Galactomannan Antigen Research Articles

Distribution of Galactomannan antigen in BAL and serum samples among Aspergillus isolates and its correlation with culture among Immunocompetent and Immunocomporomised patients in Aligarh region, a north India town

Background: Aspergillosis is one of the most common fungal infection among both general patients and immunocompromised individuals. Methods: The study was carried out on 2 groups of patients: immunocompetent and immunocompromised comprising of a total of 90 patients. Microscopy, culture, identification of isolates was done and some specialised tests on serum and BAL for antigen detection were performed. Result: Most of the patients i.e., 47 (31.3%) were between 31-40 years with a mean age of 32.5 years. The male to female ratio was 1.8:1. Galactomannan antigen was positive in 13 (28.9%) BAL samples and in 15 (33.3%) serum samples wherein the 45 BAL samples represented immunocompetent cases and 45 serum samples represented immunocompromised cases. Sensitivity and specificity of GM assay in BAL samples were found to be 100% and 97% respectively. On the other hand, sensitivity and specificity in serum samples were found to be 94.1% and 100% respectively. All patients were categorized into 4 categories as proven IPA, probable IPA, possible IPA and non-IPA. 22 (24.4%) patients were of proven invasive pulmonary aspergillosis (IPA), 7 (7.8%) of probable IPA, 2 (2.2%) belonged to possible IPA while 59 (65.5%) were of non IPA. All of the cases of proven IPA were positive on culture. Conclusion: GM assay in BAL is a very sensitive and specific marker for Aspergillosis while in case of serum samples it is somewhat lesser sensitive.

Invasive Fungal Infections in Patients with Acute Myeloid Leukemia Undergoing Intensive Chemotherapy.

Patients with hematological malignancies are severely immunocompromised and are at high risk of invasive fungal infection (IFI), particularly those undergoing remission-induction chemotherapy for acute myeloid leukemia (AML). IFIs are a major cause of morbidity and mortality in such patients. We planned to study the incidence of IFI in patients with AML undergoing intensive chemotherapy and receiving antifungal prophylaxis. We retrospectively reviewed consecutive 46 patients with non-M3 AML, who received induction chemotherapy and systemic antifungal prophylaxis. None of the patients had IFI at the time of initiation of the chemotherapy. Patients were monitored for the occurrence of IFI using high-resolution computerized tomography of the chest or para-nasal sinus and test for galactomannan antigen on serum or broncho-alveolar lavage and were followed up for 90days. Of the 46 patients on intensive chemotherapies, 41, 4 and 1 patients were started on posaconazole, amphotericin B and voriconazole prophylaxis, respectively. The occurrence of possible and probable IFI was observed in 16 and 4 patients respectively, in which 19 patients were on posaconazole and 1 patient was on amphotericin-B prophylaxis. Overall mortality in the study population was 11 (23.9%). Four out of 20 patients died with IFI but none of the death was attributable to IFI. IFI still remains a significant cause of morbidity and mortality in patients with AML despite universal use of antifungal prophylaxis. With effective pharmacotherapy, the mortality due to IFI is preventable. Appropriate antifungal prophylaxis strategy still needs to be developed through larger and prospective studies.

Monoclonal Antibody AP3 Binds Galactomannan Antigens Displayed by the Pathogens Aspergillus flavus, A. fumigatus, and A. parasiticus.

Aspergillus fumigatus and A. flavus are the fungal pathogens responsible for most cases of invasive aspergillosis (IA). Early detection of the circulating antigen galactomannan (GM) in serum allows the prompt application of effective antifungal therapy, thus improving the survival rate of IA patients. However, the use of monoclonal antibodies (mAbs) for the diagnosis of IA is often associated with false positives due to cross-reaction with bacterial polysaccharides. More specific antibodies are therefore needed. Here we describe the characterization of the Aspergillus-specific mAb AP3 (IgG1κ), including the precise identification of its corresponding antigen. The antibody was generated using A. parasiticus cell wall fragments and was shown to bind several Aspergillus species. Immunofluorescence microscopy revealed that AP3 binds a cell wall antigen, but immunoprecipitation and enzyme-linked immunosorbent assays showed that the antigen is also secreted into the culture medium. The inability of AP3 to bind the A. fumigatus galactofuranose (Galf )-deficient mutant ΔglfA confirmed that Galf residues are part of the epitope. Several lines of evidence strongly indicated that AP3 recognizes the Galf residues of O-linked glycans on Aspergillus proteins. Glycoarray analysis revealed that AP3 recognizes oligo-[β-D-Galf-1,5] sequences containing four or more residues with longer chains more efficiently. We also showed that AP3 captures GM in serum, suggesting it may be useful as a diagnostic tool for patients with IA.

