From an increase in the number of immunocompromised hosts including AIDS patients, organ transplantation, solid-organ tumor, hematological malignancy, corticosteroid use, and others underlying diseases, it leads to increasing the incidence of invasive aspergillosis (IA) as one of the most prevalent opportunistic mould infections. However, the epidemiological data are still limited. Our objective is to study the epidemiology of IA, patients’ characteristics in a tertiary-care hospital, King Chulalongkorn Memorial Hospital, Bangkok, Thailand. The retrospective study of IA as principal diagnosis in both medical and laboratory records in a tertiary-care hospital, King Chulalongkorn Memorial Hospital, from January 1, 2006 to December 31, 2011, was performed. There were 69 patients who were diagnosed as IA during 2006 till 2011. They were classified as proven (45 patients), probable (3 patients), and possible (21 patients) invasive aspergillosis following the criteria of European Organization for Research and Treatment of Cancer/Mycoses Study Group (EORTC/MSG), 2008. The numbers of patients in 2006 to 2011 were 3, 11, 12, 10, 10, and 23 respectively. Male patients were 58 percent. The age range was from 8 months to 87 years old. Most of patients were from Medicine ward. Others were derived from Pediatrics, Surgery, and Ear Nose Throat wards. The most common underlying disease was diabetes mellitus type 2 in the proven group. The main predisposing factors of patients were the history of pulmonary tuberculosis and using of immunosuppressive drugs. The sites of infection were lung (62%), sinus (28%), and brain (8%). Aspergillus fumigatus (69%) and Aspergillus flavus (15%) were common species from the isolated culture. The treatment used mostly was surgery and followed by amphotericin B or voriconazole. The case fatality rate of IA was 20 percent. From the epidemiological data, we can conclude that in this past ten years there is an incessant increase in the number of IA in the immunocompromised hosts especially from Aspergillus fumigatus, which is the most prevalent species found in IA. Diabetes mellitus and history of pulmonary tuberculosis will play the important role for IA in the future. The plan for prevention and treatment should be concerned about those underlying diseases and predisposing factors.
Aspergillus species are saprophytic filamentous fungi. This genus is characterized by the flask-shaped or cylindrical phialides on the vesicle at the apex of a conidiophore. Asexual spores or conidia are globose and various in colors. Their spores can be found in the soil, composed piles, air, animals, and humans. They can be pathogens in immunocompromised hosts such as patients with acquired immunodeficiency syndrome (AIDS), patients who had allogenic hematopoietic stem cell transplantation or solid organ transplantation, patients who received immunosuppressive drugs, patients with prolonged neutropenia, and patients with others underlying diseases. The common pathogenic Aspergillus species include A. fumigatus, A. flavus, A. niger, and A. terreus. A. nidulans can also cause infections mostly in patients with chronic granulomatous disease (CGD). There are three forms of aspergillosis: invasive aspergillosis, chronic aspergillosis, and allergic forms of aspergillosis. Chronic aspergillosis is less frequently found than the acute disease. Affected patients usually have the common underlying conditions including previous tuberculosis infection, atypical mycobacterial infection, Chronic Obstructive Pulmonary Disease (COPD), other chronic lung diseases, diabetes mellitus, and alcoholism. Allergic forms of aspergillosis include allergic sinusitis and allergic bronchopulmonary aspergillosis [
For Asian countries, an increased incidence of IA has been reported although the data are still limited [
The authors reviewed both medical and laboratory records, including culture-based methods and galactomannan enzyme immunoassay detection, retrospectively from January 1, 2006 to December 31, 2011 at King Chulalongkorn Memorial Hospital (1479-bed medical school hospital, a tertiary-care hospital in Bangkok, Thailand). The data of patients with IA were retrieved by using principal diagnosis as invasive aspergillosis (IA) with the computer system in the hospital and the clinical microbiology laboratory.
