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Recently published diagnosis articles (2008-2010)
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DNA extraction from formalin-fixed paraffin-embedded (FFPE) tissues is difficult and requires special protocols in order to extract small amounts of DNA suitable for amplification. Most described methods report an amplification success rate between 60 and 80%; therefore, there is a need to improve molecular detection and identification of fungi in FFPE tissue. Eighty-one archived FFPE tissues with a positive Gomori methenamine silver (GMS) stain were evaluated using five different commercial DNA extraction kits with some modifications. Three different panfungal PCR assays were used to detect fungal DNA, and two housekeeping genes were used to assess the presence of amplifiable DNA and to detect PCR inhibitors. The sensitivities of the five extraction protocols were compared, and the quality of DNA detection (calculated for each kit as the number of housekeeping gene PCR-positive samples divided by the total number of samples) was 60 to 91% among the five protocols. The efficiencies of the three different panfungals used (calculated as the number of panfungal-PCR-positive samples divided by the number of housekeeping gene PCR-positive samples) were 58 to 93%. The panfungal PCR using internal transcribed spacer 3 (ITS3) and ITS4 primers yielded a product in most FFPE tissues. Two of the five DNA extraction kits (from TaKaRa and Qiagen) showed similar and promising results. However, one method (TaKaRa) could extract fungal DNA from 69 of the 74 FFPE tissues from which a housekeeping gene could be amplified and was also cost-effective, with a nonlaborious protocol. Factors such as sensitivity, cost, and labor will help guide the selection of the most appropriate method for the needs of each laboratory.

Early recognition and rapid initiation of effective treatment is a prerequisite for successful management of children with invasive fungal infections. The increasing diversity of fungal pathogens in high-risk patients, the differences in the antifungal spectrum of available agents, and the increasing rates of resistance call for identification of the infecting isolate at the species level and for information on drug resistance, in order to provide state of the art patient care. Microscopy and culture of appropriate specimens remain the gold standard of mycological diagnosis, despite difficulties in obtaining appropriate and/or sufficient specimens, long time of culturing and false-negative results. Modern imaging studies, detection of circulating fungal cell wall components and DNA in blood and other body fluids or in affected tissues may improve the laboratory diagnosis of invasive mycoses.

New Aspergillus species have recently been described using multi-locus sequencing in refractory cases of invasive aspergillosis (IA). Classical phenotypic identification methods routinely used in clinical laboratories failed to identify them adequately. Some of these Aspergillus species have specific susceptibility patterns to antifungal agents and misidentification may lead to inappropriate therapy. We developed a MALDI-TOF Mass Spectrometry (MS)-based strategy to adequately identify Aspergillus spp. to the species level. A database including the reference spectra of 28 clinically relevant species from 7 Aspergillus sections (5 common and 23 unusual species) was engineered. The profiles of young and mature colonies were analyzed for each reference strain and species-specific spectral fingerprints were identified. The performance of the database was then tested on 124 clinical and 16 environmental isolates previously characterized using partial sequencing of the beta-tubulin and calmodulin genes. One hundred thirty-eight isolates out of 140 (98.6%) were correctly identified. Two atypical isolates could not be identified but no isolate was misidentified (specificity: 100%). The database, including species-specific spectral fingerprints of young and mature colonies of the reference strains, allowed identification regardless of the maturity of the clinical isolate. These results indicate that MALDI-TOF-MS is a powerful tool for rapid and accurate identification of both common and unusual species of Aspergillus. It can improve results over morphological identification in clinical laboratories.

Although Aspergillus fumigatus infection is relatively rare, it should be considered in preterm low birthweight neonates with a rapidly progressive purpuric rash. Prompt diagnosis and treatment is crucial to maximize the chances of survival.

Bronchial asthma is an inflammatory disease of the airways which may be worsened due to numerous extrinsic factors. The most common trigger is continuous exposure to allergens of which fungal agents are important factors. There is overwhelming evidence for the presence of fungal sensitization in patients with asthma. The diagnosis of fungal sensitization can be made either with skin testing with antigens derived from fungi or measuring specific IgE levels. There is also a strong association between fungal sensitization and severity of asthma. Whether this relationship is causal or just casual remains to be investigated. A variety of fungi are known to cause sensitization in asthmatics, but the most important fungal agent(s) causing severe asthma with fungal sensitization (SAFS) are currently unknown. Aspergillus species seem to be the strongest candidates as only with Aspergillus spp. does one encounter two extreme immunologic phenomena, i.e., the Aspergillus-sensitive asthma and allergic bronchopulmonary aspergillosis. The initial clinical management of SAFS should be the same as asthmatics without fungal sensitization. There is some evidence of the role of itraconazole in the management of SAFS but its routine use in SAFS requires further evaluation. This review summarizes the current evidence on the link between fungi and severe asthma.

