Invasive aspergillosis (IA) is one of the most common life-threatening fungal infections that has emerged as an important cause of morbidity and mortality in immunocom-promised patients that ranges from 70% to 90% in one year . IA is due to ubiquitous Aspergillus species that invade from the lungs into the adjacent organs if the immune system is compromised. Recently several reports have revealed a rising incidence of IA in critically ill patients admitted to the intensive care unit (ICU) in otherwise healthy individuals [2-5]. The occurrence of IA in the ICU usually entails a poor prognosis, despite major recent improvements in the diagnosis and treatment of IA in patients with hematologic diseases . Early diagnosis of IA is very important and requires diagnostic tools validated in the ICU population that show positive results in an early phase of the infection . In IA, the clinical manifestations and symptoms are generally non-speciﬁc, mechanical ventilation makes it difficult to interpret clinical signs, and radiological diagnoses are clouded by underlying lung pathologies . The significance of positive results of culture or positive ﬁndings of a direct microscopic examination of a respiratory specimen is greatly uncertain, because they have low sensitivity (50%) and speciﬁcity (20%–70%), depending on whether the patient is immunocompromised [9,10]. Histopathological examination as the golden standard method is not often practicable because of the patient’s status that prohibits invasive procedures . Therefore due to the restrictions of the aforementioned diagnostic tools, a non-culture based method, which can detect a biomarker such as galactomannan (GM) antigen, has been developed. GM is a cell wall polysaccharide component released during growth and invasion of Aspergillus in tissue that is detectable in patients with invasive aspergillosis . Studies showed a sensitivity of 61% to 71%, with a specificity of 89% to 93% for the detection of GM in serum samples . Recently, detection of GM in BAL fluid has been considered as a diagnostic approach for IPA by some authors [14-16]. On the other hand, it has not well established a consensus on optical density (OD) cut-offs in BAL samples by ELISA method for the diagnosis of IA.
According to our search in international databases, there are no available data on GM detection in BAL samples among ICU patients in the Middle East. In the current study, we aimed to assess GM detection in BAL ﬂuid with Platelia Aspergillus GM EIA as an early diagnostic tool among ICU patients who were at risk for developing IA.
Material and Methods
The Ethics Committee of Mazandaran University of Medical Sciences (code: 88-6-4) approved this research and the written informed consent was obtained from the patient or next of kin. We reviewed all adult patients suspected to have IA (host factors and clinical features) as defined by European Organization for Research and Treatment of Cancer/Mycoses Study Group (EORTC/MSG)  who hospitalized in the ICU of the University Hospitals in Mazandaran, Iran from August 2010 to September 2011. BAL samples were collected by pulmonologists twice weekly thereafter if feasible. Patients using piperacillin/tazobactam at the time of bronchoscopy and prolonged use (more than 1 week) of any antifungal drugs were excluded. Briefly, the lobe bronchus in which consolidation was imaged by chest radiograph or chest CT scan was wedged, and 20 to 50 ml of 0.9% sterile saline solution at room temperature was instilled with a syringe through the working channel of the bronchoscope. The total volume of saline solution instilled into the lung was typically 50 ml to 100 ml of BAL fluid was recovered. Subsequently, the obtained BAL fluid was sent directly unprocessed on dry ice to the department of Medical Mycology at Mazandaran University of Medical Sciences, Sari.
A total of sixty six BAL fluid samples were collected. BAL samples were analyzed by direct microscopic examination using CalcoFlour White staining, fungal culture and GM detection. Cultures for fungi were performed by inoculating clinical specimens onto Sabouraud glucose agar at 30˚C for 10 days. The fungi were identified by standard mycological techniques based upon gross cultural and microscopic morphology.
Platelia Aspergillus ELISA
The Platelia Aspergillus GM EIA (Platelia Aspergillus; Bio-Rad, Edmonds, WA) was performed for GM detection according to the manufacturer’s instructions. Briefly, 100 µl of the Platelia treatment solution was added to 300 µl of the BAL specimen, which was then heated at 100°C for 3 min, followed by centrifugation at 10,000 g for 10 min. Then, 50 µl of the supernatant and 50 µl of the horseradish peroxidase-labeled monoclonal antibody (EBA-2) were incubated at 37°C in antibody-precoated microplates for 90±5 min. The plates were washed 5 times, and then were incubated with 200 µl of substrate chromogen reaction solution for 30±5 min at room temperature in the dark. The reaction was stopped by the use of 100 µl sulfuric acid, and after 30 min, the plates were read at an optical density (OD) of the sample to that of the threshold control samples at 450 nm with a reference filter of 620 nm. An OD index of ≥1.0 in two consecutive samples was considered positive. All positive samples were rechecked and only if the repeat test was also positive they have take into accounted positive.