The patients were categorized into three groups of invasive aspergillosis diagnosis from revised definition of European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) in 2008 [
Those three categories are proven, probable, and possible diagnosis of invasive aspergillosis. Proven diagnosis of IA was defined as a culture from sterile sites with identification as Aspergillus spp. Probable diagnosis of IA was described as the combination of at least one host factor, one clinical feature, and one mycological evidence. Possible diagnosis was the host factors with clinical features excluding mycological evidence. We also categorized the response rates as complete response, partial response, relapse, and death [
All specimens were observed by using 10% KOH (Potassium hydroxide) solution and Gram stain. All specimens were cultured for at least three weeks in Sabouraud dextrose agar (BD DifcoTM, Sparks, MD, USA), Sabouraud dextrose agar with chloramphenicol (AcumediaTM, Lansing, MI, USA), MycoselTM or Sabouraud dextrose agar with chloramphenicol and cycloheximide (BD BBLTM, Sparks, MD, USA), and Sabouraud brain heart infusion agar base (BD DifcoTM, Sparks, MD, USA) at both 37˚C and 25˚C. All positive colonies were observed under light microscopes with lactophenol cotton blue staining.
The Platelia Aspergillus Ag (Bio-Rad, Redmond, WA) was used for measuring galactomannan levels following manufacturer’s instructions. Bronchoalveolar lavage (BAL) and sera samples from patients were processed and mixed well. 300 μl of each sample was added to 100 μl of sample treatment solution, heated for 6 minutes in a 120˚C heat block, and then centrifuged 10 min at 10,000 g. Meanwhile, negative controls, cut-off controls, and positive controls were treated in the same way. Supernatant (50 μl) was mixed to 50 μl of a conjugated anti-ga- lactomannan EBA-2 monoclonal antibody labeled with peroxidase in microplate. The microplate with a plate sealer was incubated at 37˚C for 90 minutes and then it was washed for 5 times with washing solution by an automated washer (Diagnostics Pasteur, Paris, France). After washing, wells were rapidly added 200 μl of chromogen tetramethylbenzidine solution and incubated at 25˚C for 30 minutes in the dark. Stopping solution (100 μl) containing 1N sulfuric acid was added to each well to stop reaction. Optical densities (ODs) at 450/620 nm were read in each well by a microplate reader (BioTek Instruments, Winooski, VT). Negative controls, cut-off controls, and positive controls were read at the same time in each assay. Results were determined as an index relative to the OD of the mean cut-off control (GM index = OD sample/mean cut-off control OD).
Statistical analysis used in this study was shown as mean, standard deviation (SD), percent, and McNemar’s test by Graph Pad Prism 5.0 software to compare risk factors leading to the death of patients. p-value at < 0.05 is significant.
This study was approved by Institutional Review Board (IRB No. 350/55), Faculty of Medicine, Chulalongkorn University.
Sixty-nine patients were diagnosed as IA from 2006 to 2011 as shown in
The leading underlying diseases were diabetes mellitus (42.6%), followed by malignancy (12.8%) including
hematological malignancy and solid organ malignancy. The main predisposing factors were the history of old pulmonary tuberculosis (53.1%), and using corticosteroid (37.5%). Lungs (62.3%) were the most common sites of infection. Thirty-six patients (52.1%) were recruited from the Medicine ward. Mean length of stay in hospital ± SD was 26.9 ± 34.7 days (range, 1 to 137 days). Demographic characteristics and clinical data are provided in
Galactomannan Enzyme Immunoassay (EIA) test was performed in twelve patients as shown in
Aspergillus fumigatus (69.2%) was the most common Aspergillus species isolated from patients with IA, followed by Aspergillus flavus (15.4%) as shown in
Characteristics | Number (%) |
---|---|
Mean age ± SD, years (range) | 48.5 ± 23.1 (8 mon-87 yr.) |
Gender Male Female | 40 (58.0) 29 (42.0) |
Underlying diseasesa Diabetes mellitus Malignancy Leukemia Lymphoma Malignant thymoma Renal diseases Systemic lupus erythematosus (SLE) Chronic granulomatous disease (CGD) Autoimmune hepatitis Chronic obstructive pulmonary disease (COPD) Others: lung transplantation, Kasabach Merritt Syndrome, aplastic anemia, anemia of chronic disease, Multiple sclerosis | 20 (42.6) 6 (12.8) 4 (8.5) 1 (2.1) 1 (2.1) 5 (10.6) 4 (8.5) 3 (6.4) 2 (4.3) 2 (4.3) 5 (10.6) |
Predisposing factorsa Old pulmonary tuberculosis Corticosteroid Chemotherapy | 17 (53.1) 12 (37.5) 3 (9.4) |
Specimensa Lungs Bronchioalveolar lavage (BAL) Sputum Tissue Sinus Pus Tissue Central nervous system Tissue Skin Pus | 43 (62.3) 3 11 29 19 (27.5) 2 17 6 (8.7) 6 1 (1.4) 1 |
Length of stay ± SD, days (range) | 26.9 ± 34.7 (1 - 137) |
Wards Medicine, general Medicine, intensive care unit (ICU) Surgery Eye Nose Throat (ENT) Pediatrics, general Pediatrics, intensive care unit (ICU) Orthopedics | 25 (36.2) 11 (15.9) 11 (15.9) 10 (14.5) 8 (11.6) 3 (4.3) 1 (1.4) |
SD: Standard deviation. aSome patients had more than one underlying diseases, predisposing factors or sites of infection.
