Multi-locus sequence typing and phylogenetics of Cryptococcus neoformans AD hybrids.

Hybrid AD strains of the human pathogenic Cryptococcus neoformans species complex have been reported from many parts of the world. However, their origin, diversity, and evolution are incompletely understood. In this study, we analyzed 102 AD hybrid strains representing 21 countries on five continents. For each strain, we obtained its mating type and its allelic sequences at each of the seven loci that have been used for genotyping haploid serotypes A and D strains of the species complex by the Cryptococcus research community. Our results showed that most AD hybrids exhibited loss of heterozygosity at one or more of the seven analyzed loci. Phylogenetic and population genetic analyses of the allelic sequences revealed multiple origins of the hybrids within each continent, dating back to one million years ago in Africa and up to the present in other continents. We found evidence for clonal reproduction and long-distance dispersal of these hybrids in nature. Comparisons with the global haploid serotypes A and D strains identified new alleles and new haploid multi-locus genotypes in AD hybrids, consistent with the presence of yet-to-be discovered genetic diversity in haploid populations of this species complex in nature. Together, our results indicate that AD hybrids can be effectively genotyped using the same multi-locus sequencing type approach as that established for serotypes A and D strains. Our comparisons of the AD hybrids among each other as well as with the global haploid serotypes A and D strains revealed novel genetic diversity as well as evidence for multiple origins and dynamic evolution of these hybrids in nature.

Laboratory exposure to Coccidioides: lessons learnt in a non-endemic country.

Coccidioides is a primary pathogenic fungus, which infects humans through highly infectious arthroconidia, causing substantial morbidity including life-threatening disseminated infections. Due to the low infectious dose, laboratory personnel might become infected during diagnostic procedures. Accordingly, coccidioidomycosis is reported as the most frequent laboratory-acquired systemic mycosis worldwide. This risk is aggravated in non-endemic countries, where the diagnosis may not be suspected. We report on an inadvertent exposure of 44 persons to Coccidioides posadasii in a clinical microbiology laboratory in Chile, the measures of containment after rapid diagnosis with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and the lessons learnt in a non-endemic setting.

Identification of Aspergillus and Mucorales in formalin-fixed, paraffin-embedded tissue samples: Comparison of specific and broad-range fungal qPCR assays.

Establishing the etiology of invasive fungal infections is important to guide therapeutic options and for epidemiologic purposes. Formalin-fixed, paraffin-embedded (FFPE) tissue specimens from patients with proven invasive fungal infections are valuable to determine the etiology of systemic fungal infections. We compared different polymerase chain reaction (PCR) amplification strategies from FFPE tissue blocks to identify agents of invasive fungal infections. We found that specific PCR assays show superior sensitivity in the identification of DNA of Mucorales and Aspergillus and mixed infections caused by both as compared to broad-range PCR assays. Shorter amplicon lengths and less detection of contaminating fungal DNA are potential factors involved. However, detection of fungal DNA by highly sensitive specific PCR assays in the absence of demonstration of fungal elements in tissue suggests that PCR results should be interpreted in the context of the histopathology and clinical findings.

Case report: A fatal case of cryptococcosis in an immunocompetent patient due to Cryptococcus deuterogattii (AFLP6/VGII).

INTRODUCTION: Cryptococcosis in immunocompetent adults is a rare disease in Europe, mostly induced by members of the Cryptococcus gattii species complex. The diagnosis can be challenging due to its rarity, unspecific symptoms and long symptomless latency. CASE PRESENTATION: A 49-year-old woman with a three weeks history of headache was admitted to the hospital due to discrete ataxia and impaired vision. Cranial magnetic resonance imaging (MRI) showed a contrast-enhancing mass in the cerebellum. Further investigations detected a slight leukocytosis and a single subpleural nodule in the right inferior lung lobe. The cerebral lesion was surgically removed, and a direct frozen section only showed an unspecific inflammation. In the course of her admission she developed non-treatable cerebral edema and died ten days after surgical intervention. Histopathological examination of the surgical specimen and postmortem evaluation of the lung and the cerebrum demonstrated fungal elements. Molecular identification of the fungal elements in formalin-fixed paraffin-embedded tissue lead to the diagnosis of cryptococcosis induced by C. gattii sensu lato. Molecular genetic analysis identified the involved cryptococcal species as genotype AFLP6/VGII, recently described as Cryptococcus deuterogattii, which is known to be endemic to the west-coast of Canada and the USA. Additional heteroanamnestic information revealed that she had spent her holidays on Vancouver Island, Canada, two years before disease onset, indicating that infection during this stay seems to be plausible. CONCLUSION: Cryptococcosis due to C. deuterogattii is a rarely encountered fungal disease in Europe, not particularly associated with immunodeficiency, and infection is likely to be contracted in endemic areas. Due to its rarity, long symptomless latency, unspecific symptoms and misleading radiological features the diagnosis can be challenging. Physicians need to be aware of this differential diagnosis in immunocompetent patients, as early adequate therapy can be lifesaving.

