Epidemiology and Antifungal Susceptibility Patterns of Invasive Fungal Infections (IFIs) in India: A Prospective Observational Study

The epidemiology of invasive fungal infections (IFI) is ever evolving. The aim of the present study was to analyze the clinical, microbiological, susceptibility, and outcome data of IFI in Indian patients to identify determinants of infection and 30-day mortality. Proven and probable/putative IFI (defined according to modified European Organization for Research and Treatment of Cancer/Mycoses Study Group and AspICU criteria) from April 2017 to December 2018 were evaluated in a prospective observational study. All recruited patients were antifungal naïve (n = 3300). There were 253 episodes of IFI (7.6%) with 134 (52.9%) proven and 119 (47%) probable/putative infections. There were four major clusters of infection: invasive candidiasis (IC) (n = 53, 20.9%), cryptococcosis (n = 34, 13.4%), invasive aspergillosis (IA) (n = 103, 40.7%), and mucormycosis (n = 62, 24.5%). The significant risk factors were high particulate efficiency air (HEPA) room admission, ICU admission, prolonged exposure to corticosteroids, diabetes mellitus, chronic liver disease (CLD), acquired immunodeficiency syndrome (AIDS), coronary arterial disease (CAD), trauma, and multiorgan involvement (p < 0.5; odds ratio: >1). The all-cause 30-day mortality was 43.4% (n = 110). It varied by fungal group: 52.8% (28/53) in IC, 58.8% (20/34) in cryptococcosis, 39.8% (41/103) in IA, and 33.9% (21/62) in mucormycosis. HEPA room, ICU admission for IC; HEPA rooms, diabetes mellitus for cryptococcosis; hematological malignancies, chronic kidney disease (CKD), sepsis, galactomannan antigen index value ≥1 for IA and nodules; and ground glass opacities on radiology for mucormycosis were significant predictors of death (odds ratio >1). High minimum inhibitory concentration (MIC) values for azoles were observed in C. albicans, C. parapsilosis, C. glabrata, A. fumigatus, A. flavus, R. arrhizus, R. microsporus, and M. circinelloides. For echinocandin, high MIC values were seen in C. tropicalis, C. guillermondii, C. glabrata, and A. fumigatus. This study highlights the shift in epidemiology and also raises concern of high MICs to azoles among our isolates. It warrants regular surveillance, which can provide the local clinically correlated microbiological data to clinicians and which might aid in guiding patient treatment.