Case and definition of positive results
The Patients with IA were classified as having probable or possible IA on the basis of modified EORTC/MSG criteria . Sensitivity, speciﬁcity, positive predictive value (PPV) and negative predictive value (NPV) were calculated according to the proportion of patients with true and false positive and negative tests. Calculations were also performed based on the certainty of IA.
Table 1 summarized and characterized the patients who were enrolled in this study. According to modified EORTC/MSG criteria, out of 43 patients, 4 cases (30.8%) were classified as possible IA and 9 (69.2%) as probable IA. In this present study, we were not able to define a proven IA case because there was an explicit refusal of the family to do biopsy or autopsy. Out of 21 BAL samples obtained from 13 patients with probable IA, 76.2% was positive for GM. Of these, 53.8% and 77% of samples had positive results in culture and direct microscopic examination methods, respectively (Table 2).
In 6 cases, culture or direct microscopic examination on BAL samples remained negative, whereas GM detection was positive. All patients with IA had at least one positive BAL GM (OD index ≥ 0.5 and > 1.0). Out of 45 BAL samples from patients without IA, 43 (95.5%) had negative GM results (Figure 1).
Table 3 shows the sensitivity, speciﬁcity, positive and negative predictive values of different BAL GM index. A cut off ≥ 0.5 yielded a 100% of sensitivity and 96% of negative predictive value (NPV) with relatively low speciﬁcity and positive predictive value (PPV). On the other hand, a cut off >2 improve of speciﬁcity and PPV.
|Characteristics||Probable IA(n=9)||Possible IA(n=4)||Non IA(n=30)||All(n=43)|
|Mean of age (years)||47.8||62||58.4||56.5|
|Sex, male, (%)||6 (66.6)||2 (50)||16 (53)||24 (58.8)|
|ICU length of stay (days)||7.3||9.75||25||19.6|
|Mechanical ventilation (days)||6.7||6.25||22||18.2|
|No. of deaths (%)||7 (77.8)||2 (50)||8 (26.7)||16 (37)|
|Underlying disease||Neutropenia (%)||7 (77.8)||0||0||8 (18.6)|
|Hematologic (%)||5 (55.6)||2 (6.7)||2 (6.7)||9 (30)|
|COPD (%)||1 (11.0)||1 (3.3)||1 (3.3)||3 (7)|
|Solid cancer (%)||2 (22.0)||5 (16.7)||5 (16.7)||8 (18)|
|Diabetes||0||5 (16.7)||5 (16.7)||6 (14)|
|Other||0||25 (83.3)||25 (83.3)||25 (58)|
|Deﬁnition of abbreviations: COPD = chronic obstructive pulmonary disease; IA = invasive aspergillosis; ICU =intensive care unit.|
|Percentage of patients|
|BAL direct examination||Positive||77||36.6||48.8|
|Deﬁnition of abbreviation: BAL= bronchoalveolar lavage; GM = galactomannan. 1Cutoff value for positivity: 1.0 ng/ml|
|Deﬁnition of abbreviation: PPV= positive predictive value; NPV= negative predictive value; GM = galactomannan|
Detections of GM in BAL samples can be consider as a method, which facilitate the early diagnosis of invasive aspergillosis-a life threatening disease in immunocompromised patients. In spite of several disadvantages, BAL or serum GM detection remains a powerful technique in the diagnosis and management of patients at high risk of invasive aspergillosis . To our knowledge, this is the first study to evaluate the utility of measuring BAL galactomannan in ICU patients in Iran. Recently, several prospective studies in ICU patients reported sensitivities, speciﬁcities and predictive values above 90% for GM detection in BAL samples [4, 14, 19]. Our results indicated that BAL GM detection with higher sensitivity (100 %) and specificity (85.7 %) at a cutoff ≥ 0.5 compared to conventional methods for the diagnosis of IPA in at-risk patients in ICU. This observation is similar to those described by previous studies [14, 19]. The high sensitivity of GM detection in the BAL may reduce the speciﬁcity due to false positive results. However, many sources of false positivity including piperacillin – tazobactam or amoxicillin – clavulanate are frequently and remarkably encountered in the ICU , the speciﬁcity was still above 85%. In our study, BAL GM detection was performed early, promptly after CT and before the start of antifungal treatment. Verweij et al, and Salonen et al, suggested that antifungal therapy can explain the somewhat lower sensitivities of GM detection in BAL ﬂuid [21, 22].
