key: cord-0740647-93tw8kk6
authors: Muthu, Valliappan; Kumar, Mohan; Paul, Raees A.; Zohmangaihi, Deepy; Choudhary, Hansraj; Rudramurthy, Shivaprakash M.; Panda, Naresh K.; Pannu, Ashok Kumar; Sharma, Navneet; Sharma, Sadhna; Chakrabarti, Arunaloke; Agarwal, Ritesh
title: Is there an association between zinc and COVID‐19–associated mucormycosis? Results of an experimental and clinical study
date: 2021-09-01
journal: Mycoses
DOI: 10.1111/myc.13365
sha: f970ef0d8594c304d14f23dd8c74a2bba848100a
doc_id: 740647
cord_uid: 93tw8kk6

BACKGROUND: The enormous increase in COVID‐19–associated mucormycosis (CAM) in India lacks an explanation. Zinc supplementation during COVID‐19 management is speculated as a contributor to mucormycosis. We conducted an experimental and clinical study to explore the association of zinc and mucormycosis. METHODS: We inoculated pure isolates of Rhizopus arrhizus obtained from subjects with CAM on dichloran rose Bengal chloramphenicol (DRBC) agar enriched with (three different concentrations) and without zinc. At 24 h, we counted the viable colonies and measured the dry weight of colonies at 24, 48 and 72 h. We also compared the clinical features and serum zinc levels in 29 CAM cases and 28 COVID‐19 subjects without mucormycosis (controls). RESULTS: We tested eight isolates of R arrhizus and noted a visible increase in growth in zinc‐enriched media. A viable count percentage showed a significantly increased growth in four of the eight isolates in zinc‐augmented DRBC agar. A time‐ and concentration‐dependent increase in the mean fungal biomass with zinc was observed in all three isolates tested. We enrolled 29 cases of CAM and 28 controls. The mean serum zinc concentration was below the reference range in all the subjects and was not significantly different between the cases and controls. CONCLUSIONS: Half of the R arrhizus isolates grew better with zinc enrichment in vitro. However, our study does not conclusively support the hypothesis that zinc supplementation contributed to the pathogenesis of mucormycosis. More data, both in vitro and in vivo, may resolve the role of zinc in the pathogenesis of CAM.

Mucormycosis is a severe infection with a high case-fatality rate and usually occurs in individuals with impaired immunity. 1, 2 Coronavirus disease 2019 has led to an enormous rise of mucormycosis cases in India, [3] [4] [5] resulting in several unanswered questions. 6 CAM is also increasingly being reported from Europe and several other countries. 7 During this unprecedented surge of COVID-19-associated mucormycosis (CAM) cases in India, several hypotheses were proposed. [8] [9] [10] Most of those assumptions were conjectural except for one study, suggesting an association between CAM and uncontrolled diabetes or inappropriate glucocorticoid therapy. 4 Interestingly, a few patients of COVID-19 managed at home and had not received glucocorticoids also developed CAM. 4, 5 Whether unproven therapies, home remedies and over-the-counter medicines contributed to the development of CAM remains unclear. One widely discussed theory in the media and the medical community was that zinc supplementation for COVID-19 management triggered the growth of Mucorales, causing CAM. 11 However, zinc supplementation was also practiced in several other countries without a significant rise in cases of mucormycosis.

Zinc is an essential micronutrient for all eukaryotes and is an important factor contributing to the growth and virulence of pathogenic fungi, including Candida, Aspergillus and others. [12] [13] [14] In one study, zinc chelator clioquinol when used with posaconazole (but not amphotericin) was shown to be effective against certain strains of Mucorales. 15 Herein, we report the results of an in vitro study evaluating the role of zinc on the growth of Rhizopus arrhizus isolated from subjects with CAM. We also supplement the experimental data with a casecontrol study evaluating serum zinc levels in COVID-19 subjects with and without mucormycosis.

We performed the current study in two parts, namely experimental and clinical, to evaluate the association of zinc with CAM. The study was performed at the Postgraduate Institute of Medical Education and Research, Chandigarh. The experiment was conducted at the Mycology laboratory (Department of Medical Microbiology), and the serum zinc was estimated at the Department of Biochemistry, as described below. The cases and controls were identified from the emergency services of our hospital. The Institute Ethics Committee approved the study protocol. We obtained written informed consent and used anonymised patient data.

We used eight randomly selected clinical isolates of Rhizopus arrhizus (Table 1) from patients with CAM. The isolates were grown on Sabouraud dextrose agar (SDA) slants supplemented with 0.02% chloramphenicol at 37℃ for 3-4 days. The sporangiospores were harvested using sterile bend glass rods and washed in sterile phosphate buffer saline (1x PBS). We used inoculums of 10 3 sporangiospores/mL measured by hemacytometer for further experiments.

To determine the effect of three different zinc concentrations on the growth of R arrhizus isolates, we enriched the basal culture media, dichloran rose Bengal chloramphenicol (DRBC) agar by adding zinc sulphate heptahydrate (ZnSO 4 , 7H 2 O). We used zinc sulphate as the source of zinc ions, and the effective zinc concentration in the solution was 22.73% mole fraction of the total 287.6 g moles of ZnSO 4 , 7H 2 O. A molar equivalent of zinc concentration (0.63 mg/L, 1 × 10 −5 M) corresponds to the normal range of serum zinc. We targeted varying concentrations of zinc (1 × 10 −5 M, 3 × 10 −5 M, and 1 × 10 −4 M) to evaluate the growth potentiating effect of zinc on R arrhizus. DRBC media without zinc were used as the negative control. A 100 µL of the inoculum containing 10 3 spores/mL of each isolate (n = 8) was spread on DRBC agar plates comprising of the test (with ZnSO 4 ) and the negative control groups (without ZnSO 4 ). We repeated the experiment thrice for each isolate. The plates were incubated at 37℃ for 24 h, and we manually counted the viable counts (CFU) at three different zinc concentrations and controls.