Introduction
Invasive fungal infections (IFIs) continue to represent a significant problem in immunocompromised individuals and a large proportion of critically ill patients [1]. However, this changing epidemiology with increasing numbers of immunocompetent hosts includes the cases following natural disasters and large iatrogenic inoculation [1,2]. On-going pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has also brought the focus back on superinfections caused by secondary IFIs [3].
Over the past few decades, incidence of IFIs has also been increasing. This is attributed primarily to the overall increase in the number of patients irrespective of severe immunosuppression such as acquired immunodeficiency syndrome (AIDS), hematological malignancies, organ transplantation, etc. or apparent immunocompetent status with diabetes mellitus, chronic obstructive pulmonary disease (COPD), etc. [1,4]. Depending upon the population cohorts, the overall IFI incidence rate varies from 3% to 20% [5][6][7][8][9][10][11]. Opportunistic pathogens such as Candida sp., Cryptococcus sp., Aspergillus sp., and Mucorales are the most common causative agents of these infections. There are other hyalohyphomycetes such as Fusarium sp. and Scedosporium sp., phaeohyphomycetes (darkly pigmented or dematiaceous fungi), and basidiomycetous yeasts (Trichosporon sp., Malassezia sp.) known to cause these infections in different populations [12]. These fungi affect various tissues throughout the body, with the respiratory system being the most common [13]. Invasive candidiasis is considered the most common IFI; however, there are shifts in epidemiology noted towards non-albicans sp. [4,14]. In hematological diseases, a predominance of invasive aspergillosis (IA) has been reported [1,4]. In critically ill patients, these infections can also present as coinfections, further complicating and delaying the diagnosis [13].
In any economic scenario, the most feasible IFI diagnostic modality-fungal culture and pathological examination-is not conducive, as it does not meet the urgent diagnosis requirement and thereby delays treatment, resulting in a high fatality rate [13]. This is further complicated by the increased occurrence of resistant species owing to a surge in antifungal prophylaxis and emergence of previously rare fungal species displaying inherent resistance to common antifungal agents used [4,15,16]. A key determinant for the outcome of IFIs is early initiation of antifungal therapy [17]. There are established guidelines for the four commonest IFIs-invasive candidiasis (IC), Cryptococcosis, invasive aspergillosis (IA), and mucormycosis-from the Infectious Diseases Society of America (IDSA), the European Society for Clinical Microbiology and Infectious Diseases, and the European Confederation of Medical Mycology (ESCMID/ECMM) [18][19][20][21]. However, uncertainty lingers about the interpretation of antifungal susceptibility testing (AST) and the significance of minimal inhibitory concentration (MIC) in predicting outcome [17]. Regardless, IFIs are a major cause of morbidity and mortality [4]. Careful consideration of local fungal epidemiology describing clinical characteristics, prognostic factors, use of diagnostic algorithms and antifungal susceptibility patterns can prove useful for overcoming these shortcomings [1].
However, there are a limited number of studies from India, which renders many aspects of IFI poorly understood. This lacuna in data prompted us to conduct this study aimed at analyzing clinical, microbiological, susceptibility, and outcome data of IFIs to support clinicians when deciding on prophylactic or empirical antifungal therapy.

Study Design and Data Collection
This was a prospective observational study to investigate IFI epidemiology from April 2017 to December 2018 conducted at the Department of Microbiology in collaboration with Departments of Hematology, Medical Oncology, Pediatrics, Sleep Disorders and Pulmonary Medicine, Otorhinolaryngology, Endocrinology, Medicine and Pathology at a tertiary care hospital, All India Institute of Medical Sciences, New Delhi, India.
Patients clinically suspected of IFI displaying at least one of the following host factors were enrolled in the study: hematologic malignancy; cancer and receiving chemotherapy within the last 3 months before admission, with or without neutropenia; chronic obstructive pulmonary disease (COPD); transplant recipient (hematopoietic/solid organ); chronic granulomatous disease (tuberculosis); other immunocompromised state (inherited immunodeficiency, child C cirrhosis, or HIV, etc.); steroid use-at least 4 mg methylprednisolone (or equivalent) a day for at least 7 days in the 3 weeks before admission or during the course of the ICU stay for at least 5 days or a cumulative dose of at least 250 mg of methylprednisolone (or equivalent) in the past 3 months before enrolment; recipient of any other immunosuppressive treatment (tacrolimus, cyclosporine, methotrexate, cyclophosphamide, etc.); diabetes mellitus with or without ketoacidosis; or microbiological evidence of Aspergillus infection during the stay in ICU (any positive culture or two positive circulating galactomannan tests) (data not shown). In addition, eligible patients could only be enrolled if they had at least two of the following three features: fever refractory to at least 3 days of appropriate antibiotics or fever relapsing after a period of defervescence of at least 48 h while still receiving antibiotics; clinical signs and/or symptoms suggestive of invasive mycosis: pleuritic chest pain or physical finding of pleural rub, or one of the following symptoms of lower respiratory tract infection (new sputum secretions, dyspnea, or hemoptysis); or development of new pulmonary infiltrates on chest X-ray. To enhance the homogeneity of the study population, only antifungal-naïve patients were included. The sole exclusion criterion was patients on antifungal prophylaxis or preexisting antifungal treatment. Baseline demographic, clinical characteristics, 30-day all-cause mortality details were recorded. Hospitalization data (general ward/high-efficiency particulate air (HEPA) units/intensive care units (ICU)) were also collected.
Ethics statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and the study was approved by the ethics committee of the institute i.e., All India Institute of Medical Sciences, New Delhi, India (Ref no. IEC/NP-25/2014RP-10/2014, OP-3/09.02.2017). The detailed procedure was as per institute guidelines: http://www.aiims.edu/aiims/academic/ethics-committee/forms%20in%20pdf/ IEC/Format_of_Institution_Ethics_Committee_15032012.pdf (accessed on 16 January 2017). The consent forms for minor/incapable participants were obtained by their LAR, i.e., legally accepted representatives (example: mother, father, children, or grandparents).

