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

Highlights

• Invasive aspergillosis (IA) is the most common fungal infection in patients with hematologic malignancies.

• The value of GM antigen in BAL and serum can be promising in early diagnosis of IA.

• Posaconazole and ravuconazole have a superiority to amphotericin B in primary therapy of IA.

• In patients with hematological malignancies and IA the most common type of coinfection is bacterial.

• The renovation in hospitals increases the risk of IA in immunosuppressed patients.

Abstract

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 $\raisebox{1ex}{$\mu g$}\!\left/ \!\raisebox{-1ex}{$ml$}\right.$ and 0.031–0.125 $\raisebox{1ex}{$\mu g$}\!\left/ \!\raisebox{-1ex}{$ml$}\right.$, 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.

Introduction

Invasive aspergillosis (IA) is being recognized as the most common acquired fungal infection in patients with hematologic malignancies or candidates for organ transplantation who have received intensive cytotoxic and immunosuppressive therapy or undergone chemotherapy with consequent neutropenic status [1]. It has been evidenced that timely diagnosis and appropriate antifungal treatments increased the life expectancy of patients with IA and decreased the risk of morbidity and mortality, but still about one-third (33%) of cases failed to respond to the treatment and eventually had died [2]. Since clinical manifestations and radiographic evidence of invasive fungal infections (IFIs) particularly mucormycosis and aspergillosis are relatively similar, their differential diagnosis is usually not reachable and as a result, timely diagnosis is usually not provided [3]. Unfortunately, in the case of patients with hematologic malignancies, the tissue debridements and biopsy for histopathological evaluation as a gold standards of diagnosis may not be achieved because of severe thrombocytopenia. By the way, considering the acuteness and wildness of the infection, establishing a timely diagnosis for prompt initiation of antifungal therapy in order to decrease the rate of mortality is highly recommended. In recent years, efforts have been directed towards developing noninvasive diagnostic techniques such as biomarkers that can be used for rapid and reliable diagnosis of IA. One such biomarker, galactomannan (GM), is a polysaccharide cell wall component of Aspergillus (specific circulating antigen), which is released by the fungal hyphae in variable quantities in the serum and adjacent fluids of infected organs (e.g. bronchoalveolar lavage (BAL)) during invasive growth [4,5] and can be detected by sandwich‐enzyme immunoassay (EIA). Early diagnosis and prompt administration of appropriate antifungal therapy have been well recognized as critical factors that increase the survival rate of patients affected by IA. Given the different management and variation of the in vitro susceptibility patterns among the pathogenic fungi, e.g. A. terreus and A. flavus that are less susceptible to routinely used antifungal agents and furthermore, to determine the epidemiological profile of the disease, the precise distinction of the etiological agents of the infection and their antifungal susceptibility patterns are crucial issues influencing the outcome of the infection [[6], [7], [8]]. Therefore, the purpose of the present study was to investigate the role of GM level in BAL and serum specimens for early diagnosis of IA. Also identification of the causal agents of IA and their antifungal susceptibility patterns in those with hematologic malignancies or candidates for organ transplantation hospitalized in Imam Khomeini hospital, Tehran, Iran is the other purposes of the current study.