Aspergillosis In High-risk Patients Research Articles

Clinico-mycological profile of suspected pulmonary aspergillosis cases

Abstract Introduction: Aspergillus is one of the most ubiquitous airborne saprophytic fungi. Humans constantly inhale numerous conidia of this fungus. The conidia are normally eliminated in the immunocompetent host by innate immune mechanisms. With increase in the number of immunocompromised patients, there has been a dramatic increase in severe and fatal invasive aspergillosis (IA). The present study was aimed to know the clinico-mycological profile of suspected pulmonary aspergillosis cases. Methods: The present study was a prospective study conducted over a period of 1 year in the department of microbiology in admitted cases with clinical suspicion of pulmonary aspergillosis. Samples such as sputum, bronchoalveolar lavage, endotracheal secretions, fine-needle aspiration or biopsy and blood (for serological analysis) were collected and processed as per standard protocol. Results: Over a period of 1 year, 89 cases were included in the study. Their mean age was 58.7 years. Majority were male (60.6%). The most common presentation was cough (89.9%), followed by chest pain (57.3%) and fever (56.2% most common chest X-ray finding was homogeneous haziness (40.4%). Fungal smear was positive in 55.1% and 61.8% showed fungal growth. Galactomannan (GM) and (1-3)-β-D glucan positivity were 56% and 83.3%, respectively. The mortality was observed in 21.4% of patients. Conclusions: The study demonstrated that GM assay and (1-3)-β-D glucan assay are useful diagnostic modalities with a high sensitivity and specificity for the detection of pulmonary aspergillosis in high-risk patients along with positive fungal culture. Thus, these tests are early markers for IA.

Evaluation of the Dynamiker® Fungus (1-3)-β-D-Glucan Assay for the Diagnosis of Invasive Aspergillosis in High-Risk Patients with Hematologic Malignancies.

IntroductionThe Dynamiker® Fungus (1–3)-β-d-glucan assay (DFA) allows the testing of samples in smaller batches compared to the well-established Fungitell® assay (FA) making the assay cost-effective in centers with small numbers of samples. Evaluations of its performance for the diagnosis of invasive aspergillosis (IA) are limited. Therefore, we compared the two assays and evaluated their clinical performance in diagnosing IA.MethodsA total of 60 adult hematology patients were screened for IA, 13 with probable IA, 19 with possible IA, and 28 with no IA. Serum specimens (n = 166) were collected twice-weekly and tested for (1–3)-β-d-glucan (BDG) using FA and DFA which were compared quantitatively with Spearman rank correlation analysis and qualitatively with the Chi-square test. Agreement and error rates were determined using FA as the reference method. Sensitivity, specificity, and positive predictive and negative predictive values in diagnosing IA were calculated.ResultsThe performance of the DFA was highly consistent with that of the FA, both quantitatively (rs = 0.913) and qualitatively (kappa = 0.725). The agreement was 85% with 8% minor, no major, and 7% very major errors (FA+/DFA−). Using a cut-off value of 20 pg/mL for DFA, very major errors were reduced to 1%, although 5% major errors were detected. BDG levels were lower with DFA than FA (slope 0.653 ± 0.031). Sensitivity, specificity, positive predictive value, and negative predictive value (NPV) was 67%, 53%, 44%, and 74% for FA, and 53%, 67%, 49%, and 71% for DFA, respectively. The optimal BDG positivity threshold calculated did not lead to significant test quality improvement for either assay. However, a higher % of patients with probable IA (62%) had ≥ 2 consecutive positive specimens compared to patients with no IA (FA-BDG 26%, p = 0.10, and DFA-BDG 10%, p = 0.01) leading to improved sensitivity and NPV (71% and 85% for DFA, and 95% and 96% for FA, respectively).ConclusionDFA could be a valuable alternative to the FA, particularly in laboratories with small numbers of samples. The results of the BDG testing should be carefully interpreted in the high-risk setting of patients with hematologic malignancies. Higher NPV was found using as criterion ≥ 2 consecutive positive samples for diagnosing IA.

Performance, Correlation and Kinetic Profile of Circulating Serum Fungal Biomarkers of Invasive Aspergillosis in High-Risk Patients with Hematologic Malignancies.