A retrospective study of 35 case reports of Aspergillus endocarditis published between 2003 and 2009 was carried out. Fifteen percent of cases presented with a new cardiac murmur, 38% with an embolus. Eighty percent of cases involved the aortic or mitral valves. Seventy-four percent of cases involved patients with a history of prior surgery, 48% of these involved a heart valve, 20% had other cardiac surgery and 32% had non-cardiac surgery. Galactomannan testing was helpful diagnostically in four out of nine cases, but PCR testing was positive in six out of six cases. Overall mortality was 68%, all eight survivors had heart valve surgery apart from one - an 8-month-old child. Seven out of eight survivors received liposomal amphotericin B, three of these in combination with other antifungals. We need to think more about the possibility of Aspergillus endocarditis, particularly in immunocompromised patients with recent surgery. Galactomannan and PCR testing may be used more vigorously. Valve replacement, or at least vegetectomy, should be carried out in all patients. Liposomal amphotericin B, 3-5mg/kg/day, for at least 4 weeks is the treatment of choice. Oral voriconazole should be used for at least 2 years. Posaconazole may be an alternative, however there have been no prior cases reported to suggest its efficacy. The value of combination antifungal therapy is uncertain, but consideration should be given to the use of a second agent in addition to liposomal amphotericin. While further case reports on this condition will be helpful, more definitive management guidelines will depend on a prospective study.

BACKGROUND:: Empiric antifungal treatment has become standard of care in children with cancer and prolonged fever and febrile neutropenia (FN), with the downside that it leads to significant over treatment. We characterized epidemiologic, clinical, and laboratory features of invasive fungal disease (IFD) in children with cancer and FN with the aim to identify risk factors for IFD that can aid in better selecting children who require antifungal treatment. METHODS:: In a prospective, multicenter study, children admitted with FN at high-risk for sepsis, in 6 hospitals in Santiago, Chile were monitored from admission until the end of the FN episode. Monitoring included periodic evaluation of clinical findings, absolute neutrophil count, absolute monocyte count (AMC), serum C-reactive protein (CRP), bacterial cultures, imaging studies, and galactomannan antigen. A diagnosis of proven, probable, and possible IFD was made after episode resolution based on European Organization for Research and Treatment of Cancer classification. RESULTS:: A total of 646 high-risk FN episodes were admitted during the study period, of which 604 were enrolled. IFD was diagnosed in 35 episodes (5.8%) of which 7 (1.2%) were proven, 10 (1.6%) probable, and 18 (3.0%) possible. Four variables obtained on day 4 were significantly more common in IFD cases, which are presence of fever, absolute neutrophil count </=500/mm, AMC </=100/mm, and CRP >/=90 mg/L. The combination of fever, AMC </=100/mm, and CRP >/=90 at day 4 provided a RR for IFD of 5.4 (99% CI, 3.2-9.2) with a sensitivity of 75%, specificity of 87%, positive and negative predictive values of 13% and 99%, respectively. CONCLUSIONS:: Fever persisting at day 4 of admission, together with AMC </=100 and CRP >/=90 significantly increased the risk for IFD in children with cancer.

Accurate species diagnosis in cases of fungal pneumonia may be hampered by environmental contamination and colonization resulting in false-positive results. Our novel approach for fungal species diagnostics combines fluorescent staining of mounted cryosections with the optical brightener Blankophor, laser capture microdissection and PCR amplification with subsequent sequencing of the first internal transcribed spacer region (ITS-1). Using clinical specimens from infected birds, we show that the procedure is suitable for species identification from single hyphae of intralesional filamentous fungi. Our data also suggest that multiple Aspergillus fumigatus strain infections may occur frequently in pulmonary aspergillosis of birds.