Role of Aspergillus Galactomannan Antigen Test for the Screening of Invasive Aspergillosis in Patients with Hematological Malignancies

Background: Invasive aspergillosis (IA) is a leading cause of death among immunocompromised patients, particularly those with hematological malignancies. The use of galactomannan (GM) antigen as a biological marker for screening of IA in high-risk patients is attractive and non-invasive tool that detect evidence of IA prior to the appearance of clinical manifestations.
 Objectives: The aim of this study was to compare the diagnostic value of the conventional blood culture technique to the serological detection of GM antigen using ELISA for screening of IA in neutropenic patients with hematological malignancies.
 Methods: Forty patients with haematological malignancies from those admitted to the Clinical Oncology Department of Menoufia University Hospitals (MUH) were enrolled and classified to have either proven (5/40; 12.5%), probable (10/40; 25%) or possible (25/40; 62.5%) invasive aspergillosis based on the clinical criteria provided by the European Organization for research and treatment of Cancer (EORTC) and Mycoses Study Group (MSG). Blood samples were collected from all participants and subjected to conventional blood culture for isolation and identification of Aspergillus spp. ELISA technique was applied for serological detection of GM antigen in the patients’ serum samples.
 Results: The sensitivity, specificity, PPV(positive predictive value) and NPV (negative predictive value of GM antigen ELISA testing were 100%, 74%, 36% and 100% respectively for both proven and probable cases. On applying the principle of test in series (the patient is positive if positive in both culture and the GM test), the results were improved to 100% sensitivity, 100% specificity, 100% PPV, 100% NPV and 100% accuracy. Galactomannan antigen testing proved excellent sensitivity (80%) compared to other clinical features and radiological criteria for diagnosis of probable aspergillosis and proved to be a good negative test.
 Conclusion: With conjunction of clinical and radiological signs, Aspergillus GM test can assist physicians in the of diagnosis of IA in patients with hematological malignancies to allow initiation of effective antifungal therapy which is ultimately important in high-risk populations.

The early diagnostic value of serum galactomannan antigen test combined with chest computed tomography for invasive pulmonary aspergillosis in pediatric patients after hematopoietic stem cell transplantation.

The purpose of our study was to evaluate the diagnostic value of serum galactomannan antigen (GM) testing combined with chest computed tomography (CT) of invasive pulmonary aspergillosis (IPA) in pediatric patients after hematopoietic stem cell transplantation. A retrospective nested case-control study was conducted in the identifying IPA among pediatric patients. A total of 141 eligible pediatric recipients with febrile neutropenia were enrolled in this study. All patients in the cases were diagnosed with proven-probable IPA(PP-IPA), while only 9 patients in the controls. GM testing was positive in 38 pediatric recipients in the cases and nine recipients in the controls with sensitivity of 62.3%, specificity of 81.8%. Among all patients with IPA, 46 patients in the cases and 9 patients in the controls had typical features of CT imaging with sensitivity of 79.3%, specificity of 85.2%. For discrimination of participants' GM testing combined with CT evaluation, the AUC of the diagnostic model was 0.887 with PPV of 0.764, and NPV of 0.872. Sensitivity was 0.793, and specificity was 0.852 in IPA. The combination methods with serum GM and CT scan might be used as a valuable marker for early diagnosis of IPA in pediatric patients after HSCT.

Epidemiology and treatment approaches in management of invasive fungal infections in hematological malignancies: Results from a single-centre study.