As conventional microbiological documentation of invasive aspergillosis (IA) is difficult to obtain, serum fungal biomarkers are important adjunctive diagnostic tools. Positivity rates and the kinetic profiles of galactomannan (GM), 1,3-β-D-glucan (BDG) and Aspergillus DNA (PCR) were studied in high-risk patients with hematologic malignancies. GM, BDG and PCR data from serial serum specimens (n = 240) from 93 adult hematology patients with probable (n = 8), possible (n = 25) and no (n = 60) IA were retrospectively analyzed. Positivity rates and sensitivity/specificity/positive/negative predictive values (NPV) of each fungal biomarker alone and in combination were estimated. The three markers were compared head-to-head and correlated with various biochemical, demographic and patient characteristics. The positivity rates for patients with probable/possible/no IA were 88%/8%/0% for GM (X2 = 55, p < 0.001), 62%/46%/35% for BDG (X2 = 2.5, p = 0.29), 62%/33%/27% for PCR (X2 = 3.9, p = 0.15), 50%/4%/0% for GM + BDG and GM + PCR (X2 = 31, p < 0.001), 50%/8%/22% for BDG + PCR (X2 = 6.5, p = 0.038) and 38%/4%/0% for GM + BDG + PCR (X2 = 21, p < 0.001). Higher agreement (76%) and negative correlation (rs = −0.47, p = 0.0017) was found between GM index and PCR Ct values. The sensitivity and NPV was 45–55% and 90–92% when biomarkers assessed alone and increased to 75–90% and 93–97%, respectively when combined. Weak significant correlations were found between GM, PCR and BDG results with renal/liver function markers (r = 0.11–0.57) with most GM+ and PCR+ samples found in the first and second week of clinical assessment, respectively and BDG later on. Different positivity rates, time profiles and performances were found for the three biomarkers advocating the combination of GM with PCR for the early diagnosis of IA, whereas the high NPV of combined biomarkerscould help excluding IA.

Molecular Tools for the Detection and Deduction of Azole Antifungal Drug Resistance Phenotypes in Aspergillus Species.

The incidence of azole resistance in Aspergillus species has increased over the past years, most importantly for Aspergillus fumigatus. This is partially attributable to the global spread of only a few resistance alleles through the environment. Secondary resistance is a significant clinical concern, as invasive aspergillosis with drug-susceptible strains is already difficult to treat, and exclusion of azole-based antifungals from prophylaxis or first-line treatment of invasive aspergillosis in high-risk patients would dramatically limit drug choices, thus increasing mortality rates for immunocompromised patients. Management options for invasive aspergillosis caused by azole-resistant A. fumigatus strains were recently reevaluated by an international expert panel, which concluded that drug resistance testing of cultured isolates is highly indicated when antifungal therapy is intended. In geographical regions with a high environmental prevalence of azole-resistant strains, initial therapy should be guided by such analyses. More environmental and clinical screening studies are therefore needed to generate the local epidemiologic data if such measures are to be implemented on a sound basis. Here we propose a first workflow for evaluating isolates from screening studies, and we compile the MIC values correlating with individual amino acid substitutions in the products of cyp51 genes for interpretation of DNA sequencing data, especially in the absence of cultured isolates.

Changes in In Vitro Susceptibility Patterns of Aspergillus to Triazoles and Correlation With Aspergillosis Outcome in a Tertiary Care Cancer Center, 1999-2015.

Azole-resistant aspergillosis in high-risk patients with hematological malignancy or hematopoietic stem cell transplantation (HSCT) is a cause of concern. We examined changes over time in triazole minimum inhibitory concentrations (MICs) of 290 sequential Aspergillus isolates recovered from respiratory sources during 1999-2002 (before introduction of the Aspergillus-potent triazoles voriconazole and posaconazole) and 2003-2015 at MD Anderson Cancer Center. We also tested for polymorphisms in ergosterol biosynthetic genes (cyp51A, erg3C, erg1) in the 37 Aspergillus fumigatus isolates isolated from both periods that had non-wild-type (WT) MICs. For the 107 patients with hematologic cancer and/or HSCT with invasive pulmonary aspergillosis, we correlated in vitro susceptibility with 42-day mortality. Non-WT MICs were found in 37 (13%) isolates and was only low level (MIC <8 mg/L) in all isolates. Higher-triazole MICs were more frequent in the second period and were Aspergillus-species specific, and only encountered in A. fumigatus. No polymorphisms in cyp51A, erg3C, erg1 genes were identified. There was no correlation between in vitro MICs with 42-day mortality in patients with invasive pulmonary aspergillosis, irrespective of antifungal treatment. Asian race (odds ratio [OR], 20.9; 95% confidence interval [CI], 2.5-173.5; P = .005) and azole exposure in the prior 3 months (OR, 9.6; 95% CI, 1.9-48.5; P = .006) were associated with azole resistance. Non-WT azole MICs in Aspergillus are increasing and this is associated with prior azole exposure in patients with hematologic cancer or HSCT. However, no correlation of MIC with outcome of aspergillosis was found in our patient cohort.

