FEATURES OF THE CANDIDA GENUS COMMUNITY PATTERN IN THE INTESTINAL BIOTOPE OF PATIENTS WITH TUBERCULOSIS

Abstract

Fungi are opportunistic microorganisms that colonize all biotopes of the human body, including intestinal. In case of emerging adverse environmental factors (HIV infection, other immunodeficiencies, antibiotic therapy), these microbial representatives begin active reproduction, which might require prescribing antimycotics. Frequent use of the latter in clinical practice induces the development of drug resistance to antifungal drugs, which may impact on effectiveness of both the treatment of fungal infections and other diseases. The purpose of the study was to assess the pattern and spectrum of drug resistance of Candida genus in the intestinal biotope of patients with respiratory tuberculosis and identify risk factors for developing total fungal drug resistance to antimycotic drugs. Material and methods. There were enrolled 21 patients with respiratory tuberculosis. Pattern of the fungal species diversity for the Candida genus isolated from faeces was evaluated, and the spectrum of relevant drug resistance to antimycotic drugs was determined. Patients (n=21) were divided into 2 groups: with (n=10) and without (n=11) total resistance to antimycotics, after which the main risk factors for its development were identified. Results. Members of the Candida genus were isolated from all patients examined, wherein pure cell cultures were characterized by high level of antimycotics resistance. Resistance to three drugs was noted in 1 culture (4.8%), to four – in 10 cultures (47.6%), also found in 10 cultures to the entire drug panel (47.6%). During statistical processing, the data were obtained on affecting formation of total resistance to antimycotics of concomitant pathology of the gastrointestinal tract, the presence of a clinically significant dyspeptic syndrome, a history of antimycotic therapy, HIV infection with severe immunodeficiency, and some decrease in the peripheral blood CD4+ lymphocyte count. Conclusions. Fungi of the Candida genus isolated from tuberculosis patients were characterized by high level of resistance to antimycotics. Total resistance was observed in 47.6% of patients. In addition, the major fungi colonizing the intestines of tuberculosis patients were found to be Candida albicans species. The risk factors for the development of total antifungal resistance included: chronic enterocolitis, dyspeptic syndrome, peripheral blood CD4+ lymphocyte count lower than 350 cells/µl, and history of antimycotic therapy.

Full Text

Fungi are present in various biotopes of the human body; their localization in the lumen of the gastrointestinal tract (GIT) causes clinical manifestations [11]. Occupying an opportunistic niche, including in the intestinal biotope, their number should not exceed diagnostic titers [10]. However, the possibility of their active reproduction is determined by a number of factors. First, the development of mycoses accompanies diseases leading to immunodeficiency, which is most important for HIV-infected patients [1, 8, 9, 15]. One of the indicators of fungal reproduction is the development of mycosis of the oral cavity, in which local antiseptics are actively used against the background of antimycotic therapy [3]. Secondly, due to the use of a large number of anti-tuberculosis drugs (ATPs) in phthisiatric practice, the balance between the obligate and conditionally pathogenic microflora of the gastrointestinal tract is disturbed, which leads to an increase in the number of its optional representatives, incl. fungi [5, 13]. Antimycotic drugs are actively used in clinical practice as the basis for effective treatment of candidiasis [4]. With each prescription, the risk of developing drug resistance (DR) of fungi to them also increases [7, 14, 12]. There is not enough data in the literature on the peculiarities of the DR of fungi to antimycotics in phthisiatric practice. This issue is especially relevant in the context of an increase in the prevalence of TB/HIV co-infection and multidrug resistance (MDR) of the causative agent of tuberculosis [2, 6], which creates the need for research in this area.
The aim of the study was to assess the structure and spectrum of drug resistance of fungi of the genus Candida in the intestinal biotope of patients with tuberculosis and to establish risk factors for the development of total DR of fungi to antimycotics.
Material and research methods. The study was carried out on the basis of the Kuzbass Clinical Phthisiopulmonological Medical Center named after I.F. Kopylova in 2021 The study included 21 patients with tuberculosis of the respiratory organs and MDR of the pathogen receiving anti-tuberculosis therapy in a round-the-clock hospital. The inclusion criteria were: the presence of respiratory tuberculosis with MDR pathogen, treatment according to the IV chemotherapy regimen with at least 30 doses of anti-TB drugs at the time of the study. The exclusion criteria were: severe immunodeficiency in HIV-infected people (the number of CD4+ lymphocytes is less than 200 cells per 1 µl). As sources of information, we used medical records of an inpatient (form 003/y), data from a microbiological study of patients' feces. In the course of the study, the structure of mycoses in the intestinal biotope of patients and the spectrum of DR of fungi of the genus Candida were evaluated. Subsequently, the patients were divided into 2 groups: the 1st group (n=10) included patients with total DR of fungi to antimycotics, the 2nd group (n=11) included patients without it.
The study used a quantitative bacteriological method. The material was taken into a sterile container and transported to the laboratory within 2 hours after collection. Then dilutions of the material were prepared from 10 -1 to 10 -9 and the pH of the material was determined, after which they were sown on selective nutrient media. Sabouraud's medium was used for fungal isolation. The selected cultures were identified according to the features of biochemical, as well as a complex of morphological, cultural and tinctorial properties. The results were expressed in lg CFU/g per gram of material. The LU spectrum was assessed using a standard disk diffusion method.
Statistical data processing was performed using the IBM SPSS program. Qualitative signs are represented by absolute and relative frequencies, expressed as a percentage, with 95% confidence intervals calculated for them using the Wilson method (rel. % [95% CI]). Quantitative data are presented in the format of median and interquartile interval (Me [25th; 75th). For ordinal variables and non-parametric scale variables, the Mann-Whitney U-test was used to compare two groups of observations. Differences in the compared groups were considered statistically significant at a p value of less than 0.05. Given the size of the sample presented, the Yates correction was used.
Research results. During the microbiological study, fungi of the genus Candida were detected in all (100%) patients included in the study. In the structure of isolated representatives, C. albicans prevailed; C. neoformans, C. kefyr, and C. glabrata were observed less frequently; C. parapsilosis and C. famata were single cases. It should be noted that in 9 cases (42.9%), 2 or more species of fungi were obtained from one patient, and the average number of representatives of Candida spp. was within 4 [3; 4.75] IgCFU/g. The structure of mycoses is shown in Figure 1.

