Skin Colonizers and Catheter Associated Blood Stream Infections in Incident Indian Dialysis Patients

Protocol for catheter insertion and manipulation

HD catheters were inserted by nephrology residents under ultrasound guidance in a dedicated surgical suite within the dialysis facility, either independently or under consultant supervision, using the modified Seldinger technique, with the right internal jugular vein being the preferred site for insertion. Standard aseptic precautions were followed for catheter insertion using skin disinfection with 2% chlorhexidine gluconate. After catheter insertion, mupirocin ointment was applied at the exit site, which was then covered with sterile gauze and transparent dressing (Tegaderm™, 3M™, St. Paul, MN).

Before, during, and after an HD session, catheter manipulation by dialysis staff was carried out under sterile barrier precautions. The catheter exit site and port caps were cleansed with 2% chlorhexidine gluconate, caps were removed, hubs were cleansed with 2% chlorhexidine gluconate, a syringe was attached to aspirate heparin lock solution, lumens were flushed with saline, following which dialysis tubing was connected. Catheter hubs were kept soaked in chlorhexidine solution during the HD sessions. At the end of HD, fresh sterile gauze and a transparent dressing were applied at the exit site. Mupirocin ointment was applied at the exit site only for the first 1 week after catheter insertion. The catheter lumen was locked with heparin solution (1000 units/ml) as per the internal volume specified by the manufacturer. No antibiotic lock solutions were used.

Patients underwent HD for 4 h, twice or thrice weekly, with a low flux polysulfone dialyzer (FX8, Fresenius Medical Care AG & Co., Bad Homburg, Germany). Dialyzers were not reused. Water purity was monitored once a month to ensure microbial contamination <200 CFU/ml and endotoxin levels <0.2 EU/ml.

Protocol for swab collection

Swabs were obtained by a single investigator (VA) from four body sites (left and right anterior nares – 1 swab, right and left axilla – 1 swab each, proposed catheter exit site in the neck – 1 swab) prior to HD catheter insertion. All swabs were pre-moistened with sterile normal saline. Nasal swabs were obtained using a standard protocol as described previously.[8] In brief, the swab was inserted into the nasal vestibule (approximately 2 cm) and rotated against the mucosa for 5 seconds while applying gentle pressure so as to lightly bend the swab. Using the same swab, the procedure was repeated for the other nostril. HD catheter exit-site swabs and axillary swabs were obtained by rolling a pre-moistened swab once, forwards and backward at the proposed exit site and bilateral axillae. Swabs were obtained from the same sites at 14-21 days (first follow-up swab) and 28-35 days (second follow-up swab) after HD catheter insertion, prior to a HD session. Swabs from the catheter exit site were obtained prior to skin cleansing of the exit site with an antiseptic solution.

The protocols for HD catheter insertion and manipulation as well as the dialysis protocol are given in the Supplementary Material.

Laboratory testing methods

After collection, all swabs were placed in the corresponding transport tube and subjected to microbiological analysis within one hour. Before inoculation onto blood agar plates, swabs were pre-incubated with nutrient broth at 37°C for 2 h. Colonies were isolated by inoculating 100 μL aliquots onto Columbia agar supplemented with 5% sheep blood. Agar plates were subsequently incubated at 37°C under ambient atmosphere for 48 h. After incubation, significant growth was identified as colonies and was characterized by using standard microbiological methods.[9] Antimicrobial susceptibility testing was performed by using the standard Kirby-Bauer disc diffusion method (HiMedia Laboratories Pvt. Ltd., Mumbai, India) as per Clinical and Laboratory Standards Institute (CLSI) recommendations. In patients who developed a CABSI due to an organism that had previously been cultured on a skin swab, or whose exit site swab grew an organism that had previously been cultured at another site, molecular characterization was done using multi-locus sequence typing (MLST) or multiplex PCR, to determine if the two strains were similar.

Protocol for diagnosis of CABSI

In all patients with clinical features suggestive of CABSI (see below), paired blood cultures (10 ml each) were obtained from the peripheral blood and venous catheter hub under sterile precautions and inoculated in culture media (BACT/ALERT^® SA, bioMérieux Inc, Durham, NC). Peripheral blood cultures were obtained from the median cubital vein or the dialysis circuit.

Definitions

Drug resistance patterns were defined as per the International Expert Proposal for Interim Standard Definitions for Acquired Resistance.[10] CABSI was defined as per the Center for Disease Control and Prevention (CDC) guidelines[11] with a diagnosis of coagulase negative staphylococcus (CoNS) CABSI being made only if both catheter and peripheral blood cultures grew the same organism, while one positive blood culture (catheter hub or peripheral blood or both) was sufficient for other organisms. Persistent Colonizers were defined as those who grew the same drug resistant organism on two successive swabs from the same site. Persistent colonizers were further classified as persistent intrinsic colonizers (those in whom the organism was identified on the baseline and consecutive follow-up swab, indicating that persistent colonization existed prior to dialysis initiation), and persistent acquired colonizers (where the organism was not identified on the baseline swab, but grew in both follow-up swabs, suggesting that the organism was acquired after initiation on dialysis). All others were termed transient colonizers.

