The Spectrum and Antifungal Drug Susceptibility Profiles of Fungi Obtained From Sputum of Tuberculosis Patients at Selected Reference Facilities in Kenya

Abstract

Background Opportunistic fungal infections and antifungal resistance represent a major global health challenge. HIV/AIDS, tuberculosis (TB), diabetes, malnutrition, cancer, and other immunosuppressive conditions are increasing fungal diseases globally. The WHO estimates that more than one million people successfully treated for TB later develop a fatal fungal infection each year. This is often left untreated because it is mistaken for a recurrence of TB and subsequently retreated. Similarly, fungal growth in TB sputum culture is disregarded as contaminants yet they could be the etiological agents in lung disease. Objective The study sought to determine the spectrum of pulmonary fungal pathogens and antifungal drug sensitivity among TB retreatment and relapse patients at selected reference facilities in Kenya. Materials and Methods The study evaluated 340 expectorated sputa samples from patients who consented to participate in the study. The samples were subjected to mycological analysis, including microscopy and isolation of fungi on Sabouraud Dextrose Agar (SDA). Moulds were identified morphologically by macroscopic and microscopic features while yeasts were inoculated on CROME-agar Candida and further identified using analytical profile index (API 20C AUX). Four antifungal drugs were tested against the isolates, namely itraconazole (ICZ), voriconazole VCZ), fluconazole (FCZ) and amphotericin B (AMB) using broth micro-dilution methods according to CLSI M38 A2 and CLSI M27 recommendations for moulds and yeast, respectively. Minimum inhibitory concentrations (MICs) were determined. Fungi contaminating TB sputum cultures on Lowenstein Jensen (LJ) media were also identified. Molecular characterization of Aspergillus spp isolates from sputum was done by sequencing of beta-tubulin (Btu) and Calmodulin (CaM) genes using specific primers. Sequences were cleaned and examined by BLAST analysis and subsequently deposited in the NCBI database. UoN, PhD, 2022 Results Diverse fungal species isolated from the sputa samples were as follows; 16% (n=53) were positive for moulds, with Aspergillus being predominant constituting 68 % (n=36) of the moulds. Among the Aspergilli, A. flavus and A. niger were the most frequently isolated constituting 23%, (n=12) and 15% (n=8), respectively. Other moulds recovered from the sputa were Paecillomyces variotii (9%, n=5), Scedosporium aspiospermum (6%, n=3), Mucor racemosus (8%, n=4) and Penicillium spp (9%, n=5). A total of 14% (n=49) of the samples were positive for yeasts. Candida albicans and C. krusei were the major yeast species isolated from sputum constituting 50% (n=24) and 20.8% (n=10) of the yeasts, respectively. C. albicans (33%, n=22) was also a predominant isolate from LJ tubes. Members of the Aspergillus spp with MICs ≥4μg/ml to some antifungal agents were noted, and all the moulds except two (n=2) isolates of S. aspiospermum exhibited MICs higher than >4μg/ml for fluconazole. The moulds generally showed greater sensitivity to AMB and VRC, while the yeasts, particularly C. albicans exhibited greater sensitivity to the four antifungal agents. There was a statistically significant difference (Chi Square; F=3.7, P=0.004) in the sensitivity pattern of moulds while yeasts exhibited no statistically significant difference (F=1.7, P=0.154>0.05). Genome sequence analysis of Aspergillus spp from sputum showed a similarity index of 98% to 100% compared to those in GenBank. Conclusion The study demonstrates a wide variety of moulds and yeasts that are potential respiratory pathogens in TB retreatment patients. The fungi could be responsible for persistent TB like symptoms despite treatment and are likely to be misdiagnosed as relapse requiring retreatment. Fungal investigation in presumptive TB relapse cases should be encouraged to reduce unnecessary retreatment, delayed antifungal intervention and poor outcomes associated with misdiagnosis. Fungi from sputum showed varied sensitivity patterns to the antifungals tested hence antifungal sensitivity testing is essential to guide treatment choices in light of possible resistant isolates.