key: cord-0819157-tt1ne9kn
authors: Rajni, Ekadashi; Singh, Ashutosh; Tarai, Bansidhar; Jain, Kusum; Shankar, Ravi; Pawar, Kalpana; Mamoria, Vedprakash; Chowdhary, Anuradha
title: A high frequency of Candida auris blood stream infections in COVID-19 patients admitted to intensive care units, North-western India: A case control study
date: 2021-09-07
journal: Open Forum Infect Dis
DOI: 10.1093/ofid/ofab452
sha: 8388c3cd211efaf9c0b55e2b20819f41fe71c1a5
doc_id: 819157
cord_uid: tt1ne9kn

BACKGROUND: The ongoing pandemic of COVID-19 has overwhelmed healthcare facilities raising an important novel concern of nosocomial transmission of Candida species in the intensive care units. METHODS: We evaluated the incidence and risk factors for development of candidemia in 2384 COVID-19 patients admitted during August 2020-January 2021 in ICUs of two hospitals (Delhi and Jaipur), India. A 1:2 case control matching was used to identify COVID-19 patients who did not develop candidemia as controls. RESULT: A total of 33 patients developed candidemia accounting for an overall incidence of 1.4% over a median ICU stay of 24 days. A two-fold increase in the incidence of candidemia in COVID-19 versus non–COVID-19 patients was observed with an incidence rate of 14 and 15/1000 admissions in two ICUs. Candida auris was the predominant species (42%) followed by Candida tropicalis. Multivariable regression analysis revealed the use of tocilizumab, duration of ICU stay (24 vs. 14 days) and raised ferritin level as an independent predictor for the development of candidemia. Azole resistance was observed in C. auris and C. tropicalis harbouring mutations in the azole target ERG11 gene. MLST identified identical genotypes of C. tropicalis in COVID-19 patients raising concern of nosocomial transmission of resistant strains. CONCLUSION: Secondary bacterial infections has been a concern with the use of tocilizumab. In this cohort of critically ill COVID-19 patients tocilizumab was associated with development of candidemia. Surveillance of antifungal resistance is warranted to prevent transmission of MDR strains of nosocomial yeasts in COVID-19 hospitalised patients.

Invasive fungal infection has been increasingly highlighted as a serious concern in critically ill patients with COVID-19 in several countries of South America, Middle East, Europe, Asia and the United States [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] . Patients with severe COVID-19 disease who are hospitalised in intensive care units (ICUs) for prolonged periods of time, often require multiple courses of broad-spectrum antibiotics, mechanical ventilation, and other invasive devices resulting in increasing exposure to, and risk of acquiring nosocomial blood stream infections (BSIs) due to Candida species (spp.) [2, 3, 14, 15] .

Recent studies originating from USA, Italy, Spain and Brazil emphasized a significant, 3-10-fold increase in incidence of ICU candidemia among COVID-19 patients as compared to non-COVID-19 cohort [2-4, 9, 16, 17-20] . These increasing reports about secondary fungal infections as complications of severe COVID-19 raises a parallel concern regarding the emergence and transmission of multi drug resistant (MDR) nosocomial Candida spp. in COVID-19 ICUs [10, 21, 22] . A recent report of secondary healthcare associated infections in COVID-19 patients from a quaternary care hospital in New York City, USA, showed that a relatively large proportion of fungal infections (15%), were primarily due to hospital-associated Candida spp [15] .

In the beginning of COVID-19 pandemic April-July, 2020, we apprised a series of candidemia cases due to nosocomial MDR yeast C. auris in critically ill COVID-19 patients hospitalized in a single center in Delhi. Notably, in the series 70% of C. auris isolates were multidrug resistant, including 30% that were resistant to 3 classes of antifungal drugs [10] . Several recent studies on Candida BSIs lack information on the species identification and antifungal susceptibilities pattern thus underestimating the burden of antifungal resistance in COVID-19 settings. Furthermore, the impact of immunomodulatory agents, such as corticosteroids and IL-6 receptor blockers, on the incidence of A c c e p t e d M a n u s c r i p t 7 Candida BSIs is largely undetermined, despite the widespread use of these agents to manage inflammatory complications of COVID-19. To get the better insight in the development of candidemia and associated risk factors in COVID-19 patients we retrospectively analysed a large set of 2384 COVID-19 patients hospitalized in the ICUs of two hospitals in Northwestern India, i.e., Delhi and Jaipur, Rajasthan for a long duration of six months period from August 2020-January 2021.