Figure 1 Characteristics of the structure of isolated fungi of the genus Candida in the intestinal biotope of the studied patients
The isolated fungi were characterized by high resistance to antimycotic drugs in vitro. Resistance to three antimycotics was observed in 1 culture (4.8%), to four in 10 cultures

The isolated fungi were characterized by high resistance to antimycotic drugs in vitro. Resistance to three antimycotics was observed in 1 culture (4.8%), to four in 10 cultures (47.6%), to all determined also in 10 cultures (47.6%). Most often, cultures were found to be resistant to ketoconazole and itraconazole, less often to cotrimoxazole, nystatin, fluconazole, and amphotericin. Data on the frequency of detection of LU are presented in Figure 2.

Figure 2 The frequency of detection of DR of fungi of the genus Candida to various antifungal drugs

It is important to note that with this picture of drug resistance of fungi of the genus Candida in 8 patients (38.1%), a clinical effect was noticeable in the treatment of oral mycosis.
The greatest practical significance is the total resistance of fungi to antimycotics in patients, the frequency of which in this study reached 47.6%. In this regard, it was necessary to identify risk factors for its development in patients with tuberculosis in conditions of high prevalence of HIV infection.
In the 1st group, 7 men (70%) and 3 women (30%) were observed, in the 2nd group - 8 (72.7%) and 3 (27.3%) people. respectively (p=0.81). The average age of patients in group 1 was 45 [34; 50] years, in the 2nd group - 47 [40; 54] years (p=0.56). According to the structure of clinical forms of tuberculosis, no statistically significant differences were obtained; in the course of their study, the prevalence of disseminated (in the 1st group 40%, in the 2nd - 36.4%) and infiltrative (in the 1st group 50%, in the 2nd th - 45.5%) of the clinical form. Also, no statistically significant differences were found when assessing the intake of anti-TB drugs included in the treatment regimen. On average, patients of the 1st group at the time of the study took 34 [25; 48] doses of anti-TB drugs, patients of the 2nd group - 40 [30; 61.25] doses (p=0.25). With a detailed description of comorbidities associated with the gastrointestinal tract, statistically significant differences were found in relation to chronic enterocolitis, which are present in 20% of patients of the 1st and none of the patients of the 2nd group (2=2.1; p=0.01) , however, the influence of other nosologies on the formation of LUs to antimycotics was not confirmed (see table 1).
In the 1st group, 6 patients (60%) with psychoactive substance dependence syndrome were observed, in the 2nd group there were 4 (36.4%) (p=0.27). 4 people suffered from alcohol dependence syndrome. (40%) 1st and 4 pers. (36.4%) of the 2nd group (p=0.90). Low social status was noted in 8 people. (80%) 1st and 3 people. (27.3%) of the 2nd group (2=5.8; p=0.01; OR=10; 95% CI [1.4-82.0]).
Dyspeptic syndrome developed in 7 people. (70%) of the 1st group and in 2 people. (18.2%) 2nd (2=5.7; p=0.01; OR=10; 95% CI [1.3-81.1]). Antimycotic therapy was previously taken by 5 people. (50%) 1st and 1 pers. (9.1%) of the 2nd group (2=7.2; p=0.008; OR=10; 95% CI [0.9-110.3]). More than one Candida spp. allocated from 5 people. (50%) 1st and 4 pers. (36.7) of the 2nd group (p=0.28).
Evaluation of the possible impact of the presence of HIV infection on the formation of total resistance of fungi to antimycotics did not lead to statistically significant results (p=0.75). However, a detailed study of the clinical and anamnestic data of patients with TB/HIV co-infection revealed a high incidence of total resistance to Candida spp. to antimycotics in persons with severe immunodeficiency - 40% versus 18.2% of patients (2=1.54; p=0.02; OR=2.5; 95% CI [1.1-9.4]). At the same time, the influence of other factors on it (adherence to ART, duration of anamnesis for HIV infection) has not been proven (p=0.88; p=0.90, respectively). The data are presented in table 2.