Definition of IFI
Three thousand three hundred consecutive patients who fulfilled European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) 2008 definitions for possible, probable, or proven IFI [22] and AspICU criteria for clinically suspected invasive aspergillosis (IA) in ICUs [23] were enrolled. However, for analysis, only the probable and proven IFIs were included, as per the new EORTC/MSG 2020 definitions [24].

Diagnosis of IFI
Samples were processed following conventional mycological procedures including direct microscopy (visualization of capsule on negative staining, budding yeast cell on grams stain, septate or aseptate hyphae on KOH mount) and growth on sabouraud dextrose agar and CHROMagar. The isolates were identified by microscopy (slide culture on Tween 80 corn meal agar, septate hyphae on lactophenol cotton blue mount, and aseptate hyphae on calcofluor mount) and morphology on CHROMagar, bird seed agar, malt extract agar, and urea hydrolysis. Galactomannan antigen (GM) assay was performed using Platelia kit (Bio-Rad, Marnes-la-Coquette, France). Serial serum samples (day 0 and day 7) were obtained for all the patients who were clinically suspected of IA for a uniform GM analysis as per the physician's recommendation. Capsular antigen of Cryptococcus was detected using latex agglutination test (LAT) of Pastorex TM Crypto Plus (Bio-Rad, Marnes-la-Coquette, France). Only the isolates difficult to speciate phenotypically were subjected to DNA sequencing, where segments of DNA comprising the ITS region were amplified with primers ITS1 (5 -TCCGTAGGTGAACCTGCGG-3 ) and ITS4 (5 -TCCTCCGCTTATTGATATGC-3 ). Invasive/sterile site samples such as cerebrospinal fluid (CSF), bronchoalveolar lavage (BAL), pleural fluid, and tissue biopsy were collected at the discretion of the attending physician, while considering the debilitated condition of thrombocytopenic patients.
The inoculum suspensions for yeasts were prepared using 0.5 McFarland [25] and for molds conidial suspensions were prepared in RPMI 1640 and adjusted to final concentration of 2.5 × 10 4 CFU/mL, as previously described [26]. The assays were incubated at 35 • C for 24/48 h except for Cryptococcus sp., where the incubation was extended for 72 h.

Statistical Analysis
Continuous variables are presented as either mean (±SD) or median, with interquartile range in case of skewed distribution. They were normally distributed and the Student's t-test was used. The categorical variables are expressed as numbers and percentages of the group from which they were derived. The chi-square test and Fisher's exact test were used to compare categorical variables as appropriate. Socio-demographic clinical characteristics and risk factors were evaluated by univariate and multivariate analysis. These were entered into a logistic regression model for calculation of unpaired and paired odds ratios (ORs). The ORs are given with 95% confidence intervals (CIs). A cutoff of p ≤ 0.05, two-tailed, was considered significant for all statistical analysis.
All statistical analysis was conducted using STATA version 9 (StataCorp. 2005. Stata Statistical Software: Release 9. College Station, TX: StataCorp LP) except for antifungal data which was statistically analyzed with Statistical Package for the Social Sciences software (version 16.0; SPSS S.L., Madrid, Spain).