Invasive fungal infections (IFIs) are a leading cause of morbidity and attributable mortality in oncohematologic patients. Timely diagnosis is essential but challenging. Herein we retrospectively describe 221 cases of antifungal treatments (AFT) administered in a monocentric real-life cohort of hematological malignancies. Between January 2010 and July 2017, 196 oncohematologic patients were treated with AFT at our Hematology Department. Diagnosis of IFIs was carried out according to EORTC/MSG-2008 guidelines.The most represented disease was acute myeloid leukemia (104 patients). Median age was 61 years; at fever onset 177 (80%) patients had a neutrophil count<0.5x109/L. Twenty-nine (13%) patients were receiving antifungal prophylaxis (26 posaconazole, 2 fluconazole, 1 itraconazole). The incidence of AFT was 13%. Serum galactomannan antigen (GM) was positive in 20% of the tested cases, while 85% of the patients had a CT scan suggestive for IFI. Twenty-one percent of these cases had a GM positive. Sixty-five out of 196 patients (33%) showed positive culture results, in particular Candida spp. were identified in 45 isolates, while Aspergillus spp. in 16 cases. Fourteen patients presented multiple positivity. Twenty-two (10%) cases were classified as proven IFIs, 61 (28%) as probable and 81 (37%) as possible, but 57 (26%) cases could not be classified. Fifty-nine percent of the patients received single agent AFT, 37% sequential AFT, 8% a combination regimen. Liposomal-amphotericin-B was the most used AFT. IFIs attributable mortality was 20%. This epidemiologic survey underlined a persistent significant use of AFT and a high mortality rate of IFIs. We suggest that further powerful diagnostic approaches should be investigated to improve the diagnostic accuracy and potential therapeutic implication.

Applicability of the Platelia EIA® Aspergillus test for the diagnosis of aspergilosis in penguins.

Even today, an effective diagnostic test for aspergillosis in penguins is unknown, being the gold standard post-mortem examinations. The fungal antigen galactomannan (GM) has been used as a biomarker of disease in humans and is detected by the Platelia Aspergillus EIA (BioRad)®, a commercial kit based on the sandwich ELISA technique. It is standardized for use in neutropenic patients, however studies have demonstrated its usefulness also possible for birds. The aim of our study was to evaluate the effectiveness of Platelia Aspergillus EIA® test (BioRad-US) in the diagnosis of aspergillosis in Magellanic penguins, determining sensitivity, specificity, and positive and negative predictive values for different cut-off points. Were included in the study, blood serum samples (n = 29) Magellanic penguins in captivity that died by aspergillosis. Detection of GM was performed following manufacturer's instructions and the GM index was obtained by dividing the average value of OD of the duplicate of the clinical sample by duplicate OD of the average value of the cut-off sample provided by the kit. Through information database results were obtained for the presence of anti-Aspergillus fumigatus antibodies detected by agar gel immunodiffusion (AGID) for all serum samples. Results were analyzed using chi-square test and Kruskal-Wallis from SPSS 20.0, IBM®. ROC curve was obtained and from this, rates of sensitivity, specificity, positive and negative predictive values were also calculated based on four different cutoff points (0.5, 1.0, 1.5 and 2.0). The serum GM index did not differ between animals of the case and control group (pkw =0.097). In determining the ROC curve for serum GM detection the value of area under the curve was 0.635. From the values ​​determined by the coordinate of the curve, four different cut points (0.5, 1.0, 1.5 and 2.0) were analyzed, resulting in sensitivity rates ranging from 86.2 to 34.5% % and specificity between 87% and 26.1%. By comparing the serum GM index in group case as the presence or absence of antibodies detected by AGID was found p=0.503. The detection of GM the Platelia Aspergillus EIA® test seems is not be useful for the diagnosis of aspergillosis in naturally infected penguins.

Diagnostic role of polymerase chain reaction in bronchoalveolar lavage fluid for invasive pulmonary aspergillosis in immunocompromised patients – A retrospective cohort study

BackgroundThis study aimed to evaluate the diagnostic role of PCR detection of Aspergillus DNA in the broncho-alveolar lavage (BAL) fluid in a large cohort of patients suspected to have invasive pulmonary aspergillosis (IPA). MethodsConsecutive immunocompromised patients who underwent bronchoscopy with BAL sampling and PCR detection of Aspergillus DNA for the diagnosis of pulmonary infiltrates were included in the study. Galactomannan (GM) antigen testing in BAL and serum and BAL fungal culture were also performed. Patients were classified as having IPA (proven/probable/possible) or no-IPA according to the EORTC/MSG diagnostic criteria. ResultsDuring 12 years (2005–2016), 1248 bronchoscopies were performed for 1072 patients. 77% had hematological malignancy, of them 40% had AML and 35.6% underwent HSCT. IPA was diagnosed in 531 patients (42.5%), 7-proven, 280-probable and 244-possible. PCR was positive in 266 cases, of them 213 had IPA, indicating a true positive rate of 80% (213/266) and a false positive rate of 20% (53/266). These results establish the diagnostic performance of PCR to have sensitivity of 40%, specificity of 93%, PPV- 80% and NPV-68%. Of 244 patients with possible IPA, 80 had positive PCR. Including PCR in the diagnostic criteria would move 80 cases from the possible group to the probable one. A combination of positive PCR and/or BAL-GM increases sensitivity to 74%, while positivity of both tests elevates PPV to 99.4%. ConclusionsInclusion PCR for the detection of Aspergillus-DNA in BAL in the mycological criteria of the EORTC/MSG definitions increases the rate and the certainty of IPA diagnosis.

