1. Background
Invasive fungal infections (IFIs) are one of the most important causes of morbidity and mortality in patients with hematological malignancies. Factors such as neutropenia, damaged mucosa, receiving high-dose chemotherapy, undergoing invasive medical procedures, and using broad-spectrum antibacterial drugs, constitute the risk factors for IFIs (1, 2). The frequency of candidemia has increased during the past few decades. The mortality rate in non-immunocompromised children hospitalized in the pediatric intensive care unit was reported to be 32% (2), whereas children with a hematologic disease displayed a mortality rate of approximately 60% (3). The mortality rate related to invasive fungal infections in children with hematologic disorders was found to be 30% in a study by Baytan et al. (4). “In children, the mortality rate for invasive aspergillosis is 2.5 to 3.5 higher than that for invasive candidiasis (respectively 70% vs. 20% and 30%)” (5).
Clinical symptoms and radiological patterns are not specific and late diagnosis may lead to delayed therapy, which is associated with a poor outcome (6). Examination commonly includes a direct smear with potassium hydroxide, culturing, and histopathological analysis, however, in some cases, this can be challenging since sampling requires invasive procedures, which are not possible in critically ill patients. Hence, the use of a non-invasive method, such as real-time PCR, for the detection of fungal infections in clinical samples could be a suitable approach in aiding early diagnosis in high-risk infected patients (7).
2. Objectives
Since the role of PCR in diagnosing fungal infections is not yet well established (8), the aim of this study was to investigate the rate of IFIs in pediatric patients with hematological disorders, using traditional (direct smear and culture) and real-time PCR methods.
3. Methods
During a 13-month period (January 2014 to February 2015), 86 pediatric patients with hematological disorders were admitted to Amir Hospital, Shiraz University of Medical Sciences, in Iran and were kept under observation for the development of IFIs. Patients with hematological disorders and clinical signs including fever, ascites, epistaxis, febrile neutropenia, chest pain, dyspnea, and dry cough as well as patients with a suspicious fungal rash were included in this study. Pediatric patients admitted due to malignancies, with the exception of hematological malignancies, were excluded from the study. In order to determine fungal colonization, clinical samples such as urine, nasal, mouth, and rectal swabs were obtained at time of admission. The samples were cultured on saboaroud dextrose agar (Merck, Germany) and incubated at 24°C for 10 days. Colonization was defined as the presence of Candida spp. in any of the body sites without any local or systemic symptoms or signs of infection. During the hospital stay, samples (e.g., plural and abdominal tap, cerebrospinal fluid, blood, urine, oropharyngeal, biopsy, and broncho-alveolar lavage samples) from patients with clinical signs of infection were examined by culturing on sabouraud dextrose agar with chloramphenicol, and direct microscopic examination. In patients suspicious to fungal infections according to clinical signs and symptoms, blood specimens were collected for PCR and cultured by bedside inoculation into BACTEC medium (Becton- Dickinson, Sparks, Md., USA), twice weekly. The clinician and an expert radiologist analyzed the imaging modalities in order to detect IFIs. The isolated molds were diagnosed by colony morphologic and microscopic characterization of species with the use of lactophenol cotton blue, whereas yeasts were identified using the API 20 C AUX system (bioMerieux, France), according to the manufacturer’s instructions. The demographic characteristics of the patients were extracted from their medical files. The results of the pathology smear were collected from the patient’s records.
Fungal DNA was extracted from the patients’ sera by using the QIAmp DNA minikit (Qiagen,Hilden, Germany), according to the manufacturer’s recommendations. To perform PCR, the suggestions of Shin et al. and Kami et al. (9, 10) were taken into consideration. The primers and a TaqMan probe (Metabion Martinsried, Germany) were used, and thermal cycling conditions for the Aspergillus and Candida real-time PCR assay were performed, as described previously (9, 10). Thermal cycling conditions (11) were carried out using the ABI 7500 FAST instrument (Applied Biosystem, Foster City, CA, USA).
Invasive fungal infections were defined according to the European Organization on research and treatment in cancer and the mycoses study group (8). To be defined as a proven infection, fungal elements in tissue (by histopathology) or isolates from etiologic agents were required to be found in cultures of samples from a normally sterile site, such as blood, CSF, and tissue in immunocompromised patients. Combination of a susceptible host, clinical signs compatible with fungal infection and mycological evidence or indirect (non-culture/non-histopathologic evidence) from non-sterile clinical samples like sputum and bronchoalveolar lavage were defined as probable IFIs.
The ethics committee of Shiraz University of Medical Sciences reviewed and approved the study, which was carried out in accordance with the 1975 declaration of Helsinki, as revised in 1983. Written consents of the patients were obtained before participating in the study.
