Elizabeth M. Johnson

The Evolution and Evaluation of a Whole Blood Polymerase Chain Reaction Assay for the Detection of Invasive Aspergillosis in Hematology Patients in a Routine Clinical Setting

BACKGROUNDnInvasive aspergillosis (IA) is associated with high mortality. Successful outcome with treatment is linked to early diagnosis. The utility of classic diagnostic methods, however, is limited.nnnMETHODSnTo aid in the diagnosis of IA, we retrospectively assessed our diagnostic service, using real-time polymerase chain reaction (PCR) and galactomannan sandwich enzyme-linked immunosorbent assay (ELISA).nnnRESULTSnA total of 203 patients at risk of invasive fungal infection were screened by PCR, and 116 of the patients were also tested by ELISA. The patient group comprised 176 patients with hematological malignancy and 28 control patients with evidence of invasive candidal infection. Consensus European Organisation for Research and Treatment of Cancer and Mycoses Study Group criteria were used to classify fungal infection, which, by definition, excluded the PCR result. The PCR method was sensitive (up to 92.3% sensitivity) and specific (up to 94.6% specificity) and had good agreement with the galactomannan ELISA (76.7%) and high-resolution computed tomography scan results.nnnCONCLUSIONSnA negative PCR result can be used to rule out IA and to limit the need for empirical antifungal therapy; thus, it has a role in diagnosing IA infections, especially in combination with antigen testing. PCR-positive cases classified as false positives regularly reflect the limitations of classic microbiological procedures or restricted use of consensus clinical methods employed to classify infection.

Management of invasive candidiasis and candidemia in adult non-neutropenic intensive care unit patients: Part I. Epidemiology and diagnosis

BackgroundInvasive candidiasis and candidemia are frequently encountered in the nosocomial setting, particularly in the intensive care unit (ICU).Objectives and methodsTo review the current management of invasive candidiasis and candidemia in non-neutropenic adult ICU patients based on a review of the literature and a European expert panel discussion.Results and conclusionsCandida albicans remains the most frequently isolated fungal species followed by C. glabrata. The diagnosis of invasive candidiasis involves both clinical and laboratory parameters, but neither of these are specific. One of the main features in diagnosis is the evaluation of risk factor for infection which will identify patients in need of pre-emptive or empiric treatment. Clinical scores were built from those risk factors. Among laboratory diagnosis, a positive blood culture from a normally sterile site provides positive evidence. Surrogate markers have also been proposed like 1,3 β-d glucan level, mannans, or PCR testing. Invasive candidiasis and candidemia is a growing concern in the ICU, apart from cases with positive blood cultures or fluid/tissue biopsy, diagnosis is neither sensitive nor specific. The diagnosis remains difficult and is usually based on the evaluation of risk factors.

Molecular identification of pathogenic fungi

Systemic fungal infections represent a major cause of morbidity and mortality in immunocompromised patients. The ever-increasing number of yeast species associated with human infections that are not covered by conventional identification kits, and the fact that moulds isolated from deep infections are frequently impossible to identify using classical methods due to lack of sporulation, has driven the need for rapid, robust molecular identification techniques. We recently developed a rapid method of preparing fungal genomic DNAs using Whatman FTA filters, which has greatly facilitated molecular identification. Mould isolates cultured from dark grain mycetomas (destructive infections of skin/subcutaneous tissues that progress to involve muscle and bone) invariably fail to produce features by which they can be identified and were taxonomic mysteries. PCR amplification and sequencing of 250 bp of the internal transcribed spacer region 1 (ITS1) allowed us to distinguish between the known agents of mycetoma, to describe three new species associated with this disease and to define phylogenetic relationships. For yeasts, 153 isolates encompassing 47 species that had failed to be identified using classical methods were unambiguously identified by conventional sequencing of 350 bp of the 26S rRNA D1D2 region. These represented 5% of the isolates examined and included common species with atypical biochemical and phenotypic profiles, and rarer species infrequently associated with infection. Our recent studies indicate that FTA extraction coupled with pyrosequencing of 25 bp of ITS2 could potentially identify most common yeast species from pure culture in half a day. Together, these data underscore the importance of molecular techniques for fungal identification.

