Early Diagnosis Of Invasive Aspergillosis Research Articles

Detection of Aspergillus Galactomannan in Blood for Early Diagnosis of Pulmonary Aspergillosis in HIV Positive Subjects with and without Tuberculosis in Calabar, Nigeria

This study was aimed at determining the prevalence of pulmonary aspergillosis using Aspergillus galactomannan detection in human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/ AIDS) subjects with and without tuberculosis. The study was a prospective cohort study carried out at the University of Calabar Teaching Hospital and Dr. Lawrence Henshaw Memorial Hospital in Calabar. Subjects were HIV-positive patients with and without tuberculosis. Blood and sputum samples were obtained from 215 subjects who consented to the study. A structured questionnaire was administered to assess demograph¬ic data and medical history. Sputum samples were subjected to direct microscopy, culture, and Ziehl-Nel¬seen test. Aspergillus galactomannan assay and CD4 counts were performed on the blood samples. The results of Aspergillus galactomannan-positive subjects were communicated to the attending Physicians for proper patient management. Aspergillus species were recovered from 35(16.3%) sputum samples. Asper¬gillus fumigatus was the most encountered isolate 18(8.4%). The most common presenting symptoms were cough 35(100%) and fatigue 30(85.7%). Out of the 35 subjects with Aspergillus isolates, 21(60.0%) had TB, 9(25.7%) had no TB and 5(14.3%) had co-morbidity of HIV and TB. All the subjects with aspergillosis had CD4 counts less than 200 cellsμl-1 and there was a statistically significant association between pulmonary aspergillosis and CD4 levels (H=7.02; p = 0.03). Galactomannan assay could be used for early diagnosis of invasive aspergillosis and timely management of patients as it is sensitive, cost-effective, and has a turna¬round time of less than 1 hour compared to culture methods.

Reliability of the galactomannan enzyme-linked immunoassay in healthy preterm infants: A prospective cohort study.

In healthy preterm infants, the galactomannan enzyme-linked immunoassay shows a high rate of positive results and should not be used for the early diagnosis of invasive aspergillosis, particularly in the very preterm age group.

Performance of the JF5-Based Galactomannoprotein EIA Compared to the Lateral Flow Device and the Galactomannan EIA in Serum and Bronchoalveolar Lavage Fluid.

Early diagnosis of invasive aspergillosis is an important factor to improve survival but remains challenging. The detection of Aspergillus antigens is included in the consensus case definitions of the European Organization for Research and Treatment of Cancer and the National Institute of Allergy and Infectious Diseases Mycoses Study Group as a criterion of "probable" invasive aspergillosis. JF5, a mouse IgG3 monoclonal antibody detecting an Aspergillus mannoprotein, has already been implemented as a lateral flow device (LFD). Now, also a JF5-based enzyme-linked immunosorbent assay (EIA) is commercialized (Aspergillus specific galactomannoprotein [GP] EIA, Euroimmun Medizinische Labordiagnostika AG). In this study, we analyzed the diagnostic performance of GP in 63 bronchoalveolar lavage fluid (BALf) samples and 224 serum samples and compared it to performance of the galactomannan (GM) (Platelia Aspergillus enzyme immunoassay (EIA) (Bio-Rad, Marnes-la-Coquette, France)) and the JF5-based LFD (AspLFD; OLM Diagnostics, Newcastle Upon Tyne, United Kingdom). The diagnostic performance of GP and GM correlated well with both having high specificity. With an optimized cutoff threshold for positivity of 0.4-deviating from the 0.5 threshold recommended by the manufacturer-sensitivity of GP in serum is not significantly different than that of GM. However, in BALf sensitivity of GP is significantly less than for GM.