Cross-sectional study of respiratory Aspergillus spp. colonization or infection in patients with various stages of chronic obstructive pulmonary disease (COPD) using culture vs non-culture based technique

Background COPD patients are now recognized to be at increased risk of colonization by Aspergillus spp. which may progress to invasive pulmonary aspergillosis (IA). Published data on the frequency of Aspergillus detection in COPD are limited. Methods A cross-sectional study was undertaken to determine Aspergillus colonization or infection in COPD patients undergoing bronchoscopy for any indication. Culture as well as galactomannan antigen (GM) and Aspergillus nucleic acid detection (PCR) were performed on bronchoalveolar lavage fluid (BAL). Results One hundred and fifty patients were included (44.7 % female, mean age 68.2 years). 21.3 % were inpatients, 74.7 % outpatients and 4 % were ICU patients. Investigation of lung masses was the most common indication (43.3 %) for bronchoscopy. Most patients (81.3 %) were either GOLD stage 1 or 2 COPD. Cancer was the most frequent co-morbidity (60.48 %). 12 % and 48.7 % were on systemic and inhaled steroids respectively. Lung mass was the most common (28.43 %) CT imaging finding. Seventeen patients (11.3 %) had a positive result for Aspergillus (Culture+Galactomannan+PCR). 76.4 % out of these seventeen were in the early stages (GOLD stage 1 or 2) of COPD. Conclusion Aspergillus sp. was detected in 3.3 % of patients by culture, which increased to 11.3 % if culture was combined with either a positive GM or PCR result. Overall the frequency of Aspergillus detection in this population of COPD patients was low which may reflect the predominance of Gold stages 1 and 2 among the study population.

Serum galactomannan antigen and Aspergillus fumigatus antibodies in the diagnosis of invasive pulmonary aspergillosis

Serum galactomannan antigen and Aspergillus fumigatus antibodies in the diagnosis of invasive pulmonary aspergillosis

Clinical features and cause analysis of false positive results of Aspergillus galactomannan assay in pulmonary cryptococcosis patients.

There have been conflicting reports of false positive galactomannan assay results in patients with systemic cryptococcosis. We sought to determine the frequency of GM positivity in patients with pulmonary cryptococcosis and confirm the source of this cross-reactivity in vitro. We conducted a retrospective study to elucidate the rate of galactomannan (GM) false positivity and cause in a cohort of 29 patients with pulmonary cryptococcal disease. The production of GM cross-reacting substances by clinical isolates and laboratory isolates of C. neoformans was tested in vitro. The mean serum GM index (Platelia Aspergillus) in patients with pulmonary cryptococcosis was 1.06, with 16 (55.2%) of patients having values above the positive cutoff value of 0.5. GM index values significantly decreased after treatment of cryptococcosis. There was no significant correlation between galactomannan and cryptococcal glucuronoxylomannan antigen (Eiken Latex test) results. Culture supernatants from clinical isolates and wild-type C. neoformans did not react in the GM assay; however, growth in the presence of 6% sodium chloride induced the production of cross-reacting GM antigens in culture supernatants from clinical isolates, wild type and a glucuronoxylomannan-deficient mutant of C. neoformans, but not in culture supernatants from a galactoxylomannan-deficient strain. Our results support the cross-reactivity of cryptococcal galactoxylomannan with the serum GM assay in vitro and in patients with pulmonary cryptococcal infection.