Analysis of the dermatophyte species isolated in the British Isles between 1980 and 2005 and review of worldwide dermatophyte trends over the last three decades

Infections of the skin, hair and nails by dermatophyte fungi are common in developed and developing countries alike. However, the species involved and the resulting clinical entities vary both geographically and with time. We have surveyed 15,333 dermatophytes obtained from primary isolations at the Mycology Reference Laboratory, Bristol, UK from 1980 through 2005. Several striking trends in dermatophyte prevalence were apparent over this period. The relative frequencies of isolations of Microsporum canis (cat and dog ringworm), Trichophyton verrucosum (cattle ringworm), T. mentagrophytes var. mentagrophytes (rodent ringworm) and Epidermophyton floccosum (a cause of human groin and foot infections) all decreased by 90%. Conversely, the contributions of T. tonsurans and T. violaceum (two anthropophilic scalp-infecting species) to total dermatophyte isolations increased by 1000% over the same period. Finally, T. rubrum and T. mentagrophytes var. interdigitale, the two common causes of foot infection comprised 80% of all dermatophytes isolated in 1980 and 90% of isolations in 2005. Similar trends in dermatophyte prevalence were evidenced throughout the British Isles, based on the voluntary reporting of isolations from a large number of British laboratories at 5-yearly intervals over the same period. The implications of these changing patterns of dermatophyte species, and the clinical entities they produce are discussed in the context of a review of worldwide dermatophyte isolations over the last three decades, with emphasis on the causal agents of tinea capitis.

Management of invasive candidiasis and candidemia in adult non-neutropenic intensive care unit patients: Part II. Treatment

BackgroundInvasive candidiasis and candidemia are frequently encountered in the nosocomial setting particularly in the intensive care unit (ICU).Objective and methodsTo review the current management of invasive candidiasis and candidemia in non-neutropenic adult ICU patients based on a review of the literature and an European expert panel discussion.Results and conclusionsEmpiric and directed treatment for invasive candidiasis are predicated on the hemodynamic status of the patient. Unstable patients may benefit from broad-spectrum antifungal agents, which can be narrowed once the patient has stabilized and the identity of the infecting species is established. In stable patients, a more classical approach using fluconazole may be satisfactory provided that the patient is not colonized with fluconazole resistant strains or there has been recent past exposure to an azole (<30xa0days). In contrast, pre-emptive therapy is based on the presence of surrogate markers.

Activities of voriconazole, itraconazole and amphotericin B in vitro against 590 moulds from 323 patients in the voriconazole Phase III clinical studies

INTRODUCTIONnFungal pathogens from the voriconazole trials were identified and tested for susceptibility at two reference laboratories.nnnMETHODSnMICs were measured using CLSI M38-A 48 h microdilution methodology.nnnRESULTSnMoulds from 29 genera and 38 species were isolated from 18 countries. Aspergillus spp. predominated (69%), followed by Scedosporium spp. (11.5%). Aspergillus fumigatus (292/590, 49.5%) was the most common species, followed by Scediosporium apiospermum (9.7%) and Aspergillus terreus (7.3%). The bronchi, lungs and sinuses yielded 45% of the isolates (57% of aspergilli), with 24% from the oropharynx/oesophagus. Other sites included blood/catheter (7.3%) and CNS (5.2%). MIC90s of itraconazole and voriconazole for Aspergillus spp. were the same (0.5 mg/L), but 17 Aspergillus isolates were itraconazole-resistant (MICs > or = 1-16 mg/L). Additionally, in 31 A. fumigatus and 23 A. terreus isolates, amphotericin MICs were > or = 2.0 mg/L. Voriconazole MICs exceeded 4 mg/L in only 5.8% (34/590) of the isolates, including one A. fumigatus (8.0 mg/L), 9/11 Scedosporium prolificans, 10/13 Fusarium solani and all 9 Zygomycetes. Most were also not susceptible to itraconazole or amphotericin B. A notable increase in MIC (more than two doubling dilutions) during voriconazole therapy was seen for one A. fumigatus isolate. The response rate of voriconazole-treated patients with isolate MICs > or = 4.0 mg/L was 38% when compared with 52% for those with MICs < 4.0 mg/L.nnnCONCLUSIONSnVoriconazole shows activity, in vitro, similar to that of itraconazole against a wide range of moulds. It is also active against some isolates not susceptible to itraconazole or amphotericin B, but not the Zygomycetes. The relationship between voriconazole MIC and clinical outcome requires further study.