[Identification of fungal pathogens in tissue samples using fluorescence in situ hybridization]. / Identifizierung von Pilzen in Gewebeschnitten mit Fluoreszenz-in-situ-Hybridisierung.

Deep fungal infections are associated with significant mortality despite the availability of new antifungal agents. The identification of causative fungi is important to define successful antifungal therapies as agents differ in the in vitro susceptibility. Characterization of tissue morphology and cultivation from tissue provide important clues to patient management. Molecular techniques such as PCR-based assays are increasingly being used to identify agents of invasive fungal infections. However, potential contamination limits the use when ubiquitous fungi are targeted. Hybridization with fluorescently labeled probes targeting the ribosomal RNA of fungi is emerging as an alternative identification strategy. Using conserved or variable regions of the rRNA as targets, group or species-specific probes can be synthesized to identify fungal pathogens and localize them in the infectious process. These techniques have been successfully applied to deep fungal infections due to different agents in various organ samples.

[Cryptococcosis: case reports, epidemiology and treatment options]. / Kryptokokkose: kasuistiken, epidemiologie und therapiestrategien.

Cryptococcosis is a fungal infection that is usually caused by Cryptococcus neoformans. Given the decreasing number of cases in HIV-infected patients in developed countries, infections in other patient populations, such as solid organ transplant recipients, patients with chronic organ diseases or even patients without immunodeficiency gain more attention. Due to a possible involvement of many organs, the clinical presentation varies from localized infections of the respiratory tract and the skin, to the characteristic meningoencephalitis or other organs after hematogenous dissemination. Sensitive laboratory tests allow a rapid diagnosis in patients with disseminated infection. Crucial therapeutic decisions depend on the underlying patient condition and the particular organ involvement. The induction therapy of disseminated infections or severe localised infections is based on amphotericin B in combination with 5-flucytosine. In non-severe localised infections and after induction therapy, antifungal treatment with fluconazole is indicated. Echinocandins are not effective in cryptococcosis.

Deciphering the aetiology of a mixed fungal infection by broad-range PCR with sequencing and fluorescence in situ hybridisation.

Simultaneous infections with multiple fungi may be misinterpreted as monomicrobial infections by current diagnostics with ramifications for the choice of antimicrobial agents that may impact patient outcomes. The application of molecular methods on tissue samples may be useful to decipher the aetiology of mixed fungal infections. We present a leukaemic patient who died from sepsis due to candidaemia. The postmortem examination documented fungal elements in lung tissue. Fungal DNA was amplified from the lung sample by broad-range PCR assays targeting the 28S ribosomal RNA gene or the internal transcribed spacer 2 (ITS-2). Fluorescence in situ hybridisation (FISH) using differentially labelled fungal probes was applied on the tissue. Sequencing identified the PCR amplicons as Aspergillus fumigatus (28S assay) and Candida tropicalis (ITS-2 assay). As a chromatogram suggested mixed amplicons, the Isentio ripseq(®) tool for in silico analysis was applied and confirmed the presence of both amplicons in the PCR products of both assays. FISH confirmed the presence of Aspergillus and Candida within the infectious process, a prerequisite for inferring a causal relationship with the infection. The combination of broad-range PCR with sequence analysis and FISH applied on tissue samples is a powerful approach to identify the aetiology of invasive fungal infections, including mixed infections.

Breakthrough zygomycosis on posaconazole prophylaxis after allogeneic stem cell transplantation.

Antifungal prophylaxis with posaconazole (POS) has been shown to decrease the mortality associated with invasive fungal infections in high-risk patients. We report on a patient, with severe graft-versus-host disease after allogeneic stem cell transplantation, who developed proven pneumonia due to Rhizopus microsporus after 40 days of POS prophylaxis (fasting serum levels: 691-904 ng/mL). Despite combination treatment with liposomal amphotericin B and POS for 39 days, the patient died from pulmonary hemorrhage. This case highlights the need for continued awareness of breakthrough zygomycosis in patients receiving POS.

Forty-one recent cases of invasive zygomycosis from a global clinical registry.

