Diagnostic implications of mycetoma derived from Madurella pseudomycetomatis isolates from Mexico.

BACKGROUND: At the dermatology service of the General Hospital of Mexico City, Mexico, two patients, father and son, with black-grain mycetoma were seen. The grains were isolated, and the cultured fungi were identified as Madurella mycetomatis based on morphology. Using the M. mycetomatis specific PCR, amplicons of a different size than that of the M. mycetomatis type strain were obtained. OBJECTIVE: To determine the causative agent of the two black-grain mycetoma cases and develop non-culture-based diagnostic tools to identify them to the species level. METHODS: The M. mycetomatis specific, the internal transcribed spacer (ITS) region, ß-tubulin (BT) and ribosomal binding protein 2 (RBP2) PCRs were used to confirm the identity of the isolates. Genetic variation was established by amplification fragment length polymorphisms. To determine the antifungal susceptibility profile, the Sensititre™ YeastOne™ assay was used. To develop a species-specific PCR primers were designed on the sequenced PCR amplicon from the M. mycetomatis specific PCR. RESULTS: By analyzing the ITS, BT and RBP2 regions the isolates were identified as Madurella pseudomycetomatis. The isolates from father and son were similar but not identical to M. pseudomycetomatis from Venezuela and one from an unknown origin. Madurella pseudomycetomatis isolates were inhibited by itraconazole, posaconazole and voriconazole but showed increased MIC values for amphotericin B and fluconazole. They were not inhibited by the echinocandins and five flucytosine. The two patients were treated with itraconazole resulting in cure for the father while the son was lost to follow-up. The species-specific PCR developed for M. pseudomyceotmatis was discriminative and specific. CONCLUSION: Madurella pseudomycetomatis is genetically diverse with same susceptibility profile as M. mycetomatis and causes eumycetoma in Latin America. The M. pseudomycetomatis specific PCR can be used to identify this causative agent to the species level; however, this needs to be validated in an endemic setting.

Detection of azole-susceptible and azole-resistant Aspergillus coinfection by cyp51A PCR amplicon melting curve analysis.

BACKGROUND: The AsperGenius® assay is a multiplex real-time PCR test that allows the simultaneous detection of Aspergillus species and identification of the most common mutations in the Aspergillus fumigatus cyp51A gene conferring resistance (TR34/L98H and TR46/T289A/Y121F) by using melting curve analysis. Mixed infections with azole-resistant and susceptible A. fumigatus have rarely been described. METHODS: The AsperGenius® multiplex real-time PCR assay (PathoNostics, Maastricht, the Netherlands) was used on bronchoalveolar lavage (BAL) samples of 91 consecutive patients with a suspected invasive Aspergillus infection at the Erasmus MC University Medical Center, Rotterdam. RESULTS: In three cases the AsperGenius® assay indicated the simultaneous presence of WT and mutant genes (two patients with TR34/L98H mutation and one patient with TR46/T289A/Y121F mutation) and therefore mixed infections with azole-susceptible and -resistant isolates. In one of the three cases, the mixed infection was confirmed by phenotypic antifungal testing of multiple A. fumigatus colonies. CONCLUSIONS: The use of a dedicated A. fumigatus cyp51A resistance PCR allowed the detection of mixed infections with azole-resistant and -susceptible Aspergillus strains. These mixed infections may remain undiagnosed with conventional phenotypic susceptibility testing.

Genotypic diversity of Pseudomonas aeruginosa in cystic fibrosis siblings in Qatar using AFLP fingerprinting.

