INTRODUCTION

COVID-19 (Coronavirus Disease-2019) is characterized by mild to moderate respiratory illness, with symptoms such as fever, cough, fatigue and breathing difficulties, and, in severe cases, results in Acute Respiratory Distress Syndrome (ARDS). In addition to the harmful effects caused by the virus itself, such as alteration of the immune response and direct damage to pulmonary and extrapulmonary tissues, may be accompanied by infections caused by other microorganisms¹. The high prevalence of morbidity and mortality in patients with COVID-19 is associated with fungal and bacterial co-infection, especially among those suffering from ARDS².

Often, critically ill patients with COVID-19 who develop ARDS are admitted to Intensive Care Units (ICUs), where invasive monitoring, such as using mechanical ventilators and intravenous catheters, can allow the entry of opportunistic pathogens³. Furthermore, the widespread use of immunosuppressive medications, such as systemic corticosteroids, and the prolonged use of broad-spectrum antibiotics, together with the tissue damage caused by SARS-CoV-2 (Coronavirus Severe Acute Respiratory Syndrome-2), increase susceptibility. of these patients to invasion by commensal yeasts, causing deep invasive fungal infections⁴ .

According to studies, invasive fungal infection (IFI) associated with COVID-19 had an incidence that varied between 4 and 27.7%, with a higher occurrence in cases admitted to ICUs, candidiasis was one of the most commonly reported IFI, representing an associated mortality rate of 40%. ^{1, 5, 6}

In healthy individuals, Candida species live as commensals, however, in hosts with a weakened immune system, they can cause infections⁷. Bronchial colonization by Candida spp. It is prevalent among patients who use mechanical ventilation (MV), being found in approximately 30% of people who use it for more than 48 hours and in 50% of those diagnosed with ventilator-associated pneumonia (VAP). Still in the study by Erami et al. (2022), the most common comorbidities among patients colonized by Candida in the respiratory tract included diabetes, renal disorders, malignancies, and cardiovascular diseases² .

Due to the complicated medical situations of COVID-19 patients and inadequate collection of clinical samples, most fungal infections in this group of patients are misidentified. Identification and diagnosis of fungal infections have been a challenge for many researchers; therefore, isolation of Candida spp. in the lower airways should be interpreted with caution as causative agents of pulmonary disease⁶ .

In this regard, secondary fungal infections can complicate the prognosis of patients with COVID-19. Therefore, it is essential to carry out a specific diagnosis as well as understand the antifungal susceptibility profile of Candida spp. The appreciation of these results, together with patients’ clinical condition, must be interpreted by the clinician, aiming for an appropriate treatment for a possible fungal co-infection with SARS-CoV-2. ^4,8

Considering the above, this study aims to compare Candida spp. incidence in the respiratory tract of patients admitted to the COVID ICU and General ICU in a teaching hospital in 2021.

METHODS

This is a descriptive, documentary and retrospective study, with a quantitative approach, carried out in a public teaching hospital located in the city of Cascavel, Paraná. This hospital has 298 beds, with 60 General ICU beds, and, during the COVID-19 pandemic, 70 beds allocated to COVID ICU.

The criteria for data collection included analysis of medical records of patients admitted to the COVID ICU (with a positive diagnosis for SARS-CoV-2) and General ICU (with a negative diagnosis for SARS-CoV-2) between January and December 2021, without sex restriction, with age including young people, adults and elderly people, who presented a positive diagnosis for yeast isolation in protected tracheal aspirate samples. Cultures with counts ≥105 CFU/mL, without the isolation of another microorganism, were considered positive. Microorganism identification was carried out by automation using VITEK^®2 (BioMérieux, France), in accordance with the manufacturer’s recommendations. Data were collected in the Philips Tasy^® electronic medical record system. The variables collected in the system were comorbidities, use of invasive procedures such as MV, MV length, tracheostomy, orotracheal tube, nasoenteral tube (NET), nasogastric tube (NGT), central venous access (CVA), peripheral venous access (PVA), indwelling urinary catheter (IUC), length of admission, use of antimicrobials and corticosteroids, in addition to sex, age, sector of origin of admission, clinical outcome, and species of Candida. Patients who did not have a confirmed or negative result by RT-PCR for SARS-CoV-2 were excluded from the research.

Microsoft Office Excel^® version 2010 was used to tabulate the data. The incidence of pulmonary candidiasis was calculated using the ratio, in which the numerator was the number of episodes of pulmonary candidiasis during the study period, and the denominator was the number of patients on MV per day in the same period, multiplying the result by 1,000. To assess the association between qualitative variables, the chi-square test was used, considering a significance level of 5%, with p < 0.05 being statistically significant. To define the factors associated with the outcome (death), a mathematical model was adjusted using the binary logistic regression method, using the criterion of p < 0.10 of the Odds Ratio being statistically equivalent to 1.