We evaluated the incidence and risk factors for development of candidemia in COVID-19 patients as compared to the matched control group of COVID-19 patients without candidemia.

Study design and data collection: A case-control study conducted in COVID-19 ICUs of two multispecialty hospitals (hospital A with 50 ICU beds and hospital B with 80 ICU beds) of northwestern India. The study included COVID-19 positive adults with more than 17 years of age admitted in the COVID-19 ICUs who developed candidemia during a period of August 01, 2020 to January 31, 2021 and were followed up for a period of thirty days. The present study is a retrospective review of candidemia in adult patients (>17 years) with PCR proven COVID-19 across COVID19-ICUs in two hospitals. Candidemia was defined as the growth of Candida spp on one or more blood culture and the date of the first positive fungal blood culture was used for calculation of all durations. Candidemia patients were identified starting from the laboratory databases of the participating hospitals, and subsequent review of clinical records. Data concerning demographics (age, gender), comorbidities, laboratory tests, SOFA score at ICU admission, treatment and outcomes (ICU admission, length of hospital stay and mortality) were collected directly from electronic health records. All patients had a diagnosis of COVID-19 confirmed by real-time reverse transcription PCR (RT-PCR) testing performed on nasopharyngeal throat swab specimens. ICU database was screened to include a control group of 70 COVID-19 (1:2 case control matching) hospitalised patients without candidemia admitted during the same time period as cases, and matched based on age (±5), and the sequential organ failure assessment (SOFA) scores available at the time of their admission to the ICUs. No sample size calculations were performed a priori for this exploratory study.

A c c e p t e d M a n u s c r i p t 8 Data was collected for demographics, risk factors for candidemia, utilization of tocilizumab, and use of steroids. For anti-inflammatory treatments for COVID-19, data were analysed based on antiinflammatory treatment used or not i.e., steroid treatment and intravenous tocilizumab (8 mg/kg single administration or repeated once). Demographic and clinical characteristics of patients are presented with number and percentage for categorical variables and median and interquartile range (IQR) for continuous variables. The processes and practices undertaken in both the hospitals are listed in supplementary data (see supplementary material).

Factors affecting the occurrence of candidemia among the COVID-19 patients were determined by binary logistic regression analysis. The dependent variable for the logistic regression analysis was presence or absence of candidemia. Multivariable logistic regression analysis was performed between the dependent variable and all the independent variables which were found to be significant in univariate logistic regression (p value <0.1). Adjusted odds ratio (OR) in multivariable and unadjusted OR in univariate logistic regression analysis with 95% confidence intervals (CI) are reported. Logistic regression was performed in EZR (Easy R, version 1.54) statistical software, which is based on R and R commander [23] . Variance inflation factor (VIF) was computed to determine the presence of multicollinearity among the independent variables in STATA software using the package "collin" which is meant for determining the multicollinearity among the categorical variables. VIF <5 confirmed the absence of multicollinearity.

Blood culture specimen collection and processing: Blood samples were obtained by using aseptic precautions. Before collecting the blood sample, the skin was disinfected with 0.5% chlorhexidine in 70% isopropyl alcohol. The antecubital fossa was the preferred sampling site and samples from central vein catheters were obtained from needleless caps that were disinfected with 0.5% chlorhexidine in 70% isopropyl alcohol. Two automated blood culture systems were used during the study period: BactecTM FX (Becton Dickinson, Sparks, Maryland, USA) and Bact/Alert®3D (bioMérieux, Marcy l'Etoile, France). Blood cultures A c c e p t e d M a n u s c r i p t 9 were incubated in instrument for up to 5 days. Bottles flagged positive were streaked onto blood and Sabouraud agar plates. Blood culture bottles that did not show visible microorganism were also sub-cultured after 5 days. After incubation at 37 °C for up to 48 h yeast were identified to the species level.

Yeast identification: Candida species isolated from blood cultures were identified by matrixassisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF Bruker Biotyper OC version 3.1, Daltonics, Bremen, Germany, https://www.bruker.com) using the ethanol-formic acid extraction protocol [24] . In addition, C. auris species identification was confirmed by amplification and sequencing of the internal transcribed spacer region of ribosomal DNA and of the D1/D2 domain of the large subunit ribosomal DNA as described previously [25] .