Pseudomonas aeruginosa is one of the primary pathogens in patients with cystic fibrosis (CF) and a major cause of morbidity and mortality. Reports of the spread of epidemic or transmissible strains of P. aeruginosa within and across CF centers raised the possibility of clonal spread among siblings with CF. This work reports the genotypic relatedness of P. aeruginosa in CF patients with the CFTR I1234V mutation, and to determine whether the genotypes are identical among CF siblings and among different families with the same CFTR mutation. Sixty-six P. aeruginosa isolates were obtained from sputa/deep-pharyngeal swabs from 27 CF patients belonging to 17 families. Genotypic relatedness was assessed using amplified fragment-length polymorphism (AFLP) fingerprinting. Twenty-three distinct genotypes of P. aeruginosa were identified. Eleven families each had one distinct genotype. In the other 6 families more than one genotype was observed; 3 families each showed two genotypes, 2 families each had three genotypes and 1 family had four genotypes of P. aeruginosa. In several cases, siblings with CF from the same family harbored the same strain of P. aeruginosa, which were different from the genotypes in other families. On the other hand, there was an overlap in P. aeruginosa between closely related families. Some patients show persistent colonization with the same genotype of P. aeruginosa over the longitudinal period. The presence of the same genotypes in siblings of the same family and closely related families suggests cross-transmission of P. aeruginosa or acquisition from common environmental exposure.

Identification and nomenclature of the genus Penicillium.

Penicillium is a diverse genus occurring worldwide and its species play important roles as decomposers of organic materials and cause destructive rots in the food industry where they produce a wide range of mycotoxins. Other species are considered enzyme factories or are common indoor air allergens. Although DNA sequences are essential for robust identification of Penicillium species, there is currently no comprehensive, verified reference database for the genus. To coincide with the move to one fungus one name in the International Code of Nomenclature for algae, fungi and plants, the generic concept of Penicillium was re-defined to accommodate species from other genera, such as Chromocleista, Eladia, Eupenicillium, Torulomyces and Thysanophora, which together comprise a large monophyletic clade. As a result of this, and the many new species described in recent years, it was necessary to update the list of accepted species in Penicillium. The genus currently contains 354 accepted species, including new combinations for Aspergillus crystallinus, A. malodoratus and A. paradoxus, which belong to Penicillium section Paradoxa. To add to the taxonomic value of the list, we also provide information on each accepted species MycoBank number, living ex-type strains and provide GenBank accession numbers to ITS, ß-tubulin, calmodulin and RPB2 sequences, thereby supplying a verified set of sequences for each species of the genus. In addition to the nomenclatural list, we recommend a standard working method for species descriptions and identifications to be adopted by laboratories working on this genus.

Phylogeny, identification and nomenclature of the genus Aspergillus.

Aspergillus comprises a diverse group of species based on morphological, physiological and phylogenetic characters, which significantly impact biotechnology, food production, indoor environments and human health. Aspergillus was traditionally associated with nine teleomorph genera, but phylogenetic data suggest that together with genera such as Polypaecilum, Phialosimplex, Dichotomomyces and Cristaspora, Aspergillus forms a monophyletic clade closely related to Penicillium. Changes in the International Code of Nomenclature for algae, fungi and plants resulted in the move to one name per species, meaning that a decision had to be made whether to keep Aspergillus as one big genus or to split it into several smaller genera. The International Commission of Penicillium and Aspergillus decided to keep Aspergillus instead of using smaller genera. In this paper, we present the arguments for this decision. We introduce new combinations for accepted species presently lacking an Aspergillus name and provide an updated accepted species list for the genus, now containing 339 species. To add to the scientific value of the list, we include information about living ex-type culture collection numbers and GenBank accession numbers for available representative ITS, calmodulin, ß-tubulin and RPB2 sequences. In addition, we recommend a standard working technique for Aspergillus and propose calmodulin as a secondary identification marker.

A search strategy for detecting duodenoscope-associated infections: a retrospective observational study.

