Applicability of the Platelia EIA® Aspergillus test for the diagnosis of aspergilosis in penguins / Aplicabilidade do Platelia EIA® Aspergillus como teste diagnostico da aspergilose em pinguins

Abstract 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.

Resumo Ainda hoje, um teste diagnóstico eficaz para aspergilose em pinguins não é conhecido, sendo o padrão-ouro os exames post-mortem. O antígeno fúngico galactomanana (GM) tem sido utilizado como biomarcador da doença em humanos, sendo detectado pelo Platelia Aspergillus EIA (BioRad)®, um kit comercial que se baseia na técnica ELISA sanduíche. É padronizado para utilização em pacientes neutropênicos, no entanto estudos tem demonstrado sua possível utilidade também para aves.O objetivo de nosso estudo foi avaliar a eficácia do teste Platelia Aspergillus EIA® (BioRad-US) no diagnóstico da aspergilose em pinguins-de-Magalhães, determinando sensibilidade, especificidade e valores preditivos positivos e negativos em diferentes pontos de corte. Foram incluídas no estudo, amostras de soro sanguíneo (n=29) de pinguins-de-Magalhães em cativeiro que vieram a óbito por aspergilose. A detecção de GM foi realizada seguindo instruções do fabricante e o índice de GM foi obtido dividindo o valor da média da DO da duplicata da amostra clínica pelo valor da média da DO da duplicata da amostra de cut-off fornecida pelo kit. Através de informações em banco de dados foram obtidos resultados sobre a presença de anticorpos anti-Aspergillus fumigatus, detectada por Imunodifusão em gel de ágar (IDGA) em todas as amostras séricas. Os resultados foram analisados utilizando-se teste de qui-quadrado e Kruskal-Wallis a partir do programa estatístico SPSS 20.0, IBM®. Curva ROC foi obtida e a partir desta, taxas de sensibilidade, especificidade, valores preditivo positivo e negativo foram igualmente calculados considerando quatro diferentes pontos de corte (0.5, 1.0, 1.5 e 2.0). O índice de GM sérica não diferiu entre os animais do grupo caso e controle (pKW = 0.097). Na determinação da curva ROC para detecção de GM sérica o valor da área sobre a curva foi de 0.635. A partir dos valores determinados pelas coordenadas da curva, quatro diferentes pontos de corte (0.5, 1.0, 1.5 e 2.0) foram analisados, resultando em taxas de sensibilidade variando de 86.2% a 34.5%, e de especificidade entre 87% e 26.1%. Ao comparar o índice de GM sérica nos animais do grupo caso quanto a presença ou não de anticorpos detectados pela IDGA foi encontrado p=0.503. A detecção de GM pelo teste Platelia Aspergillus EIA® não parece ser útil para o diagnóstico da aspergilose em pinguins naturalmente infectados.

Aspergilosis pulmonar invasora en pacientes reumatológicos: presentación de dos casos / Invasive pulmonary aspergillosis in rheumatological patients: presentation of two cases

La aspergilosis pulmonar invasora (API) es una infección causada por hongos del género Aspergillus que afecta principalmente a pacientes inmunocomprometidos y corresponde a la forma más grave de aspergilosis. Se asocia a una alta morbi-mortalidad, siendo fundamental un diagnóstico y tratamiento oportuno. Las manifestaciones clínicas son inespecíficas, por lo que un estudio adecuado es importante para el diagnóstico, principalmente en pacientes con factores de riesgo poco habituales. En la actualidad se han establecido categorías diagnósticas que consideran factores del hospedero, laboratorio micológico tradicional y biomarcadores como galactomanano. Éstos, junto a la mejor comprensión e interpretación de las imágenes tomográficas permiten ofrecer un manejo adecuado. En este artículo, se presentan dos casos clínicos de API en pacientes reumatológicos, y se discute la utilidad de los métodos diagnósticos.