The discussion of the results. Thus, fungi of the genus Candida in the intestinal biotope were isolated in all examined patients with respiratory tuberculosis, while in 42.9% of patients more than two of their cultures were identified, which is explained by the high proportion of patients with TB/HIV co-infection among them [3] . It is important to note that the isolated cultures were characterized by high resistance to antimycotic drugs, incl. and total in 47.6% of patients. Despite the presented frequency of resistance, 38.1% of patients showed clinical efficacy of antifungal therapy based on an examination of the oral cavity. This phenomenon can be explained by the use of local antiseptic therapy with chlorhexidine solutions in patients [6]. Previously, the scientific community has not evaluated risk factors for the development of total drug resistance of Candida fungi in patients with tuberculosis. In the course of this study, the influence on its formation of such factors as the presence of chronic enterocolitis, clinically significant dyspeptic syndrome, severe immunodeficiency in patients with HIV infection, and antimycotic therapy in history was shown.
conclusions
1. In the structure of isolated representatives of the fungal microflora, C. albicans prevailed in 85.7%.
2. Fungi of the genus Candida were isolated in the intestinal biotope of tuberculosis patients receiving anti-tuberculosis therapy in 100% of cases, and were characterized by a high level of resistance to antimycotic drugs, incl. total in 47.6% of cases.
3. Risk factors for the development of total resistance of fungi to antimycotics were: clinically significant dyspeptic syndrome (OR=10), the number of CD4+ lymphocytes is less than 350 cells. in 1 µl. (OR=2.5), presence of comorbid pathology of the gastrointestinal tract in the form of chronic enterocolitis (p=0.01), history of antimycotic therapy (OR=10).

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About the authors

Artyom A. Kholodov

Kemerovo State Medical University

Email: artyomkass96@gmail.com
ORCID iD: 0000-0001-5249-8822

Clinical Resident, Department of Phthisiology

Russian Federation, 650056, Kemerovo region - Kuzbass region, Kemerovo, st.

Julia V. Zakharova

Kemerovo State Medical University

Author for correspondence.
Email: artyomkass96@gmail.com
ORCID iD: 0000-0002-3475-9125

Doctor of Medical Sciences, Associate Professor, Associate Professor of the Department of Microbiology, Immunology and Virology

Russian Federation, Russia, 650029, Kemerovo, Voroshilov street 22 A, tel.: 8-384-2-54-56-51

Larisa Yu. Otdushkina

Kemerovo State Medical University

Email: artyomkass96@gmail.com
ORCID iD: 0000-0003-4126-4312

Assistant of the Department of Microbiology, Immunology and Virology

Russian Federation, 650056, Kemerovo region - Kuzbass region, Kemerovo, st. Voroshilova, 22 a

Catherine O. Bryukhacheva

Kemerovo State Medical University

Email: artyomkass96@gmail.com
ORCID iD: 0000-0001-5212-9234

assistant of the department of phthisiology

Russian Federation, 650056, Kemerovo region - Kuzbass region, Kemerovo, st. Voroshilova, 22 a

Tatiana V. Pyanzova

Kemerovo State Medical University

Email: artyomkass96@gmail.com
ORCID iD: 0000-0002-4854-5734

Doctor of Medical Sciences, Associate Professor, Head of the Department of Phthisiology

Russian Federation, 650056, Kemerovo region - Kuzbass region, Kemerovo, st. Voroshilova, 22 a

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