Antifungal susceptibility testing was performed by using the Clinical and Laboratory Standards Institute broth microdilution method M27-A3/S4 [26, 27] . Antifungals tested were and C. parapsilosis ATCC 22019 were used as quality control strains. The geometric mean (GM) MICs with 95% CIs, MIC50, MIC90, medians and ranges were calculated using Prism version 6.00 (GraphPad Software). Further, all azole resistant yeast isolates obtained from candidemia were subjected to azole target ERG 11 gene sequencing as detailed previously [28] . To determine the genotypes of C. tropicalis prevalent in COVID-19 ICUs, we performed A c c e p t e d M a n u s c r i p t 10 MLST using six housekeeping genes: ICL1, MDR1, SAPT2, SAPT4, XYR1, and ZWF1α as described previously by Tavanti et al [29] . Allelic profiles and the diploid sequence type (DSTs) of the six gene sequences were obtained from the C. tropicalis MLST sequence-type database (http://pubmlst.org/ctropicalis/). Phylogenetic analysis of the isolates was 

ICUs during the 6-months span of the study period. Overall, 33 patients with candidemia were identified accounting for an incidence of 1.4% over a median ICU stay of 24 days. 

The present study, demonstrate a high incidence rate of candidemia i.e., 14 and 15/1000 admissions in critically ill patients with COVID-19 in two ICUs in India, which is about two-fold higher than (table 3) .

Interestingly, in the five studies originating from New York, New Jersey and Georgia, USA the prevalence of candidemia in COVID-19 patients showed a wide range i.e. 0.07 -8.9% [1, 2, 14, 15, 18] . The high prevalence of 8.9% was observed among 89 COVID-19 adult patients who were admitted to the ICU for worsening disease status [14] . A study investigating the incidence of bacterial and fungal coinfections in 836 hospitalized patients across two London hospitals during the first UK wave of COVID-19 reported that BSIs due to Candida spp. presented as late-onset infection accounting for 0.4% of secondary infections [30] . In contrast a national, multicentred study applying an enhanced testing strategy to diagnose invasive fungal disease in COVID-19 intensive care patients in Wales UK, identified 12.6% incidence of invasive yeast infection, mainly (93.8%) due to Candida spp. [7] .

The wide ranges of incidence of candidemia occurring in COVID-19 patients in the above mentioned studies may be attributed to calculations of incidence based on all patients or extrapolating the incidence to entire population to determine a total disease burden. This strategy may not accurately identify the burden of BSIs due to Candida spp. as these infections occurs primarily in intensive care settings.

A three to 8-fold increase in the incidence of candidemia in COVID-19 patients versus non-COVID-19 patients has been reported in studies originating from New York, USA, Rio de Janeiro and southern Brazil, Wales, UK and Milan, Italy [2-4, 9, 31] . Interestingly, 8-fold increase in the incidence of candidemia in COVID-19 patients has been observed in two hospitals of Southern Brazil [4] . In the present study a 2-fold increase in candidemia in two hospitals was recorded with C. auris being the predominant agent of candidemia (42%). In fact 64% of candidemia in the present study were due to non-albicans Candida spp i.e., C. auris and C. tropicalis. In contrast studies from USA (table 3) showed that C. albicans contributed to 25-54% of candidemia whereas 18% of candidemia in the present study was due to C. albicans [2, 14, 15, 16] . Also, C. albicans was the predominant agent of candidemia in COVID-19 patients in European countries including Italy, Spain and UK [7, 9, 17, 19, 20, 30] .

A c c e p t e d M a n u s c r i p t 14 As anticipated earlier in the COVID-19 pandemic, BSIs due to C. auris has been recently recognised widespread in critically ill COVID-19 patients [32] [33] [34] [35] [36] [37] . Interestingly, COVID-19 associated C. auris candidemia has been recognised in countries that had not previously recorded this yeast [37] . The first outbreak of C. auris occurred during a COVID-19 pandemic in a tertiary-care center in Lebanon [37] .

Similarly outbreak of C. auris was recently highlighted in a COVID-19 hospital in Monterrey, Mexico that started in non-COVID-19 patient and during the transition of the hospital to the exclusive COVID-19 facility later spread to 12 patients in COVID-19 ICU [34] . Additionally, increasing reports of transmission of C. auris among COVID-19 patients has been observed in those geographic regions where this MDR yeast was already prevalent in the hospital environment [32] [33] [34] 38] . Candida auris outbreak in a COVID-19 specialty care unit in Florida, July-August 2020, identified three C. auris

the COVID-19 unit 52% were colonised with C. auris [38] . Several factors including health care personnel using multiple gown and glove layers, extended use of the underlayer of PPE, lapses in cleaning and disinfection and adherence to hand hygiene likely contributed to widespread C. auris transmission [38] .