Role of galactomannan assay in bronchoalveolar lavage fluid in the diagnosis of invasive aspergillosis in high risk patients with hematological disorders

Introduction: Invasive aspergillosis (IA) is the most frequent invasive fungal infection occurring in high risk patients with haematological disorders and also an important cause of morbidity and mortality in them. Lungs comprise the primary site of IA in the vast majority of cases, and the most frequently occurring form of IA. Early diagnosis followed by early initiation of therapy improves outcome. The current culture based diagnostic methods are insensitive and slow. Detection of Aspergillus galactomannan (GM) in serum using the platelia Aspergillus enzyme immunoassay (GM EIA) represents sensitive, non-culture-based tool for the early diagnosis of IA. Recent studies have shown that during IPA, GM is released earlier and in higher concentration in the BAL fluid than in serum. Thus, an early and accurate diagnosis of IPA can be reached by detecting antigen in BAL samples. Materials and Methods: Thirty consecutive high risk patients with haematological disorders satisfying the inclusion and exclusion criteria according to EORTC-MSG diagnostic criteria and undergoing indoor treatment were recruited between November 2013 and December 2014 into the study. In all the patients, bronchoalveolar lavage (BAL) fluid GM and serum GM assays were assayed. Results: The diagnostic accuracy of the two tests was measured by calculating the sensitivity, specificity, predictive values and receiver operating characteristic curve (ROC). The optimal BAL GM index for diagnosing probable and possible IPA was >1.00 OD, resulting in a sensitivity and specificity of 93.75% and 78.57% respectively. As per ROC curve, when the optimal BAL GM index for the diagnosis of probable and possible invasive pulmonary aspergillosis (IPA) was OD >0.5, it resulted in sensitivity and specificity of 50% and 71.43% respectively. BAL GM was thus found to have a significantly higher sensitivity as compared to serum GM. Conclusion: In all high-risk patients with haematological disorders where the clinical and radiological findings favour the possibility of invasive pulmonary aspergillosis, conducting a GM assay in BAL fluid sample is far more rewarding than carrying out the traditional serum GM assay. BAL GM has a high diagnostic accuracy when correlated with the EORTC/MSG diagnostic criteria, and this high diagnostic yield is useful in obviating the unnecessary widespread use and abuse of empirical antifungal therapy in patients with IPA.

Combined real‐time PCR and galactomannan surveillance improves diagnosis of invasive aspergillosis in high risk patients with haematological malignancies

Invasive aspergillosis (IA) is a leading complication of intensive treatment for haematological malignancies. Earlier diagnosis should facilitate effective antifungal therapy and prevent progression to invasive disease, which is often lethal. Polymerase chain reaction (PCR) assays, targeting the 28S and ITS ribosomal gene regions respectively, were evaluated for early detection of Aspergillus DNA and for diagnostic utility in patients receiving treatment in two busy haematopoietic stem cell transplant centres. Patients undergoing intensive chemotherapy, autologous or allogeneic transplant were eligible for inclusion in the study. EDTA blood and serum samples for circulating Aspergillus biomarkers, including galactomannan (GM), were collected twice-weekly on a prospective basis from all study patients who were categorized according to international consensus criteria for defining invasive fungal disease (IFD). Of 278 patients recruited there were 44 probable IA cases and only one proven case. Moderate sensitivity and specificity, poor positive predictive value (50-80%), but good negative predictive value (>80-90%) were common to both PCR assays. Overall biomarker performance could be improved by combining positive results of either PCR assay with GM taken within a 12-d period. The addition of PCR to GM monitoring in high-risk patients with haematological malignancies provides greater diagnostic accuracy in invasive aspergillosis.