Галактоманановий антиґен як один із маркерів діаґностики інвазійного аспергільозу

Галактоманановий антиґен як один із маркерів діаґностики інвазійного аспергільозу

Prevalence of pulmonary aspergillosis among chronic obstructive pulmonary disease patients presented with acute exacerbation

Introduction Acute exacerbation of chronic obstructive pulmonary disease (AECOPD) is a major event through the life of all chronic obstructive pulmonary disease (COPD) patients. This study was designed to assess the prevalence of pulmonary aspergillosis among COPD patients presented with acute exacerbation. Patients and methods A cross-sectional study was withheld and recruited 20 patients diagnosed with AECOPD who were admitted in the respiratory care unit (RCU). Sociodemographic data were recorded. All patients had undergone chest radiography together with serum galactomannan (GM) assay in order to detect pulmonary aspergillosis. Results Twenty patients diagnosed with AECOPD were admitted to the RCU, among which 30% of them died along their course of RCU stay. Of these patients, one-third was found to have pulmonary aspergillosis using serum GM. Serum GM showed no difference among patients who died compared with survived subjects; however, it was significantly higher among patients diagnosed with pulmonary aspergillosis. Using steroids in the past 3 months was not a risk factor for positive aspergillosis, whereas patients with the increased number of exacerbations showed an increased risk for aspergillosis. Conclusion Pulmonary aspergillosis is considered as one of the risk factors of AECOPD being detected among 20% of all studied patients. Frequent exacerbations are associated with pulmonary aspergillosis among COPD patients. Serum GM antigen is used as a marker of positive aspergillosis; however, it did not differ much among the patients who died; therefore, it could not be used as a marker of mortality among AECOPD patients.

Late Immune Reconstitution and Its Relationship with Infections in Patients with Fanconi Anemia and Bone Marrow Transplantation

BackgroundFanconi anemia (FA) is a condition characterized by congenital malformations, low height, and progressive bone marrow failure during childhood, genomic instability and hypersensitivity to DNA cross-linking agents. Bone marrow transplantation (BMT), is currently the only treatment capable to restore normal hematopoiesis and improve survival of these patients. To achieve a successful allogeneic BMT, a normal T-cell immunity reconstitution is required.1,2ObjectiveTo describe the kinetics of immune reconstitution in six patients with FA after BMT, having used a reduce intensity conditioning regimen, well as associated infections during the post-transplant process.MethodsWe describe the distribution in peripheral blood of CD3+ T lymphocytes, CD16 +/CD56+ NK cells, CD4+ helper T lymphocytes, CD8+ cytotoxic T lymphocytes and CD19+/CD20+ B cells, in six patients with FA, after BMT from an HLA-matching sibling and complete chimerism, on days +90, +120, +150, +180, +210 and +360. The conditioning regimen employed consisted of fludarabine (Flu) 150 mg/m2, cyclophosphamide 20 mg/kg and rabbit anti-thymocyte globulin (r-ATG) 20 mg/kg. Infection was determined by a positive reaction of the DNA polymerase chain to Cytomegalovirus (CMV), Epstein-Barr virus, Adenovirus and BK virus (BKV), as well as galactomannan antigen and antibodies against Candida sp, or positive bacterial cultures.ResultsWe observed different kinetics regarding the recovery of different lymphocyte subpopulations, starting from day +90. CD16+/CD56+ NK cells recovered first, between days 90 and 120, followed by CD8+ T lymphocytes between day +120 and +150, CD19+/CD20+ B cells between day +180 and +210, and finally, CD4+ T lymphocytes starting from day 210. Five patients presented infection in the post-BMT stage. Four patients before +90, developed CMV infection, based on positive reaction of the CMV polymerase chain. All patient responses to ganciclovir therapy and no CMV disease were documented. The fifth patient presented with meningitis due to Lysteria monocitogenes and BKV hemorrhagic cystitis at day +153; this patient had the diagnosis of chronic-graft versus host disase, and was treated with ampicillin, rifampicin and intravenous immunoglobulin, evolving satisfactorily. All patients remain alive and well.DiscussionMost patients in our report developed CMV infection, as being reported with FLU and anti-thymocyte globulin combination. We observed a later reconstitution in comparison with patients in whom lower doses of r-ATG (<10 mg/kg) have been used3. In our group of patients, reconstitution of NK cells and CD8+ T lymphocytes, helped to reduce the frequency of asymptomatic viral infections, as reconstitution of innate immunity and cytotoxic CD8+ T lymphocytes can achieve effective antiviral effector responses, allowing for the control of viral infections, until reconstitution of CD4+ T lymphocytes were fully achieved. The infectious complications associated with this prolonged state of profound immunosuppression and late immune reconstitution, can be attenuated with a closely follow-up for these infectious agents through PCR-mediated detection techniques, which allows an early diagnosis and prompt treatment.ConclusionImmunological reconstitution after a BMT in FA patients is determinant for morbidity and mortality, mainly due to opportunistic infections and GVHD. We observed that recovery kinetics of different lymphocyte populations is different in cases in which higher doses of r-GAT are used, also an early detection and prompt treatment of opportunistic infections may be determinant for patient's survival.Conflict-of-interest disclosure: The authors declare they have nothing to disclose.BibliographyOgonek J, Kralj Juric M, Ghimire S, Varanasi PR, Holler E, Greinix H, et al. Immune reconstitution after allogenic hematopoietic stem cell transplantation. Front immunol. 2016; 7 (507):1-15.Smith AR, Wagner JE. Current clinical management of Fanconi anemia. Expert Rev Hematol. 2012; 5 (5): 513-522.Perlingeiro-Beltrame M, Malvezzi M, Bonfim C, Tadeu Covas D, Pasquini R. Immune reconstitution in patients with Fanconi anemia after allogeneic bone marrow transplantation. Cytotherapy. 2014; 0: 1e14. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Prevalence and Clinical Characteristics of False-Positive Aspergillus Galactomannan Test in Healthy Subjects and Patients with Hematologic Malignancies