Evaluation of (1,3)--d-glucan assay in diagnosis of invasive fungal infections with Aspergillus

Invasive fungal infections caused by Aspergillus are a significant problem in immunocompromised and critically ill patients and associated with increased morbidity and mortality. Early diagnosis of invasive aspergillosis is still a big clinical and diagnostic challenge. Conventional methods are not sensitive enough, and therefore, there is a need for rapid, more sensitive methods for early diagnosis of invasive fungal infections with Aspergillus. The aim of this study was to evaluate the diagnostic performance, sensitivity and specificity of serological panfungal (1,3)-b-D-glucan marker compared to conventional method for diagnosis of invasive fungal infections with Aspergillus. Material and methods: Specimens of 125 patients divided into 4 groups (group I - immune deficiency, group II - prolonged ICU stay, group III - chronic aspergillosis, group IV - cystic fibrosis), classified according to clinical diagnosis and EORTC/MSG criteria, were analyzed at the Institute of Microbiology and Parasitology, with conventional and serological methods, during a period of two years. Results: A total of 71 isolates of Aspergillus were confirmed in this study. Four isolates were recovered from bloodculture of patients with primary immune deficiency. With BAL culture, Aspergillus was detected in the group of chronic aspergillosis (63.33%), followed by the groups of cystic fibrosis (56.67%), primary immune deficiency (51.43%), and the group with prolonged ICU stay (43.33%). Sensitivity and specificity of BAL culture were: 64.29% and 100%, 59.09% and 100%, 54.55% and 12.5%, 100% and 54.17%, in I, II, III and IV group, respectively. In 79.1% (53/67) from positive BAL cultures in all groups, A. fumigatus was confirmed, of which, 32.1% (17/53) in group III, followed by group I – 26.42% (14/53) and group IV – 26.42% (14/53), and 15.1% (8/53) in group II. Other species confirmed in BAL were A. flavus 16.42% (11/67) and A.terreus 4.48% (3/67). Sensitivity and specificity of the serological panfungal (1,3)-b-D-glucan (BDG) marker were: 64.71% and 85.71%, 50% and 87.5%, 36.36% and 50%, in groups I, II and III, respectively. No positive findings of the panfungal (1,3)-b-D-glucan (BDG) marker were found in the group with cystic fibrosis. Conclusion: The results obtained in this study have demonstrated that a positive (1,3)-b-D-glucan assay highlights the value of this test as a diagnostic adjunct in the serodiagnosis of invasive fungal infections with Aspergillus, and along with the results from conventional mycological investigation, helped in reaching a timely antifungal treatment with a favorable clinical outcome.

Role of Nasal Lavage Fluid Galactomannan Levels For Diagnosis of Invasive Aspergillosis in Patients with Leukemia

Background: Invasive aspergillosis (IA) is the most prevalent invasive infection with high mortality among patients with leukemia. Early diagnosis of IA has been a challenging topic in this group of patients. Objectives: In this study, we evaluated the galactomannan levels of nasal lavage fluid (NALF) as a possible auxiliary method for IA diagnosis in patients with leukemia. Methods: In a prospective study, 32 adult patients with leukemia who were taking induction and/or consolidation chemotherapy with fever and neutropenia were included. In all patients, galactomannan (GM) levels of serum and NALF, and mycological examinations were evaluated before the first dose of antifungal therapy. Results: Fourteen (43.7%) patients had NALF GM ≥ 0.5; however, in 16 (50%) patients the level of serum GM was ≥ 0.5. The elevated level of NALF GM had a significant association with the proven IA cases (P = 0.048). The GM level of NALF with a cut-off value of 0.45 (by receiver operating characteristic curve analysis) had 78% sensitivity and 64% specificity for the diagnosis of invasive aspergillosis (P = 0.033). Conclusions: Due to its non-invasive nature, GM level of NALF may be contributory to be used as part of the diagnostic work‐up of IA particularly in leukemic patients with thrombocytopenia which prohibits acquiring bronchoalveolar lavage.

Comparison of β-D-Glucan and Galactomannan in Serum for Detection of Invasive Aspergillosis: Retrospective Analysis with Focus on Early Diagnosis.

