Molecular Characterization and Antifungal Susceptibility Testing of Aspergillus spp Isolated from Sudanese Patients with Underlying Lung Diseases

Abstract

Pulmonary aspergillosis is lung infection caused by Aspergillus spp, most of which are harmless, but a few can cause serious illnesses when people with weakened immune systems, underlying lung disease or asthma inhale their spores. The incidence of this infection has increased, primarily due to the increasing number of immunosuppressed patients with the advent of solid organ and bone marrow transplantation, the increased use of corticosteroids and other immune-modulating drugs, and the infection with the human immunodeficiency virus (HIV). This is descriptive cross sectional study conducted at different hospitals in Khartoum State during the period from 2016 to 2019, which aimed to detect Aspergillus spp from Sudanese patients with underlying pulmonary infections by conventional cultural method, to perform antifungal susceptibility of isolated Aspergillus spp using E test and to study molecular characterization by PCR and sequencing. Three hundred and eighty four patients with pulmonary underlying diseases distributed as follows , chronic pulmonary infection 219 (57.0%) , asthma 77 (20.1%), cystic fibrosis 34 (8.9%) , pulmonary tuberculosis 29 (7.6%), pleural effusions 12 (3.1%) , malignancy 9 (2.3%) , emhysema , hemoptysis and lung abscess 4 (1.0%) were included in this study. Three hundred forty one sputum and forty three bronchoalveolar lavage samples were collected from them. The age of patients ranged from 9 to 90 years, with mean age of 42 ± 16.14 (SD) years distributed on the following level: 2 (0.5%) age group less than ten years, 258 (67.2%) , age group (10-49 years) and 124 (32.3% ) 50 years and older. Of whom 233 (61.0%) were males, while females were 151 (39.0%). The sensitivity of the direct microscopy in this study was (28%) while the specifity was (99.4%). Twenty eight (7.4%) Aspergillus spp out of three hundred eighty four and 4/348 (1%) of Candida spp were isolated distributed as follows: 15 (46.9%) from patients with chronic pulmonary infections, 7 (29.1%) from asthmatic patients, 3 (9.4%) from patients with cystic fibrosis .3 (9.4%) from pulmonary tuberculosis patients and the 4 (12.4%) Candida spp were isolated from patients with malignant diseases and chronic pulomonary infections , with a statistically significant relationship (P value = 0.003) between underlying lung disease and pulmonary aspergillosis. The isolated fungal organisms by conventional cultural techniques was 11(34.4%) A. flavus, 9 (28.1%) A.fumigatus, 7 (21.9%) A. terreus, 1 (3.1%) A.niger and 4 (12.5%) Candida spp. The results of the E test for Itraconazole against Aspergillus spp showed : 3(33.3%) A. fumigatus were sensitive, 2(22.3%) intermediate and 4 (44.4%) resistant, While 6 (54.5%) A. flavus were sensitive , 4 (36.4%) intermediate and 1 (9.1 %) was resistant, 5 (71.4%) A. terrus reflected sensitive, 1 (14.3%) intermediate and 1 (14.3%) resistant and A. niger was resistant to Itraconazle. E test for Voriconazole against Aspergillus spp: 7 (77.8%) A. fumigats were sensitive, 1(11.1%) intermediate and 1 (11.1%) was resistant, While 11(100%) A. flavus were sensitive and 5 (83.3%) of A.terrus were sensitive, 1 (26.7%) was resistant. A.niger was intermediate for voriconazole. Sequencing of interspace transcribed region was determined for 17 isolates of Aspergillus spp after performing conventional PCR, with gene product of 535-613 bp and the obtained sequences were aligned with reference strains in Gene bank. The sequence similarity was greater than 99% between reference Aspergillus strains from Gene bank and clinical isolates. The Phylogenetic analysis was performed to compare the genetic distances and evolutionary lineage for 17 isolates with well-characterized reference isolates from Genebank and this study indicated that the isolated Apergillus spp was related to several strains worldwide that are far from Sudan (China, Korea, Malaysia, Netherland, India, Egypt, Zimbabwe, South Africa ,Brazil ,Nigeria). In conclusion: The significant association between pulmonary aspergillosis and underlying lung diseases indicate that these diseases might be the risk factors for infection by Aspergillus. Furthermore, the high activity of voriconazole against A.flavus which was the predominant isolate from Sudanese patients with pulmonary underlying diseases in this study may make it the treatment of choice.