Mucormycosis in Cairo, Egypt: review of 10 reported cases

Medical Mycology, 2014, 52, 73–80 doi: 10.3109/13693786.2013.809629 Advance Access Publication Date: 15 July 2013 Original Article Original Article Mucormycosis in Cairo, Egypt: review of 10 reported cases Sherif M. Zaki1,∗ , Iman M. Elkholy2 , Nadia A. Elkady1 and Khayria. Abdel-Ghany1 1 Microbiology Department, Faculty of Science and 2 Clinical Pathology Department, University Specialized Hospital, Ain Shams University, Cairo, Egypt *To whom correspondence should be addressed. E-mail: Received 5 January 2013; Revised 11 April 2013; Accepted 21 May 2013 Abstract We report on 10 cases of mucormycosis, as defined by The European Organization for Research and Treatment of Cancer and Mycoses Study Group (EORTC/MSG) standards of invasive fungal diseases, among patients with a recent history of neutropenia, prolonged use of corticosteroids and treatment with immunosuppressants. They were all observed at the Ain Shams University Specialized Hospital in Cairo, Egypt, during the year 2010. These cases were categorized as 50% proven and 50% probable, with none considered to be possible mucormycosis. The median age of the patients discussed in this report was 50 years (range 22–68 years), of which 80% were male and 20% were female. Uncontrolled diabetes with ketoacidosis was noted in 60% of cases, while 40% of the patients had undergone liver transplantations. Pulmonary mucormycosis was the predominant presentation as it was noted in 80% of cases, but there was only 20% sinus involvement. Members of the genus Lichtheimia were the most common etiologic agents (40% of all cases), whereas Rhizopus ssp. were recovered from 30% of cases, Syncephalastrum spp. in 20%, and 10% of patients were infected with Rhizomucor. Liposomal formulation of amphotericin B (LAMB) was successfully used to treat all the cases described in this report. We concluded that the incidence of mucormycosis was relatively high during the study period in this one-center study and that additional studies looking into the diagnosis and the control of mucormycosis in Egypt are required. Key words: mucormycosis, epidemiology, ITS1-5.8s-ITS2, Egypt. Introduction Mucormycosis is considered the third most common invasive fungal disease after candidiasis and aspergillosis [1] and all such diseases are important causes of morbidity and mortality [2]. Risk factors for mucormycosis include corticosteroid and deferoxamine therapy, diabetic ketoacidosis, hematologic malignancy, solid organ transplant, penetrating trauma or burns, and complications of health care pro- cedures [3]. The clinical presentations of mucormycosis fall in six major forms which include rhinocerebral, pulmonary, cutaneous, gastrointestinal, disseminated and uncommon or rare forms [1]. Members of the order Mucorales are the causative agents of mucormycosis worldwide and are composed of thermotolerant fungi which are ubiquitous in nature and generally found on decaying organic matter. Members of the genera  C The International Society for Human and Animal Mycology 2013. All rights reserved. For permissions, please e-mail: . 73 74 Materials and methods hyphae are seen accompanied by evidence of associated tissue damage. Alternatively, a proved case can be described upon recovery of a mold by culture of a specimen obtained by a sterile procedure from a normally sterile and clinically or radiological abnormal site consistent with an infectious disease process. Probable IFD cases require presence of a host factor, clinical features, and mycological evidence consistent with the IFD, whereas a possible invasive infection has the same features accept for the absence of mycological evidence [2]. Sampling, culturing and strain identification The collected lung aspiration fluid and sinus biopsy tissue samples were directly cultured on Sabouraud dextrose agar (SDA) and potato dextrose agar (PDA). Part of the fluid and the biopsy tissue was sent to the pathology laboratory for the preparation of tissue slides stained with Hematoxylin-and-Eosin and periodic acid-Schiff procedures. Sputum samples were concentrated and cultured on SDA and PDA. The obtained isolates were identified through examination of micro- and macro-morphologic features in accord with standard morphological criteria [3,4,7]. Molecular identification was used by comparing the ITS1-5.8S-ITS2 rDNA region sequence data of the isolated strains with reference strains data deposited in GenBank. Study population This study included patients hospitalized from January 2010 to December 2010 in Cairo at the Ain Shams Specialized Hospital. Those included had diabetic ketoacidosis or solid organ transplants with host factors such as recent history of neutropenia ([<500 neutrophils/mm] for >10 days), prolonged use of corticosteroids in previous 90 days, and those treated with immunosuppressant in previous 30 days prior to the study period. The investigations described in this report adhered to the ethical principles for medical research of Helsinki Declaration. The microbiology and pathology laboratories records were reviewed daily. The corresponding medical records were reviewed and the clinical data analyzed included demographic characteristics such as the site of infection, host factors and the type of underlying disease at the time of diagnosis of infection. Case definition We applied the criteria of the (EORTC/MSG) for proven, probable or possible invasive fungal disease (IFD) [2]. As such, classifying a case as a proven IFD requires histopathologic, cytopathologic, or direct microscopic examination of a specimen obtained by needle aspiration or biopsy in which Extraction of DNA Fungal isolates were grown on PDA and DNA extraction was conducted in accord with the instructions provided by Fermentas Genomic DNA Purification Kit #K0512 (Thermo Fischer Scientific, EU). Briefly, a sufficient inoculum was suspended in 200 µl TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) in a 2.2 ml Eppendorf tube, the tubes were boiled for 3 min and then placed in ice water for 10 min. Lysis solution (400 µl) was added, the tubes heated to 65◦ C for 30 min and then 600 µl of chloroform were added and mixed carefully. The aqueous phase containing DNA was separated by centrifugation for 10 min at 12,000 rpm at 4◦ C and mixed with 800 µl precipitation solution by several inversions at room temperature for 1 min each. The tubes are then centrifuged for 10 min at 12,000 rpm at 4◦ C. The DNA pellets were dissolved in 100 µl of 1.2 M NaCl solution by gentle vortexing. Icecold isopropanol (500 µl) was added to the solution, the tubes were incubated for 15 min at − 20◦ C and then centrifuged for 10 min at 12,000 rpm at 4◦ C. The DNA pellet was washed with 1 ml ice cold 70% ethanol, dried and resuspended in sterile TE buffer. Rhizopus, Lichtheimia and Mucor are most often recovered from clinical specimens while other Mucorales genera, such as Rhizomucor, Cunninghamella, Syncephalastrum, Saksenaea and Apophysomyces, a (...truncated)