BACKGROUND: Duodenoscope-associated infections (DAIs) are exogenous infections resulting from the use of contaminated duodenoscopes. Though numerous outbreaks of DAI have involved multidrug-resistant micro-organisms (MDROs), outbreaks involving non-MDROs are also likely to occur. Detection challenges arise as these infections often resolve before culture or because causative strains are not retained for comparison with duodenoscope strains. AIM: To identify and analyse DAIs spanning a seven-year period in a tertiary care medical centre. METHODS: This was a retrospective observational study. Duodenoscope cultures positive for gastrointestinal flora between March 2015 and September 2022 were paired with duodenoscope usage data to identify patients exposed to contaminated duodenoscopes. Analysis encompassed patients treated after a positive duodenoscope culture and those treated within the interval from a negative to a positive culture. Patient identification numbers were cross-referenced with a clinical culture database to identify patients developing infections with matching micro-organisms within one year of their procedure. A 'pair' was established upon a species-level match between duodenoscope and patient cultures. Pairs were further analysed via antibiogram comparison, and by whole-genome sequencing (WGS) to determine genetic relatedness. FINDINGS: Sixty-eight pairs were identified; of these, 21 exhibited matching antibiograms which underwent WGS, uncovering two genetically closely related pairs categorized as DAIs. Infection onset occurred up to two months post procedure. Both causative agents were non-MDROs. CONCLUSION: This study provides crucial insights into DAIs caused by non-MDROs and it highlights the challenge of DAI recognition in daily practice. Importantly, the delayed manifestation of the described DAIs suggests a current underestimation of DAI risk.

Proposal for a screening protocol for Candida auris colonization.

BACKGROUND: Candida auris is an emerging multidrug-resistant yeast which can cause severe infection in hospitalized patients. Since its first detection in 2009, C. auris has spread globally. The control and elimination of this pathogen in a hospital setting is particularly challenging because of its ability to form biofilms, allowing for long-term patient colonization and persistence in the environment. Identification of C. auris from cultures is difficult due to the morphologic similarities to other yeasts, its slow growth, and the low culture sensitivity when using standard agars and temperatures. AIM: We have developed a screening protocol for C. auris colonization using an in-house-developed polymerase chain reaction (PCR), combined with confirmatory culture in optimized conditions. METHODS: C. auris-specific primers and probe were developed, targeting the internal transcribed spacer (ITS) region, and specificity was confirmed in silico using the BLAST tool. The PCR was validated using a panel of 12 C. auris isolates and 103 isolates from 22 other Candida species and was shown to be 100% accurate. The limit of detection of the assay was determined at approximately four cells per PCR. FINDINGS: C. auris screening was introduced on February 15th, 2023, and was used for patients who had been admitted to a healthcare facility abroad in the two months prior to admission to our hospital. The screening protocol included swabs from nose, throat, rectum, axilla, and groin. In the first eight months, 199 patients were screened and seven were found positive (4%). CONCLUSION: Our proposed screening protocol may contribute to control C. auris in hospitals.

Schizophyllum commune as an emerging fungal pathogen: a review and report of two cases.

We report Schizophyllum commune as the aetiological agent of one case each of allergic broncho-pulmonary mycosis (ABPM) and pulmonary fungal ball, and present a literature review. The fungus was characterised by clamp connections, hyphal spicules, and formation of basidiocarps with basidiospores. The phenotypic identification was confirmed by sequencing of the ITS region. To-date, ABPM and pulmonary fungal ball to S. commune have been reported exclusively from Japan and North America respectively. Of the 71 globally reported cases due to S. commune, 45 (63%) were bronchopulmonary, 22 (31%) sinusitis and 4 extrapulmonary. Taken together, cases of bronchopulmonary disease and sinusitis numbered 67 (94%), indicating the respiratory tract as the primary target of disease. Concerning the country-wise distribution, Japan topped the list with 33 cases (46%), followed by Iran - 7 cases (10%), U.S.A. - 6 cases (9%), and a lower prevalence of 1.4-6% for the remaining 12 countries. The preponderance of the disease in Japan may be attributed to its greater awareness vis-à-vis that in other countries rather than to any geographical/climatic factors. We believe that the burden of S. commune-incited disease is currently underestimated, warranting comprehensive prospective studies to determine its prevalence.

