geometric mean MIC of 0.15 and 64 mg/L, respectively). The activity of the drugs was dependent on the incubation period. A significant increase in MICs was noted between 24 and 48 h of incubation. On the other hand, the two methods used for MIC determination (visual and spectrophotometric readings) showed good agreement. These results suggest that the zygomycetes are a heterogeneous group for antifungal susceptibility. Some of the conventional and new antifungals are effective in vitro; their efficacies in vivo remain to be determined. The spectrophotometric method appears to be a valuable alternative to the visual method for MIC determination for zygomycetes.

In vitro drug interaction modeling of combinations of azoles with terbinafine against clinical Scedosporium prolificans isolates.

The in vitro interaction between terbinafine and the azoles voriconazole, miconazole, and itraconazole against five clinical Scedosporium prolificans isolates after 48 and 72 h of incubation was tested by a microdilution checkerboard (eight-by-twelve) technique. The antifungal effects of the drugs alone and in combination on the fungal biomass as well as on the metabolic activity of fungi were measured using a spectrophotometric method and two colorimetric methods, based on the lowest drug concentrations showed 75 and 50% growth inhibition (MIC-1 and MIC-2, respectively). The nature and the intensity of the interactions were assessed using a nonparametric approach (fractional inhibitory concentration [FIC] index model) and a fully parametric response surface approach (Greco model) of the Loewe additivity (LA) no-interaction theory as well as a nonparametric (Prichard model) and a semiparametric response surface approaches of the Bliss independence (BI) no-interaction theory. Statistically significant synergy was found between each of the three azoles and terbinafine in all cases, although with different intensities. A 27- to 64-fold and 16- to 90-fold reduction of the geometric mean of the azole and terbinafine MICs, respectively, was observed when they were combined, resulting in FIC indices of <1 to 0.02. Using the MIC-1 higher levels of synergy were obtained, which were more consistent between the two incubation periods than using the MIC-2. The strongest synergy among the azoles was found with miconazole using the BI-based models and with voriconazole using the LA-based models. The synergistic effects both on fungal growth and metabolic activity were more potent after 72 h of incubation. Fully parametric approaches in combination with the modified colorimetric method might prove useful for testing the in vitro interaction of antifungal drugs against filamentous fungi.

In vitro synergistic interaction between amphotericin B and pentamidine against Scedosporium prolificans.

To develop new approaches for the treatment of invasive infections caused by Scedosporium prolificans, the in vitro interaction between amphotericin B and pentamidine against 30 clinical isolates was evaluated using a checkerboard microdilution method based on the National Committee for Clinical Laboratory Standards M38-P guidelines. The interaction between the drugs was analyzed using fractional inhibitory concentration index (FICI) analysis and response surface modeling. Amphotericin B alone was inactive against all the isolates. The geometric mean MIC for pentamidine was 57 micro g/ml (range, 8 to 256 micro g/ml; MIC at which 50% of the isolates tested were inhibited [MIC(50)], 64 micro g/ml; MIC(90), 128 micro g/ml). The combination was synergistic against 28 of 30 isolates (93.3%) by FICI analysis and 30 of 30 (100%) by response surface modeling analysis. Antagonism was not observed.