Piperacillin/tazobactam (TazocinTM) seems to be no longer responsible for false-positive results of the galactomannan assay

Galactomannan (GM) testing is extremely useful for diagnosing invasive aspergillosis in high-risk patients, but false-positive results have been reported in patients treated with piperacillin/tazobactam. The aims of this study are to test if the recent piperacillin/tazobactam (Tazocin™; Pfizer) preparation still contains GM, and if serum GM positivity in haematopoietic stem cell transplant (HSCT) recipients receiving piperacillin/tazobactam can be attributed to this treatment. Serum samples obtained from 1 October 2009 to 31 October 2010 from HSCT recipients for GM testing were analysed. The difference in the rate of positive results (defined as GM ≥ 0.5) in patients receiving and not receiving piperacillin/tazobactam was evaluated. Piperacillin/tazobactam vials from randomly selected batches were tested. Of 1606 samples drawn in the absence of piperacillin/tazobactam therapy, 25 (1.6%) tested positive for GM versus 10 of 394 samples (2.5%) drawn while on piperacillin/tazobactam (P = 0.18). The median GM result of samples drawn on piperacillin/tazobactam was slightly higher than that of samples drawn in the absence of piperacillin/tazobactam (0.141 versus 0.122; P < 0.001). All 90 piperacillin/tazobactam vials from 30 randomly selected batches tested negative for GM, with a median GM value of 0.057 (range: 0.011-0.320). Although some residual GM might still be present in piperacillin/tazobactam, currently available brand piperacillin/tazobactam preparations seem no longer responsible for false-positive GM results.

TLR3 essentially promotes protective class I–restricted memory CD8+ T-cell responses to Aspergillus fumigatus in hematopoietic transplanted patients

AbstractAspergillus fumigatus is a model fungal pathogen and a common cause of severe infections and diseases. CD8+ T cells are present in the human and murine T-cell repertoire to the fungus. However, CD8+ T-cell function in infection and the molecular mechanisms that control their priming and differentiation into effector and memory cells in vivo remain elusive. In the present study, we report that both CD4+ and CD8+ T cells mediate protective memory responses to the fungus contingent on the nature of the fungal vaccine. Mechanistically, class I MHC-restricted, CD8+ memory T cells were activated through TLR3 sensing of fungal RNA by cross-presenting dendritic cells. Genetic deficiency of TLR3 was associated with susceptibility to aspergillosis and concomitant failure to activate memory-protective CD8+ T cells both in mice and in patients receiving stem-cell transplantations. Therefore, TLR3 essentially promotes antifungal memory CD8+ T-cell responses and its deficiency is a novel susceptibility factor for aspergillosis in high-risk patients.

Detection of laboratory biomarkes in haematological and pulmonology patients at high risk for aspergillosis

During the past decades a dramatic increase in the incidence of invasive fungal diseases, especially invasive aspergillosis has been observed. Undiagnosed and diagnosed late invasive aspergillosis is followed by lethality of up to 90%. Detection of early laboratory biomarkers (galactomannan and anti-Aspergillus antibodies) contributes to early diagnosis and is used for screening, as well as for monitoring therapy of aspergillosis. The aim was to evaluate usefulness of "non-culture" methods (galactomannan and anti-Aspergillus antibodies IgA, IgM and IgG) for early diagnosis of aspergillosis in high-risk patients. Prospective two-year study involved 262 high-risk patients for aspergillosis. In pulmonology and haematology patients (adults and children) blood samples were tested on galactomannan and anti-Aspergillus antibodies. Early laboratory biomarkers were statistically significantly higher in pulmonology patients (p = 0.00033). However, in haematological patients galactomannan was a more frequently positive finding, while in pulmonology patients it was the finding of anti-Aspergillus antibodies. It is interesting that, despite the possible immunosuppression, in about 1/3 of haematological patients anti-Aspergillus antibodies were confirmed. Early diagnosis and treatment of aspergillosis represent both clinical and laboratory problem. Nowadays, the application of new "non-culture" methods is of particular importance for the outcome of aspergillosis. Clinical features, laboratory findings of biomarkers and their correct interpretation significantly increase the possibility of timely implementation of appropriate therapy. In this regard, the new organization of reference laboratory for medical mycology has significantly improved the outcome of aspergillosis in high-risk patients in our country. However, further investigations, implementation of European standards and introduction of new diagnostic methods are necessary in this field.