First environmental isolation of Cryptococcus gattii, genotype AFLP5, from India and a global review.

We report the first environmental isolation from India of Cryptococcus gattii, genotype amplified fragment length polymorphism 5 (AFLP5), which is one of the rarely reported genotypes of this pathogen. It originated from decayed wood inside a trunk hollow of Manilkara hexandra, a native tree in Delhi. We investigated 101 isolates of C. gattii, originating from 556 samples of decayed wood inside trunk hollows of 311 heterogeneous tree species and their surrounding soil. Of these, only a solitary isolate proved to be AFLP5, the remainder belonged to AFLP4. Antifungal susceptibility testing showed a low MIC90 (0.25 µg ml(-1) ) of the new azoles posaconazole and isavuconazole for these environmental isolates. Genotype AFLP5 has been mainly reported from environmental sources in Colombia and from clinical sources in California (USA), where it seems to be endemic. Phylogenetic analysis of multi-locus sequence typing data showed that the Indian AFLP5 C. gattii isolate had a distinct profile compared with a cluster of mainly Colombian and Californian C. gattii AFLP5 isolates. As molecular typing of human pathogenic fungi is still in its infancy and not accessible to many countries, our current knowledge cannot be taken as reflective of the true geographic distribution of C. gattii AFLP5 or its other rarely reported molecular types.

Outbreak of invasive aspergillosis after major heart surgery caused by spores in the air of the intensive care unit.

BACKGROUND: Outbreaks of invasive aspergillosis (IA) are believed to be caused by airborne Aspergillus conidia. Few studies have established a correlation between high levels of Aspergillus fumigatus conidia and the appearance of new cases of IA or have demonstrated matching genotypes between clinical isolates and those from the environment. METHODS: We detected an outbreak of IA (December 2006 through April 2008) in the major heart surgery intensive care unit (MHS-ICU) of our institution. Our local surveillance program consists of monthly environmental air sampling in operating rooms and ICUs for quantitative and qualitative identification of filamentous fungi. During the study period, we obtained 508 environmental samples from 3 different periods: 6 months before the outbreak, during it, and 6 months after it. Available environmental and clinical strains were genotyped according to the short tandem repeats assay. RESULTS: Seven patients developed proven or probable IA (5 with lung infection, 1 with mediastinitis, and 1 with lung infection and mediastinitis). A. fumigatus was involved in 6 cases. The underlying conditions of the patients were heart transplantation (n = 3), corticosteroid-dependent conditions (n = 2), and diabetes mellitus (n = 2). The mortality rate was 85.7%. Before and after the outbreak (±6 months), the median airborne A. fumigatus conidia levels were 0 colony-forming units (CFUs) per cubic meter, and no cases of IA occurred during these periods. However, during the outbreak period, the occurrence of the 6 cases of IA caused by A. fumigatus was linked to peaks of abnormally high A. fumigatus airborne conidia levels (175, 50, 25, 20, 160, and 400 CFUs/m(3)) in the MHS-ICU, whereas counts in the air of both operating rooms remained negative. Matches between A. fumigatus genotypes collected from the air of the MHS-ICU and from representative clinical samples were found in 3 of the 6 patients. The outbreak abated when high-efficiency particulate air filters were installed in the affected areas. CONCLUSIONS: Our study revealed that abnormally high levels of airborne A. fumigatus conidia correlated with new cases of IA, even in patients who were not severely immunocompromised. The demonstration of matches between air and clinical genotypes reinforces the role of environmental air in the acquisition of IA during the period following MHS. Environmental monitoring of Aspergillus spores in the air of postoperative units is mandatory, even when these units receive nonimmunocompromised patients undergoing major surgery.

Molecular identification tools for sibling species of Scedosporium and Pseudallescheria.