Invasive pulmonary aspergillosis (IPA) is an infection caused by fungi of the genus Aspergillus that mainly affects immunocompromised patients and corresponds to the most severe form of aspergillosis. It is associated with high morbidity and mortality, and diagnosis and timely treatment are essential. Clinical manifestations are nonspecific, so an adequate study is important for diagnosis, mainly in patients with unusual risk factors. At present, diagnostic categories have been established that consider factors of the host, traditional mycological laboratory and biomarkers such as galactomannan. These, together with the better understanding and interpretation of the tomographic images, allow us to offer an adequate management. In this article, two clinical cases of API in rheumatological patients are presented, and the usefulness of the diagnostic methods is discussed.

Teste de galactomanana para o diagnóstico de aspergilose invasiva: uma revisão / Galactomannan test for the diagnosis of invasive aspergillosis: a review

As doenças fúngicas invasivas têm sido um problema crescente em ambientes hospitalares, sobretudo nas últimas duas décadas. A aspergilose invasiva (AI), ocasionada pelo gênero Aspergillus, está entre as principais causas de morte em pacientes gravemente imunocomprometidos, com mortalidade que varia de 70 a 90%. O padrão de referência para o diagnóstico de AI é o cultivo do micro-organismo e a análise histopatológica dos órgãos afetados. Estes procedimentos são dificilmente realizados na maioria dos casos, e apresentam baixa sensibilidade (<50%), além de as amostras serem habitualmente obtidas em estados avançados da infecção. O teste de detecção de galactomanana tem sido objeto de estudo para o diagnóstico de AI, por representar uma promissora ferramenta e por ser uma técnica sorológica rápida e não invasiva. A presente revisão tem por objetivo fazer levantamento de estudos que utilizaram o teste de galactomanana em amostras de pacientes com quadros clínicos distintos, porém com suspeita e/ou com comprovada AI, bem como as atuais tendências de conhecimento, aplicação e utilidade do ensaio laboratorial.

Invasive fungal diseases represent an increasing problem in the hospital environments, predominantly in the last two decades. The invasive aspergillosis (IA), induced by Aspergillus species, has been the main cause of death in severely immunocompromised patients, with mortality varying from 70 to 90%. Difficulties are found for diagnosing the IA. In vitro culture of biological material shows low sensitivity (<50%), besides the positivity usually occurs at the advanced stages of the infection. The test for detecting galactomannan has been the object of the present study, seeing that it represents a promising diagnostic tool, as a fast and non-invasive serological procedure. The objective of the present review is to survey the studies which have been performed by using methods for detecting galactomannan in samples from patients with distinct clinical pictures. Patients presenting suspicion and/or confirmed IA were also included, as well as the up-to-date trends in knowledge, application and utility of the test.

Evaluación del Aspergillus lateral flow device para el diagnóstico de aspergilosis invasora, experiencia en un hospital universitario / Evaluation of the lateral flow Aspergillus assay for the diagnosis of invasive aspergillosis, experience in a university hospital

Resumen Introducción: El diagnóstico de aspergilosis invasora (AI) se realiza mediante criterios clínicos y microbiológicos los que incluyen marcadores séricos. Recientemente, el test inmunocromatográfico Aspergillus lateral flow device (LFD), ha sido evaluado como método para diagnóstico de AI. Objetivo: Evaluar el desempeño de este test para el diagnóstico de AI. Material y Método: Estudio transversal en que se evaluaron muestras de suero y lavado bronco-alveolar (LBA) procesadas para galactomanano provenientes de pacientes adultos con sospecha de AI, atendidos en el Hospital Clínico de Red de Salud UCCHRISTUS. Resultados: Se procesó un total de 142 muestras de 98 pacientes, correspondientes a AI probada 5,6%, AI probable 41,5%, AI posible 12,7% y ausencia de AI 40,1%. Al confrontar los resultados con las categorías diagnósticas según criterios EORTC/MSG se obtuvo una sensibilidad y especificidad de LFD para diagnóstico de AI de 70,9 y 53,5% para muestras de suero y 83,3 y 38,5% para muestras de LBA. La concordancia entre galactomanano y LFD fue de 62,4% (54,1-69,9) con un índice Kappa de 0,202 (0,03682-0,3669). Conclusiones: Aspergillus LFD presentó una adecuada sensibilidad; sin embargo, la especificidad fue baja por lo que un resultado positivo requiere ser confirmado.