Data were analyzed by descriptive statistical methods using the SPSS statistical package (SPSS, Chicago, IL, USA).
4. Results
The female-to-male ratio was 38:48, and the mean age of the patients was 6 years (standard deviation: 3.9). The most common hematological disorders in these patients were acute lymphoblastic leukemia (ALL) in 50 patients (58%), acute myeloid leukemia (AML) in 21 patients (24%), followed by Hodgkin’s lymphoma, Burkit lymphoma, megaloblastic anemia, and aplastic anemia. The mean white blood cell count was 3522 (standard deviation: 3300).
Candida colonization was seen in 42 patients (48.8%) and the most frequent sites of colonization were oral cavity 53.3%, rectum 33.3%, nose 6.7% and urine 6.7%. The etiologic agents were found to be C. albicans in 73.3% of the cases, followed by C. krusei, C. tropicalis, C. dubliniensis and C. famata. Oral candidiasis was diagnosed in 7 (8%) patients (C. albicans was grown in 5 cases, C. tropicalis in 1 and C. krusei in 1 case).
A total of 225 clinical specimens consisting of blood, urine, cerebrospinal fluid, pleural and abdominal tap, bronchoalveolar lavage, abscess secretion, and sputum were examined for fungal infection. Candidemia was detected in 4 patients. According to clinical, radiological and mycological data, proven and probable IFIs were detected in 14 of the 86 patients (16.3%), using pathological, conventional and molecular diagnostic methods. Demographic and clinical characteristics of the pediatric patients are presented in Table 1. The etiologic agents based on the culture results from clinical specimens consisted of C. albicans in 5 cases, A. flavus in 3, and A. fumigatus, fusarium, Alterneria and Mucor, each in 1 case. In 2 cases with liver abscess, only the Candida PCR was positive. Blood culture results in these 2 cases were negative and sampling from the abscess was not done. Lungs and sinuses were the most infected sites (Table 1). The detection limit of real-time PCR was 10 copy numbers per ml. Real-time PCR was positive in 10 patients (5 of 8 proven, 5 of 6 probable). Despite appropriate antifungal therapy, 50% (7/14) of the patients unfortunately passed away.
Table 1. Characteristics of Pediatric Patients with Proven and Probable Invasive Fungal Infections
| No | Background | Criteria | Culture Result | Serum PCR result | WB Ccount | Treatment | Site of Infection (Sample) | Outcome |
|---|---|---|---|---|---|---|---|---|
| 1 | ALL | Proven | C. albicans | A: negative | 22000 | Amphotericin | Blood | Survival |
| C: positive | ||||||||
| 2 | ALL | Proven | C. albicans | A: negative | 1800 | Amphotericin | Blood | Survival |
| C: positive | ||||||||
| 3 | ALL | Proven | Fusarium | A: negative | 228 | Amphotericin | Lung (tissue) | Death |
| C: negative | ||||||||
| 4 | ALL | Proven | C. albicans | A:negative | 900 | Amphotericin | Blood | Survival |
| C: positive | ||||||||
| 5 | ALL | Proven | Alterneria | A: negative | 400 | Amphotericin | Sinus (Tissue) | Death |
| C: negative | ||||||||
| 6 | ALL | Proven | C. albicans | A: negative | 1400 | Amphotericin | Sinus (Tissue) | Death |
| C: positive | ||||||||
| 7 | ALL | Proven | C. albicans | A:negative | 22000 | Amphotericin | Blood | Survival |
| C: positive | ||||||||
| 8 | ALL | Proven | Aspergillus flavus | A:negative | 900 | Voriconazole | Sinus (Tissue) | Death |
| C:negative | ||||||||
| 9 | ALL | Probable | Mucor | A:negative | 6900 | Posaconazole | Lung (sputum) | Death |
| C:negative | ||||||||
| 10 | AML | Probable | Aspergillus flavus | A: positive | 1700 | Voriconazole | Lung (sputum) | Death |
| C:negative | ||||||||
| 11 | AML | Probable | Aspergillus flavus | A: positive | 18800 | Voriconazole | Lung (sputum) | Survival |
| C:negative | ||||||||
| 12 | ALL | Probable | No sample | A:negative | 500 | Amphotericin | Liver Abscess | Survival |
| C: positive | ||||||||
| 13 | ALL | Probable | Aspergillus fumigatus | A: positive | 13600 | Amphotericin Voriconazole | Lung (sputum) | Survival |
| C:negative | ||||||||
| 14 | ALL | Probable | No sample | A: negative | 1200 | Amphotericin | Liver Abscess | Death |
| C: positive |
Abbreviations: ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; A, Aspergillus PCR in serum; C, Candida PCR in serum; EORTIC/MSG, European organization on research and treatment in cancer and the mycoses study group.