The aim of this study was to develop molecular identification tools for currently recognized species of Pseudallescheria and Scedosporium through the use of species-specific primers and RFLP, so as to enhance rapid differentiation of clinically relevant species. The variability of species was established in a set of 681 Internal Transcribed Spacer (ITS) and 349 ß-tubulin (BT2) sequences. Amplified Fragment Length Polymorphism profile clustering matched with BT2 results, whereas ITS grouping was less detailed. ITS was sufficient for the differentiation of most haplotypes of clinically relevant species (P. apiosperma, P. boydii, S. aurantiacum, S. dehoogii, and S. prolificans) and of environmental species (P. minutispora and Lophotrichus fimeti) when Restriction Fragment Length Polymorphism (RFLP) were applied. For the identification of P. apiosperma and P. boydii species-specific BT2 primers were needed. Pseudallescheria fusoidea, P. ellipsoidea and P. angusta remained difficult to distinguish from P. boydii.

Environmental isolation and characterisation of Cryptococcus species from living trees in Havana city, Cuba.

Cryptococcus isolates from Cuban patients were identified as C. neoformans var. grubii. Although this species has since long been associated with bird droppings, a recent genotyping study provided strong evidence for additional origins of exposure. We sampled different species of trees in Havana, Cuba to identify other potential sources of exposure to this fungus. A total of 662 samples were collected from 331 trees and cacti from Havana, Cuba. Initial selection of the isolates was carried out by conventional techniques. Isolates were further characterised using a combination of AFLP analysis and DNA sequence analysis. Identification by conventional methods yielded 121 C. neoformans and 61 C. gattii isolates. Molecular analyses showed that none of these isolates was C. gattii and only one isolate proved to be C. neoformans var. grubii. A total of 27 different other species were identified. The most prevalent species was C. heveanensis (33%). Sixty-five unidentifiable isolates segregated into ten potentially novel species. Conventional cultivation methods have a low specificity for C. neoformans complex and molecular analyses need to be applied to confirm identification of isolates from environmental sources. Environmental niches responsible for most of human cryptococcal infections in Cuba remain to be identified.

The Q fever epidemic in The Netherlands: history, onset, response and reflection.

The 2007-2009 human Q fever epidemic in The Netherlands attracted attention due to its magnitude and duration. The current epidemic and the historical background of Q fever in The Netherlands are reviewed according to national and international publications. Seroprevalence studies suggest that Q fever was endemic in The Netherlands several decades before the disease was diagnosed in dairy goats and dairy sheep. This was in 2005 and the increase in humans started in 2007. Q fever abortions were registered on 30 dairy goat and dairy sheep farms between 2005 and 2009. A total of 3523 human cases were notified between 2007 and 2009. Proximity to aborting small ruminants and high numbers of susceptible humans are probably the main causes of the human Q fever outbreak in The Netherlands. In general good monitoring and surveillance systems are necessary to assess the real magnitude of Q fever.

Infection and colonisation due to Scedosporium in Northern Spain. An in vitro antifungal susceptibility and molecular epidemiology study of 60 isolates.

Since the latest taxonomical changes in the genus Scedosporium by Gilgado et al. in 2010, no species-specific studies on epidemiology and antifungal susceptibility patterns (AFSP) have so far been published. This study aimed to provide qualitative epidemiological data of Scedosporium spp. isolated from cystic fibrosis (CF) patients and immunocompromised patients from Northern Spain. Isolates were identified by using amplified fragment length polymorphism (AFLP), and species-specific AFSP were generated for all currently available antifungal compounds. AFLP was a useful tool for identification to species-level and for the discrimination of inter- and intra-patient isolates. Scedosporium prolificans represents the most prevalent species in the respiratory tract of CF patients and immunocompromised patients in Northern-Spain, followed by Pseudallescheria boydii, P. apiosperma, and P. ellipsoidea. CF patients were exclusively colonised with either P. boydii or S. prolificans. Patients were colonised over years exclusively with isolates affiliated to one species, but some patients were colonised with multiple strains with different AFSP. The sum of those co-colonising strains in one patient, may appear in vitro and in vivo as a multi-resistant S. prolificans isolate, as strains are morphologically identical and might therefore be regarded as only one strain. A majority of Scedosporium strains (with exception of S. prolificans) were found susceptible for voriconazole and micafungin.