Dectin-1 Y238X polymorphism associates with susceptibility to invasive aspergillosis in hematopoietic transplantation through impairment of both recipient- and donor-dependent mechanisms of antifungal immunity

AbstractThe C-type lectin receptor Dectin-1 plays a pivotal role in antifungal immunity. In this study, the recently characterized human DECTIN1 Y238X early stop codon polymorphism leading to diminished Dectin-1 receptor activity was studied in relation to invasive aspergillosis susceptibility and severity in patients receiving hematopoietic stem cell transplantation. We found that the presence of the DECTIN1 Y238X polymorphism in either donors or recipients of hematopoietic stem cell transplantation increased susceptibility to aspergillosis, with the risk being highest when the polymorphism was present simultaneously in both donors and recipients (adjusted hazard ratio = 3.9; P = .005). Functionally, the Y238X polymorphism impaired the production of interferon-γ and interleukin-10 (IL-10), in addition to IL-1β, IL-6, and IL-17A, by human peripheral mononuclear cells and Dectin-1 on human epithelial cells contributed to fungal recognition. Mechanistically, studies on preclinical models of infection in intact or bone marrow-transplanted Dectin-1 knockout mice revealed that protection from infection requires a distinct, yet complementary, role of both donor and recipient Dectin-1. This study discloses Dectin-1 deficiency as a novel susceptibility factor for aspergillosis in high-risk patients and identifies a previously unsuspected role for Dectin-1 in antifungal immunity that is the ability to control both resistance and tolerance to the fungus contingent on hematopoietic/nonhematopoietic compartmentalization.

A prospective, randomised study on the use of well-fitting masks for prevention of invasive aspergillosis in high-risk patients

The problem of inhalation of Aspergillus spores outside rooms with high-efficiency particulate air (HEPA) filtration has not been resolved as yet. Well-fitting masks are used in industrial and health care settings to protect from inhaling particles of 0.3–0.5μm size. To investigate the efficacy and tolerability of well-fitting masks in high-risk patients, we conducted a prospective, randomised, multicentre study comparing standard hospital hygiene procedures with or without wearing masks in adults undergoing chemotherapy for acute leukaemia or allogeneic haematopoietic stem-cell transplantation (aHSCT). Forty-one patients were randomly assigned to wearing masks and 39 to the control group. In all, 76% of patients were treated in laminar airflow or HEPA-filtered rooms, 84% received oral polyenes, and three aHSCT recipients were given fluconazole. Duration of neutropenia was similar in both treatment groups. Invasive fungal infections were diagnosed in eight patients in either study arm. One patient in each arm died from proven invasive aspergillosis. There was no difference in the use of systemic antifungals. Of patients in the mask group, 65% described the comfort as acceptable, 26% as unpleasant, and 9% as intolerable. This first randomised study on the use of well-fitting masks failed to show a reduction of invasive fungal infections.

Prevention of Invasive Aspergillosis in High-Risk Patients: Universal Versus Preemptive, Targeted Treatment.

Invasive aspergillosis (IA) is associated with increased morbidity and mortality, and there is a need for better preventative and therapeutic approaches. Successful treatment of documented IA remains difficult, often because of the inability to detect disease at an early stage. An important, recent advance in the management of aspergillosis is the availability of the newer broad-spectrum azoles, voriconazole and posaconazole, which have good activity against Aspergillus spp. In addition, newer diagnostic modalities including Aspergillus galactomannan, β-glucan, and polymerase chain reaction are more readily available. These diagnostic and treatment options have made new strategies possible for the management of IA. Prophylaxis and empirical therapy for high-risk patients have been popular for decades, and now a preemptive, targeted approach to IA management has become more attractive. This article reviews strategies for the prevention and management of IA and compares and contrasts universal prophylaxis to the preemptive, targeted approach for IA in high-risk patients.