5. Discussion
Acute leukemia (myeloid, lymphoid) was the most frequent underlying disease among the patients, as was similarly seen in other studies (12). Candida colonization in this study was reported in 48.8% of all patients and 100% of patients with invasive candidiasis. There was a significant correlation between invasive candidiasis and Candida colonization (PV < 0.05) in patients with hematologic malignancy. The rate of Candida colonization was reported to be 55.5% in two other studies, with 107 patients out of 197 in the first study and 35 patients out of 63 in the second study (13, 14). Candida albicans was the most prevalent etiologic agent in both this study and other earlier studies (13, 14). Candida colonization may be a pre-requisite for systemic candidiasis in patients with hematologic malignancy.
In the present study, the rates of IFIs among pediatric patients with hematological disorders were found to be 16.3%. The rate of proven IFIs in an autopsy study over a 15-year period (Chamilos et al.) revealed to be 31% in patients with with hematologic malignancies (15) and the incidence rate of invasive aspergillosis and systemic candidiasis in the pediatric patients was shown to be 27.4% and 12.9%, respectively (13, 16). Since many studies do not have the possibility to perform autopsies or molecular assays, lower incidence rates were found, including 13% (17), 6.9% (18), and 4.6% (19). Unfortunately, despite starting the antifungal agents, 50% (7/14) of the patients died. These patients were infected with Mucor, Fusarium, Alternaria, A. flavus, and C. albicans. The mortality rate of IFIs was reported to be 48.2% (18). In a review study on 29 episodes of IFIs, Candida spp. was the leading pathogen followed by the Aspergillus species (19). In a large epidemiological survey of fungal infections in patients with hematologic malignancies conducted by Ramirez et al. over half of the fungal infections were caused by molds, especially Aspergillus spp. followed by yeast infections including candidemia (20). The highest IFI-attributable mortality rates were associated with zygomycosis followed by fusariosis, aspergillosis, and candidemia (21).
The most common infected region presented in our proven and probable patients was the respiratory tract system, specifically 3 sinuses and 5 lungs (57.1%), followed by fungemia 4/14 (28.6%) and the liver 2/14 (14.3%). In a study by Kobayashi et al., the most common infected region in 334 patients with hematologic and malignant diseases were “lung in 14 patients, liver in 5, brain in 3, fungemia in 2, kidney in 1, and endophthalmitis in 1 patient” (18).
Culture of clinical specimens is the gold standard test for detection and identification of IFIs, however, the method is time consuming and sample collection through biopsy in some hematological patients requires invasive procedures. Blood cultures are rarely positive in infected patients with mold fungi. In this study, no positive blood culture for mold fungi was observed and 4 patients had a positive result for candidemia. If other samples such as tissue, bronchoalveolar fluid, sputum, needle aspiration and peritoneal fluid could be obtained from the patients, then, culture and direct smear (pathology smear) would serve as the best method for diagnosis of infections. Since such specimens are not available in some cases, it would be better to use non-invasive methods like PCR in sera. In this study, we selected real-time PCR as a facilitating and rapid screening test for medically important fungi in sera, and a feasible approach that enables culture-independent screening of fungal infections and helps identify infected patients at an early stage. The real-time PCR assay provides a high sensitivity and specificity for the detection of fungal DNA and rapidly identifies most of clinically relevant Aspergillus species (20). Many studies have reported the correlation between the presence of proven, probable, and possible criteria of IFIs according to the EORTC-MSG criteria and PCR results of the respective patients’ sera (17, 21, 22). The results of the present study show 5/8 patients with proven and 5/6 patients with probable criteria. In proven patients, when the specific primers (Aspergillus and Candida) were used, PCR proved suitable for diagnosis. In probable patients, where clinical sampling is difficult or impossible (in patients with liver abscess), this method could help the clinicians with diagnosing the infection. As revealed in this study, aspergillosis was confirmed by PCR in patients with negative blood culture results, in accordance with the results by Schabereiter-Gurtner al. (23). The limitation of this study was the lack of using real-time PCR for the detection of fungal pathogens like Fusarium, Alternaria, and Mucor spp.
The rate of invasive fungal infections in our study was high and Aspergillus and Candida were the most frequently encountered fungi. Some new non-culture based methods such as PCR would be useful for early and accurate detection of IFIs resulting in a better outcome for the infected pediatric patients.
