β-Glucan Antigenemia Assay for the Diagnosis of Invasive Fungal Infections in Patients With Hematological Malignancies: A Systematic Review and Meta-Analysis of Cohort Studies From the Third European Conference on Infections in Leukemia (ECIL-3)

This Third European Conference on Infections in Leukemia meta-analysis of high-quality hemato-oncological cohort studies shows that 2 consecutive positive 1,3-β-D-glucan assays have high specificity and both positive and negative predictive values but low sensitivity for diagnosis of invasive fungal infectio

Desenvolvimento e validação de um método por CLAE-EM/EM para quantificação de gliotoxina em plasma humano para diagnóstico precoce da aspergilose

Orientador: Prof. Dr. Roberto PontaroloCo-orientadora: Profa. Dra. Francinete Ramos CamposDissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências da Saúde, Programa de Pós-Graduação em Ciências Farmaceuticas. Defesa: Curitiba, 22/02/2013Bibliografia: fls. 117-125Resumo: A aspergilose é uma infecção oportunista causada por fungos do gênero Aspergillus. É uma das principais complicações que acometem pacientes imunossuprimidos, podendo atingir índices de mortalidade de até 80%. O principal agente causador dessa infecção é o A. fumigatus, também considerada a principal espécie produtora de gliotoxina. Esta toxina é um produto do metabolismo secundário fúngico, apresenta comprovada toxicidade frente às células do sistema hematopoético e já foi detectada em soro de pacientes com diagnóstico positivo de aspergilose, podendo ser considerada um potencial biomarcador para o diagnóstico da doença. Atualmente o método de escolha para o diagnóstico da aspergilose é imunoenzimático, mas oferece apenas um indicativo da infecção, sendo necessários três resultados positivos consecutivos para que ela seja confirmada. A cromatografia liquida de alta eficiência acoplada à espectrometria de massas sequencial (CLAE-EM/EM) surge como uma nova alternativa para o diagnóstico, uma vez que é capaz de fornecer resultados com alta precisão e exatidão, e excelente sensibilidade. Nesse sentido, esse trabalho visa o desenvolvimento de um método por CLAE-EM/EM para detecção de gliotoxina em plasma de pacientes imunossuprimidos com suspeita de aspergilose. Para isso, alíquotas de plasma branco foram fortificadas com gliotoxina e submetidas ao processo de extração liquido-liquido, utilizando uma solução de éter etílico/acetato de etila (50:50, v/v), agitada em vórtex por 3 min. A fase orgânica foi recuperada e, em seguida, evaporada. O resíduo obtido foi resuspendido em uma solução de ACN/H2O (98:2, v/v) contendo 0,1% de ácido fórmico (AFO). As análises foram realizadas utilizando um cromatógrafo Agilent 1200 acoplado ao espectrômetro de massas do tipo triplo quadrupolo API 3200 da ABSciex, provido de fonte de ionização por electrospray (ESI), operado no modo negativo de ionização. O experimento de Multiple Reaction Monitoring (MRM) foi utilizado para monitorar as transições dos íons de m/z 325'243 e m/z 325'261 referente à gliotoxina e m/z 301'179 e m/z 301'151 referentes ao padrão interno (quercetina). A separação cromatográfica foi obtida em uma coluna C18 XBridge (150 x 2,1 mm i.d; 5 ?m) e pré-coluna (XBridge C18, 10 x 2,1 mm i.d; 5 ?m). A fase móvel foi constituída de solução de NH4HCO2 1mM (A) e ACN contendo 5% de solução 1mM de NH4HCO2 (B) submetidos ao seguinte gradiente de eluição: t0-0,10 min.: 65%-0% A; t0,10-0,70 min.: 0% A; t0,7-0,71 min.: 0-65% A; t0,71-4,00 min.: 65% A. O tempo de corrida foi de 4 min. em um fluxo de 0,45 mL/min. O volume de injeção foi de 20 ?L. O método desenvolvido foi validado, considerado sensível, seletivo, linear (r>0,99) na faixa de 10 a 120 ng/mL, preciso, exato e livre de efeitos residuais e de matriz. O método foi aplicado com sucesso a 30 amostras reais de pacientes com suspeita de aspergilose, com 70% de concordância com os resultados obtidos pelo método de ELISA. O presente trabalho confirmou que é possível quantificar a gliotoxina em plasma através da técnica de CLAE-EM/EM e sua aplicação como diagnóstico é uma possibilidade real e muito promissora.Abstract: Aspergillosis is an opportunistic infection caused by fungi of the genus Aspergillus. It is a major complication of immunocompromised patients and its mortality can reach up to 80%. The main causative agent of this infection is A. fumigatus, which is also the main gliotoxin-producing species. This toxin is a product of fungal secondary metabolism and has been shown toxicity against hematopoietic system cells. It has already been detected in sera samples of patients with positive diagnosis of aspergillosis and, for this reason, can be considered as a potential biomarker for diagnosis of this disease. Imunoenzimatic assay is the currently gold standard method for diagnosing Aspergillosis but it only provides an indicative of infection, requiring three consecutive positive results to confirm the diagnosis. The high performance liquid chromatography coupled tandem mass spectrometry (HPLC-MS/MS) appears as a new alternative for the diagnosis of aspergillosis, since it is capable to provide high precision and accuracy results with excellent sensitivity. On this way, this work aims to develop an HPLC-MS/MS method for the detection of gliotoxin in plasma of patients with suspected infection by Aspergillus. For this, aliquots of blank plasma were spiked with gliotoxin and submitted to liquid-liquid extraction, using a solution of ethyl ether / ethyl acetate (1:1, v /v). The organic phase was recovered and then evaporated. The residue obtained was resuspended in a solution of ACN/H2O (98:2, v /v) with 1 mM of NH4HCO2. Analyses were performed using an Agilent 1200 liquid chromatograph coupled to mass spectrometer API 3200 triple quadrupole ABSciex provided with electrospray ionization source (ESI) operated in the negative ion mode. The experiment Multiple Reaction Monitoring (MRM) was used to monitor the transition of the ions of m/z 325' 243 and m/z 325 ' 261 corresponding to gliotoxin and m/z 301 ' 179 and m/z 301' 151 related to internal standard (quercetin). The chromatographic separation was obtained in a XBridge C18 column (150 x 2.1 mm id; 5 mm) and guard column (XBridge C18, 10 x 2.1 mm id, 5 mm). The mobile phase was composed of 1 mM NH4HCO2 solution (A) and MeCN containing 5% NH4HCO2 1mM (B) eluted by the following gradient: t0-0, 10 min: 65% -0% A; t0 10 -0.70 min: 0% A; t0 0.7 to 0, 71 min: 0-65% A; t0 0.71 to 4, 00 min: 65% A. The run time was 4 min at a flow rate of 0.45 mL / min. The injection volume was 20 ?L. The method was fully validated, being considered linear (r> 0.99) in the range of 10 to 120 ng/ mL, sensitive, selective, precise, accurate and free of matrix and carry over effects. The method was successfully applied to real samples from 30 patients with suspected aspergillosis, reaching 70% of agreement with ELISA results. This study confirmed that it is possible to quantify gliotoxin in plasma using the technique of HPLC-MS/MS and the present method can be potentially applied for aspergillosis diagnosis