sponse came from surgery alone (33.3%), surgery combined with antifungal agents (8.7%), and antifungal agents alone (26.0%). About a half of patients with complete response among antifungal agents alone received medications as voriconazole alone and voriconazole combined with other antifungal agents. Fourteen patients died from IA. The sites of infection from those fourteen patients came from lung (10 patients), brain (3 patients), and sinus (1 patient). Six patients who died from IA received amphotericin B alone. Two patients who died from
Galactomannan test | Culture for Aspergillus spp. | |
---|---|---|
Positive | Negative | |
Positive | 4 | 6 |
Negative | 0 | 2 |
Characteristics | Number (%) |
---|---|
Treatmentsa Surgery Voriconazole Amphotericin B Caspofungin Anidulafungin Micafungin | 33 (44) 19 (25.3) 17 (22.7) 4 (5.3) 1 (1.3) 1 (1.3) |
Outcomes Complete response Partial response Relapse Death | 48 (69.6) 2 (2.9) 5 (7.2) 14 (20.3) |
aSome patients had more than one treatment.
Factors | 95% CI | p-value* |
---|---|---|
Using corticosteroid Diabetes Brain infection Lung infection | 0.542 to 6.837 0.274 to 1.464 0.969 to 20.46 0.046 to 0.535 | 0.4227 0.3447 0.0614 0.0009 |
*p-value at < 0.05 is significant.
IA received voriconazole alone and another two patients received echinocandin (caspofungin or anidulafungin) alone. The remainder received combined therapy of amphotericin B and other antifungal agents.
According to the previous studies in North America [
For the diagnostic strategies of IA, the gold standard is the tissue biopsy but it is very invasive and could cause other complications such as bleeding in high-risk patients. Positive blood culture for IA is rare and the main difficulty of interpretation the positive culture from respiratory and other specimens is the lack of sensitivity and difficulty in distinguishing between infection and colonization. Galactomannan EIA test is an assay to detect galactomannan (GM) antigen, which is the main component of Aspergillus cell wall, using a rat anti-GM monoclonal antibody, EB-A2. It will recognize the 1,5-β-D-galactofuranoside sidechains. This test shows high specificity (>90%) and variable sensitivity (70% - 97%) for the detection of IA [
Aspergillus species isolated from patients with bone marrow transplant and solid organ transplant in North America were generally Aspergillus fumigatus [
For the treatment options of IA, it is dependent on many factors including host factors, underlying diseases, concomitant infections, and degree and duration of immunosuppression [
Crude mortality rate for IA in North America was about 58 percent in 1995-1999 [
This study has some limitations. There could be some missing data from the misdiagnosis. It also had small sample size that might not represent the Thai population. Further studies that combine the data from every tertiary-care hospital in Thailand are necessary to be performed to understand the IA situation in Thai population.
In conclusion, the epidemiological data of Thai patients were similar to other countries. Although in this study, the vast majority of underlying disease was diabetes mellitus, it could not definitely conclude that diabetes mellitus was the only patients’ risk factor to IA. This is because more than 790 people per 100,000 Thai population had diabetes mellitus as underlying disease in 2011 [
A.T. would like to express our special thanks to Dr. Nitipong Permpalung and Nicholas R. Waldner for their great suggestion and Department of Microbiology and Department of Medicine, Faculty of Medicine, Chulalongkorn University for providing the data.