The Prevalence of Bacterial and Fungal Coinfections among Critically Ill COVID-19 Patients in the ICU in Jordan

Background Secondary bacterial and fungal coinfections have been reported among critically ill coronavirus disease-19 (COVID-19) patients and are associated with increased disease severity and mortality incidence (MI) rates. Aims This study aimed to track bacterial and fungal coinfections among COVID-19 patients in the intensive care unit (ICU) and to assess the impact of these infections on disease prognosis and patient outcomes in Jordan. Materials and Methods This was a single-center study that enrolled 46 ICU patients diagnosed with COVID-19. Microbiological and antimicrobial susceptibility results and inflammatory biomarker data were retrospectively analyzed. Results The MI rate attributed to bacterial and fungal coinfections was 84.8%, and the highest rate was reported among patients older than 70 years (66.7%). The MI rate related to bacterial coinfections was 95.2%, whereas that of fungal coinfections was 4.8%. The most commonly isolated bacterium in the blood was a coagulase-negative staphylococcus (41%), followed by Klebsiella pneumoniae in nasopharyngeal swabs (34%) and Acinetobacter baumannii in sputum samples (31%). Candida species were the sole cause of fungal coinfections in the studied population. In particular, Candida albicans was isolated from 3% of patients with bacteremia, whereas Candida glabrata was isolated from 8% of nasopharyngeal swabs. Klebsiella pneumoniae was considered the major cause of upper respiratory tract infections (34%). Multifactorial infection was significantly associated with increased MI (p value <0.001). Conclusion COVID-19 MI is associated with respiratory bacterial/fungal coinfections. The ability to predict bacterial and fungal coinfections in ICU patients may be crucial to their survival and prognosis.


Introduction
A large proportion of patients with respiratory viral infections develop secondary bacterial and/or fungal coinfections, leading to increased severity and mortality due to the synergistic interaction of microbial pathogenesis and the host immune system [1]. Te risk of death among COVID-19 patients increases by more than twofold in the presence of bacterial and fungal coinfections [2]. Additionally, several studies have reported low rates of confrmed bacterial coinfections in COVID-19 patients, which were attributed to the lack of prompt diagnosis and administration of broadspectrum empirical antibiotics in COVID-19 patients at the time of hospital administration [3].
Te exact mechanism by which COVID-19 predisposes patients toward coinfections with other microorganisms is not yet fully understood. However, there are diferent hypotheses to explain this relationship; one of them is that infected respiratory cells are induced to release anti-infammatory cytokines [4]; these cytokines inhibit the link between the adaptive and innate immune systems, leading to delayed or inhibited bacterial clearance [4]. Another hypothesis is that the production of viral enzymes such as neuraminidase and sialidase promotes bacterial and fungal colonization [5].
Te prevalence of COVID-19 coinfection varies in different situations. Te highest prevalence of coinfections has been detected among hospitalized immune-suppressed patients (who were exposed to central lines and mechanical ventilators) and patients who had an underlying history of diabetes or other chronic diseases [6,7]. Such infection may be caused by a single microbe or multiple microbes [8,9], with various complications such as severe pneumonia [8], epidermal signs [10], alterations in the gastrointestinal microbiome [11], bacteremia [12], and hospital-acquired bacterial infections in the intensive care unit (ICU) [13]. Additionally, the presence of multidrug-resistant bacteria creates an additional crisis in the treatment of critically ill COVID-19 patients [14]. Complications of coinfections in COVID-19 patients depend on the etiology of coinfected microorganisms, viral load, "severity of viral infection," and the host immune response to the infection [1].
A computerized tomography (CT) scan plays a vital role among critically ill COVID-19 patients admitted to the ICU because it is used in the monitor of patients, evaluating patient disease severity and prognosis, informing the treating physician about laboratory tests required, and classifying patients into diferent risk groups [15,16].
Several studies have reported that clinicians tend to administer empirical broad-spectrum antibiotics to treat or avoid suspected bacterial coinfection [17,18]. Tis, in turn, results in many adverse efects that change the normal microfora, leading to the emergence of new antibiotic-resistance mutations [19]. Terefore, it is important to begin empirical therapies to ensure patient survival and then to apply narrower-spectrum antibiotics after receiving microbiological lab results [20,21]. Terefore, this study aimed to investigate isolated bacterial and fungal coinfections among COVID-19 patients in the ICU and to assess the impact of such coinfections on disease prognosis and patient outcomes in a private hospital in Amman, Jordan.