1,3-β-d-Glucan Antigenemia for Early Diagnosis of Invasive Fungal Infections in Neutropenic Patients with Acute Leukemia

Background. Invasive fungal infections (IFIs) are life-threatening complications in neutropenic patients with hematological malignancies. Because early diagnosis of IFI is difficult, new noninvasive, culture-independent diagnostic tools are needed to improve clinical management. Recent studies have reported that detection of 1,3-β-d-glucan (BG) antigenemia may be useful for diagnosis of IFI. The aim of the present prospective study was to evaluate the usefulness of monitoring BG in patients undergoing chemotherapy for acute leukemia. Methods. BG antigenemia was measured by a colorimetric assay twice weekly in the absence of fever and daily in the presence of fever. IFIs were classified according to the criteria of the European Organization for Research and Treatment of Cancer/Mycoses Study Group. Results. During 190 consecutive neutropenic episodes (median duration, 22 days; range, 7-113 days) in 95 patients, 30 proven or probable IFIs (13 aspergillosis, 15 candidiasis, and 2 mixed IFIs) were diagnosed. Sensitivity, specificity, positive predictive value, negative predictive value, and efficiency of 2 consecutive BG values ⩾7 pg/mL for diagnosis of proven or probable IFI was 0.63 (95% confidence interval, 0.44-0.79), 0.96 (95% confidence interval, 0.89-0.98), 0.79 (95% confidence interval, 0.57-0.92), 0.91 (95% confidence interval, 0.84-0.95), and 0.89, respectively. The time interval between onset of fever as first sign of IFI and BG antigenemia was significantly shorter than the time to diagnosis of IFI by clinical, microbiological, radiological, and/or histopathological criteria (P50 pg/mL were observed in only 2 patients, both of whom experienced failure of antifungal therapy. Conclusion. Monitoring of BG antigenemia is a useful noninvasive method for early diagnosis of IFI in patients with acute leukemi