To prevent transmission of C. auris in the COVID-19 care facilities, enhanced vigilance and essential screening of patients is warranted. Furthermore, it is also pertinent to emphasise that patients who have been hospitalised and recover from severe COVID-19 may remain colonised by C. auris for prolong periods. Thus, screening of patients for C. auris needs to be undertaken in patients that require repeated admission for long term sequalae in the post COVID-19 facilities. A study from Delhi, India, undertaking screening of C. auris colonisation among chronic respiratory diseases patients that required repeated admissions in healthcare identified that 9.5% of patients were colonised at the time of admission and 75% remained colonised till discharge [39] . Also, C. tropicalis is of particular importance as it is a major cause of nosocomial candidemia particularly in the Asia-Pacific region [40] [41] [42] [43] [44] . In the present study, identical C. tropicalis genotypes infected the patients in ICUs suggesting patient-to-patient transmission. Antifungal resistance was observed in both C. auris and in a single isolate of C. tropicalis raising concern of nosocomial transmission of resistant isolates [14] . Similarly, another study from New York, USA showed that the ICU length of stay prior to the development of candidemia was significantly longer in the COVID-19 group (19 days vs. 5 P =.001) as compared to non-COVID-19

patients who developed candidemia [2] .

Although management strategies for COVID-19 have progressively been evolving through the pandemic, therapies with immune-modulating properties such as IL-6 receptor antagonists and corticosteroids has been commonly used in severe disease. Tocilizumab, an interleukin (IL)-6 receptor blocker, is used in the treatment for severe, progressive COVID-19 infection. However secondary infections has been a concern with the use of tocilizumab [16, 45, 46] . Although a clear association with (HAIs) primarily bacterial infections. Although, 11 cases of BSIs due to Candidia spp. were recorded in the study. It was observed that tocilizumab was associated with increased risk of HAIs (OR 5.04, 95% CI 2.4-10.6, p < 0.001) [16] . Morena et al studied clinical characteristics and outcome of 51 patients hospitalized with severe COVID-19 pneumonia treated with tocilizumab and late complications were serious bacterial and fungal infections of the bloodstream in 27% cases [46] .

Further concern of candidemia with use of tocilizumab has been raised in a report from Milan, Italy.

The authors observed that during an eleven-day period 43 patients with severe COVID-19 pneumonia were treated with tocilizumab and 3 patients (6.9%) developed candidemia, with one patient developing endophthalmitis and endocarditis [47] .

In the present study receipt of tocilizumab was more likely among candidemia cohort (67% versus 20% without candidemia) comprising a high percentage of patients requiring mechanical ventilation (64% vs 34% without candidemia) and significantly raised severity parameters (serum ferritin levels 82% vs 26% without candidemia) suggesting severe immunosuppression. IL-6 is a pro-inflammatory cytokine involved in the regulation of multiple aspects of innate immune response. Therefore, blockade of IL-6 may impair B-cell proliferation and T-cell differentiation and cytotoxicity, essential for immune control of infections [48] . Interestingly, severe impairment of the macrophage and neutrophil response to Candida infection was observed in IL-6-deficient mice. These mice were more susceptible to systemic C. albicans infection, had a decreased survival and an increased fungal load in their organs when compared with IL-6 positive controls [49, 50] . Further, an ex vivo whole blood stimulation assay with C. albicans lysate revealed an impaired response of COVID-19 patients toward C. albicans. COVID-19 patients showed an attenuated monocyte CD80 upregulation and abrogated release of IL-6, TNF, IL-1a, and IL-1b toward C. albicans suggesting an increased susceptibility for C. albicans infection in critically ill COVID-19 patients [51] .

Finally it should be considered that our sample size of candidemia patients was limited (n = 33) and other limitations of the present study are its retrospective nature. Moreover, variable infrastructure of 

The authors declare no conflict of interest A c c e p t e d M a n u s c r i p t 19 M a n u s c r i p t 21 M a n u s c r i p t 23 M a n u s c r i p t 26 A c c e p t e d M a n u s c r i p t 27 

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A c c e p t e d M a n u s c r i p t 22 M a n u s c r i p t 28 C. catenulata [ A c c e p t e d M a n u s c r i p t 29