The early diagnosis of invasive aspergillosis (IA) relies mainly on computed tomography imaging and testing for fungal biomarkers such as galactomannan (GM). We compared an established ELISA for the detection of GM with a turbidimetric assay for detection of the panfungal biomarker β-D-glucan (BDG) for early diagnosis of IA. A total of 226 serum specimens from 47 proven and seven probable IA cases were analysed. Sensitivity was calculated for samples obtained closest to the day of IA-diagnosis (d0). Additional analyses were performed by including samples obtained during the presumed course of disease. Most IA cases involved the respiratory system (63%), and Aspergillus fumigatus was the most frequently isolated species (59%). For proven cases, sensitivity of BDG/GM analysis was 57%/40%. Including all samples dating from –6 to +1 weeks from d0 increased sensitivities to 74%/51%. Sensitivity of BDG testing was as high as or higher than GM testing for all subgroups and time intervals analysed. BDG testing was less specific (90–93%) than GM testing (99–100%). Combining BDG and GM testing resulted in sensitivity/specificity of 70%/91%. Often, BDG testing was positive before GM testing. Our study backs the use of BDG for diagnosis of suspected IA. We suggest combining BDG and GM to improve the overall sensitivity.

Study on invasive aspergillosis using galactomannan enzyme immunoassay and determining antifungal drug susceptibility among hospitalized patients with hematologic malignancies or candidates for organ transplantation

The incidence of invasive aspergillosis (IA) has dramatically increased during the last decade. This infection is associated with high morbidity and mortality, ranging from 30% to 70%, especially in immunocompromised patients. Delay in diagnosis and treatment is usually associated with high mortality rates. This study was aimed to assess the diagnostic value of Galactomannan EIA (GM) for early diagnosis of aspergillosis in hospitalized patients with underlying conditions. Also, the antifungal drug susceptibility profiles of causative agents were investigated. In this descriptive cross-sectional study, during the period of 18 months starting from September 2017 until February 2019, 22 bronchoalveolar lavage (BAL) and 13 biopsies from infected sinuses were obtained from a total of 150 patients suffering from different types of hematologic malignancies. All the samples were subjected to microscopic examination and fungal culture. Also, serum specimens were obtained from all patients (n = 135). 22 serum and 17 BAL specimens were tested for the GM level. Fungal identified were confirmed through the PCR-sequencing of the β-tubulin gene. The susceptibility to amphotericin B, itraconazole, voriconazole, posaconazole, ravuconazole, and caspofungin was evaluated according to the Clinical and Laboratory Standards Institute document M38-A2 (CLSI M38-A2) broth microdilution protocol. The results showed that the incident rate of IA was 23.33% and 35 patients with IA (12 proven cases and 23 probable cases) were diagnosed according to the European Organization for Research and Treatment of Cancer and Mycoses Study Group criteria. The 35 patients with IA in the current study comprised 19 men (54.29%) and 16 women (45.71%) with the median age of 42 years. AML (31.5%) was documented as the most prevalent risk factor among our subjects with IA and Aspergillus flavus (65.7%) was the most prevailing causal agent in this study. Among patients with IA, ague (71%) and cough (60%) were the most common symptoms. In the present study, a sensitivity of 94% and a specificity of 98% was reported for GM ELISA in BAL specimens. Also, a sensitivity of 58% and a specificity of 98% was reported for GM ELISA in serum samples. Among 6 tested antifungal drugs, the lowest minimum inhibitory concentration (MIC) values were observed for posaconazole and ravuconazole which showed the range of 0.008–0.0062 μgml and 0.031–0.125 μgml, respectively. The current study has demonstrated that determining the value of GM investigation in BAL and serum specimens can be promising in early diagnosis of IA, also molecular identification of the agents causing IA and their antifungal susceptibility patterns are essential issues for the targeted antifungal therapy and outcome improvement of patients with this life-threatening disease.

Evaluation of Commercially Available Real-Time Polymerase Chain Reaction Assays for the Diagnosis of Invasive Aspergillosis in Patients with Haematological Malignancies.

