Vancomycin resistant enterococci gut Colonization and its associated factors among HIV infected patients on anti-Retro viral therapy in Ethiopia

Background The emergence of vancomycin resistant Enterococci (VRE) has alarmed the global infectious diseases community due to its tendency for colonization of the gastrointestinal tract. Human Immunodeciency Virus (HIV) patients are colonized by vancomycin resistant Enterococci than other groups. The aim of this study was to determine the prevalence of vancomycin resistant Enterococci gut colonization and its associated factors among HIV infected patients on Anti-Retroviral Therapy (ART). Methods Institution based cross sectional study was conducted among HIV infected patients on ART at from June 1 to August 30, 2020. Socio-demographic and clinical data were collected by pre-tested structured questionnaire. Stool sample was collected and processed by standard microbiological techniques. Kirby Bauer Disc diffusion method was used to perform antimicrobial susceptibility testing. Data were entered by Epi data version 4.6.0.2 and analyzed by SPSS version 25. P-value <0.05 was considered as signicant.


Abstract Background
The emergence of vancomycin resistant Enterococci (VRE) has alarmed the global infectious diseases community due to its tendency for colonization of the gastrointestinal tract. Human Immunode ciency Virus (HIV) patients are colonized by vancomycin resistant Enterococci than other groups. The aim of this study was to determine the prevalence of vancomycin resistant Enterococci gut colonization and its associated factors among HIV infected patients on Anti-Retroviral Therapy (ART).

Methods
Institution based cross sectional study was conducted among HIV infected patients on ART at from June 1 to August 30, 2020. Socio-demographic and clinical data were collected by pre-tested structured questionnaire. Stool sample was collected and processed by standard microbiological techniques. Kirby Bauer Disc diffusion method was used to perform antimicrobial susceptibility testing. Data were entered by Epi data version 4.6.0.2 and analyzed by SPSS version 25. P-value <0.05 was considered as signi cant.

Conclusion
High prevalence of vancomycin resistant Enterococci gut colonization was found. Previous exposure to antibiotics for more than two weeks and previous hospitalization for more than six months were signi cant factors for vancomycin resistant Enterococci colonization. The isolated Enterococci had variable degrees of resistance to commonly prescribed antibiotics. Therefore, periodic surveillance on antimicrobial resistance pattern, adhering to rational use of antibiotics and implementing infection prevention protocols may reduce colonization by VRE.

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A major problem with the Enterococci is that they are very resistant to antibiotics and have ability to survive in harsh environments in community and persist in hospital settings (1). Because of this, they become important in health facility based settings (2). According to World Health Organization (WHO) report in 2017, vancomycin resistant Enterococci (VRE) is one of the most resistant bacteria in their "Global Priority list of antibiotic-resistant bacteria" (3). In the same manner, the Center for Disease Control and Prevention (CDC) has classi ed Enterococci among bacteria with a threat level of serious (4).
Currently, VRE are the cause of one-third and one fth of all health care associated infections in the United States and in some European countries respectively (5). VRE are well known in causing different enterococcal infections such as infective endocarditis, bacteremia, urinary tract infection, intra-abdominal and pelvic infections, surgical wound infections, and very rarely Central nervous system (CNS) infection (6,7). Many of these infections originate from intestinal ora of colonized individuals (8).
Enterococci are enteric bacteria that are commonly recovered in feces collected from humans and from a variety of mammals as well as birds, reptiles and insects. The relative importance of Enterococcus as a pathogen has increased with the occurrence of high-level resistance to multiple antimicrobial drugs, such as ampicillin and vancomycin (9)(10)(11)(12)(13).
Vancomycin is the primary alternative drug to penicillin (plus an aminoglycoside) for treating enterococcal infections (13). It is active against most Gram positive bacteria and is considered as a drug of 'last resort'. However, nowadays Enterococcus spp that are resistant to vancomycin are emerged and spreading all over the world (12).
Vancomycin resistant Enterococci was rst encountered in clinical isolates in England and France in 1986 as E. faecium, followed the next year by isolation of Vancomycin Resistant Enterococcus faecalis in the United States (15). They have become an important cause of serious invasive healthcare-associated infections globally (6,7).
The emergence of VRE has alarmed the global infectious diseases community due to its tendency for colonization of the gastrointestinal (GI) tract and transfer of its resistance gene to Methicillin resistant Staphylococcus aureus (MRSA) to form a vancomycin-resistant Staphylococcus aureus (VRSA) isolate. Besides, VRE have different selection pressures for proliferation and rapid expansion of its resistant populations (16). Furthermore, few options are left for management of diseases caused by VRE, and hence causing increased mortality on infected individuals (12,14). VRE now represent approximately one third of Enterococcus isolates (17).
Asymptomatic VRE gut colonization precedes infection with susceptible hosts. Such susceptible hosts are patients who are exposed to multiple and prolonged courses of antimicrobial agents like Human Immunode ciency virus (HIV) infected individuals, severely ill, hospitalized for long lengths of stay (LOS), living in a long-term care facility, located in close proximity to another colonized or infected patient, or hospitalized in a room previously occupied by a patient colonized with VRE (2,18,19). Colonization is often obtained by vulnerable hosts in an environment with increased rate of patient colonization with VRE (20).
Vancomycin-resistant enterococci can persist in the environment for elongated periods (> 1 week), can contaminate almost any surface, and can be transferred from colonized or infected individual to another by health care workers (9,13). Transmission of VRE can occur through direct contact with colonized or infected patients, indirect contact via the hands of healthcare workers, via contaminated personal protective equipment (PPE), environmental surfaces or from animals to human through food chain (21,22). Usually colonization with VRE precedes infection, and the duration of colonization may be extremely extended ranging between 7 weeks and 3 years (22).
Colonization with VRE increases the risk of developing infection up to 5-10 folds (20). Whether VRE colonization leads to infection depends on the health status of the patient. Whereas immunocompetent patients colonized with VRE are at low risk for infection, weakened hosts such as HIV infected patients, patients with hematologic disorders, transplant recipients, or severely ill patients have an increased likelihood of developing infection following colonization (19,20).
Compromised immune system of HIV infected patients increases the chances of acquiring various opportunistic infections (23). These individuals are at increased risk of developing infections, including infections caused by resistant bacteria pathogens (24). VRE is an important opportunistic bacterial pathogen causing signi cant morbidity and mortality in immunocompromised individuals like HIV patients (8,25).
The prevalence of VRE was reported in Europe, Asia, Australia, South America and some African countries (1,5,24,26). However, there is no su cient data available on the prevalence and risk factors of VRE in developing countries like Ethiopia. Therefore, this study was conducted with the aim of determining the prevalence of vancomycin resistant enterococci gut colonization and its associated factors among HIV infected patients on Anti-Retroviral Therapy (ART).