An Atypical Case of Fatal Zygomycosis: Simultaneous Cutaneous and Laryngeal Infection in a Patient with a Non-neutropenic Solid Prostatic Tumor

We describe what we believe is the first reported case of simultaneous highly invasive cutaneous and laryngopharyngeal zygomycosis in a non-neutropenic, nondiabetic but immunosuppressed patient with prostate cancer. An invasive fungal process was not suspected until late in the patient's hospital course; when it was, a tracheotomy and direct laryngoscopic biopsies were performed. Unresectable invasive zygomycosis with Rhizopus rhizopodiformis was diagnosed. The patient was managed with liposomal amphotericin B initially and later with palliative medical therapy until he died. This case emphasizes the need for a rapid and specific diagnosis with timely introduction of appropriate antifungal management, particularly now that voriconazole is frequently used as empiric prophylaxis against aspergillosis in high-risk patients.

Preventative use of antifungal drugs in patients treated for cancer.

Prophylactic use of antifungal compounds has more or less become standard clinical practice for patients who are treated for a haematological malignancy. However, apart from the prevention of infections by Candida species in bone marrow transplant recipients and a possible reduction in invasive aspergillosis in high-risk patients, there is little evidence to justify this approach. Antifungals ought to be administered to patients on their perceived individual risk and better studies should be conducted to provide a more rational basis for our clinical decisions. Results of studies in specific populations should not be used to create guidelines for other patient groups or general populations. Antifungals are potentially toxic and overuse might be associated with unnecessary direct and indirect drug-related costs.

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

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

Invasive aspergillosis. Disease spectrum, treatment practices, and outcomes. I3 Aspergillus Study Group.

A review of representative cases of invasive aspergillosis was conducted to describe current treatment practices and outcomes. Eighty-nine physicians experienced with aspergillosis completed case forms on 595 patients with proven or probable invasive aspergillosis diagnosed using modifications of the Mycoses Study Group criteria. Pulmonary disease was present in 56%, with disseminated infection in 19%. The major risk factors for aspergillosis were bone marrow transplantation (32%) and hematologic malignancy (29%), but patients had a variety of underlying conditions including solid organ transplants (9%), AIDS (8%), and pulmonary diseases (9%). Overall, high antifungal failure rates occurred (36%), and complete antifungal responses were noted in only 27%. Treatment practices revealed that amphotericin B alone (187 patients) was used in most severely immunosuppressed patients while itraconazole alone (58 patients) or sequential amphotericin B followed by itraconazole (93 patients) was used in patients who were less immunosuppressed than patients receiving amphotericin B alone. Response rate for patients receiving amphotericin B alone was poor, with complete responses noted in only 25% and death due to or with aspergillosis in 65%. In contrast, patients receiving itraconazole alone or following amphotericin B had death due to or with Aspergillus in 26% and 36%, respectively. These results confirm that mortality from invasive aspergillosis in severely immunosuppressed patients remains high even with standard amphotericin B. Improved responses were seen in the less immunosuppressed patients receiving sequential amphotericin B followed by itraconazole and those receiving itraconazole alone. New approaches and new therapies are needed to improve the outcome of invasive aspergillosis in high-risk patients.

Specific detection of Aspergillus species in blood and bronchoalveolar lavage samples of immunocompromised patients by two-step PCR.

The increasing incidence of aspergillosis, a life-threatening infection in immunocompromised patients, emphasizes the need to improve the currently limited diagnostic tools. We developed a two-step PCR assay that specifically amplifies a region of the 18S rRNA gene that is highly conserved in Aspergillus species. A number of primers with the least homology to equivalent human or Candida gene sequences were screened for the pairs that gave the highest sensitivity and specificity. No cross-reaction with the wide range of fungal and bacterial pathogens so far tested was observed. This assay allows direct and rapid detection of down to 10 fg of Aspergillus DNA corresponding to 1 to 5 CFU per ml of blood. A total of 315 blood and bronchoalveolar lavage samples from 140 subjects, including 93 patients at risk for invasive fungal disease, were screened. The result was a 100% correlation between positive histology, culture, or high-resolution computed tomography findings and PCR results. The test specificity was 89%. Our data point to the considerable potential clinical value of this simple, specific, rapid, and inexpensive PCR assay for improving the means of early diagnosis of systemic aspergillosis in high-risk patients.