Low Effective Dispersal of Asexual Genotypes in Heterogeneous Landscapes by the Endemic Pathogen Penicillium marneffei

Long-distance dispersal in microbial eukaryotes has been shown to result in the establishment of populations on continental and global scales. Such “ubiquitous dispersal” has been claimed to be a general feature of microbial eukaryotes, homogenising populations over large scales. However, the unprecedented sampling of opportunistic infectious pathogens created by the global AIDS pandemic has revealed that a number of important species exhibit geographic endemicity despite long-distance migration via aerially dispersed spores. One mechanism that might tend to drive such endemicity in the face of aerial dispersal is the evolution of niche-adapted genotypes when sexual reproduction is rare. Dispersal of such asexual physiological “species” will be restricted when natural habitats are heterogeneous, as a consequence of reduced adaptive variation. Using the HIV-associated endemic fungus Penicillium marneffei as our model, we measured the distribution of genetic variation over a variety of spatial scales in two host species, humans and bamboo rats. Our results show that, despite widespread aerial dispersal, isolates of P. marneffei show extensive spatial genetic structure in both host species at local and country-wide scales. We show that the evolution of the P. marneffei genome is overwhelmingly clonal, and that this is perhaps the most asexual fungus yet found. We show that clusters of genotypes are specific to discrete ecological zones and argue that asexuality has led to the evolution of niche-adapted genotypes, and is driving endemicity, by reducing this pathogens potential to diversify in nature.

Association of Mannose-Binding Lectin Deficiency with Acute Invasive Aspergillosis in Immunocompromised Patients

BACKGROUNDnInvasive aspergillosis is a devastating infection with attributable mortality of 40% despite antifungal therapy. In animal models of aspergillosis, deficiency of mannose-binding lectin (MBL), a pattern recognition receptor that activates complement, is a susceptibility factor. MBL deficiency occurs in 20%-30% of the population. We hypothesized that MBL deficiency may be a susceptibility factor for invasive aspergillosis in humans.nnnMETHODSnSerum MBL concentrations were measured by enzyme-linked immunosorbent assay in 65 patients with proven or probable acute invasive aspergillosis and 78 febrile immunocompromised control subjects. MBL concentrations and the frequency of MBL deficiency were compared.nnnRESULTSnThe median serum MBL level was significantly lower in patients with aspergillosis than in control subjects (281 ng/mL vs 835 ng/mL; P = .007). MBL deficiency (MBL concentration, <500 ng/mL) was significantly more common in patients with aspergillosis than control subjects (62% vs 32%; P < .001). Frequency of MBL deficiency was similar among patients with aspergillosis irrespective of response to antifungal therapy (P = .10).nnnCONCLUSIONSnThis study is the first, to our knowledge, to show an association between MBL deficiency and acute invasive aspergillosis in humans. Further study is required to investigate the causal nature of this association and to define whether diagnosis of MBL deficiency may identify immunocompromised patients at increased risk of invasive aspergillosis.