Materials and Methods
Eighty critically ill patients were admitted to the ICU between January 2020 and June 2021 in a private hospital in Amman, Jordan. All of them were assessed for COVID-19 infection by real-time reverse transcription-polymerase chain reaction (RT-PCR). Only 56 were diagnosed with COVID-19, and they were included in the study. Based on the defnition of coinfection, namely, infections that occur ≤48-72 h after hospital admission [22], only 46 patients fulflled the criterion and were included in the study. In addition, 10 patients were excluded from the study owing to missing data regarding the date of administration and the date of bacterial and fungal growth (Figure 1).
At the time of ICU administration, required clinical, radiological, and microbiological assessments were performed. Bacterial isolates were identifed based on colony characteristics and biochemical tests using a VITEK 2 system (bioMérieux) ® (USA); fungal isolates were determined by colony morphology and colony characteristics. A galactomannan test to detect bacteremia caused by aspergillosis infections was not performed. Data collected included age, sex, patient outcome (died or survived), treatment provided, coinfection outcomes (upper respiratory infection, lower respiratory infection, bacteremia, and meningitis), and antimicrobial susceptibility results. Additionally, data on infammatory biomarkers, including C-reactive protein (C-RP), ferritin, d-dimer, procalcitonin (PCT), globulin, white blood cell count (WBC), neutrophil count, and lymphocyte count, were selected to be included in the study to indicate 80 patients were administrated to emergency department with chest pain, high body temperature, sore throat, and general pain.

Results
A total of 46 ICU COVID-19 patients were included in this study. Te types of isolated microorganisms for each and patient demographic data (age, sex, and patient outcomes) are presented in Table 1. Te major isolated infectious bacteria were Klebsiella pneumoniae, Staphylococcus aureus, Enterococcus faecalis, Serratia marcescens, Pseudomonas aeruginosa, Acinetobacter baumannii, coagulase-negative staphylococci, Coryneform bacilli, and Staphylococcus epidermidis. Among the isolated fungi, only two species of Candida were detected: Candida glabrata and C. albicans.

International Journal of Microbiology
Te patterns of bacterial and fungal coinfections based on the type of specimen where the microorganism was isolated are illustrated in Figure 2. Te most numerous bacterial types were isolated from blood; coagulase-negative staphylococci had the highest proportion (41%), followed by Klebsiella pneumoniae (14%), Staphylococcus aureus (11%), and Staphylococcus epidermidis (8%). In sputum, the highest proportion of isolated bacteria was Acinetobacter baumannii (31%), followed by Pseudomonas aeruginosa and Klebsiella pneumoniae (23% for each). Candida glabrata was the sole cause of fungal coinfection in lower respiratory tract infections, with an 8% prevalence in the studied population. Regarding nasopharyngeal swabs, Klebsiella pneumoniae was considered the major isolated microorganism, with a 34% prevalence, followed by Staphylococcus aureus and Enterococcus faecalis (22% for each). Table 3 shows the microorganism distribution among diferent specimen types based on sex. In females, the highest proportion of isolated microorganisms in sputum was Klebsiella pneumoniae, Serratia marcescens, Acinetobacter baumannii, and coagulase-negative staphylococci (25% for each), whereas coagulase-negative staphylococci had the