▪Background: Monitoring of the Aspergillus galactomannan (GM) antigen is an useful tool for early diagnosis of aspergillosis, especially in patients with hematologic malignancies. However, one of the major limitations of this assay is false-positivity, which has been described in pediatric patients, patients with graft-versus-host disease, and those taking dietary GM or certain antibiotics. Only a few studies have shown high incidence of false-positive GM test results in patients with multiple myeloma (MM). Here we investigated the prevalence of false-positive GM test results in healthy subjects and in patients with hematologic malignancies.Methods: First, we analyzed the prevalence and distribution of false-positive GM test results in healthy subjects. Next, we retrospectively analyzed the prevalence and clinical characteristics of false-positive GM test results in consecutive patients with symptomatic MM diagnosed between 2006 and 2018 at Kameda Medical Center, Kamogawa, Japan, and compared these findings with those of our historical cohort of patients with hematologic malignancies other than plasma cell disorders (consecutive patients with non-Hodgkin lymphoma [NHL] and myelodysplastic syndrome [MDS]). Patients with clinical and/or imaging findings consistent with aspergillosis (e.g. sinopulmonary infection) were excluded from the analysis. Serum GM was measured using the double-sandwich enzyme-linked immunosorbent assay (Platelia, Bio-Rad, France) in all subjects before any use of antibiotics or anticancer agents. Serum GM test results were considered positive for optical index value of ≥0.5 in duplicate tests.Results: We enrolled 572 healthy subjects. The median age was 46 years (interquartile range [IQR]: 30-55 years), and 267 (46.7%) were men. We found a positive correlation between the GM antigen levels and age; GM antigen levels tended to be higher in older patients (Figure 1A), and the patients with false-positive GM results were significantly older than those without (median: 54 and 43 years, respectively; P<0.001). We also found that the GM levels were significantly higher in men than in women (P<0.001, Figure 2A); false-positive GM test results were more prevalent in men than in women (21.0% and 9.8%, respectively; P<0.001, Figure 2B). The number of patients with MM, NHL, and MDS was 244, 187, and 112, respectively. There was no inter-group difference in age across the three groups (median: 73, 73, and 74 years, respectively; P=0.95), whereas male patients were significantly more prevalent among patients with MDS (67.9%) and less prevalent among those with MM (46.7%) (P=0.031). Nevertheless, the patients with MM showed significantly higher GM antigen levels and a higher frequency of false-positive GM test results compared to those with NHL or MDS (Figure 3A and 3B). We further investigated the myeloma-related factors, which were associated with the high incidence of the false-positive GM test results in MM, and found that patients with IgG-type MM had significantly higher GM antigen levels and a significantly higher frequency of the false-positive GM test results than those with IgA-type or light-chain-only MM (Figure 4A and 4B). Among the patients with IgG-type MM, IgG levels in patients with positive GM test results were higher than those with negative GM test results, although the difference was not statistically significant (median: 5092.0 and 4559.5 mg/dL, respectively; P=0.10, Figure 5A), whereas no difference in IgG levels was detected between patients with positive and negative results on GM tests among non-IgG-type MM patients (Figure 5B). IgG levels in patients with positive GM test results were significantly higher than in those with negative results among both patients with NHL and MDS (P=0.013 and 0.041, respectively; Figure 6).Conclusions: Our findings revealed significantly high frequencies of false-positive GM test results in the elderly and in men among healthy subjects. We also found a significantly higher frequency of false-positive GM test results in patients with MM, especially in those with IgG-type myeloma, when compared with patients having lymphoid and myeloid neoplasia. These results indicated that careful interpretation of the GM test results may be required in these populations in the assessment of possible aspergillosis. Further studies are required to validate our results and elucidate the mechanisms of these phenomena. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Utility of serum galactomannan antigen testing combined with chest computed tomography for early diagnosis of invasive pulmonary aspergillosis in patients with hematological malignancies with febrile neutropenia after antifungal drug treatment.