Background: The incidence of invasive aspergillosis is increasing. Its diagnosis is based on clinical and microbiological criteria which include the determination of serological markers such as galactomannan. Recently, the Aspergillus lateral flow device, an inmunocromatograph assay has been described for its diagnosis. Aim: To evaluate the performance of the lateral flow device for the diagnosis of invasive aspergillosis (IA) in adult patients. Material and Method: In this cross-sectional study, frozen samples that had been previously evaluated for galactomannan from patients classified with proven/probable/possible or no AI according to the EORTC/MSG criteria were selected. Results: A total of 142 samples from 98 patients were processed, corresponding to proven AI 5.6%, probable IA 41.5%, possible IA 12.7% and no-IA 40.1%. The sensitivity and specificity of the Aspergillus lateral flow was 70.9% and 53.5% for serum samples and 83.3% and 38.5% for BAL samples. The concordance between the galactomannan and Aspergillus lateral flow was 62.4% (54.1 - 69.9) with a Kappa index of 0.202 (0.03682 - 0.3669). Conclusions: We observed a good sensitivity but low specificity, a positive result need a confirmatory test.

Utilidad de la prueba Aspergillus-LFD para el diagnóstico de aspergilosis: primera experiencia en Chile / Utility of Aspergillus-LFD: first experience in Chile

Introduction: Invasive fungal diseases (IFD) by filamentous fungi are a common cause of morbidity and mortality in immunocompromised patients, especially those with myeloid leukemia. In 2011 a protocol for the rapid diagnosis of IFD by filamentous fungi was implemented in Valparaiso Region. Objectives: To describe cases of IFD by filamentous fungi of the Valparaíso Region, since the implementation of rapid diagnosis and to compare results with the period 2004-2009. Materials and Method: Descriptive and prospective study conducted in two public hospitals: Carlos van Buren at Valparaiso and Gustavo Fricke at Viña del Mar. We selected patients with a diagnosis of filamentous fungal diseases considering the EORTC/MSG criteria. Demographics, underlying diseases, risk factors for EFI, galactomannan (GM) results in blood and bronchoalveolar lavage, cultures and biopsies, treatment and overall lethality rates at 30 days were registered. Results: Eighteen patients were detected, 6 with proven and 12 probable IFD. Nine were diagnosed by GM, 8 by culture and two with both methods. In cases which the agent (9/18) was isolated from Rhizopus oryzae was the most frequent. When comparing overall lethality with the period 2004-2009, there was a reduction of 47.8%, which was statistically significant. Conclusions: Compared to data previously published in the region, demographic and comorbidities of patients with IFD caused by filamentous fungi are similar, however the currently rapid diagnosis protocol has improved survival of patients and lethality experienced overall decrease.

Introducción: la enfermedad fúngica invasora (EFI) por hongos filamentosos es una causa frecuente de morbilidad y mortalidad en pacientes inmunocomprometidos, en especial en aquellos con leucemia mieloide. En el 2011 se implementó en la Región de Valparaíso un protocolo de diagnóstico rápido de la EFI por hongos filamentosos. Objetivos: describir los casos de EFI por hongos filamentosos de la Región de Valparaíso, desde la implementación del diagnóstico rápido y compararlos con el período 2004-2009. Materiales y Método: Estudio descriptivo y prospectivo realizado en los hospitales públicos Carlos van Buren de Valparaíso y Gustavo Fricke de Viña del Mar. Se seleccionaron aquellos pacientes con diagnóstico de EFI por hongos filamentosos considerando los criterios EORTC/MSG. Se obtuvieron datos demográficos, enfermedad de base, factores de riesgo para EFI, resultados de galactomanano (GM), cultivos y biopsias, tratamiento y letalidad global a 30 días. Resultados: Se identificaron 18 pacientes, seis con EFI probadas y 12 probables. Nueve fueron diagnosticados con galactomanano, ocho con cultivos y uno con los dos métodos. En los casos en que se aisló el agente (9/18), Rhizopus oryzae fue el más frecuente. Al comparar la letalidad global con la del período 2004-2009, hubo una reducción de 47,8%, la cual fue estadísticamente significativa. Conclusiones: En relación a lo publicado anteriormente en la región, se conservan las características demográficas y de co-morbilidad de los pacientes con EFI por hongos filamentosos; sin embargo, la introducción del nuevo protocolo de diagnóstico rápido se asoció a una disminución en la letalidad global.