Study design, period and setting
The study was conducted in Arba Minch General Hospital from June 1 to August 30, 2020. The hospital is located in Arba Minch town Gamo zone, 505 km South of Addis Ababa. The hospital has ART clinic where it provides different services for HIV-infected patients. The total population of Arba Minch town from 2007 central statistical agency (CSA) census report was 74,879, of whom 39,208 were men and 35,671 women (27). The inclusion criteria is all HIV infected patients on ART at Arba Minch General Hospital, ART clinic during the study period. Exclusion criteria are those HIV infected patients who don't have Parent or guardian assent if they are < 18 years old and those HIV infected patients who were critically ill and unable to respond.

Sample size determination and sampling technique
The required sample size was calculated for prevalence and associated factors. For prevalence, the sample size was determined by using single population proportion formula. The following assumption was considered with 7.8% VRE prevalence from Gondar, Ethiopia (18), 95% con dence level and marginal error which is half of the VRE prevalence (7.8%/2 = 3.9%) since VRE prevalence is less than 10% (28).
However, when factors analysis was considered from previous studies such as previous antibiotic treatment for more than two weeks (51.7%) (18) and (6.8%) (2), level of hemoglobin (57.1%) (16) and history of hospitalization (3.18%) (2), we found a sample size of 48, 54, 44 and 48 respectively; all of which are less than the sample size calculated by prevalence of VRE colonization. Therefore, sample size for the prevalence was taken after compensation for 10% non-response rate: n= (sample size determined before addition of non-response rate * 0.1) + sample size determined before addition of non-response rate = (182*0.1) + 182 = 18 + 182 = 200. Hence, the nal sample size of this study was 200.

Sampling technique
Systematic random sampling technique was used to select the study participants. Based on the 2020 three months (June-August, 2020) data obtained during COVID-19 pandemic from Arba Minch General Hospital ART clinic, N = 366 (N = total number of study participants) HIV infected patients has visited the ART clinic. Assuming the same number of HIV-infected patients for the study period (June-August, 2020) and taking sample size of 200, the k th value (k = N/n = 366/2002) is found to be 2. The rst one is selected with lottery method from 1st and 2nd patients and found to be 2nd patient. Then every 2nd patient was included.
Dependent variable was VRE gut colonization and independent variables were: Age, Sex, CD4 count, Level of hemoglobin, Previous antibiotic treatment for > 2 weeks, Current visiting status (Inpatient or Outpatient), History of hospitalization in the last six months, Urinary catheterization, Co-morbid conditions (diabetes and renal failure).