ObjectiveTo investigate the value of serum galactomannan antigen (GM) testing combined with chest computed tomography (CT) as a noninvasive method for early diagnosis of invasive pulmonary aspergillosis (IPA) in patients with hematological malignancies with febrile neutropenia after antifungal drug treatment.MethodsWe retrospectively analyzed the data of 376 patients with febrile neutropenia from January 2015 to August 2017. All patients were given broad-spectrum antibiotics and divided into the control group (effective antibiotic treatment, no antifungal drugs given) and the observational group (ineffective antibiotic treatment, antifungal drugs given). The serum GM testing, chest CT, and microbiological examination findings were compared between the two groups.ResultsThe false-positive rates of GM testing for IPA in the control and observational groups were 4.04% and 8.65%, respectively, and the false-negative rates in the two groups were 1.10% and 9.62%, respectively. Sixty-five patients in the observational group and 11 in the control group had typical features of CT imaging.ConclusionClinical weekly screening of serum GM and chest CT may be an effective combined approach to the early diagnosis of IPA in patients with febrile neutropenia, even if they have undergone antifungal treatment.

APPLICATION OF 3-HYDROXYBUTYRATE MEASUREMENT AND PLASMA PROTEIN ELECTROPHORESIS IN THE DIAGNOSIS OF ASPERGILLOSIS IN AFRICAN PENGUINS ( SPHENISCUS DEMERSUS).

New alternative laboratory means are needed to improve the options for antemortem diagnosis of avian aspergillosis. In this study, 3-hydroxybutyrate was measured in plasma samples collected from a cohort of African penguins ( Spheniscus demersus) maintained under human care. Results were interpreted in combination with those of protein electrophoresis and compared with anti- Aspergillus antibody and galactomannan antigen detection. Overall, 3-hydroxybutyrate levels were found significantly increased in Aspergillus-diseased cases versus the control penguin group ( P = 0.002). Mean absolute concentration of β-globulins was increased >20% in samples from infected birds, and α2-globublins were also found to be significantly increased versus clinically normal controls ( P < 0.001 and P = 0.001 respectively). Of note, the α2-globulins were also significantly increased versus penguins with inflammatory (non-aspergillosis) diseases ( P = 0.001). The specificity of 3-hydroxybutyrate, β-globulins, and α2-globulins for aspergillosis was 78.6%, 79.6%, and 92.2%, respectively. Using these measures in tandem resulted in high specificity (>90%) and negative predictive value (≥80%). In contrast, anti- Aspergillus antibody and galactomannan antigen did not distinguish between infected cases and controls ( P > 0.05). This study demonstrates that basic testing in tandem with the new biomarker 3-hydroxybutyrate may provide reliable evidence for the diagnosis of aspergillosis in penguins.

Diagnosis and treatment of histoplasmosis in solid organ transplant patients.

Unlike immunocompetent hosts, solid organ transplant (SOT) recipients with posttransplant histoplasmosis (PTH) often present with disseminated disease and have an attributable mortality of approximately 10%. In this review, we discuss currently available diagnostic tests and treatment strategies in PTH. None of the available tests have a 100% diagnostic accuracy. Histoplasma antigen assays are the most sensitive commercially available tests. However, crossreactivity of histoplasma antigen with aspergillus galactomannan and false positive histoplasma antigen tests because of rabbit antithymocyte globulin may cause difficulty in interpreting positive test results in transplant recipients. Molecular assays such as amplification and sequencing of 'panfungal' portions of the 28S ribosomal RNA from clinical specimens appear to be promising.Lipid formulations of amphotericin B and itraconazole are the drugs of choice in the treatment of PTH. Other extended spectrum azoles also appear to be effective, but, like itraconazole, problems with drug interactions and prolongation of the QTc interval (except for isavuconazole, which shortens the QTc interval) remain. Mycophenolate therapy is associated with severe disease and should be stopped during active disease and, if feasible, calcineurin inhibitors and steroids should be reduced. A combination of various tests (culture, antigen tests, nucleic amplification tests, etc.) should be used to optimize diagnostic yield. The role of unbiased next generation sequencing for early diagnosis and newer azoles in the treatment needs to be further explored.

