Study on patients with Clostridioides difficile infection during the COVID-19 pandemic in Bulgaria

Abstract One of the most pressing public health problems is the misuse of antibiotics, especially in industrialized countries. In addition to antibiotic resistance, a disease associated with this abuse is Clostridioides difficile infection (CDI). It is also a serious problem in long-term hospital treatment, as it often causes intra-hospital infections. The coronavirus disease 2019 (COVID-19) pandemic has also led to widespread misuse of antibiotics and prolonged hospitalizations. The aim of our study was to compare two groups of patients who developed CDI before and during the COVID-19 pandemic, respectively. A total of 246 CDI patients with an average age of 58 years were studied for the period of March-August 2021, who also recovered from COVID-19. They were compared with another group of CDI patients who had become ill in the period before COVID-19. We found an increase in CDI patients for the period of the COVID-19 pandemic by 21.95%. Patients with CDI associated with COVID-19 were older, with higher rates of neurologic, tumor and cardiovascular disease, and with metabolic syndrome. In the group with COVID-19 CDI, there was higher use of proton pump inhibitors, use of antibiotics and significantly higher use of corticosteroids. All these factors lead to the conclusion that increased caution is needed in patients with COVID-19 and that mandatory testing should be done for C. difficile infection at the onset of the diarrheal syndrome.


Introduction
The coronavirus disease 2019 (COVID- 19), caused by SARS-CoV-2, was first detected in China in December 2019 and was declared a pandemic by the World Health Organization (WHO) on March 11, 2020.In the first months since the start of the pandemic, key questions about the pathogenesis of COVID-19 were still unspecified.The catarrhal nature of the infection primarily affecting the upper and subsequently the lower respiratory tract with the development of pneumonia and respiratory failure of varying severity led to the wide empiric use of antibiotics.In addition to primary lung damage, not infrequently, COVID-19 also occurs with intestinal inflammation with diarrhea of varying intensity.Currently, these phenomena are mainly explained by the presence of receptors for angiotensin-converting enzyme 2, which allows the virus to enter the cell [1][2][3].A number of authors believe that the intestinal inflammation caused by SARS-CoV-2 may favor co-infection with a number of enteric pathogens, mainly C. difficile [2][3][4].
C. difficile is gram-positive, anaerobic and spore-forming.It is the most significant pathogen that causes hospital-acquired diarrhea.Clostridioides difficile infection (CDI) is the cause for nearly one third of antibiotic-associated diarrhea and almost nine out of ten cases of pseudomembranous enteritis [5,6].CDI affects over 400,000 cases annually, about 29,000 with a fatal outcome, according to the Centers for Diseases Control and Prevention (CDC) in the United States.As highlighted by Kola et al. [5], epidemiological surveillance data have shown a concerning increase in the CDI cases in North America and Europe since 2000, with considerable morbidity, mortality and attributable costs [5][6][7].Many recent outbreaks of CDI in these two regions have been associated mainly with a new hypervirulent PCR ribotype 027, leading to an increase in the incidence and the number of cases per 10,000 patient-days per hospital [4][5][6][7].
CDI is one of the widespread and serious problems directly related to the long-term misuse of antibiotics.It is also a serious problem with prolonged hospital treatment, i.e. C. difficile is a common cause of nosocomial infections.The rapid development of the COVID-19 pandemic and the lack of clarity about the pathogenesis of the disease led to heterogeneous empirical approaches to treatment; hence, the multiple hospitalizations, the lack of beds and health personnel led to two significant problems: the inappropriate use of multiple antibiotics in a disease of viral origin and non-compliance with some of the recommendations for limiting nosocomial infections.Currently, there is insufficient clarity about the severity and incidence of CDI caused by the chaotic management of the COVID-19 pandemic in different regions of the world [1,4,6].Perhaps due to the strict anti-epidemic measures taken in place, some authors report a slight decrease or a relatively slight increase in the incidence of СDI during the COVID-19 pandemic.According to the thesis of Bentivegna et al. [7], an increase in the incidence of HA-CDI is observed only in 2020.
The objectives of our study were to assess the incidence, severity and predisposing factors of healthcare-associated CDI (HA-CDI) and CDI associated with long-term antibiotic use in the context of hospitalization with COVID-19 infection in Bulgaria.We also aimed to compare some characteristics of patients with CDI before the onset of the COVID-19 pandemic and during the pandemic.