Severe prosthetic joint infection in an immunocompetent male patient due to a therapy refractory Pseudallescheria apiosperma.

Prosthetic joint infections (PJI) are rarely due to fungal agents and if so they are mainly caused by Candida strains. This case represents a PJI caused by a multi-drug resistant Pseudallescheria apiosperma, with poor in vivo response to itraconazole and voriconazole. This case differs also by the way of infection, since the joint infection did not follow a penetrating trauma. In the majority of cases, Scedosporium extremity infections remain local in immunocompetent individuals. We report a persistent joint infection with multiple therapeutic failures, and subsequent amputation of the left leg. Detailed clinical data, patient history, treatment regime and outcome of a very long-lasting (>4 years) P. apiosperma prosthetic knee infection in an immunocompetent, 61-year-old male patient are presented with this case. The patient was finally cured by the combination of multiple and extensive surgical interventions and prolonged antifungal combination therapy with voriconazole and terbinafine.

Prevalence of and risk factors for extended-spectrum beta-lactamase genes carriership in a population-based cohort of middle-aged and elderly.

INTRODUCTION: Increasing resistance to beta-lactam antibiotics is an alarming development worldwide. Fecal carriership of TEM, SHV, CTX-M and CMY was studied in a community-dwelling population of middle-aged and elderly individuals. PATIENTS AND METHODS: Feces was obtained from individuals of the Rotterdam Study. Carriership of the TEM, SHV, CTX-M and CMY genes was determined using real-time polymerase chain reaction (qPCR). Possible associations were investigated between carriership of these genes and several risk factors, such as the use of antimicrobial drugs, diabetes mellitus, protein pump inhibitor (PPI) use, travelling, the composition of the gut microbiota, and intake of certain foods. RESULTS: The most prevalent gene was TEM (53.0%), followed by SHV (18.4%), CTX-M (5.4%) and CMY (3.6%). Use of penicillins with extended spectrum was associated with TEM carriership, whereas use of macrolides and lincosamides was associated with TEM and SHV carriership. Interestingly, use of PPIs was associated with a higher prevalence of carriership of TEM, SHV and CMY (TEM: odds ratio [OR] 1.34; 95% confidence interval [CI] 1.05-1.77; SHV: OR 2.17; 95%CI 1.55-2.87; CMY: OR 2.26; 95%CI 1.23-4.11). Furthermore, associations were found between the richness and composition of the gut microbiota and TEM and SHV carriership. CONCLUSIONS: The prevalence of carriership of TEM was substantial, but the prevalence of carriership of the extended-spectrum ß-lactamase gene, CTX-M and the AmpC ß-lactamase gene, CMY was relatively low in this community-dwelling, population-based cohort. The composition of the microbiota might play a role in the retention of resistance genes, but future studies are necessary to further elucidate this relationship.

Association of environmental surface contamination with hand hygiene and infections in nursing homes: a prospective cohort study.

