Triazole Resistance and Sequence Analysis of Aspergillus Species from Fungicide Naïve and Experienced Soils in Naivasha Sub-County and Nairobi County

Abstract

New triazole antifungals are recommended for the management of Aspergillosis infections. The emergence of Aspergillus fumigatus strains resistant to azole have been identified in some countries ascribed to either previous antifungal treatment, prophylaxis or triazoles use in agriculture. Azole based fungicides use is robust horticulture in Kenya is a significant risk factor for antifungal resistance. The objective of the study was to isolate, characterize and determine triazole resistance amongst Aspergillus species from fungicide naïve and experienced soils compared to those from clinical sources. A total of 250 naïve soils, 252 experienced soils, and eleven clinical samples were analyzed. Dry top surface soils were sampled from the study site. Each soil sample was processed and approximately 100µl of the preparations plated onto SDA containing no drug, 1µl/ml itraconazole and 1µl voriconazole. Aspergillus species were identified by MALDI TOF MS and ITS genes amplification. Absence or presence of TR34 in the promoter region was determined by A. fumigatus DNA amplification using AFCYPPR and AFCYPPF primers. While L98H the mutation was determined by amplification of isolates DNA using AFCYP98R and AFCYP98F primers. Gel electrophoresis was run to detect DNA amplicon Aspergillus species isolated were as follow: Nairobi experienced soils 22/45 (48.89%) A. fumigatus, 20/45 (44.44%) A. niger, 3/45 (6.67%), A. flavus, 0/51 (0%) A. terreus. Naivasha experienced soils. 23/42 (54.76%) A. fumigatus, 12/42 (28.57%) A. niger, 3/42 (7.14%) A. flavus, 3/42 (7.14%) A. terreus. In naïve soils from Nairobi 26/40 (65%) A. fumigatus, 12/40 (30%) A. niger, 0/40 (0%) A. flavus, 2/40 (5%) A. terreus while in Naivasha 21/45 (46.67%) A. fumigatus, 23/40(51.11%) A. niger, 0/45 (0%), A. flavus, 0/45 (0%), A. terreus and 1/45 (2.2%) A. ochraceus were isolated. The resistance of Aspergillus species isolated from Nairobi experienced soils were 3/45 (6.7%), 5/45 (11.1) and 27/45 (60%) against itraconazole, voriconazole, and posaconazole respectively. Naivasha experienced soils had a resistance of 3/42 (7.1%) against Itraconazole, 4/42 (9.5%) against voriconazole and 32/42 (76.2%) against posaconazole. Naïve soils form Nairobi had a resistance of 6/40 (15%) against posaconazole compare to 3/45 (6.7%) from Naivasha. Aspergillus fumigatus with TR34 in their promoter regions yielded 139bp amplicon and wild type (L98H) yielded DNA fragments of 279bp. Hence the study detected TR34/L98H mutation in all the sequenced isolates. The finding is worrisome and we must strive for more surveillance of environmental and clinical source to detect azole resistance in Aspergillus species.