BACKGROUND: The annual prevalence of Aspergillus colonization (AC) and allergic bronchopulmonary aspergillosis (ABPA) has recently increased in pediatric patients with cystic fibrosis (CF). The reasons remain unclear although a number of factors have been suggested to be involved. This study was set up to investigate the association between potential predisposing factors, including new therapies recommended in CF, and the occurrence of AC or ABPA in children with CF. METHODS: The medical records of 85 children monitored regularly in the Pediatric Reference Centre for Cystic Fibrosis Care (RCCFC) of the University Hospital of Marseille (France) were analyzed from the first time they attended the RCCFC until either the occurrence of an end event, or their last visit to the RCCFC. Risk factors for AC or ABPA were analyzed by univariate and multivariate logistic regression. RESULTS: Eight children developed ABPA and 18 had AC. In univariate analysis, ABPA was significantly associated with RhDNase therapy, sensitization to Alternaria and Candida, and a low body mass index (BMI). Multivariate analysis identified an independent association between low BMI and ABPA (OR = 10.6, 95% CI [2.2-51.8], P = 0.004), and for the first time, between long-term azithromycin therapy and AC (OR = 6.4, 95% CI [2.1-19.5], P = 0.001). This latter association might be explained by the inhibitory effect of azithromycin on both the recruitment and the activation of neutrophils, which represent the first-line defenses against Aspergillus. CONCLUSIONS: The risk factors associated with AC and ABPA in children with CF identified in this comprehensive exploratory study now need to be confirmed in further prospective studies.

Traditionally, the patients believed to be at highest risk of invasive aspergillosis (IA) are those who are neutropenic due to chemotherapy for hematological malignancy or those undergoing allogeneic hematopoietic stem cell transplantation. However, emerging data show that other patients are vulnerable to IA, even though some are not classically immunocompromised. These include: solid organ transplant recipients; patients with TB, chronic obstructive pulmonary disease and patients in the intensive care unit for other reasons. The conference highlighted the diagnostic and therapeutic challenges facing physicians treating this diverse group, not least of which include the unreliable estimates of IA incidence due to poor surveillance and inadequate data collection. Moreover, although there is now considerable experience of IA in neutropenic patients, much less is known about the management of those who are non-neutropenic. Nevertheless, approaches that have proven effective in neutropenic patients may also benefit others in this growing population. The meeting, attended by more than 500 delegates from almost 50 countries, also provided the opportunity to hear how basic scientific research may improve the understanding of the pathogenic mechanisms and therapy of IA.

Earlier diagnosis articles

We present two cases of aspergillus infection confirmed by blood culture and review 30 other cases of genuine aspergillus fungemia and 34 cases of aspergillus pseudofungemia. Multiple different media and blood culture systems were used to isolated Aspergillus. The median time to positive blood culture was 8.5 days (range, 1-27 days) in the genuine cases. Genuine aspergillus fungemia was observed more often after cardiac surgery (n = 11 [34%]) or during neutropenia (n = 9 [28%]) than in other settings. In a recent series of fungemia during neutropenia, 7.6% of all episodes were due to Aspergillus. Other patients at risk for aspergillus fungemia were similar to those at risk for invasive aspergillosis, including patients with AIDS. Seven (44%) of 19 patients who were treated survived. In the group of patients with aspergillus pseudofungemia, there were no deaths, and cultures of additional specimens from the same patient were not positive. Criteria that may be applied to ascertain whether the isolation of Aspergillus from blood cultures is clinically significant are put forward.

Subhaemolytic amounts of saline extracts of homogenized, disrupted A. fumigatus sensitized tanned erythrocytes for agglutination to high titres (8,192) by an antiserum to A. fumigatus. Extracts of 5 different strains of A. fumigatus were equally active. Erythrocytes sensitized by similarly prepared extracts of P. notatum and an unclassified Aspergillus strain gave low titres (64 and 128), while extracts of some other fungi and bacteria had no sensitizing activity in tests with the antiserum. Results of absorption and inhibition experiments using the various preparations indicated that the sensitizing antigen(s) in extracts of A. fumigatus may be species specific. Results obtained with 441 human sera showed good correlation between high titres and presence of aspergillosis. Titres above 128 are highly indicative of active disease.