BACKGROUND: Little is known about the presence of infections in nursing home residents, the causative micro-organisms, how hand hygiene (HH) influences the presence of infections in residents, and the extent to which environmental contamination is associated with the incidence of infection among residents. AIMS: To establish if environmental contamination can be used as an indicator for HH compliance, and if environmental contamination is associated with the incidence of infection. METHODS: Environmental surface samples (ESS) were collected in an exploratory study as part of a HH intervention in 60 nursing homes. ESS results from three distinct surfaces (nurses' station, communal toilet and residents' shared living area) were compared with nurses' HH compliance and the incidence of infection among residents. Real-time polymerase chain reaction assays were used to detect norovirus genogroup I and II, rhinovirus and Escherichia coli. HH compliance was measured by direct observation. The incidence of infection was registered weekly. FINDINGS: Rhinovirus (nurses' station: 41%; toilet: 14%; living area: 29%), norovirus (nurses' station: 18%; toilet: 12%; living area: 16%) and E. coli (nurses' station: 14%; toilet: 58%; living area: 54%) were detected. No significant (P<0.05) associations were found between HH compliance and the presence of micro-organisms. An association was found between E. coli contamination and the incidence of disease in general (P=0.04). No other associations were found between micro-organisms and the incidence of disease. CONCLUSION: Rhinovirus, norovirus and E. coli were detected on surfaces in nursing homes. No convincing associations were found between environmental contamination and HH compliance or the incidence of disease. This study provides reference data about surface contamination.

Apophysomyces elegans: epidemiology, amplified fragment length polymorphism typing, and in vitro antifungal susceptibility pattern.

Apophysomyces elegans is an emerging pathogen in India. We planned the present study to analyze the clinical pattern of the disease, to perform molecular strain typing, and to determine the in vitro activities of eight antifungal drugs against A. elegans. A total of 16 clinical and two environmental A. elegans isolates were included in the study. The clinical histories of the patients were noted. MICs or minimum effective concentrations (MECs) were determined for antifungal drugs by microdilution testing in accordance with CLSI standard M38-A2 guidelines. Of 16 patients, seven had rhino-cerebral, five had cutaneous, and three had renal zygomycosis. One patient had osteomyelitis. Uncontrolled diabetes was observed in 63% of the patients. Amplified fragment length polymorphism (AFLP) analysis divided the strains into two clearly different clades. The fingerprints of the environmental strains (including the type strain) were clearly different from those of the clinical strains. The MIC50s and MIC90s for amphotericin B, itraconazole, posaconazole, and isavuconazole were 2 and 4, 1 and 2, 0.5 and 1, and 2 and 4 µg/ml, respectively. The strains had high MICs for fluconazole, voriconazole, and echinocandins. The study indicates a possible change in the clinical pattern of zygomycosis due to A. elegans in India. The fungus caused not only cutaneous or subcutaneous infection but also other deep-seated infections, and the disease is commonly associated with uncontrolled diabetes. The AFLP patterns show a clear difference between environmental and clinical strains. Posaconazole is the most active drug against the isolates, followed by itraconazole. The MICs of amphotericin B against A. elegans were higher than those of the other drugs.

Interlaboratory reproducibility of a single-locus sequence-based method for strain typing of Aspergillus fumigatus.

Seven international laboratories tested the recently proposed single-locus typing strategy for Aspergillus fumigatus subtyping for interlaboratory reproducibility. Comparative sequence analyses of portions of the locus AFUA_3G08990, encoding a putative cell surface protein (denoted CSP), was performed with a panel of Aspergillus isolates. Each laboratory followed very different protocols for extraction of DNA, PCR, and sequencing. Results revealed that the CSP typing method was a reproducible and portable strain typing method.

MLST versus microsatellites for typing Aspergillus fumigatus isolates.

In recent years, there has been a clear and growing tendency to use exact typing methods for discrimination between microbial isolates. Exact typing methods that yield an unambiguous typing result offer a number of advantages over conventional methods in the generation of typing data that is reproducible, portable and exchangeable. Two such methods are multi-locus sequence typing (MLST) and microsatellite-based typing. Here I will discuss the basic principles of both methods and compare them from a practical and performance point of view with respect to typing Aspergillus fumigatus isolates. Microsatellites offer the best available typing option by outperforming MLST in terms of speed, throughput, costs and discriminatory power. This latter advantage of microsatellites is a direct consequence of their inherent instability. This (in)stability of individual microsatellite markers and alleles should be taken into account in the interpretation of microsatellite-based typing data.