Data collection and laboratory processing
A pretested well designed structured questionnaire was used to collect data from the study participants.
The questionnaire was designed in English and translated in to Amharic language. The tool had two sections and was adapted by reviewing different literatures. The rst section consisted of socio demographic questions and the second section embraced clinical data.
Upon the arrival of each study participant at ART clinic, written assent/consent was obtained. Sociodemographic and clinical data were collected by two nurses using a pretested well designed structured questionnaire through face-to-face interview. Moreover, recent CD4 count and hemoglobin level of respondents have been taken from ART clinic logbook.

Isolation and Identi cation of Enterococci
Each patient was instructed how to collect stool specimens, and informed to collect about 2 gm of the faecal specimen in a sterile wide-mouth screw capped container labelled with the unique sample number, date, and time of collection. The collected stool specimens were transported to Arba Minch University College of Medicine and Health Sciences Microbiology and Parasitology laboratory.
Immediately after delivery, the transported stool specimens was streaked on Bile Esculin Azide agar (BEAA) (Park Scienti c Limited, 24 Low Farm Place, Moulton Park, Northampton, NN3 6HY) and incubated for 24 hours at 37 °C. Plates were observed for appearance of characteristic growth with brown-black colored colonies in the medium and dark halo centers. Typical characteristic colonies were selected randomly for characterization and presumptive identi cation of Enterococci by Gram stains, Catalase test, Salt tolerance test and Heat tolerance test (29) (Fig. 2).

Antimicrobial Susceptibility Testing
The antimicrobial susceptibility testing was performed using Kirby Bauer disc diffusion method according to Clinical Laboratory Standards Institute guidelines (CLSI). After a pure colony was obtained, a loop full of bacteria were taken, transferred to a tube containing 5 ml of sterile normal saline (0.85% NaCl) and mixed gently until it formed a homogenous suspension.
The turbidity of the suspension was determined by comparison with 0.5 McFarland standards. A sterile swab was dipped into the suspension, and excess suspension was removed by pressing the swab against the wall of the tube. The entire surface of Muller Hinton Agar plate was uniformly ooded with suspensions and allowed to dry for about 3-5 minutes.
The antimicrobial impregnated disks were placed by using sterile forceps at least 24 mm away from each other to avoid the overlapping zone of inhibition. The disks were placed on agar plates and allowed to stand for 30 minutes to dissolve the antibiotics in the media (16). The plates were then inverted and incubated at 37 o c for 16-24 hours depending on the type of the antibiotic used and inhibition zone was measured using a ruler. Antimicrobial susceptibility patterns of Enterococci were assessed against the following antibiotic discs: Penicillin (10 IU), Ampicillin (10 µg), Tetracycline (30 µg), Doxycycline (30 µg), Cipro oxacin (5 µg), Vancomycin (30 µg), Erythromycin (15 µg) and Chloramphenicol (30 µg), and were interpreted according to CLSI M100-2018 (30). Antibiotics were selected based on CLSI recommendation, local availability (in health facility) and feasibility (cost and method of antimicrobial susceptibility test).

Data Quality Assurance
The reliability of the study ndings were guaranteed through performing a pretest of 5% (n = 10) on HIV infected individuals at Arba Minch Town Shecha Health Center, and implementing the standard quality measures through the entire process of data collection and laboratory work. Accordingly, one day training was given for data and sample collectors concerning on the research objective, data collection tools, sample collection procedures, and infection prevention protocols to be taken related with COVID-19 Pandemic.
The data were checked for completeness, accuracy, clarity, and consistency by the principal investigator on a daily basis. Standard Operating Procedures (SOP) for each procedure were strictly followed. It was veri ed that the media and reagents met expiration date and quality control parameters per CLSI guideline. All culture media were prepared following the manufacturer's instruction and sterility of the culture media was tested by incubating 5% of the batch at 35-37 °C overnight for evaluation of possible contamination.
Moreover, E. faecalis ATCC 29212 and Staphylococcus aureus ATCC 25923 standard control strains obtained from Ethiopian Public Health Institution (EPHI) were used as a quality control for biochemical tests and agar plates including Mueller-Hinton agar with vancomycin disc to ensure testing performance of the potency of the disc as per CLSI 2018 guidelines (30).