Our study had several limitations; rare case of IA and small sample size which may have limited our power to detect differences between the groups, bronchoscopy procedures is not usually performed in the evaluation of IA in ICU patients due to the critical conditions of patients, and unless with abnormal respiratory signs at CT finding and autopsy process from patients suspected IA, has not been reported cases of proven IA. Moreover, OD measurements are dependent on the concentration of GM antigen, which being dependent on several factors such as the area that is washed, the amount of saline instilled during bronchoscopy procedure, and the amount of fluid retrieved. In our study, instilled either 20 or 50 ml each time, this difference may add variability to the results. The amount of sterile saline instilled varied significantly (40 to 150 ml) between studies [15, 16, 23, 24] suggesting that the results although similar may not be comparable or extrapolated to other centers. Therefore, a standardized method of collection should also be established before results can be comparable between centers. Finally, we cannot provide data on the inﬂuence of antifungal therapy on the performance of GM detection in BAL ﬂuid.
Since the diagnosis of IA is a big challenge, early diagnosis is crucially important because early antifungal therapy improves surviva1. Our data have revealed that the sensitivity of GM detection in BAL was better than that of conventional tests. It seems that GM detection in BAL is beneficial to establish or exclude of early diagnosis of IA in ICU patients. Therefore, a prospective screening of BAL GM is an essential role for early diagnosis of IA, especially when it is combined with other diagnostic tools. The validity of the data needs to be conﬁrmed in other ICUs and examine various aspects in ICU Patients.
- Kousha M, Tadi R, Soubani AO. Pulmonary aspergillosis: a clinical review. Eur Respir Rev. 2011; 20(121):156-74.
- Meersseman W, Lagrou K, Maertens J, Wijngaerden EV. Invasive Aspergillosis in the Intensive Care Unit. Clin Infect Dis. 2007; 45(2):205-16.
- Dimopoulos G, Frantzeskaki F, Poulakou G, Armaganidis A. Invasive aspergillosis in the intensive care unit. Ann N Y Acad Sci. 2012; 1272:31-9.
- Sipsas NV, Kontoyiannis DP. Invasive fungal infections in patients with cancer in the Intensive Care Unit. Int J Antimicrob Agents. 2012; 39(6):464-71.
- Dutkiewicz R, Hage CA. Aspergillus infections in the critically ill. Proc Am Thorac Soc. 2010; 7(3):204-9.
- Hope WW, Walsh TJ, Denning DW. Laboratory diagnosis of invasive aspergillosis. Lancet Infect Dis. 2005; 5(10):609-22.
- Meersseman W, Wijngaerden EV. Invasive aspergillosis in the ICU: an emerging disease. Intensive Care Med. 2007; 33:1679-1681.
- Nucci M, Nouér SA, Cappone D, Anaissie E. Early diagnosis of invasive pulmonary aspergillosis in hematologic patients: an opportunity to improve the outcome. Haematologica. 2013; 98(11):1657-60.
- Tarrand JJ, Lichterfeld M, Warraich I, Luna M, Han XY, May GS. Diagnosis of invasive septate mold infections. A correlation of microbiological culture and histologic or cytologic examination. Am J Clin Pathol. 2003; 119(6):854-8.
- Perfect JR, Cox GM, Lee JY, Kauffman CA, de Repentigny L, Chapman SW. The impact of culture isolation of Aspergillus species: a hospital-based survey of aspergillosis. Clin Infect Dis. 2001; 33:1824-33.
- Guarner J, Brandt ME. Histopathologic Diagnosis of Fungal Infections in the 21st Century. Clin Microbiol Rev. 2011; 24(2):247-280.