Study design and patients
We conducted a retrospective cohort study of patients from two tertiary infectious disease hospitals in Bulgaria.Each of the hospitals had dedicated frontline COVID-19 areas.These areas were in both cases in separate buildings, treated only patients with COVID-19 and were served by separate medical staff.Each patient was diagnosed by real-time polymerase chain reaction (RT-PCR) from a nasopharyngeal swab made in a reference laboratory.The study included patients with healthcare-associated CDI (HA-CDI), i.e.CDI acquired in these healthcare facilities after treatment for COVID-19.We defined HA-CDI as a case of CDI that started at least 48 h after admission and up to 10 days after discharge from the COVID-19 unit.We did not include patients with onset of diarrheal symptoms in the community within 4 weeks of discharge, as accepted [6,8].The diagnosis of CDI was based on clinical symptoms (presence of at least three loose stools in 24 h according to ESCMID guidelines) [6,8] and culture for C. difficile, detection of the enzyme glutamate dehydrogenase (gDH) and/or rapid immunochromatographic test (EIA) for toxins A/B.Patients in whom stool collection was impossible due to staff failure (overload) or who refused to participate in the study were not included.
Demographic and clinical data of the patients are described.All patients enrolled in the study were classified according to the ATLAS severity score (age, antibiotic treatment, white blood cell count, albumin levels, and body temperature).The severity of CDI was also assessed according to the Society for Health Epidemiology of America (SHEA), as defined by the presence of leukocytosis and serum creatinine above a certain level [3,9].
All stools before and during the COVID-19 period were tested at the patient's bedside with a rapid immunochromatographic test for C. difficile A/B toxins.Each sample was taken in a container by a medical person (from a diaper or when defecating in a medical potty).About 20% of fecal samples from patients in the pre-COVID-19 period were also tested for the enzyme and cultured.Almost all fecal samples from patients with diarrheal syndrome during the COVID-19 period underwent the same treatment.
Feces were treated using the alcohol shock method to inhibit non-sporulating organisms and thereby improve the isolation of C. difficile.A pea-sized portion of the sample was transferred to 300 mL of absolute alcohol, and the suspension was allowed to stand at room temperature for 30 min.Then, 100-150 µL of the suspension was inoculated onto Brain Heart Agar, 10% sheep blood and was incubated at 37 °C for 48 h under anaerobic conditions (gas pack from Tsveti Company, Sofia).Recovered isolates were identified as C. difficile by performing gram stain and enzyme immunoassay (EIA) tests for simultaneous detection of C. difficile glutamate dehydrogenase antigen and toxins A and B (quick-check Complete, Alere North America, LLC) in accordance with the manufacturer's instructions.

Statistical analysis
The collected data were analyzed using the statistical analysis software package SPSS 23.0 (IBM SPSS, Chicago, IL, USA).Differences between groups were analyzed using the Mann-Whitney U-test for continuous variables and the chi-square test or Fisher's exact test.Differences were considered statistically significant at the p < 0.05 level.

Results
This study included a total of 246 patients with CDI from two medical facilities, for the period of March-August 2021, who also suffered from COVID-19.The average age was 58 years (20-92 years).This cohort was compared with another group of CDI patients admitted to these hospitals between March and August 2018, which included 52 patients with a mean age of 67 years (35-89 years).These are all patients with laboratory-proven CDI in both hospitals.We found an increase in CDI patients for the period of the COVID-19 pandemic by 21.95% compared to the period before COVID-19.Thirty-eight cases of CDI (73%) diagnosed in the pre-COVID-19 group were first episodes of CDI, while 221 (89.8%) first episodes of CDI were diagnosed in the COVID-19 group (p = 0.07).It is proper to take into account certain limits in the study indicated at the end of the text.
According to the baseline patient demographic and clinical-laboratory characteristics (Table 1), the age of the patients with CDI associated with COVID-19 was higher.The comorbidity rates were similar in the patients with COVID-19-associated CDI, although there were higher rates of neurological, tumor and cardiovascular disease and metabolic syndrome.Nerological and oncological premorbidities were more prevalent in the group of COVID-19 CDI patients than in the group of CDI patients from the pre-COVID-19 period (p < 0.001).The patients with CDI in the pre-COVID-19 period had more often been hospitalized for another problem within two months before the CDI hospitalization (p = 0.03).In the group with COVID-19 CDI, there was higher use of proton pump inhibitors (p < 0.001), use of antibiotics (41% of patients reported taking two or more antibiotics) and significantly higher use of corticosteroids (p < 0.001).In both groups, the predominant antibiotic was cephalosporin.In the COVID-19 CDI group, macrolides and azithromycin ranked second, followed by quinolones.For CDI in the pre-COVID-19 period, quinolones were the second most frequently used group of antibiotics, followed by glycopeptides.According to the SHEA definition of CDI severity, there was no statistically significant difference in those infected with C. difficile in the pre-COVID-19 and COVID-19 period.The data with Median ATLAS scores were similar.
However, it is worth noting that in CDI in the pre-COVID-19 period we reported one death (1.72%) and in the COVID-19 period three deaths of CDI patients (4.76%), one of whom developed toxic megacolon and was transferred to a specialized surgical clinic.There was also a statistically significant difference in the length of hospitalization of patients with CDI in the COVID-19 period as compared to those before the pandemic.The average stay of the former was almost twice as long (p = 0.03).The patients in the CDI group with COVID-19 had a significantly higher rate of CDI recurrence (p = 0.01).Four patients had to be hospitalized three times, 38 twice, and the majority of relapses were treated on an outpatient basis.
In the COVID-19 CDI group, diarrhea due to CDI coincided with hospital admission (at least 48 h after