Discussion
Bacterial coinfection was common during previous respiratory viral pandemics. It was associated with a poor prognosis of the viral disease and considered a risk factor for death [23]. Te MI of bacterial and fungal coinfection in COVID-19 patients was 50%, with more concern for antibiotic-resistant bacteria [3,5]. Te present data revealed that coinfections among COVID-19 in the ICU resulted from diferent types of bacteria and fungi, confrming what have been reported in other studies. Overall, coinfection among ICU COVID-19 patients results from bacteria [3], fungi [3], or other respiratory viruses [24]. In agreement with our fndings, Silva et al. (2021) reported that the MI among COVID-19 patients in the ICU was 50.47% (83.14% being coinfected by fungi and/or bacteria) [25]. In addition, the present results found that the MI as a result of bacterial coinfection was 94.9%, 13.0% for upper respiratory infection, 24.1% for lower respiratory infection, and 63.0% for bacteremia. Tese results confrm what had been reported by Hughes et al., i.e., that bacteremia is considered a serious complication of bacterial superinfection and a risk factor for mortality in COVID-19 patients [10]. Bacteremia among COVID-19 patients has been reported worldwide with various percentages: 1.8% in Michigan City [26], 6% in New York City [27], 7.1% in the United Kingdom [10], 25% in Pennsylvania [28], and 37.0% in Milan [12], with mortality rates ranging between 9.8% and 47.5%. Te worst-case scenario for critically ill COVID-19 patients is the development of polymicrobial coinfections [17], especially if one of the bacteria is multidrug-resistant, with a signifcant risk of complicating multiple organ failure and septic shock [8]. Te main infuence of bacterial coinfection in COVID-19 patients is mainly attributed to septicemia, multiorgan failure, septic shock, and respiratory, cardiac, kidney and liver dysfunction [27,29,30].
Te present study shows an increase in infammatory biomarkers, including PCT, C-RP, D-dimer, and ferritin, among patients who did not survive, in line with previous fndings [27] and correlating signifcantly with respiratory failure distress, extended mechanical ventilation, and an increased MI rate [31]. Lymphocytopenia among our participants can be explained by interleukin upregulation and cytokine storms that cause lymphocyte apoptosis [7,9], which have been considered risk factors for coinfection during the COVID-19 pandemic [6].
Antibiotic resistance was detected among approximately half of the patients who died (51.3%), which creates additional challenges in controlling patient outcomes and prognosis [14]. Te prophylactic use of broad-spectrum antibiotics among such patients must be taken into consideration to avoid the drawbacks of long-term broadspectrum antibiotic misuse [28]. Te protocols regarding broad-spectrum antibiotic use are diferent at the national level; for example, the Chinese Institutes of Health do not recommend the use of broad-spectrum antibiotics even in critically ill patients without bacterial coinfection [17], whereas in the Netherlands, empirical antibiotics are recommended for all ICU and mechanically ventilated patients [32].
Among the study strengths, this study is the frst in Jordan and included accurately identifed bacterial and fungal isolates that caused coinfection among COVID-19 patients in the ICU, and it is the frst study to clinically characterize ICU COVID-19 patients in the Middle East and North Africa region (MENA). Additionally, the samples were obtained from diferent types of specimens. Among limitations, this was a single-center study, and the present fndings should be considered with caution. Te small sample size is attributed to the nature of the studied population, namely, ICU patients.
In conclusion, bacterial/fungal coinfections hurt critically ill COVID-19 patients and may lead to worse disease prognosis and increased MI. Timely prediction of bacterial and fungal coinfection among critically ill ICU patients through monitoring of infammatory biomarkers before microbiological culture and antibiotic susceptibility results may play a critical role in improving patient prognosis and increasing the survival rate.

Data Availability
Te database analyzed for the current study is available from the corresponding author upon reasonable request.

Ethical Approval
Te study protocol was carried out following the principles of the Declaration of Helsinki as revised in 2000. Ethical approval for this research was obtained from the Ethics Committee for Scientifc Research (ECSR) at Zarqa University by the requirement of the protection of human subjects and the ethical principles related to research studies (no. 2/8/2021).

Conflicts of Interest
Te authors declare no conficts of interest.

Authors' Contributions
All the authors have taken responsibility for the entire content of this manuscript and authorized its submission. 6 International Journal of Microbiology