Investigation of antifungal compounds against Aspergillus species from respiratory infections of immunocompromised patients

Abstract

The rapid emergence of invasive fungal infections correlates with the increasing population of immunocompromised individuals, with many cases leading to death. The progressive increase in the incidence of Aspergillus isolates is even more severe due to clinical challenges in treating invasive respiratory infections in immunocompromised patients. Azoles are the drugs of choice for the prevention and treatment of Aspergillus infections however, azole resistance in Aspergillus-related infections is an increasing concern, especially against Aspergillus fumigatus. This situation is further complicated by the use of azoles in agriculture, as increasing resistance has been associated with increased use in agriculture. Rapid initiation in detection, diagnosis and appropriate antifungal therapy is needed to reduce mortality among individuals with invasive Aspergillus-related infections. Improvements in all aspects of azole resistance management, from identification and antifungal therapy to the discovery of novel non-toxic drugs, impact clinical success. Hence, this study assessed the effect of routine and salvage drug therapy on Aspergillus respiratory infections and investigated the potential of new drugs in combination therapy in light of increasing antifungal resistance in the management of invasive fungal infections. Aspergillus species isolated from immunocompromised patients with respiratory infections at the Inkosi Albert Luthuli Hospital in Kwa-Zulu Natal were investigated. Conventional morphology identification methods were assessed for reliability and compared to molecular identification. In the search for reliable and rapid alternatives in fungal identification to impact diagnosis, phenotypic microarray (Biolog) for fungal species identification was explored. Phenotypic microarray (Biolog) was also used to analyse the isolates' nutritional patterns and drug sensitivity profiles to investigate the potential for new compounds in drug therapy. In addition, gliotoxin expression, which has been linked to increased pathogenesis, was quantified and analysed using high-performance liquid chromatography. This was investigated in vitro to correlate gliotoxin production as a possible virulence factor in azole resistance. The effect of pathogen-associated molecular patterns (PAMPS) in bacterial coinfection was also investigated, especially since the species were isolated from respiratory infections where co-infection is common. Conventional morphological techniques were generally similar to 18S rRNA identification, assigning twenty-six Aspergillus fumigatus species, eight A. niger and two A. flavus. Biolog technology only identified isolates correctly up to genus level and had no significant similarity matches at species level due to the Biolog fungal database not having adequate reference clinical species however, an updated database makes this technology a good alternative when reliability and speed in identification are considered. Antifungal profiles showed that 6% of the 36 isolates were resistant to the routine azole voriconazole, with 61% having moderate susceptibility. All isolates resistant to the salvage therapy drug, posaconazole pose a serious concern. Significantly, A.niger was the only species resistant (25%) to voriconazole and has recently been reported as the species isolated from patients with COVID-19-associated pulmonary aspergillosis (CAPA). Mutations in the cpy51A gene representing common mechanisms for azole resistance in clinical Aspergillus isolates were highlighted in the azole susceptibility profile. Phenotypic microarray showed that 83% of the isolates were susceptible to the 24 new compounds. Berberine and blasticidin hydrochloride were selected and further investigated for combination drug therapy, considering their non-toxicity upon oral administration currently with thirty of the thirty-six isolates showing susceptibility to the new agents. Carbon profiles demonstrated the consistent assimilation of alternate monosaccharides and disaccharides by all three Aspergillus species, accomplished by secreted enzymes which very likely contribute to nutrient acquisition during infection, impact persistent growth and survival to influence infection maintenance and disease progression. Of the twenty-six A. fumigatus isolates, 58% produced gliotoxin during the 48-hour incubation at 37℃. Forty per cent of those gliotoxin-producing isolates showed increased gliotoxin production after exposure to PAMPS, with the highest concentration of 2.6 mg/L and a mean of 0.6 mg/L. Compared to similar investigations, the gliotoxin concentrations for invasive aspergillosis were higher than for colonization in respiratory patients. This study found that different identification strategies in azole resistance treatment management are required for both developed and resource-challenged situations to influence diagnosis. Drug resistance in azole monotherapy was demonstrated. Thus this study supports the search for new agents in drug regimen strategy and found potential in novel compounds, berberine and blasticidin hydrochloride, for combination therapy.