Invasive Candidiasis
All Candida infections were bloodstream. Multiple drugs, single and combinational, were used for treatment with liposomal amphotericin B being used in 35% (19/53) of cases, followed by a combination of fluconazole with an echinocandin in 20.8% (11/53) of cases ( Figure 2). Irrespective of the class of drug, the duration of treatment ranged from 3 to 28 days (median, 11 days; IQR, 7 days). Overall 30-day mortality was 52.8% (28/53

Cryptococcosis
Other than two pulmonary cases (5.6%, 2/34), all had cerebral presentation (94.4%, 32/34). A total of 24 (70.6%, 24/34) were culture positive with Cryptococcus neoformans, whereas 10 (29.4%) only showed budding round yeast cell with halo on india ink staining. Latex agglutination testing for the capsular antigen was carried out for all patients (data not shown). Clinical characteristics noted are shown in Figure 3.

Mucormycosis
Fifty percent (31/62) of cases were rhino-orbital with sinus involvement. Overall site and tissue involvement is shown in Figure 5. All were direct microscopy positive with 87% (54/62) of culture growth. Surgical debridement was performed in 71% (44/62) of cases, with L-amphotericin B (43/62, 69%) as the most common antifungal used. Overall Table 3.

Antifungal Susceptibility Testing
All experiments were performed in duplicates, and the MIC values of quality control strains fell within the established ranges published by CLSI methodologies. Table 4 summarizes the in vitro susceptibility value ranges, geometric mean, mode, MIC 50 , and MIC 90 values of all the isolates to the antifungals tested. Irrespective of genera, all isolates were susceptible to amphotericin B. Based on breakpoints for different fungi, high MIC values (intermediate and resistant combined) were recorded for fluconazole in 3 C. albicans, 4 C. parapsilosis, and 1 C. guillermondii; for voriconazole in 3 C. albicans, 3 C. parapsilosis, and 5 C. glabrata; for itraconazole in 4 C. albicans, 4 C. parapsilosis, 3 C. glabrata, 1 A. flavus, 1 A. fumigatus, 1 A. niger, 12 R. arrhizus, 3 R. microsporus, and 1 Mucor circinelloides; for posaconazole in 15 R. arrhizus, and 7 R. microsporus; for caspofungin in 1 C. albicans, 2 C. tropicalis, 4 C. glabrata, and 2 A. fumigatus; and for micafungin in 3 C. tropicalis, 2 C. guillermondii, and 4 C. glabrate (Table 5).
In this study, species identification revealed that Candida albicans and Candida parapsilosis were the most common cause of invasive candidiasis (IC), which is incongruent with the listing of Candida tropicalis as the most common cause of IC from India [46,47]. However, fungal isolations of Cryptococcus neoformans, Aspergillus flavus, and Rhizopus arrhizus were in accordance with previously published literature on respective IFIs [27,[48][49][50].
Similar to global data in our invasive candidiasis isolates, azole resistance was noted in C. albicans, C. parapsilosis, and C. glabrata, whereas echinocandin resistance was noted in C. albicans and C. glabrata [51][52][53][54][55]. It is significant that for echinocandins, in vitro susceptibility tested resistance is known to translate into treatment failures owing to FKS mutations [52,53,56]. Previous invasive candidiasis data from our center listed <6% resistance to fluconazole and 100% sensitivity to amphotericin B [57]. Global data support the CLSI C. albicans clinical breakpoints for fluconazole, whereas lacking the similar acceptance for C. glabrata, the most isolates of this non-albicans species fall in the intermediate category [17]. To overcome these shortcomings, susceptibility testing research has broadened. Natural oils from algae such as Ruta graveolans or north Sardinia plants have been evaluated for their efficacy (fungistatic and fungicidal). They have been found active against multidrug-resistant Candida sp. [58,59].
Although once known to be rare, cryptococcosis has occurred at a high frequency in India in the past two decades, as envisaged in a recent multicenter study [50]. It is one of the AIDS-defining infections and is responsible for about 15% of AIDS-related deaths [60]. However, in the current study, 50% of cases were seen with renal involvement, and only 29.4% were AIDS-related. The decrease in AIDS-related secondary cryptococcal infection may be owing to highly active antiretroviral therapy (HAART) therapy [60].