Data collection from patient records occurred after the study was approved by the local Ethics Committee, under Certificate of Presentation for Ethical Consideration (CAAE - Certificado de Apresentação para Apreciação Ética) 65827722.2.0000.0107 and favorable Opinion 5.798.336. The research was conducted in accordance with the required ethical standards (Resolutions 466/2012, 510/2016, 580/2018 of the Ministry of Health).

RESULTS

From January to December 2021, protected tracheal aspirate cultures were performed from 556 patients diagnosed with SARS-CoV-2 admitted to the COVID ICU and from 260 patients admitted to the General ICU (with a negative diagnosis for SARS-CoV-2), as, of these, 38 and 10 had positive cultures for Candida spp., respectively. The incidence of pulmonary candidiasis was 68.3/1000 patients per day in the COVID ICU and 38.5/1000 patients per day in the General ICU.

In the COVID ICU, 23 (60.5%) were male, and 15 (39.5%) were female. The age of these patients ranged from 39 to 83 years, with a median age of 64.5 years. The interaction time ranged from 3 to 55 days (median = 18). Considering the clinical picture evolution, 24 (63.2%) of patients died and 14 (36.8%) were discharged. In the General ICU, 7 (70%) of patients were male, and 3 (30%) were female, aged between 24 and 75 years (median = 65.5 years). The duration of admission ranged from 13 to 48 days (median = 21.5 days). According to the clinical outcome, 6 (60%) of patients were discharged and 4 (40%) died.

The majority of patients had underlying risk factors, 81.6% from COVID ICU and 100% from General ICU. The most prevalent were systemic arterial hypertension and diabetes mellitus in both wards analyzed. Other comorbidities that were equally present among patients were alcohol consumption, smoking, dyslipidemia, obesity, among others (Figure 1).

Table 1 describes the invasive procedures to which patients were subjected. It is observed that invasive devices were widely used, resulting in 100% use by patients. The most frequent were orotracheal tube (OTT), NET, CVA, IUC and MV, whose time ranged from 3 to 40 days (median = 15.5) in COVID ICU and from 10 to 29 days in General ICU (median = 18.5).

Table 2 demonstrates predictive factors for the outcome of death. The variables considered significant by the logistic regression method were admission days, days of MV and tracheostomy. Among these factors, it was observed that patients with more days on MV were 3.74 times more likely to die (p = 0.007), and patients admitted to the COVID ICU were 38.2 times more likely to die than those admitted to the General ICU.

The percentage of deaths comparing the number of positive and negative cases of Candida spp. among patients admitted to COVID ICU and General ICU it was 6.83% and 3.46%, respectively, not being statistically significant (p = 0.0540). However, a trend towards significance was observed, with the COVID ICU tending to present more positive cases of Candida spp. than the General ICU (Table 3).

Regarding the patients who presented tracheal secretion culture with isolation of Candida, all (100%) used antimicrobials. Among the most prescribed were piperacillin/tazobactam, ceftriaxone, azithromycin, meropenem and linezolid (Table 4).

Regarding antifungal medications, the frequency of use in COVID ICU patients was 31.6%, with fluconazole being the most prescribed drug, with 91.7%, followed by echinocandins (anidulafungin and micafungin 8.3%, respectively). Meanwhile, in the General ICU, the frequency was 50%, with anidulafungin present in the majority of prescriptions (80%), followed by fluconazole, with 40%.

Regarding the use of steroidal anti-inflammatory drugs, 100% of patients admitted to the COVID ICU used these medications. Dexamethasone was prescribed to 100% of patients, followed by hydrocortisone, 10.5%, prednisone, 5.3%, and methylprednisolone, 7.9%. In the Adult ICU, 40% of patients used steroidal anti-inflammatory drugs, with hydrocortisone prescribed in 75% of cases, followed by methylprednisolone and prednisone, with 25% both. Dexamethasone, contrary to what was observed in the COVID ICU, was not prescribed in this unit.

Regarding the isolated yeasts, it was observed that C. albicans was the prevalent species in both wards, representing 60.5% in the COVID ICU and 40% in the General ICU. In COVID ICU, there was greater diversity among the isolated species, with five different species, such as C. tropicalis, C. lusitaniae, C. dubliniensis and C. parapsilosis. In the Adult ICU, the isolated species were C. tropicalis, C. glabrata, C. parapsilosis and C. famata (Table 5).

DISCUSSION

Although there are few studies on the lung microbiome, growing evidence indicates that the fungal microbiota is altered in critically ill patients; however, in patients with COVID-19, lung fungal colonization/infection represents a major concern⁹. Although microbial colonization is an important factor in the development of secondary infections, Candida pneumonia is rarely reported in ICUs. In the present study, it was observed an incidence of pulmonary candidiasis of 68.3/1000 patients per day in the COVID ICU and 38.5/1000 patients per day in the Adult ICU.