Discussion
During the COVID-19 pandemic, healthcare systems around the world faced almost identical problems.
Overcrowding of healthcare facilities needs more serious isolation measures and a lack of medical staff [1,2].In Bulgaria, hospitals relatively quickly introduced individual protective equipment such as latex gloves, masks, helmets and protective clothing, and patients were accommodated in separate departments, wings, preferably buildings, in rooms with their own bathroom, and in most cases up to 3 beds.Relatives were not allowed to visit the patients.Surfaces were regularly cleaned with appropriate anti-viral agents.
Unfortunately, in an effort to protect themselves and patients from the nosocomial spread of airborne infection, healthcare professionals in some locations might have been downplaying the protective measures for other common nosocomial pathogens.
The initial uncertainties about the COVID-19 pathogenesis led to massive empirical use of broad-spectrum antimicrobials for a disease of viral aetiology, in violation of the international guiding principles for the use of antibiotics.In this regard, we observed an increase in CDI during the COVID-19 pandemic, and these observations are shared by a number of other authors from Europe and other parts of the world [3,8,10].
In our study, we observed a significantly increased use of antibiotics in CDI during the COVID-19 pandemic, but there is also evidence of high antibiotic use in CDI patients from the pre-COVID-19 period.
Clearly, antibiotic misuse is a major risk factor for CDI, independent of COVID-19, but during the pandemic period, this dependence increased significantly (p = 0.03).Similar conclusions are drawn by other authors [10][11][12].In our study, only 12 (4.87%)patients with COVID-19 had a superimposed bacterial infection.However, 201 (81.07%) of the COVID-19 CDI group were treated with one or more antibiotics, mostly broad-spectrum agents.Corticosteroid use increased significantly during the COVID-19 pandemic, apparently leading to immunosuppression in patients, which could most likely explain the statistically significant increase in HA-CDI we observed.The use of proton inhibitors, which also increased sharply during the pandemic months with a view to protecting the gastric mucosa from corticosteroids, gave significant rates of increase in CDI and was an important risk factor [8,[11][12][13].
One of the serious challenges during the COVID-19 pandemic was treatment in multimorbid, mostly elderly patients.Bulgaria ranks among the first countries in the European Union in terms of cardiovascular diseases, obesity and cancer [14][15][16].Many reports, as well as our empirical experience, show the aggravated course of the COVID-19 infection in such patients [4,13,14,16].Our study showed that there was a very high correlation between patients with neurological and malignant diseases and the occurrence of HA-CDI (p < 0.001).Albeit to a lesser extent, a similar statistical dependence was also observed in patients with metabolic syndrome or obesity and with cardiovascular disease [2,[17][18][19][20].
Although we did not find a more severe course of CDI associated with COVID-19 compared to data from the pre-epidemic period, we did observe a more prolonged diarrheal syndrome in HA-CDI associated with a longer hospital stay (p = 0.03).We also observed more relapses (p = 0.01).All patients with HA-CDI were discharged with long-term oral treatment at home and/ or compliance with strict diets, including the intake of probiotics containing all Bimuno ingredients (glucose, galactose and lactose) in certain proportions [13,[18][19][20][21][22].
For HA-CDI, we found no statistically significant differences in the onset of diarrhea (Table 2).Those infected with C. difficile manifested the diarrheal  [9,17,[21][22][23][24].Some of the limitations of our work are the fact that we studied patients from two tertiary care hospitals, although the patients were managed similarly.It is possible that some of the COVID-19 patients who stayed at the two hospitals were hospitalized for C. difficile infection at another facility or were only treated as outpatients.Among the CDI patients in the pre-COVID-19 group, some patients were diagnosed only with rapid immunochromatographic tests for C. difficile A/B toxins.It is likely that some patients remained undiagnosed.In the COVID-19 CDI group, most fecal samples were tested by all three methods (immunochromatographic assays, enzyme assays and microbiological cultures).Unfortunately, at the beginning of the COVID-19 pandemic in our country, there was a serious shortage of kits for molecular diagnostics, and mass rapid tests were done at home, which did not show the real picture of the incidence of the new coronavirus.At the same time, the regulations of our health authorities do not require mandatory reporting of CDI patients.These limitations do not allow us to make a global epidemiological review of the problem in Bulgaria.

Conclusions
As this is the first such study in our country, much more research is needed to elucidate the intimate mechanism by which SARS-CoV-2 impairs intestinal pathophysiology, and to establish uniform protocols for the treatment of COVID-19 infection, especially regarding the indiscriminate use of antibiotics and corticosteroids.Extensive work focusing on patients with COVID-19 without CDI, compared with patients similar to ours, is also needed, perhaps revealing some unsuspected predisposing factors for co-infection.

Table 1 .
Demographic and clinical data for patients before and during the coViD-19 pandemic.

Table 2 .
treatment of patients with cDi from the coViD-19 period.