Early diagnosis of invasive aspergillosis (IA) is a challenge. Non-specific clinical and radiologic findings, as well as difficulties in conventional diagnostic method application, may delay correct diagnosis. Nowadays, nucleic acid-based assays have reduced the need for conventional antigen detection and culture-based methods and provided new opportunities for patient care. Aspergillus PCR is now included in the latest European Cancer Research and Treatment Organization/Mycosis Study Group definition updates. We evaluated the performance of commercial real-time polymerase chain reaction (PCR) MycAssay Aspergillus PCR and Artus Aspergillus RG PCR assays and compared the results with galactomannan enzyme immunoassay. During 41 febrile neutropenic episodes, 168 serum samples were collected from 32 patients with haematological malignancies. IA diagnosis was established according to the revised guidelines of the European Organization for Research and Treatment of Cancer/Mycoses Study Group. Twenty-one probable episodes were identified. There were no proven IA cases in the study. In 20 episodes, patients did not fulfil the established criteria for the IA diagnosis. Artus Aspergillus RG PCR assay had a sensitivity of 47.6% and specificity of 100%, while those of MycAssay Aspergillus PCR were 61.9% and 100%, respectively. Two different PCR assays were used in this study. Although there are many studies that evaluated MycAssay Aspergillus PCR, data regarding Artus Aspergillus RG PCR assay are scarce. We found moderate sensitivity and high specificity in the diagnosis of IA in patients with haematological malignancy in both PCR methods. Our results demonstrated that commercial PCR assays can be applied for the early diagnosis and pre-emptive treatment of IA.

Polymerase chain reaction blood tests for the diagnosis of invasive aspergillosis in immunocompromised people

This is an update of the original review published in the Cochrane Database of Systematic Reviews Issue 10, 2015.Invasive aspergillosis (IA) is the most common life-threatening opportunistic invasive mould infection in immunocompromised people. Early diagnosis of IA and prompt administration of appropriate antifungal treatment are critical to the survival of people with IA. Antifungal drugs can be given as prophylaxis or empirical therapy, instigated on the basis of a diagnostic strategy (the pre-emptive approach) or for treating established disease. Consequently, there is an urgent need for research into both new diagnostic tools and drug treatment strategies. Increasingly, newer methods such as polymerase chain reaction (PCR) to detect fungal nucleic acids are being investigated. To provide an overall summary of the diagnostic accuracy of PCR-based tests on blood specimens for the diagnosis of IA in immunocompromised people. We searched MEDLINE (1946 to June 2015) and Embase (1980 to June 2015). We also searched LILACS, DARE, Health Technology Assessment, Web of Science and Scopus to June 2015. We checked the reference lists of all the studies identified by the above methods and contacted relevant authors and researchers in the field. For this review update we updated electronic searches of the Cochrane Central Register of Controlled Trials (CENTRAL; 2018, Issue 3) in the Cochrane Library; MEDLINE via Ovid (June 2015 to March week 2 2018); and Embase via Ovid (June 2015 to 2018 week 12). We included studies that: i) compared the results of blood PCR tests with the reference standard published by the European Organisation for Research and Treatment of Cancer/Mycoses Study Group (EORTC/MSG); ii) reported data on false-positive, true-positive, false-negative and true-negative results of the diagnostic tests under investigation separately; and iii) evaluated the test(s) prospectively in cohorts of people from a relevant clinical population, defined as a group of individuals at high risk for invasive aspergillosis. Case-control and retrospective studies were excluded from the analysis. Authors independently assessed quality and extracted data. For PCR assays, we evaluated the requirement for either one or two consecutive samples to be positive for diagnostic accuracy. We investigated heterogeneity by subgroup analyses. We plotted estimates of sensitivity and specificity from each study in receiver operating characteristics (ROC) space and constructed forest plots for visual examination of variation in test accuracy. We performed meta-analyses using the bivariate model to produce summary estimates of sensitivity and specificity. We included 29 primary studies (18 from the original review and 11 from this update), corresponding to 34 data sets, published between 2000 and 2018 in the meta-analyses, with a mean prevalence of proven or probable IA of 16.3 (median prevalence 11.1% , range 2.5% to 57.1%). Most patients had received chemotherapy for haematological malignancy or had undergone hematopoietic stem cell transplantation. Several PCR techniques were used among the included studies. The sensitivity and specificity of PCR for the diagnosis of IA varied according to the interpretative criteria used to define a test as positive. The summary estimates of sensitivity and specificity were 79.2% (95% confidence interval (CI) 71.0 to 85.5) and 79.6% (95% CI 69.9 to 86.6) for a single positive test result, and 59.6% (95% CI 40.7 to 76.0) and 95.1% (95% CI 87.0 to 98.2) for two consecutive positive test results. PCR shows moderate diagnostic accuracy when used as screening tests for IA in high-risk patient groups. Importantly the sensitivity of the test confers a high negative predictive value (NPV) such that a negative test allows the diagnosis to be excluded. Consecutive positives show good specificity in diagnosis of IA and could be used to trigger radiological and other investigations or for pre-emptive therapy in the absence of specific radiological signs when the clinical suspicion of infection is high. When a single PCR positive test is used as the diagnostic criterion for IA in a population of 100 people with a disease prevalence of 16.3% (overall mean prevalence), three people with IA would be missed (sensitivity 79.2%, 20.8% false negatives), and 17 people would be unnecessarily treated or referred for further tests (specificity of 79.6%, 21.4% false positives). If we use the two positive test requirement in a population with the same disease prevalence, it would mean that nine IA people would be missed (sensitivity 59.6%, 40.4% false negatives) and four people would be unnecessarily treated or referred for further tests (specificity of 95.1%, 4.9% false positives). Like galactomannan, PCR has good NPV for excluding disease, but the low prevalence of disease limits the ability to rule in a diagnosis. As these biomarkers detect different markers of disease, combining them is likely to prove more useful.