Some studies have reported that the development of VAP is independent of Candida colonization in the airways. Therefore, the meaning of Candida colonization in the airways remains controversial, requiring caution when interpreting many clinical conditions² .

With regard to gender, there was a higher prevalence of males in admissions, corresponding to 60.5% in the COVID ICU and 70% in the General ICU, corroborating findings from other studies. A study carried out in a university hospital in Italy also showed a predominance of males, compared to females. This occurrence may be related to the greater number of men admitted to hospital in these sectors⁹.

According to patients’ age, the median was 64.5 years for COVID ICU patients and 65.5 for General ICU patients, which were close to the study by Viciani et al. (2022), demonstrating that the median age was 64 years for patients without COVID-19 and 68 for individuals with COVID-19⁹ .

According to Taylor (2021), hospital admission rates for COVID-19 were higher in patients over 65 years of age, requiring ICU admission. This predominance can be justified by the fact that older patients are more vulnerable to complications when subjected to prolonged stays, medical interventions, immune system imbalance, or when they have diseases or comorbidities. As the population ages, the frequency of older patients with health problems requiring treatment in the ICU also increases. ^{10, 11, 12}

Related to the period of stay in the ICU, it was found that COVID ICU patients had a median stay of 18 days, and General ICU patients had a median of 21.5 days. These data were close to what was presented in a retrospective study, carried out in a tertiary hospital in Spain, where the median length of stay for patients was 20 days.¹³ Studies have shown that increased hospital and ICU stays increase the risk of co-infections. The combination of factors associated with treatment, prolonged stays in the ICU, medical interventions, such as the use of MV and intravenous catheters, increase the risk of candidiasis⁷ .

Considering the comorbidities presented among patients, systemic arterial hypertension and diabetes mellitus were the most prevalent. These data coincide with a cross-sectional study carried out in a tertiary hospital in Egypt, which analyzed patients with COVID-19 under MV admitted to the ICU, which presented hypertension (62.4%) and diabetes mellitus (56.3%) as the main underlying diseases. In addition to these, obesity, hypothyroidism, lung disease and heart disease were observed in both the present study and the one mentioned. The presence of cancer/malignancy was also observed in other studies. ^{7, 14} Patients with underlying chronic diseases and advanced age tend to be more likely to acquire infections due to their weaker immune conditions. ¹⁵

In this study, the invasive devices most used by patients were MV, OTT, CVA, IUC, NET and PVA. Patients admitted to the ICU often require central venous catheterization and parenteral nutrition, while those for whom MV is not necessary receive peripheral venous catheters. In recent decades, urinary catheters have been identified as risk factors for the development of invasive candidiasis.^7,5 These data coincide with those of another study carried out in a university hospital in Spain, where patients positive for Candida spp. were admitted to the ICU and required orotracheal intubation. The treatment received was with parenteral nutrition and nasogastric catheters and central and bladder access routes³.Using these medical devices provides a direct route into the host by penetrating the skin barrier. Candida species can form biofilms on these devices and act as physical barriers to protect antifungal treatment and host immune system defenses, already weakened due to COVID-19⁷ .

In our study, patients with more days on MV were 3.74 times more likely to die (p = 0.007), and patients admitted to the COVID ICU were 38.2 times more likely to die than those admitted in General ICU. However, this result must be interpreted with caution, as the value obtained for “p” is not significant (p = 0.07), but indicates a tendency towards significance. Isolation of Candida spp. via the respiratory tract is associated with longer periods of MV and ICU admission, with unfavorable outcomes⁷. As in the present study, Meawed et al. (2021) found, in their research, that longer duration of MV proved to be a highly significant risk factor for candidiasis (p < 0.001). ¹⁴

In this research, antibiotic therapy was present in 100% of prescriptions in ICUs, with all patients using more than one class of antibiotics. Patients with COVID-19 are more likely to acquire bacterial co-infections, and the most commonly prescribed antibiotics were ceftriaxone and azithromycin, observed in a single-center retrospective analysis, similar to the current study⁸. In another retrospective observational study, carried out in a tertiary hospital in Spain, ceftriaxone was also the most used antibiotic among patients as well as piperacillin/tazobactam, carbapenems, linezolid and levofloxacin, corroborating the data from this research. ¹³

Prolonged use of broad-spectrum antibiotics is associated with microbiota imbalance, creating a favorable environment for the proliferation and transformation of commensal to pathogenic Candida morphogenesis.¹⁵ The dysbiosis caused by using these medications allows Candida spp. overcome other microorganisms, providing their colonization and dissemination⁷ .