Ultra-rapid preparation of total genomic DNA from isolates of yeast and mould using Whatman FTA filter paper technology – a reusable DNA archiving system

Conventional methods for purifying PCR-grade fungal genomic DNA typically require cell disruption (either physical or enzymatic) coupled with laborious organic extraction and precipitation stages, or expensive column-based technologies. Here we present an easy and extremely rapid method of preparing yeast and mould genomic DNAs from living cultures using Whatman FTA filter matrix technology. Aqueous suspensions of yeast cells or hyphal fragments and conidia (in the case of moulds) are applied directly (or after freeze-thawing) to dry FTA filters. Inoculated filters are then subjected to brief microwave treatment, to dry the filters and inactivate the organisms. Filter punches are removed, washed rapidly, dried and placed directly into PCR reactions. We show that this procedure inactivated all of the 38 yeast and 75 mould species tested, and generated PCR-grade DNA preparations in around 15 minutes. A total of 218 out of 226 fungal isolates tested liberated amplifiable DNA after application to FTA filters. Detection limits with yeast cultures were approximately 10 colony-forming units per punch. Moreover, we demonstrate that filter punches can be recovered after PCR, washed and used in fresh PCR reactions without detectable cross-contamination. Whatman FTA technology thus represents a cheap, ultra-rapid method of fungal genomic DNA preparation, and also potentially represents a powerful fungal DNA archiving and storage system.

Comparative Pathogenicity of United Kingdom Isolates of the Emerging Pathogen Candida auris and Other Key Pathogenic Candida Species

The incidence of invasive candidiasis, which includes candidemia and deep tissue infections, continues to rise and is associated with considerable mortality rates. Candida albicans remains the most common cause of invasive candidiasis, although the prevalence of non-albicans species has increased over recent years. Since its first description in 2009, Candida auris has emerged as a serious nosocomial health risk, with widespread outbreaks in numerous hospitals worldwide. However, despite receiving considerable attention, little is known concerning the pathogenicity of this emerging fungal pathogen. Here, using the Galleria mellonella insect systemic infection model, we show strain-specific differences in the virulence of C. auris, with the most virulent isolates exhibiting pathogenicity comparable to that of C. albicans, which is currently accepted as the most pathogenic member of the genus. ABSTRACT Candida auris, first described in 2009, has since emerged as an important, multidrug-resistant, nosocomial agent of candidemia, with large outbreaks reported worldwide and high mortality rates associated with therapeutic failure. The current study employed C. auris isolates from a variety of centers in the United Kingdom to evaluate the pathogenicity of this emerging pathogen compared to that of other common pathogenic yeast species in the invertebrate Galleria mellonella infection model. We showed that C. auris isolates differ in their growth characteristics in vitro, with a proportion of isolates failing to release daughter cells after budding, resulting in the formation of large aggregates of cells that cannot be physically disrupted. Our results also demonstrate strain-specific differences in the behavior of C. auris in G. mellonella, with the aggregate-forming isolates exhibiting significantly less pathogenicity than their nonaggregating counterparts. Importantly, the nonaggregating isolates exhibited pathogenicity comparable to that of C. albicans, which is currently accepted as the most pathogenic member of the genus, despite the fact that C. auris isolates do not produce hyphae and produce only rudimentary pseudohyphae either in vitro or in G. mellonella. IMPORTANCE The incidence of invasive candidiasis, which includes candidemia and deep tissue infections, continues to rise and is associated with considerable mortality rates. Candida albicans remains the most common cause of invasive candidiasis, although the prevalence of non-albicans species has increased over recent years. Since its first description in 2009, Candida auris has emerged as a serious nosocomial health risk, with widespread outbreaks in numerous hospitals worldwide. However, despite receiving considerable attention, little is known concerning the pathogenicity of this emerging fungal pathogen. Here, using the Galleria mellonella insect systemic infection model, we show strain-specific differences in the virulence of C. auris, with the most virulent isolates exhibiting pathogenicity comparable to that of C. albicans, which is currently accepted as the most pathogenic member of the genus.