Early Diagnosis of Invasive Aspergillosis in Neutropenic Patients. Comparison between Serum Galactomannan and Polymerase Chain Reaction

Background Invasive aspergillosis (IA) is a major cause of morbidity and mortality in profoundly neutropenic patients, so early diagnosis is mandatory. Aim Consecutive patients with hematological malignancies undergoing intensive chemotherapy were screened for IA with two different methods which were compared. Methods From October 2000 to August 2003 we tested 1311 serum samples from 172 consecutive patients with a polymerase chain reaction assay and between April 2005 and April 2008 we tested 806 serum samples from 169 consecutive patients with a Galactomannan (GM) test. Bronchoalveolar (BAL) samples were obtained whenever the patient's condition allowed and tested with either method. Results: The serum PCR assay had a sensitivity of 75.0% and a specificity of 91.9% and the serum GM assay had a sensitivity of 87.5% and a specificity of 93.1%, ( P > 0.05). The presence of two or more consecutive positive serum samples was predictive of IA for both assays. BAL GM/PCR was positive in some patients without serum positivity and in patients with 2 or more positive serum GM/PCR. Conclusions: No significant differences between the 2 serum tests were found. The GM assay has the advantage of being standardized among several laboratories and is incorporated in the criteria established by the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycosis Study Group (EORTC/MSG), however is much more expensive. BAL GM and PCR sampling aids in IA diagnosis but needs further validation studies to differentiate between colonization and true infection in cases where serum GM or PCR are negative

Diagnosis of invasive fungal infections in immunocompromised children

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

Improving Mobile Phone Banking Usefulness, Usability, Risk, Cost, and Intention to Adopt

Millions of people use mobile phone banking daily, and business leaders should understand the factors influencing mobile phone banking adoption among users. Based on the theory of technology acceptance model and the innovation diffusion theory, the purpose of this correlational study was to examine the relationship between usefulness, ease of use, risk, cost, and mobile phone banking adoption in Burkina Faso. One hundred and six mobile phone banking users living in the city of Ouagadougou completed the online survey created to measure consumers understanding of mobile phone banking. Results of the multiple linear regression analysis indicated a statistically significant relationship between the predictor variables and mobile phone banking adoption, F(5,101) = 36.07, p \u3c .001. Three of the predictors contributed significantly to the model, with usefulness recording the highest beta value (Ã? = .692), cost the next highest beta value (Ã? = .225), and ease of use the next highest beta value (Ã? = .173). The 4th predictor, risk, did not contribute significantly to the model, recording a negative beta value (Ã? = -.058). Results may enhance local business leaders\u27 understanding of mobile phone banking adoption, which could result in more effective business strategies to increase the affordability, availability, and quality of mobile banking services for Burkina Faso residents. Development of the mobile phone banking industry could enable business leaders to foster access to affordable financial services for individuals and contribute to the development of Burkina Faso\u27s local economy and trade

Future challenges and chances in the diagnosis and management of invasive mould infections in cancer patients.

Diagnosis, treatment, and management of invasive mould infections (IMI) are challenged by several risk factors, including local epidemiological characteristics, the emergence of fungal resistance and the innate resistance of emerging pathogens, the use of new immunosuppressants, as well as off-target effects of new oncological drugs. The presence of specific host genetic variants and the patient's immune system status may also influence the establishment of an IMI and the outcome of its therapy. Immunological components can thus be expected to play a pivotal role not only in the risk assessment and diagnosis, but also in the treatment of IMI. Cytokines could improve the reliability of an invasive aspergillosis diagnosis by serving as biomarkers as do serological and molecular assays, since they can be easily measured, and the turnaround time is short. The use of immunological markers in the assessment of treatment response could be helpful to reduce overtreatment in high risk patients and allow prompt escalation of antifungal treatment. Mould-active prophylaxis could be better targeted to individual host needs, leading to a targeted prophylaxis in patients with known immunological profiles associated with high susceptibility for IMI, in particular invasive aspergillosis. The alteration of cellular antifungal immune response through oncological drugs and immunosuppressants heavily influences the outcome and may be even more important than the choice of the antifungal treatment. There is a need for the development of new antifungal strategies, including individualized approaches for prevention and treatment of IMI that consider genetic traits of the patients. Anticancer and immunosuppressive drugs may alter the ability of the immune system to fight invasive mould infections and may be more important than the choice of the antifungal treatment. Individualized approaches for prevention and treatment of invasive mold infections are needed