DNA microarray for identification of fungal pathogens causing invasive fungal infections [IFIs] in neutropenic patients

Opportunistic invasive fungal infections [IFIs] remain as important cause of morbidity and mortality. Candida and Aspergillus species are the most common fungi that cause disease in immunocompromised patients and transplant recipients. This study was designed to identify Candida and AspergilIus spp. as possible causes of IFIs in neutropenic patients with different hematological diseases, using high multiplexing capacity of DNA microarray [species identification array]. Twenty eight patients admitted to Hematology unit-Ain Shams University Hospitals with provisional diagnosis of lFl were enrolled in this study. Venous blood samples were collected to detect Candida and Aspergillus spp. using DNA microarray. Nineteen out of 28 studied patients [67.9%] were infected with Candida and Aspergillus spp. Invasive aspergillosis constituted 13/19[68.4%] distributed as follows: A.fumigatus 6/19 [31.6%], A.flavus 4/19 [21%] and A.niger 3/19 [15.8%]. On the other hand, Fl with Candida spp. constituted 6/19 [31.6%] distributed as follows; C.glabrata 3/19 [15.8%], C.tropicalis 2/19 [10.5%] and C.albicans 1/19 [5.3%]. Duration of hospital stay [mean +/- SD = 30.8 +/- 4 days] was statistically significant among the infected group in comparison to other patients [mean +/- SD = 22.7 +/- 2.3 days]. Nineteen out of 28 studied patients [67.9%] were infected with Candida and Aspergillus spp. Invasive aspergillosis constituted 13/19 [68.4%], while Candidal infection constituted 6/19 [31.6%]. DNA microarray represents a reliable method of potential use in clinical laboratories for parallel one-shot detection and identification of the most common fungal pathogens at the species level for prompt management of infection with tailored antifungal treatments

Comparación de métodos de extracción de ADN de sangre para detectar ADN fúngico por PCR / Comparison of different methods of DNA extraction from blood to detect fungal DNA by PCR

La infección fúngica invasora (IFI) está asociada a un alto índice de mortalidad, que alcanza el 50% debido a la frecuente falla en el tratamiento antifúngico. Existen dificultades para realizar un diagnóstico micológico rápido y certero dada la baja sensibilidad de los métodos convencionales, especialmente en pacientes neutropénicos y con SIDA. Numerosos métodos para diagnosticar infecciones micóticas basados en el estudio del ADN fúngico están actualmente en desarrollo. Nosotros evaluamos la utilidad de dos procedimientos de extracción y purificación del ADN fúngico presente en sangre para su posterior detección por PCR. Ambos métodos resultaron igualmente eficientes para obtener ADNs de óptima calidad y para realizar la técnica de PCR con los iniciadores universales para hongos ITS 1 e ITS 4.

Invasive fungal infections (IFI) are associated with high mortality by reaching levels of 50%, and also with a significant failure in antifungical treatments. This fact mostly obeys to difficulties in obtaining a fast and accurate mycologic diagnosis due to the low sensitivity of conventional methods, mainly in neutropenic and AIDS patients. Various methods based on fungal DNA study are currently being used for the diagnosis of mycotic infections. We herein evaluated two procedures of extraction and purification of fungal DNA in blood for their use in PCR detection. Both of them showed equal efficiency in obtaining high performance DNA with universal primers ITS 1and ITS 4 as target.

Evaluation of polymerase chain reaction for the detection of Paracoccidioides brasiliensis DNA on serum samples from patients with paracoccidioidomycosis

The aim of this study was to demonstrate the DNA of Paracoccidioides brasiliensis in human serum samples of patients with paracoccidioidomycosis using the polymerase chain reaction (PCR). The diagnosis of paracoccidioidomycosis (PCM) was defined by microscopic observation of the fungus on direct exam or histopathology, culture, and serological positivity. DNA from serum of 33 patients with PCM was extracted and submitted to nested-PCR using primers from the gp 43 gene. Only one sample was positive on nested-PCR. We conclude that the prevalence of fungemia in patients with different clinical forms of PCM is low, limiting the use of serum DNA detection as an alternative diagnostic tool.