Regarding the use of steroidal anti-inflammatory drugs, it was observed that, in COVID ICU, there was 100% use of these medications. Patients with COVID-19 develop a cytokine storm syndrome that is characterized by an increase in pro-inflammatory cytokines and a decrease in anti-inflammatory cytokines. In this sense, systemic corticosteroids are frequently used as treatment, such as dexamethasone, which is a well-known medication used to reduce the dysregulation of the inflammatory state⁵.

Although this glucocorticoid reduces the risk of a hyperinflammatory response in patients with COVID-19, its use is a risk factor for the development of opportunistic fungal co-infection, as the hyphae are protected from phagocytic attack. A study demonstrated that treatment with corticosteroids is associated with a 3.33 times greater risk of developing an IFI, when compared to other patients who did not receive this type of medication. ¹⁰

In this study, the mortality rate of patients diagnosed with COVID-19 and admitted to the COVID ICU (63.2% 24/38) was significantly higher than that observed among negative patients admitted to the Adult ICU (40% 4/10), similar to the study by Calderaro et al. (2021), who analyzed infectious agents in lower respiratory tract samples from patients positive and negative for SARS-CoV-2 admitted to the ICU of a tertiary hospital located in Parma, Italy. According to study reports, infections caused by Candida and other fungal species tend to affect patients with severe viral infections, and may be associated with increased morbidity and mortality. ^17,18

Regarding the yeasts isolated, C. albicans was the most frequently isolated species in both wards. Candida spp. are recognized as opportunistic microorganisms that cause serious infections in immunocompromised individuals. An important characteristic of Candida virulence is biofilm formation, which involve the ability to adhere to tissues and surfaces, and are extremely resistant to routine antifungals. Biofilm formation involves the adhesion of yeast cells, which promotes their proliferation, extracellular matrix material accumulation and yeast cell dispersion, which can establish new biofilms.¹⁹ The extracellular matrix assists in fungal colonization and invasion, by acting as a protective barrier, increasing adhesion and production of hyphae; thus, it protects the cell from immune attack by increasing resistance to antifungal agents⁷ .

These data corroborate data from the study by Erami et al. (2022), in which C. albicans was the prevalent species, isolated from bronchoalveolar lavage (BAL) samples from patients with COVID-19 pneumonia who used MV for more than four days, admitted to a hospital in Iran² .

The exact pathogenesis of candidiasis associated with COVID-19 is not clear, however several factors have been proposed to explain how patients with COVID-19 are more predisposed to Candida infection. SARS-CoV-2 infection can cause a reduction in lymphocytes and, consequently, the impairment of immune defense against fungal agents, including Candida. Elevated blood lactate and acidosis is associated with greater severity of COVID-19, allowing restructuring of the yeast cell wall to mask β-glucans and escape host immunological recognition. Additionally, Candida’s ability to form biofilms may be triggered through oxidative stress and pH imbalance observed in COVID-19 patients¹ . In our study, although the outcome of death and positive tracheal secretion culture for Candida is not statistically significant, the COVID ICU tended to present more positive cases of candidiasis in relation to the General ICU. Delisle et al. (2008) showed, based on regression analysis, that there is a significant association between respiratory tract colonization by Candida and hospital mortality.²⁰

Disseminated candidiasis has become more prevalent as COVID-19 has progressed². However, isolation of Candida spp. should be valued in all critical patients, due to the potential virulence factors produced by this microorganism, which corroborate the successful colonization or invasive infection of the host’s tissues. ²¹ As there are controversies regarding the interpretation through the isolation of yeasts in samples from the respiratory tract, since the diagnostic criteria for candidiasis for this site are not well established⁶ . Therefore, it is important to question whether it is possible Candida pneumonia or just colonization, with more laboratory and clinical interaction studies needed to determine cut-off points and establish the use of antifungal prophylaxis in a population at risk.

We conclude that patients admitted to the ICU for COVID-19 share some risk factors and underlying diseases, such as chronic respiratory diseases, corticosteroid therapy and invasive devices. To our knowledge, this is the first case series reported of a possible correlation of respiratory tract candidiasis after COVID-19 in critically ill patients. More studies are needed to understand the association between isolation of Candida in the respiratory tract and its clinical importance. An accurate and rapid diagnosis of candidiasis will provide adequate treatment for patients, in addition to representing an improvement in the mortality rates resulting from these infections not only in patients with severe COVID-19, the target of this study, but also in other critical patients mainly in ICUs.

Acknowledgments

To the Pharmaceutical Residency Program in Clinical Analysis of the Hospital Universitário do Oeste do Paraná, for the vast source of data provided to carry out this research, and to the Coordination for the Improvement of Higher Education Personnel (CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior).

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