BACKGROUND: Invasive zygomycosis accounts for a significant proportion of all invasive fungal diseases (IFD), but clinical data on the clinical course and treatment response are limited. PATIENTS AND METHODS: Fungiscope-A Global Rare Fungal Infection Registry is an international university-based case registry that collects data of patients with rare IFD, using a web-based electronic case form at www.fungiscope.net. RESULTS: Forty-one patients with invasive zygomycosis from central Europe and Asia were registered. The most common underlying conditions were malignancies (n = 26; 63.4%), diabetes mellitus (n = 7; 17.1%) and solid organ transplantation (n = 4; 9.8%). Diagnosis was made by culture in 28 patients (68.3%) and by histology in 26 patients (63.4%). The main sites of infection were the lungs (n = 24; 58.5%), soft tissues (n = 8; 19.5%), rhino-sinu-orbital region (n = 8; 19.5%) and brain (n = 6; 14.6%). Disseminated infection of more than one non-contiguous site was seen in six patients (14.6%). Mycocladus corymbifer was the most frequently identified species (n = 10, 24.4%). A favourable response was observed in 23 patients (56.1%). Overall survival was 51.2% (n = 21). At diagnosis, four patients (9.8%) were on continuous antifungal prophylaxis with itraconazole (n = 1; 2.4%) or posaconazole (n = 3; 7.3%). Initial targeted treatment with activity against zygomycetes was administered to 34 patients (82.9%). Liposomal amphotericin B was associated with improved response (P = 0.012) and survival rates (P = 0.004). CONCLUSIONS: Pathogen distribution and, consequently, drug susceptibility seem to vary across different geographic regions. Furthermore, protection from invasive zygomycosis for patients on posaconazole prophylaxis is not absolute. Our findings indicate that the use of liposomal amphotericin B as first-line treatment for patients diagnosed with zygomycoses merits further investigation, preferably in the form of a clinical trial.

First case of successful allogeneic stem cell transplantation in an HIV-patient who acquired severe aplastic anemia.

We report on the first successful allogeneic stem cell transplantation (SCT) in an HIV-infected patient with severe aplastic anemia (SAA) per- formed at a tertiary care institution. Highly active antiretroviral therapy (HAART) was administered until transplantation and restarted 34 days later with sustained virological response. The patient did however develop a rapid rise in HIV load during the interruption of HAART associated with an acute febrile illness. Due to the extended period between the onset of SAA until SCT, the posttransplant course was complicated by bacterial infections. Stage two skin GvHD, but no AIDS-defining opportunistic diseases were experienced. Neutrophils recovered to >0.5/nL on day +18 and the CD4 count reached 250/microL on day +71 and >500/microL on day +182. The patient is in good condition with an ECOG score of 0 twelve months after transplantation. This report demonstrates the feasibility of allogeneic stem cell transplantation in the HIV setting.

Successful treatment of disseminated mucormycosis with a combination of liposomal amphotericin B and posaconazole in a patient with acute myeloid leukaemia.

The combination of resection of infected tissue and antifungal therapy is the treatment of choice in mucormycosis. In disseminated mucormycosis, where surgery is impossible, the mortality is almost 90%. We report the first case of disseminated mucormycosis that was cured with a combination therapy of liposomal amphotericin B and posaconazole without surgical intervention.

Diagnosis of invasive aspergillosis and mucormycosis in immunocompromised patients by seminested PCR assay of tissue samples.

Aspergillosis and mucormycosis are the most common mold infections in patients with hematological malignancies. Infections caused by species of the genus Aspergillus and the order Mucorales require different antifungal treatments depending on the in vitro susceptibility of the causative strain. Cultures from biopsy specimens frequently do not grow fungal pathogens, even from histopathologically proven cases of invasive fungal infection. Two seminested PCR assays were evaluated by amplifying DNA of zygomycetes and Aspergillus spp. from organ biopsies of 21 immunocompromised patients. The PCR assays correctly identified five cases of invasive aspergillosis and six cases of mucormycosis. They showed evidence of double mold infection in two cases. Both assays were negative in five negative controls and in two patients with yeast infections. Sequencing of the PCR products was in accordance with culture results in all culture-positive cases. In six patients without positive cultures but with positive histopathology, sequencing suggested a causative organism. Detection of fungal DNA from biopsy specimens allows rapid identification of the causative organism of invasive aspergillosis and mucormycosis. The use of these PCR assays may allow guided antifungal treatment in patients with invasive mold infections.

PCR based identification and discrimination of agents of mucormycosis and aspergillosis in paraffin wax embedded tissue.