Triacetylfusarinine C: A urine biomarker for diagnosis of invasive aspergillosis

Early diagnosis of invasive aspergillosis (IA) remains challenging, with available diagnostics being limited by inadequate sensitivities and specificities. Triacetylfusarinine C, a fungal siderophore that has been shown to accumulate in urine in animal models, is a potential new biomarker for diagnosis ofIA. We developed a method allowing absolute and matrix-independent mass spectrometric quantification of TAFC. Urine TAFC, normalized to creatinine, was determined in 44 samples from 24 patients with underlying hematologic malignancies and probable, possible or no IA according to current EORTC/MSG criteria and compared to other established biomarkers measured in urine and same-day blood samples. TAFC/creatinine sensitivity, specificity, positive and negative likelihood ratio for probable versus no IA (cut-off ≥ 3) were 0.86, 0.88, 6.86, 0.16 per patient. For the first time, we provide proof for the occurrence of TAFC in human urine. TAFC/creatinine index determination in urine showed promising results for diagnosis of IA offering the advantages of non-invasive sampling. Sensitivity and specificity were similar as reported for GM determination in serum and bronchoalveolar lavage, the gold standard mycological criterion for IA diagnosis.

Improving the outcome of invasive fungal diseases: Early diagnosis of invasive Aspergillosis

Improving the outcome of invasive fungal diseases: Early diagnosis of invasive Aspergillosis

Galactomannan antigen detection as a screening tool for early diagnosis of invasive aspergillosis: Experience of Turkish adult patients

Galactomannan antigen detection as a screening tool for early diagnosis of invasive aspergillosis: Experience of Turkish adult patients

Galactomannan use in clinical practice: providing free testing is not the full answer

IntroductionInvasive aspergillosis is a condition associated with a high mortality rate mostly due to difficulties in performing an early diagnosis. In recent years, galactomannan detection has markedly improved the diagnosis of invasive aspergillosis, but very little is known on how physicians deal with this test in clinical practice. MethodsThis cross-sectional study aimed to analyze the indications for the use of serum galactomannan in a large Brazilian hospital, between 2015 and 2016. No specific protocol was in place for GM request. We reviewed the medical records of adult (>18 years-old) patients who were tested for galactomannan due to one the following indications: screening, diagnosis, or treatment follow-up. Additional variables included demographic data, underlying diseases, presence of neutropenia, and use of previous antifungal (anti-Aspergillus) drugs. ResultsThe mean age of the patients was 51 years-old (sd±15.8), and 63.3% of patients were male. Patients with hematological malignancies accounted for 60.1% of the cases, mostly acute myeloid leukemia (19.6%). Galactomannan testing was positive in 12.2% of patients, including 1.6% of occasions in which the test was used for screening purposes, 13.2% for diagnosis, and 32.4% during follow-up. Median time for chest imaging request was two days before GM testing. Previous antifungal therapy was reported for 35.1% of patients, mostly amphotericin B (57.1%). ConclusionThe correct use of galactomannan testing is essential for an early diagnosis of invasive aspergillosis, which may improve the prognosis of the disease. We demonstrated that clinicians usually ask for galactomannan tests to confirm imaging findings in patients who frequently were on antifungal drugs, something that could be improved by medical education. We observed a low frequency of galactomannan use for preemptive antifungal therapy (25.7%), which is worrying considering the well-known beneficial use of GM testing in this scenario.