Local retrospective analysis of galactomannan cut-off values in bronchoalveolar lavage fluids for diagnosis of invasive aspergillosis.

Galactomannan antigen (GM) testing has been used for decades to screen immunocompromised patients for invasive aspergillosis (IA). Recent publications suggested that using a higher cut-off value than 0.5 in bronchoalveolar lavage fluid (BALF) could be more discriminant for hematology patients. We retrospectively analyzed the values of GM in BALF over 7years (from 2010 to 2016). Performance indicators of the GM in BALF, according to three different cut-off values (0.5, 0.8, 1.5), were calculated using Stata 14.1. IA classification for hematology patients was based on European Organization for Research and Treatment of Cancer/Mycoses Study Group (EORTC/MSG) criteria, as defined in 2008. A number of 716 GM were performed on BALF from 2010 to 2016 (597 patients) and 66 were positive (> 0.5). Among these 597 patients, 27 IA were diagnosed, 13 with a positive GM in BALF, 9 with a negative GM in BALF, and 5 unclassified IA (ICU patients). The analysis of performance indicators, based on our local data, did not demonstrate any significant difference using a higher cut-off value of GM in BALF. This result may be explained by the local recruitment of patients and by pre-analytic variations during BALF realization.

Use of Galactomannan Antigen and Aspergillus DNA Real-time Polymerase Chain Reaction as Routine Methods for Invasive Aspergillosis in Immunosuppressed Children in Greece

PurposeInvasive aspergillosis (IA) remains a critical issue in immunosuppressed patients. Detection of galactomannan antigen (GM) in serum samples is included as a criterion of IA by the European Organization for the Research and Treatment of Cancer/Mycoses Study Group. Nevertheless, Aspergillus DNA detection by polymerase chain reaction (PCR) has not yet been included because clinical data validation is lacking. The present study describes the simultaneous performance of GM and PCR tests as routine methods for IA diagnosis. MethodsDuring the period January 2012 to December 2017, a total of 156 white children hospitalized in a tertiary children’s hospital of Athens (97 boys and 59 girls; age range, 5 months–14 years) were examined as possible cases of IA. Patients were classified into 4 groups based on their underlying diseases: hematologic malignancies (107 of 156 [68.6%]), solid tumors (16 of 156 [10.2%]), primary immunodeficiency (12 of 156 [7.7%]), and hereditary immunodeficiency (21 of 156 [13.5%]). GM detection was made with the Platelia Aspergillus Ag kit (Bio-Rad Laboratories, Hercules, California). Sera with a cut-off index ≥0.5 on at least 2 separate blood collections were considered positive. Serum detection of Aspergillus DNA was conducted with real-time PCR MycAssay Aspergillus assay (Myconostica Ltd, Cambridge, United Kingdom). PCR positivity was determined by using a threshold of 38 cycles in at least 1 serum sample. Four or more successive samples per patient were tested. FindingsOverall, 28 of 156 patients (53 of 744 serum samples) were found positive. Eleven patients were positive using both methods (24 samples). Four children were positive only by PCR (6 samples), whereas 13 (23 samples) were positive only with GM in consecutive samples. Agreement of both methods, GM(+)/PCR(+) or GM(–)/PCR(–), was found in 139 patients (90% of total patients) and 715 samples (96.1% of total samples). The agreement of both methods was found: (1) 85% in patients with hematologic malignancies; (2) 100% in patients with solid tumors; (3) 97.5% in patients with primary immunodeficiency; and (4) 98.8% in patients with hereditary immunodeficiency. Overall disagreement was observed in 17 patients, in which the positive result in any of the 2 methods was estimated as true positive in conjunction with radiologic and other clinical findings. ImplicationsThe combination of GM and PCR, provided high diagnostic accuracy in consecutive samples (twice a week). Clinical, radiologic, and other laboratory findings should be taken into consideration in the evaluation of GM and PCR.