Statistical analysis
Data were checked for its completeness, entered by Epi data version 4.6.0.2 software, and analyzed by SPSS version 25. Descriptive statistics like frequency, mean and percentage were used. The tness of the model was checked by Hosmer-Lemeshow goodness of t test.
Bivariable and multivariable logistic regression model was used to analyze the association between dependent and independent variables. Those variables with P-value < 0.25 in bivariate analysis were considered as candidate for further multivariable analysis. P-values in the multivariable analysis, adjusted odds ratio (AOR) and 95% con dence interval (CI) were used to determine the strength of association. P-value < 0.05 was considered as statistically signi cant. Finally, the study ndings were explained in words, gures and tables.

Socio-Demographic Characteristics of the Study Participants
A total of 200 study participants were enrolled in this study. Among these the ratio of male to female was 1:1. The mean age and standard deviation was 38.68 ± 10.97 ranging from 18-67 years. More than three fourth of the study participants 154 (77.0%) came from urban settings (Table 1).   (Fig. 1).

Antimicrobial Resistance Pro le of Isolated Enterococci
Among 123 Enterococci isolates tested for commonly prescribed antimicrobial agents the highest rate of resistance was observed against ampicillin in which more than two thirds 86 (69.9%) of the isolates were resistant (Fig. 2).

Factors Associated with Vancomycin Resistant Enterococcus Gut Colonization
Factors independently associated with VRE gut colonization on bivariable analysis, and t into a multivariable logistic regression model were sex, visiting status, history of catheterization, previous antibiotics treatment, history of hospitalization in the last six months and presence of Diabetes Mellitus (Tables 4 and 5).

Discussion
The hasty emergence and the increasing incidence of colonization with VRE have become challenging healthcare problems that have caused serious concern to health care providers and health authorities (2).
However, the prevalence of VRE in our study was lower than the report from Ireland ( (17). The lower prevalence in the present study might be due to the variation in study settings in which most of the previous countries have been using oral and intravenous vancomycin massively for human diseases (34,35), and variation in study participants in which in the previous studies most of the study participants were hospitalized patients and critically ill patients in ICU who were frequently exposed to different antibiotics and experienced insertion of external devices like catheter (7).
On the other hand, this prevalence is higher than the report from USA (4.7%) (36), Nigeria (4.07%) (37) and Ethiopia (5.9%) (16). The gradual increase in the prevalence of VRE might have contributed to this higher prevalence as supported by other studies (32,38). Moreover, the present study also showed that 49.6% of Enterococci isolates were multidrug resistant. This result is lower than the ndings in Iraq (85.7%) (17) and Ethiopia 80.8% (41). In contrary, the result is higher than the nding in Ethiopia 29.5% (16). The discrepancy of the result might be due to variation in geographical distribution of strain, trend and frequency of antibiotic prescription, community drug usage practice.
Regarding associated factors, the current study revealed that HIV patients who were exposed to antibiotics for more than two weeks previously were seven times more likely to be colonized with VRE as compared with HIV patients who never exposed to antibiotics previously [AOR = 7.35; 95% CI: (1.21-44.64); P-value = 0.030]. This result is in agreement with other studies conducted in Brazil (1), South Korea (43), Egypt (44), and Gondar and Ethiopia (2). The reason might be a prior exposure to antibiotics for a prolonged duration can cause VRE colonization due to the fact that the antibiotics exert selective pressure to Enterococci and alter the competing microbial ora in the GI tract allowing VRE to predominate as evidenced by other studies (7,34).
Moreover, the present study also showed that HIV patients who had history of hospitalization in the last six months were six times more likely to be colonized with VRE as compared with HIV patients who didn't have history of hospitalization in the last six months [AOR = 5.68; 95% CI: (1.09-29.74); P-value = 0.040].
The nding is consistent with previous studies done in USA (36), South Korea (43) and Ethiopia (2). The reason might be VRE have been isolated from virtually every object within patient rooms since they are intrinsically resistant to several commonly used antibiotics in hospital and have ability to acquire resistance genes. Besides, they are ubiquitous in their presence and have high survivability on dry surfaces, thereby causing high VRE transmission rates within healthcare facilities (7,9).

Limitations Of The Study
The isolated Enterococci were not identi ed to species level and no molecular characterization done due to resource limitation.

Conclusions
The prevalence of VRE gut colonization among HIV patients on ART was relatively high. Previous exposure to antibiotics for more than two weeks and previous hospitalization for more than six months were signi cant factors for vancomycin resistant enterococci gut colonization. The study also showed that the isolated Enterococci had variable degrees of resistance to commonly prescribed antibiotics.
Therefore periodic surveillance on antimicrobial resistance pattern of Enterococcus species is important for proper treatment, health professionals should strictly follow infection prevention protocols and further studies should be conducted on species identi cation and molecular characterization of Enterococci. Zones of Inoculation for Stool Sample from HIV Infected Patients on ART at Arba Minch General Hospital, Southern Ethiopia, 2020