Utility of the serum galactomannan assay for the diagnosis of invasive aspergillosis in children with acute lymphoblastic leukemia

Invasive aspergillosis (IA) is an important cause of mortality and morbidity in children with hematological malignancies. The monitoring of serum galactomannan (GM) antigen is considered useful in the diagnosis of IA . The aim of this study was to determine the utility of serum GM monitoring in the early diagnosis of IA and the role of positive antigenemia in the management of children with acute lymphoblastic leukemia (ALL). The cases of 141 children who were being treated for ALL in the Division of Pediatric Hematology of the Medical School of Ege University between January 2006 and February 2015 were reviewed retrospectively. Cases of proven and probable IA were defined according to the European Organization for the Research and Treatment of Cancer/Mycoses Study Group (EORTC/MSG) criteria. The incidence of proven and probable IA was 3.5% (5/141). The incidence of positive GM antigenemia among 3264 serum samples was 5.5% (n=179). Of the cases detected, 21.7% were true-positive, 52.1% were false-positive, and the remaining 26.1% were classified as 'undetermined.' An increase in the incidence of true-positive tests and induction of antifungal therapy was determined through multiple consecutive positive tests. GM may be detected in the serum before the clinical signs of IA appear, but its sensitivity and specificity are variable. False-positivity is a significant disadvantage, and consecutive positive GM must be taken into account in the case of clinical and imaging findings that are relevant to IA.

Development and evaluation of enzyme-linked immunosorbent assay of nucleic acid sequence-based amplification for diagnosis of invasive aspergillosis.

Invasive aspergillosis (IA) is a life-threatening infection in immunocompromised patients, rapid and sensitive detection of Aspergillus from clinical samples has been a major challenge in the early diagnosis of IA. An enzyme-linked immunosorbent assay of nucleic acid sequence-based amplification (NASBA-ELISA) was developed to fulfil the need for the efficient diagnosis of these infections. The primers targeting 18S rRNA were selected for the amplification of Aspergillus RNA by the isothermal digoxigenin (DIG)-labeling NASBA process. The DIG-labeled RNA amplicons were hybridized with a specific biotinylated DNA probe immobilized on streptavidin-coated microtiter plate. The hybrids were colorimetrically detected by the addition of an anti-DIG antibodies linked to ALP and substrate (disodium 4-nitrophenyl phosphate). The detection limit of the Aspergillus NABSA-ELISA system was 1 CFU and the RNA in non-target bacteria or fungus was not amplified. The performance of this NASBA-ELISA compared to RT-PCR and galactomannan (GM) was evaluated by testing blood samples from 86 patients at high risk for IA. The sensitivity of NASBA-ELISA, RT-PCR and GM-ELISA was 80.56 % (95 % CI 63.98–91.81), 72.22 % (95 % CI 54.81–85.80), 58.33 % (95 % CI 40.76–74.49), respectively, and the specificity was 80.00 % (95 % CI 66.28–89.97), 84.00 % (95 % CI 70.89–92.83), 82.00 % (95 % CI 68.56–91.42). The efficiency of the three methods in various combinations was also evaluated. Combination of NASBA-ELISA and GM-ELISA testing achieved perfect specificity (100 %; 95 % CI 92.89–100) and perfect positive predictive value (100 %; 95 % CI 83.16–100). The best sensitivity (97.22 %; 95 % CI 85.47–99.93) and the highest Youden index (0.652) were obtained by testing with both NASBA and RT-PCR in parallel. In conclusion, the NASBA-ELISA assay consists of an alternative process for large-scale samples detection with semi-quantitative results and provides good clinical performance without resorting to expensive equipment. This assay makes it possible for the NASBA based RNA diagnosis to become a routine work in laboratories in less developed countries with fewer resources.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-016-0266-0) contains supplementary material, which is available to authorized users.

Detection of a Serum Siderophore by LC-MS/MS as a Potential Biomarker of Invasive Aspergillosis.