BACKGROUND: Invasive fungal infections are often diagnosed by histopathology without identification of the causative fungi, which show significantly different antifungal susceptibilities. AIMS: To establish and evaluate a system of two seminested polymerase chain reaction (PCR) assays to identify and discriminate between agents of aspergillosis and mucormycosis in paraffin wax embedded tissue samples. METHODS: DNA of 52 blinded samples from five different centres was extracted and used as a template in two PCR assays targeting the mitochondrial aspergillosis DNA and the 18S ribosomal DNA of zygomycetes. RESULTS: Specific fungal DNA was identified in 27 of 44 samples in accordance with a histopathological diagnosis of zygomycosis or aspergillosis, respectively. Aspergillus fumigatus DNA was amplified from one specimen of zygomycosis (diagnosed by histopathology). In four of 16 PCR negative samples no human DNA was amplified, possibly as a result of the destruction of DNA before paraffin wax embedding. In addition, eight samples from clinically suspected fungal infections (without histopathological proof) were examined. The two PCR assays detected a concomitant infection with Absidia corymbifera and A fumigatus in one, and infections with Rhizopus arrhizus and A fumigatus in another two cases. CONCLUSIONS: The two seminested PCR assays described here can support a histopathological diagnosis of mucormycosis or aspergillosis, and can identify the infective agent, thereby optimising antifungal treatment.

The Protease Inhibitor Transfer Study (PROTRA 1): abacavir and efavirenz in combination as a substitute for a protease inhibitor in heavily pretreated HIV-1-infected patients with undetectable plasma viral load.

OBJECTIVES: The aim of the study was to evaluate the safety and efficacy of abacavir (ABC) and efavirenz (EFV) instead of a protease inhibitor (PI) in HIV-1-infected subjects treated with two nucleoside reverse transcriptase inhibitors (NRTIs) and one PI with undetectable viral loads (< 50 HIV -1 RNA copies/mL). To be eligible for inclusion, patients had to have a history of viral load < 400 copies/mL for at least 3 months and had to be naive to treatment with nonnucleoside reverse transcriptase inhibitors (NNRTIs) and ABC, but multiple pretreatment and treatment failure were allowed. DESIGN: An open-label, single-centre pilot study of duration 48 weeks was conducted. ABC was added to the original treatment with two NRTIs and one PI at baseline, and at week 6 the PI was replaced by EFV. At each study visit, CD4 cell count, viral load [measured by polymerase chain reaction (PCR)] and clinical chemistry were measured. Fasting blood samples were taken at baseline and at weeks 12, 24, 36 and 48 to measure levels of cholesterol [high-density lipoprotein (HDL)/low-density lipoprotein (LDL)], triglycerides, insulin and C-peptide. Additionally, an oral glucose tolerance test (OGTT) was performed. A bioelectric impedance analysis (BIA) and a single slice abdominal and mid-thigh computed tomography (CT) scan were carried out to assess changes in body composition. RESULTS: Thirty patients were included in the study. Three patients experienced ABC-hypersensitivity and one patient demonstrated virological failure caused by nonadherence. At week 48, all remaining patients had viral loads < 50 copies/mL with stable CD4 counts. The fasting metabolic parameters and abdominal fat distribution remained unchanged. CONCLUSIONS: In heavily pretreated patients, ABC and EFV in combination provide an effective, simplified and well-tolerated alternative to PI treatment.

[Clinical presentation and management of the severe acute respiratory syndrome (SARS)]. / Klinik und Behandlung des schweren akuten respiratorischen Syndroms.

BACKGROUND AND OBJECTIVE: In February 2003, a newly emerged infectious disease was described, the etiology of which was initially unknown. It is referred to under the term SARS. In the beginning, it spread in some regions South-East Asia. Import infections appeared in many other parts of the world. Based on the first cases in Germany, this report illustrates the clinical appearance, the diagnostic results and the management of this new disease. PATIENTS AND METHODS: We analysed the data of two patients with SARS and one suspected patient. The results of radiological, laboratory, microbiological and physical examinations were abstracted and compared with the data obtained in other regions. RESULTS: Two of the three patients under our care developed SARS disease. This is characterised by fever of sudden onset lasting for more than 5 days, rapidly changing consolidations in chest x-ray not affected by antimicrobial therapy, leuko-, lympho- as well as thrombopenia with a compromised pulmonary function later in the course. Close contacts with SARS patients does not regularly result in full development of the disease. Secretion of a coronavirus could be detected in respiratory samples during the febrile phase and in feces for a longer time. It is still an open question whether bedrest and antibiotic prophylaxis by themselves or an additional administration of ribavirin and corticosteroids can improve the outcome. CONCLUSION: SARS is a new and highly contagious lung disease. It is crucial to be able to recognize the clinical appearance and the diagnostic features of this disease at an early stage, in order to prevent a further dissemination of the disease.