Variability in Galactomannan detection by platelia Aspergillus EIA™ according to the Aspergillus species / Variabilidade na detecção de galactomanana pelo Platelia Aspergillus EIA® de acordo com a espécie de Aspergillus

Here we investigate the extent to which different Aspergillus species release galactomannan (GM) in vitro. Marked variability was observed in GM reactivity between and within Aspergillus species, with A. terreus strains showing the highest GM indexes. The in vivo significance of these findings remains to be determined.

O estudo objetivou investigar a liberação in vitro de galactomanana (GM) em distintas espécies patogênicas de fungos do gênero Aspergillus. Grande variabilidade foi detectada tanto intra quanto inter espécies, sendo as cepas da espécie A. terreus relacionadas aos maiores índices de GM detectados. O significado in vivo destes achados permanece em aberto, porém merece investigação.

Galactomannan detection from piperacillin-tazobactam brands available in the Brazilian market

Piperacillin-tazobactam is a broad spectrum antimicrobial agent that can cause false-positive results in the commercial Platelia Aspergillus EIA test. So far, no study has been performed in Latin America to evaluate the clinical implication of this finding. Here we studied the potential for galactomannan detection in piperacillin-tazobactam batches commercialized in the Brazilian market. Five batches from distinct laboratories were tested in duplicate in the Platelia Aspergillus EIA according to the manufacturer's instructions. Only one drug showed crossreaction at a cut-off of 0.5. Human serum was spiked with this particular drug aiming to mimic achievable piperacillin-tazobactam concentrations in the serum. Results were all negative for galactomannan detection, even at high drug concentrations. Results from this pilot study suggest that piperacillin-tazobactam might not be a clinically significant cause of false-positive results in the Platelia Aspergillus EIA test in Brazil.

Mannan antigen detection in the diagnosis of patients with invasive candidiasis.

BACKGROUND & OBJECTIVES: With the increase in the incidence of invasive candidiasis (IC) in recent years, there is a need to improve the sensitivity of diagnosis. A conventional technique like blood culture is positive in nearly 50 per cent of cases. To improve the diagnostic efficiency in invasive candidiasis mannan antigen detection holds promise. Hence mannan antigen detection was evaluated in patients with suspected invasive candidiasis in the Paediatric Intensive Care Unit (PICU). METHODS: A prospective study, involving 186 consecutive patients admitted to the PICU of the Advanced Paediatric Center at the Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh between March 1999 and November 1999 were followed up for possible invasive candidiasis. Sixty five children clinically suspected to have sepsis and at risk for developing IC and or who stayed in the hospital more than 5 days were further evaluated for the diagnosis of IC by collecting two blood culture samples 48 h apart and by mannan antigen and anti-mannan antibody detection. Both antigen and antibody were detected by in-house standardized techniques: antibody by whole cell agglutination (WCA) and antigen by sandwich enzyme linked immunosorbent assay (ELISA). RESULTS: Twenty of 65 patients (30.7%) were positive by blood culture. Mannan antigen was positive in all 20 patients and significant anti-mannan antibody titre (> or = 128) was present in 12 (60%) patients. Mannan antigen was detected in 15 more patients, who were negative for isolation of Candida from blood. Twelve of these patients had febrile episode not responding to antibacterial therapy but responding to fluconazole/itraconazole therapy indicating that the patients possibly had IC. Mannan concentration was also found to be significantly higher in the patients with Candida isolated from blood. INTERPRETATION & CONCLUSION: Thus, the present study confirms the earlier claim that mannan antigen detection possibly improves the diagnostic efficiency of IC.