Invasive aspergillosis (IA) is a life-threatening systemic mycosis caused primarily by Aspergillus fumigatus. Early diagnosis of IA is based, in part, on an immunoassay for circulating fungal cell wall carbohydrate, galactomannan (GM). However, a wide range of sensitivity and specificity rates have been reported for the GM test across various patient populations. To obtain iron in vivo, A. fumigatus secretes the siderophore, N,N',N"-triacetylfusarinine C (TAFC) and we hypothesize that TAFC may represent a possible biomarker for early detection of IA. We developed an ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for TAFC analysis from serum, and measured TAFC in serum samples collected from patients at risk for IA. The method showed lower and upper limits of quantitation (LOQ) of 5 ng/ml and 750 ng/ml, respectively, and complete TAFC recovery from spiked serum. As proof of concept, we evaluated 76 serum samples from 58 patients with suspected IA that were investigated for the presence of GM. Fourteen serum samples obtained from 11 patients diagnosed with probable or proven IA were also analyzed for the presence of TAFC. Control sera (n = 16) were analyzed to establish a TAFC cut-off value (≥6 ng/ml). Of the 36 GM-positive samples (≥0.5 GM index) from suspected IA patients, TAFC was considered positive in 25 (69%). TAFC was also found in 28 additional GM-negative samples. TAFC was detected in 4 of the 14 samples (28%) from patients with proven/probable aspergillosis. Log-transformed TAFC and GM values from patients with proven/probable IA, healthy individuals and SLE patients showed a significant correlation with a Pearson r value of 0.77. In summary, we have developed a method for the detection of TAFC in serum that revealed this fungal product in the sera of patients at risk for invasive aspergillosis. A prospective study is warranted to determine whether this method provides improved early detection of IA.

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.

Multiple myeloma as a major cause of false-positive galactomannan tests in adult patients with cancer

The galactomannan (GM) test is a useful method for early diagnosis of invasive aspergillosis. Recently, multiple myeloma has newly been suggested to be related to false-positive results of GM. We performed a case-control study to validate this finding. Electronic medical records were reviewed for patients admitted March through June 2014. Patients with false-positive GM results were selected as cases and those with negatives as controls. To verify the results of the four-month analysis, additional analysis was performed in multiple myeloma patients over a three-year period. There were 30 false-positive and 316 negative cases during the four-month period. Among the factors evaluated, multiple myeloma was the only significant factor in the adjusted analysis (OR=3.59, CI 1.28-10.04). In the three-year analysis of 145 multiple myeloma patients, 25.5% showed false-positive results, which was 3 times higher than overall. GM false-positivity was not related to serum monoclonal protein level or type of immunoglobulin. GM optical density indexes (ODIs) in all false positives were lower than 3.0. Multiple myeloma was a major cause of GM false-positivity in adult cancer patients. GM was false-positive in 25.5% of multiple myeloma patients with GM ODIs lower than 3.0.

Polymerase chain reaction blood tests for the diagnosis of invasive aspergillosis in immunocompromised people.