For Cryptococcosis, the drugs of choice are described in detail [19]. Amphotericin B (and its lipid formulations) with flucytosine is indicated as induction therapy in HIVinfected individuals, organ transplant recipients, non-HIV, and non-transplant patients, with differences in dosage and duration. The maintenance and consolidation therapy is fluconazole. For patients with CD4 count >100 cells/µL and undetectable viral load for >3 months, a minimum of 1 year of antifungal therapy is recommended [19]. From India, high MICs against fluconazole and flucytosine have been reported [61][62][63]. However, from our center in the current and another multicenter study [19], 100% sensitivity was noted for all the drugs.
In Western countries, local epidemiology highlights the predominance of A. fumigatus in invasive aspergillosis (IA) cases [64,65], whereas from India, A.flavus is most commonly isolated. Voriconazole is the drug of choice for primary therapy (especially with cases of invasive pulmonary aspergillosis) [20,66]. However, triazole (itraconazole, voriconazole, and posaconazole) drug resistance has been previously reported [64,67,68]. In this study, only three strains showed high MICs to itraconazole, of which one was A. fumigatus. In the Western world, Aspergillus fumigatus azole resistance (ARAF) has been extensively researched for its clinical implications [69][70][71][72], whereas from India, there are few sporadic reports of clinical and environmental ARAF strains [28,[73][74][75].
Another life-threatening IFI that was noted in high numbers in this study was mucormycosis. It presented in its most common form, rhino-orbital, and with the usual predisposing conditions of renal involvement and ketoacidosis [48]. The increasing trends of this infection hint towards breakthrough infections [76,77]. Antifungal treatment strategies are generally associated with surgical intervention for these cases. The focus is on the roles of amphotericin B formulations, posaconazole, combination therapies, and newer therapeutic approaches [78]. It is important to identify the genus, or if possible the species, since Cuninghamella, Lichtheimia, and Rhizopus oryzae can be resistant to posaconazole, which usually shows susceptible MIC profiles [79,80]. The standard treatment is liposomal amphotericin B dose according to the localization and extent of infection. The role of posaconazole is that it can be used as salvage therapy along with amphotericin B [80,81]. Incongruent with amphotericin B susceptibility data from India, in this study all strains were susceptible [82][83][84]. However, about 70% of Rhizopus species were susceptible to posaconazole, which is similar to previously published data [82][83][84].
Novel antifungal therapies and strategies can aid in the management of IFIs. In highrisk patients (neutropenic, etc.), antifungal prophylaxis is also recommended. However, the benefits associated with antifungal therapy (prophylactic/empirical) need to be evaluated with respect to local epidemiology and cost effectiveness. The treatment modalities are still unavailable/unaffordable to many patients in a developing nation such as ours.
The study was limited by its clinical suspicion inclusion bias and unexpectedly low numbers of probable IFIs, which may be due to the lack of invasive sampling owing to the poor condition of patients. There was one Taleromyces marneffi recovered from an AIDS patient, limiting the overall picture of the burden of rare pathogens among these infections.

Conclusion
In conclusion, the local epidemiology of IFIs in this study was significantly different from elsewhere. The predictors of infection or mortality were found similar to global data. However, these considerations underscore the importance of understanding both the epidemiology and resistance profile of the invasive fungal isolates that are commonly seen in both immunocompromised and immunocompetent populations. An active surveillance of invasive fungal infections, along with multidrug susceptibility testing of isolates to monitor the extent of the problem and develop feasible local diagnostic algorithms, will provide the database that might aid in future treatments to limit the emergence of resistance and alleviate the fatality rate. Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and the study was approved by the ethics committee of the institute i.e., All India Institute of Medical Sciences, New Delhi, India (Ref no. IEC/NP-25/2014RP-10/2014, OP-3/09.02.2017). The detailed procedure was as per institute guidelines: http://www.aiims.edu/aiims/ academic/ethics-committee/forms%20in%20pdf/IEC/Format_of_Institution_Ethics_Committee_15 032012.pdf (accessed on 16 January 2017).

Informed Consent Statement:
The consent forms for minor/incapable participants were obtained by their LAR, i.e., legally accepted representatives (example: mother, father, children, or grandparents).