Prevalence of Aspergillus fumigatus and other fungal species in the sputum of adult patients with cystic fibrosis.

Aspergillus fumigatus is often found in the respiratory tract secretions of patients with cystic fibrosis (CF), although the role of the fungus for progression of pulmonary disease remains unclear. This study aimed to investigate the frequency of A. fumigatus and other fungi in sputum of adult CF patients using different methods for culture and microscopy. Results from the analysis of 369 samples from 94 patients showed that A. fumigatus could be isolated in 45.7% of patients. Other moulds were rare, but the yeast Candida albicans was another frequent isolate, detected in 75.5% of patients. A comparison of different culture media showed no difference between a selective medium developed to specifically inhibit Pseudomonas aeruginosa and a standard fungal culture medium for growth of A. fumigatus, although both were more efficient for detection of fungi than other bacterial culture media. Fluorescent microscopy with calcofluor white was more sensitive for detection of fungal hyphae in undiluted sputum than standard methylene blue staining. This study shows that A. fumigatus and C. albicans have a high frequency in adult CF patients. Microbiological analysis should routinely include methods for specific identification of fungi to monitor for potential complications arising from fungal disease in these patients.

Rapid PCR-based diagnosis of disseminated histoplasmosis in an AIDS patient.

Disseminated histoplasmosis is an unusual opportunistic infection in patients with advanced HIV infection living outside endemic areas. Diagnosis usually is made on the basis of isolation of Histoplasma capsulatum from clinical specimens or histologic examination. Reported is the case of an HIV-infected Columbian individual in whom the diagnosis of histoplasmosis was established within 24 h of collection of an adequate bronchoalveolar lavage specimen. The diagnosis was made by detection of specific fungal DNA and confirmed by isolation of Histoplasma capsulatum from blood, bone marrow and respiratory specimens 10 days later. The patient recovered under antifungal treatment and remained asymptomatic up to the last follow-up visit 6 months later. The polymerase chain reaction assay might be a powerful and rapid diagnostic tool for the diagnosis of non-European invasive fungal infections and should be further evaluated.

[Risk factor for invasive zygomycosis in patients with hematologic malignancies]. / Risikofaktoren für invasive Zygomykosen bei Patienten mit hämatologischen Neoplasien.

Zygomycosis (mucormycosis) is a relatively uncommon infection in immunocompromised patients most often diagnosed in patients with haematological malignancies and neutropenia. Postmortem series demonstrate a high mortality rate up to 80%. Pulmonary involvement mimicking the more frequently diagnosed invasive aspergillosis is the typical clinical presentation. Other risk factors for the development of zygomycosis that have been described in other patient populations include diabetic ketoacidosis, iron overload, use of deferoxamine and steroids. If these factors are also associated with zygomycosis in patients with haematological malignancies has not been described. In a retrospective case-control study including 13 patients with zygomycosis and 13 control patients with the same underlying diseases, without zygomycosis we determined the frequency of various risk factors. Patients with zygomycosis experienced a longer period of neutropenia (17 vs. 13 days) and lymphopenia (23 vs. 20 days). A relapse of their underlying disease was diagnosed more frequently in patients with zygomycosis (7/13 vs. 3/13) as were a diagnosis of diabetes mellitus (6/13 vs. 3/13) and a cardiovascular disease (6/13 vs. 1/13). The previous use of steroids was more frequent in patients with zygomycosis (8/13 vs. 4/13) as was a systemic antifungal prophylaxis with itraconazole (9/13 vs. 4/13). Knowledge of these risk factors may be of benefit in diagnosing and monitoring zygomycosis in patients with haematological malignancies.

[Molecular biological identification of Cuninghamella spec]. / Molekularbiologische Identifizierung von Cunninghamella spec.

Among the genus Cunninghamella, so far C. bertholletiae is known to be the only clinically relevant species. Correct identification of C. bertholletiae is not possible with classical methods. PCR and sequencing of the internal transcribed spacer (ITS) region was used to identify seven of nine clinical isolates as C. bertholletiae and two as C. echinulata. Also an isolate of the surrounding area of one patient infected with C. echinulata could be identified as C. echinulata. High homology in the ITS region was found within the isolates of C. bertholletiae. Within the species C. echinulata and C. elegans a differentiation on subspecies level was achieved by an analysis of restriction fragment length polymorphism of the ITS amplicons after incubation with TaqI and HinfI. Similar results were obtained by PCR fingerprinting of the complete DNA with the single microsatellite DNA primers (GTG)5 and (GAC)5. For the first time C. echinulata could be identified as agent of zygomycosis in humans.