Invasive aspergillosis (IA) is the most common life-threatening opportunistic invasive mould infection in immunocompromised people. Early diagnosis of IA and prompt administration of appropriate antifungal treatment are critical to the survival of people with IA. Antifungal drugs can be given as prophylaxis or empirical therapy, instigated on the basis of a diagnostic strategy (the pre-emptive approach) or for treating established disease. Consequently there is an urgent need for research into both new diagnostic tools and drug treatment strategies. Newer methods such as polymerase chain reaction (PCR) to detect fungal nucleic acids are increasingly being investigated. To provide an overall summary of the diagnostic accuracy of PCR-based tests on blood specimens for the diagnosis of IA in immunocompromised people. We searched MEDLINE (1946 to June 2015) and EMBASE (1980 to June 2015). We also searched LILACS, DARE, Health Technology Assessment, Web of Science and Scopus to June 2015. We checked the reference lists of all the studies identified by the above methods and contacted relevant authors and researchers in the field. We included studies that: i) compared the results of blood PCR tests with the reference standard published by the European Organisation for Research and Treatment of Cancer/Mycoses Study Group (EORTC/MSG); ii) reported data on false-positive, true-positive, false-negative and true-negative results of the diagnostic tests under investigation separately; and iii) evaluated the test(s) prospectively in cohorts of people from a relevant clinical population, defined as a group of individuals at high risk for invasive aspergillosis. Case-control studies were excluded from the analysis. Authors independently assessed quality and extracted data. For PCR assays, we evaluated the requirement for either one or two consecutive samples to be positive for diagnostic accuracy. We investigated heterogeneity by subgroup analyses. We plotted estimates of sensitivity and specificity from each study in receiver operating characteristics (ROC) space and constructed forest plots for visual examination of variation in test accuracy. We performed meta-analyses using the bivariate model to produce summary estimates of sensitivity and specificity. Eighteen primary studies, corresponding to 19 cohorts and 22 data sets, published between 2000 and 2013 were included in the meta-analyses, with a median prevalence of IA (proven or probable) of 12.0% (range 2.5 to 30.8 %). The majority of people had received chemotherapy for a haematological malignancy or had undergone a hematopoietic stem cell transplant. Several PCR techniques were used among the included studies. The sensitivity and specificity of PCR for the diagnosis of IA varied according to the interpretative criteria used to define a test as positive. The mean sensitivity and specificity were 80.5% (95% CI; 73.0 to 86.3) and 78.5% (67.8 to 86.4) for a single positive test result, and 58.0% (36.5 to 76.8) and 96.2% (89.6 to 98.6) for two consecutive positive test results. PCR shows moderate diagnostic accuracy when used as screening tests for IA in high-risk patient groups. Importantly the sensitivity of the test confers a high negative predictive value (NPV) such that a negative test allows the diagnosis to be excluded. Consecutive positives show good specificity in diagnosis of IA and could be used to trigger radiological and other investigations or for pre-emptive therapy in the absence of specific radiological signs when the clinical suspicion of infection is high. When a single PCR positive test is used as diagnostic criterion for IA in a population of 100 people with a disease prevalence of 13.0% (overall mean prevalence), three people with IA would be missed (sensitivity 80.5%, 19.5% false negatives), and 19 people would be unnecessarily treated or referred for further tests (specificity of 78.5%, 21.5% false positives). If we use the two positive test requirement in a population with the same disease prevalence, it would mean that six IA people would be missed (sensitivity 58.0%, 42.1% false negatives) and three people would be unnecessarily treated or referred for further tests (specificity of 96.2%, 3.8% false positives). Galactomannan and PCR have good NPV for excluding disease but the low prevalence of disease limits the ability to rule in a diagnosis. The biomarkers are detecting different aspects of disease and the combination of both together is likely to be more useful.

Nonspecific elevation of serum Aspergillus galactomannan antigen levels in patients with rheumatoid arthritis.

Infections are an important cause of morbidity and mortality in patients with rheumatoid arthritis. Patients receiving immunosuppressive or anti-tumor necrosis factor (TNF) agents are vulnerable to fungal infections, including those derived from Aspergillus species. Detection of the Aspergillus galactomannan antigen in serum is useful for the early diagnosis of invasive aspergillosis in patients with hematological malignancies. However, its usefulness for detecting early invasive aspergillosis in rheumatoid arthritis patients remains unestablished. Galactomannan antigen levels were measured in 340 patients (311 female patients). For patients who exhibited galactomannan antigen levels ≥0.5 during the initial examination, a second examination was performed 3-6 months later. Conventional blood tests and chest radiography were also performed. Elevated galactomannan antigen levels (≥0.5) were observed in 62 (18.2%) of 340 patients during the initial examination. A second examination was performed in 56 of 62 patients, 50 of whom exhibited elevated antigen levels. Elevated antigen levels were not associated with the use of any drug including anti-TNF agents. Serum galactomannan antigen levels were correlated with the albumin/globulin ratio (r=-0.19, p<0.001), γ-globulin (%; r=0.17, p=0.001), and hemoglobin concentration (r=-0.15, p=0.005). No patient was clinically diagnosed with invasive aspergillosis during the study period. Serum galactomannan antigen levels are frequently elevated in a nonspecific manner in patients with rheumatoid arthritis.