- Pinel C, Fricker-Hidalgo H, Lebeau B, Garban F, Hamidfar R, Ambroise-Thomas P. Detection of circulating Aspergillus fumigatus galactomannan: value and limits of the Platelia test for diagnosing invasive aspergillosis. J Clin Microbiol. 2003; 41(5):2184-6.
- Clancy CJ, Jaber RA, Leather HL, Wingard JR, Staley B, Wheat LJ. Bronchoalveolar Lavage Galactomannan in Diagnosis of Invasive Pulmonary Aspergillosis among Solid-Organ Transplant Recipients. J Clin Microbiol. 2007; 45(6):1759-1765.
- Meersseman W, Lagrou K, Maertens J, Wilmer A, Hermans G, Vanderschueren S. Galactomannan in bronchoalveolar lavage fluid: a tool for diagnosing aspergillosis in intensive care unit patients. Am J Respir Crit Care Med. 2008; 177(1):27-34.
- Musher B, Fredricks D, Leisenring W, Balajee SA, Smith C, Marr KA. Aspergillus galactomannan enzyme immunoassay and quantitative PCR for diagnosis of invasive aspergillosis with bronchoalveolar lavage fluid. J Clin Microbiol. 2004; 42(12):5517-22.
- Husain S, Paterson DL, Studer SM, Crespo M, Pilewski J, Durkin M. Aspergillus galactomannan antigen in the bronchoalveolar lavage fluid for the diagnosis of invasive aspergillosis in lung transplant recipients. Transplantation. 2007; 83(10):1330-6.
- De Pauw B, Walsh TH, Donnelly JP, Stevens DA, Edwards JE, Calandra T. European Organization for Research and Treatment of Cancer/ Invasive Fungal Infections Cooperative Group; National Institute of Allergy and Infectiou Diseases Mycoses Study Group (EORTC/MSG) Consensus Group: Revised definitions of invasive fungal disease from the European Organization for Research and Treatment of Cancer/ Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infections Diseases Mycoses Study Group (EORTC/MSG) Consensus Group. Clin Infect Dis. 2008; 46(12):1813-2.
- D'Haese J, Theunissen K, Vermeulen E, Schoemans H, De Vlieger G, Lammertijn L. Detection of Galactomannan in Bronchoalveolar Lavage Fluid Samples of Patients at Risk for Invasive Pulmonary Aspergillosis: Analytical and Clinical Validity. J Clin Microbiol. 2012; 50(4):1258-1263.
- Guo YL, Chen YQ, Wang K, Qin SM, Wu C, Kong JL. Accuracy of bronchoalveolar lavage galactomannan in diagnosing invasive aspergillosis: a bivariate meta- analysis and systematic review. Chest J. 2010; 138(4):45-55.
- Zou M, Tang L, Zhao S, Zhao Z, Chen L, Chen P. Systematic Review and Meta- Analysis of Detecting Galactomannan in Bronchoalveolar Lavage Fluid for Diagnosing Invasive Aspergillosis. PLoS One. 2012; 7(8):e43347.
- Verweij PE, Latge JP, Rijs AJ, Melchers WJ, De Pauw BE, Hoogkamp-Korstanje JA. Comparison of antigen detection and PCR assay using bronchoalveolar lavage ﬂuid for diagnosing invasive pulmonary aspergillosis in patients receiving treatment for hematological malignancies. J Clin Microbiol. 1995; 33(12):3150-3153.
- Salonen J, Lehtonen OP, Terasjarvi MR, Nikoskelainen J. Aspergillus antigen in serum, urine and bronchoalveolar lavage specimens of neutropenic patients in relation to clinical outcome. Scand J Infect Dis. 2000; 32(5):485-490.
- Maertens J, Maertens V, Theunissen K, Meersseman W, Meersseman P, Meers S. Bronchoalveolar lavage fluid galactomannan for the diagnosis of invasive pulmonary aspergillosis in patients with hematologic diseases. Clin Infect Dis. 2009; 49(11):1688-93.
- Nguyen MH, Jaber R, Leather HL, Wingard JR, Staley B, Wheat LJ. Use of bronchoalveolar lavage to detect galactomannan for diagnosis of pulmonary aspergillosis among nonimmunocompromised hosts. J Clin Microbiol. 2007; 45(9):2787-92.