Three clinical isolates of R arrhizus were used for evaluating the effect of zinc on fungal biomass production. Briefly, we seeded 100 µl of TA 

We compared the serum zinc levels in subjects of COVID-19 with (cases) and without (controls) mucormycosis. The clinical details and laboratory values of the study participants were obtained from the medical records of the patients attending the emergency services of our institute. Only hospitalised subjects with a confirmed diagnosis (by reverse transcriptase PCR for SARS-CoV-2 in nasopharyngeal or oropharyngeal swabs) of COVID-19 were included. Both the cases and controls were recruited during the same study period. We classified CAM as subjects wherein mucormycosis was diagnosed concurrently or within eight weeks of COVID-19. We defined confirmed mucormycosis (cases) in subjects with a consistent clinical and radiological feature for mucormycosis and microbiology or histopathology evidence of aseptate hyphae (with or without culture reports showing growth of Mucorales) in the tissue or respiratory specimens. 16 The control subjects were hospitalised subjects with COVID-19 without mucormycosis and followed up for at least eight weeks.

We obtained the following information from the records: (1) 

We estimated zinc on archived samples using the atomic absorption spectrophotometer (Agilent AA-200 Analyser). We used a minimum of 600 µL serum sample as per the manufacturer's recommendation.

The measuring range was 50-700 µg/dL, and values above the measuring range were retested after the required dilution. The precision (CV) of the instrument was 0.5%-5.8%. Before analysis, the serum sample was briefly diluted with 2% HNO3 in a 1:10 ratio in 'flame mode'. Zinc ions in the serum sample were converted to atomic states using a flame. The atomised element absorbed light from the hollow cathode lamp, elevating it to the excited states. The amount of light energy absorbed was proportional to the number of analyte atoms in the light path. We also tested 10% of the samples in duplicate, and the results were consistent.

We used the commercially available statistical software package 

We found the colonies from all the eight isolates on the zincsupplemented plates to be larger, profluent and more cottony than the control media where zinc was not added (Figure 1 ). The visual assessment suggested a significant increase in the size of the colonies;

F I G U R E 1 Viable counts of Rhizopus arrhizus determined at three different concentrations of zinc. R1 and R2 two representative clinical isolates (of the eight tested) of Rhizopus arrhizus from CAM patients showing an increased number of colonies and more cottony growth with zinc supplementation however, this was not quantifiable due to the profuse growth. In addition, we observed a significant increase in growth (viable CFU/mL) of R arrhizus in four of the eight isolates incubated in zinc-enriched culture media compared to those without ( Figure 2 ). There was no increase in the viable counts with increasing zinc concentrations ( Figure 2 ).

We tested three isolates. Two isolates were those showing significantly increased CFU counts (P489, P582), while one isolate did not significantly grow with zinc (P503). In all three, we observed a temporal and concentration-dependent increase in the mean fungal biomass. The fungal biomass production in the presence of zinc was higher compared to matched controls; the effect was more pronounced at higher concentrations of zinc and at 72 h (Table 1 ).

During the study period, we identified 31 confirmed cases of CAM, of which two were excluded due to inadequate details. Finally, we enrolled 29 cases and 28 controls ( 

We found that half of the R arrhizus isolates obtained from CAM patients showed a significant increase in growth in zinc-enriched fungal culture medium. In addition to the increased colony count, we also observed that the colonies were larger and had higher fungal biomass in zinc-supplemented media. However, there was no increase in the colony numbers with increasing zinc concentrations.

We did not find a significant difference in serum zinc levels among CAM cases and COVID-19 controls.

Pathogenic fungi (Aspergillus, Candida and others) grow better in the presence of zinc. 13, [17] [18] [19] During infections, the human host makes zinc unavailable to the pathogens, causing apparent zinc deficiency. 20 In experimental studies, zinc chelators have shown promising results against Aspergillus and several species of Mucorales. 15, 21 We found zinc promoted the in vitro growth in half of the Rhizopus arrhizus isolates from CAM subjects. The growth did not increase significantly in all others, possibly due to strain-to-strain variation, the availability of other nutritional elements and differences in zinc transporters. Strain-dependent variation was also noted in an experimental study evaluating zinc chelators and antifungal agents. 15 While the effect of zinc on Rhizopus arrhizus is not universal, the isolates that responded to zinc enrichment showed significant and unequivocal growth. In an experimental study, investigators found that zinc acted as a catalyst in promoting the in vitro growth of Rhizopus nigricans (now Rhizopus stolonifer). 22 The authors found more effi- Conclusive proof of zinc in the pathogenesis of invasive mucormycosis requires further research. 

Data are available on request with the corresponding author.

Shivaprakash M. Rudramurthy https://orcid. org/0000-0002-9097-9253

Ritesh Agarwal https://orcid.org/0000-0003-2547-7668

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Is there an association between zinc and COVID-19-associated mucormycosis? Results of an experimental and clinical study