Clinico-mycological profile and antifungal susceptibility patterns of zygomycosis during the COVID-19 pandemic at a tertiary care center

References

1. method for filamentous fungi. The antifungal agents tested were:
2. Amphotericin B
3. Posaconazole
4. Stock preparation Antifungal stock solutions were prepared at concentrations of ≥1280 µg/mL, depending on drug solubility. For example, 16 mg of amphotericin B was dissolved in 10 mL of 100% DMSO to yield a 1600 µg/mL solution. Aliquots were stored in 2 mL snap-cap tubes at –70°C.
5. Reading and interpretation MICs were read after 46–50 hours of incubation. The Minimum Inhibitory Concentration (MIC) was defined as the lowest drug concentration that completely inhibited visible fungal growth.
6. Results
7. Demographics The study included 57 patients with suspected zygomycosis, aged between 10 and 70 years, with the highest incidence in the 50–60-year age group. Males constituted 74% (42/57) and females 26% (15/57). A history of COVID-19 was noted in 38 patients (66.7%), while 19 (33.3%) had no prior COVID- 19 infection. A total of 84% of our patients were diabetic (48/57) and almost 92% of our patients were diabetic patients affected with COVID infection.
8. Sample distribution Out of the 57 clinical specimens, 42 (73.7%) were culture- positive and 15 (26.3%) were KOH-positive but culture- negative. Histopathological examination was performed in 42 cases, all of which confirmed the presence of fungal elements.
9. Source of clinical specimens The majority of samples were from paranasal sinus tissue (41 cases; 72%), followed by bronchoalveolar lavage/bronchial wash (6 cases; 11%), infected wound tissue (5 cases; 9%), 374 Subramaniyan et al. / Indian Journal of Microbiology Research 2025;12(3):372–375 nasal swabs (2 cases; 4%), lung tissue (2 cases; 4%), and brain tissue (1 case; 1%).
10. Clinical presentation Rhino-orbitocerebral zygomycosis was the most common presentation, seen in 44 patients (77%), followed by pulmonary (8 cases; 14%) and cutaneous zygomycosis (5 cases; 9%).
11. Fungal species identified Rhizopus spp. was the predominant fungal isolate, followed by other members of the order Mucorales:
12. Antifungal susceptibility patterns Antifungal susceptibility testing was performed for all 42 culture-positive isolates against amphotericin B, posaconazole. The MIC interpretive ranges for the antifungal agents tested are summarized in Table 1. Amphotericin B is likely to be effective for most isolates, with MICs ranging from 0.5– 2 µg/mL. Isolates with MICs ≤1 µg/mL were considered likely to be susceptible, while those with MICs ≥4 µg/mL indicated reduced susceptibility. Posaconazole showed good in vitro activity, with MICs ranging from 0.125–4 µg/mL in most isolates. MICs ≤1 µg/mL were considered likely to be susceptible, whereas values ≥4 µg/mL suggested resistance. Itraconazole and voriconazole were ineffective against all isolates, with MICs >8–16 µg/mL; no susceptibility breakpoints exist for these drugs, and MICs in this range are consistent with their intrinsic resistance against Mucorales. (Table 2).
13. Discussion Zygomycosis has become a prominent opportunistic infection in India, especially during the COVID-19 pandemic. The high prevalence of uncontrolled diabetes mellitus, compounded by the indiscriminate use of corticosteroids for COVID-19 treatment, has created a high- risk population vulnerable to invasive fungal infections. 1,4,5 Our study reinforces this link, as the majority of zygomycosis cases were seen in patients with a history of COVID-19 and diabetes, particularly those with rhino-orbitocerebral involvement. Rhizopus spp. was the dominant isolate, which aligns with both national and global epidemiological patterns. 6,7 However, the identification of less common species such as Apophysomyces variabilis and Cunninghamella bertholletiae emphasizes the importance of molecular methods for accurate species-level identification, particularly in atypical or resistant cases. 10
14. Antifungal resistance: Mechanisms and consequences Amphotericin B, a polyene antifungal, showed the highest efficacy overall, but resistance (MIC ≥8 µg/mL) was noted in a small subset of isolates. Resistance to amphotericin B is generally attributed to alterations in ergosterol biosynthesis or reduced ergosterol content in the fungal cell membrane, which compromises drug binding and efficacy. 11 This can have serious clinical implications, especially in severe infections where delayed or ineffective treatment can be fatal. Posaconazole, a second-generation triazole, demonstrated good activity against most isolates. However, elevated MICs in a few cases suggest the possibility of efflux pump overexpression or mutations in the CYP51A gene, which reduce drug binding affinity. 10,14 This underlines the importance of therapeutic drug monitoring and individualized dosing strategies, particularly in salvage therapy. 12 Table 1: Distribution of fungal species isolated and their MIC values for amphotericin B and posaconazole Isolate Number (n = 42) Amphotericin B MIC Range Posaconazole MIC Range Rhizopus spp. 36 0.5–16 µg/mL 0.125–8 µg/mL Apophysomyces variabilis 3 0.5–8 µg/mL 0.125–0.5 µg/mL Cunninghamella bertholletiae 1 16 µg/mL Not tested Lichtheimia corymbifera 2 1 µg/mL and 16 µg/mL 0.12 and 0.5 µg/mL There were 18 cases diagnosed solely by histopathology that were not sent for culture. Table 2: Identification of the isolates and their susceptibility pattern Isolate Number MIC value for amphotericin B and posaconazole Rhizopus arrhizus 19 0.5-16 µg /ml 0.125-8 µg /ml Rhizopus microsporus 9 Rhizopus delmar 8 Apophysomyces variabilis 3 0.5 -8 µg /ml 0.125-0.5 µg /ml Cunninghamella bertholletiae 1 16 µg /ml Subramaniyan et al. / Indian Journal of Microbiology Research 2025;12(3):372–375 375 Itraconazole exhibited inconsistent activity, likely due to limited in vitro efficacy against Mucorales and poor tissue penetration in some anatomical sites. Voriconazole showed universally high MICs, which is expected given the intrinsic resistance of zygomycetes to this azole due to lack of target binding affinity. 2,13 The frequent empirical use of voriconazole, especially in centers with limited diagnostic capacity, risks treatment failure and delayed intervention. The clinical consequence of antifungal resistance is profound: it can lead to delayed response, progression of tissue necrosis, higher rates of surgical intervention, prolonged hospitalization, and increased mortality. In high- burden settings, such outcomes also place a considerable strain on healthcare resources.
15. Conclusion This study highlights the predominance of Rhizopus spp. as the leading etiological agent of mucormycosis in our patient cohort, particularly among individuals with diabetes and recent COVID-19 infection. A smaller proportion of cases were caused by non-Rhizopus Mucorales, which exhibited variable antifungal susceptibility patterns, underscoring the need for precise species-level identification. Amphotericin B remained the most likely antifungal to be effective, while posaconazole served as a reliable second-line or adjunct therapy probably to be efficacious, especially in isolates showing resistance or in patients with drug intolerance. Importantly, routine antifungal susceptibility testing, combined with molecular confirmation, proved essential in tailoring appropriate therapy and improving patient management. The integration of these diagnostic and therapeutic strategies into routine clinical practice is strongly recommended to enhance treatment precision and outcomes in mucormycosis cases.
16. Source of Funding None.
17. Conflict of Interest None.
18. Acknowledgment We would like to thank the Department of Microbiology and ENT, along with the technical staff of the laboratory, for their invaluable support and assistance throughout this study. References
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