Despite progress with diagnostic criteria, the type and timing of laboratory tests used to diagnose infective endocarditis (IE) have not been standardized. This is especially true with serological testing. Patients with suspected IE were evaluated by a standard diagnostic protocol. This protocol mandated an evaluation of the patients according to the modified Duke criteria and used a battery of laboratory investigations, including three sets of blood cultures and systematic serological testing for Coxiella burnetii, Bartonella spp., Aspergillus spp., Legionella pneumophila, and rheumatoid factor. In addition, cardiac valvular materials obtained at surgery were subjected to a comprehensive diagnostic evaluation, including PCR aimed at documenting the presence of fastidious organisms. The study included 1,998 suspected cases of IE seen over a 9-year period from April 1994 to December 2004 in Marseilles, France. They were evaluated prospectively. A total of 427 (21.4%) patients were diagnosed as having definite endocarditis. Possible endocarditis was diagnosed in 261 (13%) cases. The etiologic diagnosis was established in 397 (93%) cases by blood cultures, serological tests, and examination of the materials obtained from cardiac valves, respectively, in 348 (81.5%), 34 (8%), and 15 (3.5%) definite cases of IE. Concomitant infection with streptococci and C. burnetii was seen in two cases. The results of serological and rheumatoid factor evaluation reclassified 38 (8.9%) possible cases of IE as definite cases. Systematic serological testing improved the performance of the modified Duke criteria and was instrumental in establishing the etiologic diagnosis in 8% (34/427) cases of IE.

No abstract. First paragraph: Fungi of the genus Aspergillus are ubiquitous saprophytes in nature. They are filamentous fungi producing air-borne spores which are found throughout the year. Noble and Clayton have recently shown that, in London, there is approximately a hundred-fold increase in the air count of Aspergillus fumigatus spores between the months of October and February.

PURPOSE: To assess the radiographic, computed tomographic (CT), and pathologic findings in invasive aspergillosis of the airways. MATERIALS AND METHODS: The study included nine consecutive patients (aged 17-65 years [median, 49 years]) with pathologically proved invasive aspergillosis of the airways. All nine underwent chest radiography and seven underwent CT within 3 days of diagnosis. RESULTS: The radiographic findings include normal parenchyma (n = 1), unilateral consolidation (n = 1), bilateral consolidation (n = 5), and ill-defined nodules (n = 2). The main findings at CT included lobar consolidation (n = 1), bilateral predominantly peribronchial consolidation (n = 3), ground-glass attenuation (n = 1), and centrilobular nodules less than 5 mm in diameter (n = 2). At pathologic examination, the peribronchial infiltrates represented bronchopneumonia and the nodules represented Aspergillus bronchiolitis with a variable degree of peribronchiolar organizing pneumonia and hemorrhage. CONCLUSION: Radiographic findings of invasive aspergillosis of the airways consist of consolidation or ill-defined nodules. At CT, the consolidation can be seen to be peribronchial and the nodules centrilobular.

The incidence of invasive aspergillosis is increasing rapidly in the developed world with two Aspergillus spp., A. fumigatus and A. flavus, causing the majority of infections (85-90% and 5-10%, respectively). The major risk factors are profound neutropenia (</=1000 x 10(6) cells/L), prolonged neutropenia, neutrophil function deficits, and corticosteroid therapy. Useful diagnostic techniques include sputum culture, CT scan, bronchoscopy with microscopy and culture, percutaneous lung biopsy, open lung biopsy and serology. Invasive aspergillosis has an almost 100% mortality rate if untreated. Amphotericin B is the usual first-line therapy although it is associated with a high failure rate. Itraconazole (>/=400 mg daily) is a useful alternative and surgical resection may be life saving in some cases. The efficacy of the initial therapy is critical for improving mortality rates.

PURPOSE: To define the role of lower-respiratory-tract cultures in the diagnosis of invasive pulmonary aspergillosis (IPA) in immunocompromised hosts. METHODS: Immunocompromised patients with a positive, nonbiopsy, lower-respiratory-tract culture for Aspergillus species were classified as having definite, probable, indeterminate, or no IPA. Culture data, positive predictive values (PPVs), correlation with clinical and radiographic findings, and the relationship between the number of specimens submitted and the likelihood of recovering Aspergillus were assessed. RESULTS: Definite or probable IPA was diagnosed in 72% of episodes from patients with hematologic malignancy, granulocytopenia, or bone-marrow transplant; in 58% of those with solid-organ transplant or using corticosteroids; and in 14% of those with human immunodeficiency virus infection. The PPV of cultures ranged from 14% in the latter group to 72% in the first group (bone-marrow-transplantation subgroup, 82%). Fungal cultures were more often positive than were routine cultures (P < 0.001). Clinical and radiographic findings suggestive of IPA were present more frequently in infected than uninfected patients (59% versus 24%, P < 0.025); and 73% versus 6%, (P < 0.0001, respectively). Infected patients with > or = 1 positive node had more cultures submitted than a control group of patients with no positive cultures (5.8 +/- 4.7 versus 2.1 +/- 2.2 cultures, P < 0.001). CONCLUSION: Recovery of Aspergillus species from high-risk patients is associated with invasive infection. Clinical and radiographic correlations help to separate true- from false-positive cultures. At least 3 sputum specimens should be submitted for fungal culture whenever fungal infection is suspected.

BACKGROUND: Invasive Aspergillus is an important cause of morbidity and mortality among lung transplant recipients. The diagnosis can be difficult and treatment is often unsuccessful so many centers preemptively treat all Aspergillus airway isolates to prevent invasive disease. This approach is untested as little is known about the relationship between Aspergillus airway colonization and invasive disease. This study was undertaken to evaluate the incidence of Aspergillus airway colonization after lung transplantation and the risk of invasive disease after colonization. DESIGN: All cultures and histologic specimens obtained from a consecutive series of 151 lung transplant cases were reviewed for the presence of Aspergillus and compared with clinical data. RESULTS: Aspergillus was isolated from the airway in 69 (46%) of 151 transplant recipients. Invasive disease occurred in five cases and was uniformly fatal, accounting for 13% of all posttransplant deaths. Results of cytologic examination of BAL fluid were normal in all cases of invasive disease and cultures were positive in only one of five patients prior to invasion. Invasive disease occurred exclusively in patients who died or were colonized with Aspergillus fumigatus within the first 6 months posttransplant. Patients growing A. fumigatus from the airway during the first 6 months were 11 times more likely to develop invasive disease relative to those not colonized. CONCLUSION: Aspergillus airway colonization after lung transplantation is common and in most cases, transient. In contrast, invasive Aspergillus disease is less common, but fatal. Bronchoscopy with cytologic examination and fungal culture are not sensitive or timely predictors of invasive disease. Invasive Aspergillus occurred only in patients initially colonized with A. fumigatus within the first 6 months posttransplant. A trial of empiric anti-Aspergillus therapy limited to the first 6 months posttransplant may be warranted.

Bronchoalveolar lavage (BAL) material is commonly received in cytopathology for the exclusion of microorganisms. When crystalline material suggestive of calcium oxalate is present in the specimen, a search for fungal elements should be undertaken. Aspergillus niger is the hyaline mold associated with the presence of oxalate crystals. Commonly fragments of hyphae and occasionally entire conidiophores may be present in BAL specimens from patients with aspergillosis. We report a case of a patient with saprophytic colonization of a bullous/cavitary lesion. The BAL consisted of abundant acute inflammation, crystalline material suggestive of oxalate, and darkly pigmented conidia. Although an extensive search was undertaken, hyphal fragments could not be found. The suspicion of an A. niger infection was confirmed by culture. We believe that even in the absence of hyphal fragments, darkly pigmented, occasionally rough-walled conidia are sufficient evidence to be highly suspicious of an A. niger infection in patients with pulmonary oxalosis.

A 23-year-old man, intensively treated for acute lymphoblastic leukaemia in relapse and with documented pulmonary aspergillosis, was admitted to the intensive care unit for acute respiratory failure. The diagnosis of invasive tracheal aspergillosis was made by bronchoscopy and biopsy. The lesions consisted of extensive necrotizing bronchitis with transmural and peribronchial extension associated with tracheal and bronchial obstruction due to the presence of pseudomembranes almost entirely composed of fungal hyphae. Despite treatment with amphotericin B and itraconazole, mechanical ventilation and bronchoscopy, the patient died 3 weeks later of massive bleeding.

(N.B. The Aspergillus website used to maintain a bibliographic database which was compiled from Medline and Web of Science (GRAsp), but as all users now have access to the former free of charge via the NCBI website and most will have access to Web of Science via their own libraries this resource is currently not being updated. It contains papers dating up to 7th October 2002. Search the GRASp Database here.)