Statistical analysis

Categorical variables were summarized using counts and percentages. Quantitative variables were summarized using mean and standard deviation or median and IQR. Chi-square test was used to compare the proportions between categorical variables. The Independent t-test was used to compare the means between two groups for normally distributed continuous variables and the Mann–Whitney U test was used for skewed variables. Changes in proportions were compared using the N-1 Chi-square test (available at https://www.medcalc.org/calc/comparison_ of_proportions.php). A 5% level of significance was considered significant. Statistical analysis was carried out using STATA v. 13.1 (StataCorp. College Station, TX)

Proportion of patients colonized with MDR bacteria prior to HD initiation

At least one organism was cultured from 96.5% of the swabs taken at baseline, and 47.4% swabs grew MDR organisms. The majority of colonizing organisms were gram-positive (91.2%), with methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) being the most common (28.3% and 26.7% of all organisms cultured, respectively). MDR bacterial colonization, MRSA colonization, and MDR gram-negative colonization at one or more sites on the baseline set of swabs was seen in 76.7%, 69.7%, and 9.3% patients respectively [Table 2]. In 30 patients with MRSA colonization, MRSA was isolated from both nasal and axillary swabs in 13 (43.3%), axillary alone in 12 (40%), nasal alone in 4 (13.3%), and nasal and proposed exit site in 1 patient (3.3%). MDR gram-negative colonization was seen only in the axilla.

Factors associated with colonization by MDR bacteria prior to HD initiation

Females, diabetics, those with history of broad-spectrum antibiotic use in the last 3 months and those with higher comorbidity index showed a trend towards colonization by MDR organisms prior to HD initiation, but these factors did not reach statistical significance [Table 3].

Change in colonization pattern over time

Supplementary Tables 1 and 2 show the colonization pattern observed on swabs obtained 14-21 days and 28-35 days after the baseline swab. The only significant change seen was a decrease in patients colonized by methicillin-sensitive coagulase-negative staphylococcus (MSCoNS) between baseline and 14-21 days [P = 0.03, Table 4]. In terms of organisms cultured on swabs, methicillin-resistant CoNS (MRCoNS) on swabs increased significantly from baseline to 14-21 days (P = 0.02) and baseline to 28-35 days (P = 0.001), with a consequent fall in colonization by MSCoNS [p < 0.001, Supplementary Table 3]. There was no significant change in patients colonized by, or swabs positive for, MRSA over time.

Persistent vs. transient colonization by MDR bacteria

A total of 29 patients had at least two consecutive sets of swabs obtained. Of these, 23 patients (79.3%) were classified as persistent colonizers. Of the persistent colonizers, 19/23 (82.6%) were persistent intrinsic colonizers. The most common MDR organism associated with persistent colonization was MRSA. Of the 38 sites at which persistent colonization was documented in the 23 persistent colonizers, MRSA was cultured at 25/38 sites (65.7%), while MRCoNS was cultured in the rest. The most common site of persistent colonization was the axilla (n = 19, 50%). In persistent acquired colonizers (n = 4), who formed a subgroup of the persistent colonizers, the most common organism cultured was MRCoNS in 75%, and the most common site was the axilla (100%). There was no persistent colonization by MDR gram-negative bacteria. Four patients acquired MRSA at the exit site, and in all four cases the strain was the same as that which had colonized the axilla previously.

Correlation between colonizing bacteria and bacteria causing CABSI

Six patients developed a CABSI during the follow-up period, accounting for a CABSI incidence rate of 3.7 per 1000 patient days. Of these 6 patients, 3 were persistent colonizers (3/3 with MRSA, 2/3 with MRCoNS); however, neither of these organisms caused the CABSI in each case. Instead, 6 of CABSIs were caused by gram-negative organisms (Klebsiella pneumoniae– 2, Enterobacter – 2, Pseudomonas aeruginosa– 1, MRCoNS – 1). In only 1/6 cases (16.6%) was the strain of bacteria (Klebsiella pneumoniae) causing CABSI identical to that which had transiently colonized the skin on a previous swab. The median time between last swabs taken and diagnosis of CABSI was 13.5 days. Persistent colonization was not associated with CABSI (P = 0.24).

Strengths and limitations

This study provides the first estimate of skin colonization by MDR organisms among incident HD patients in the subcontinent. We adopted rigorous inclusion criteria, our subjects hailed from all parts of India (85% hailed from other states), we obtained swabs from the nares and extra-nasal sites, followed patients prospectively, and adhered to established standards for swab collection, processing and culture. Our study results are therefore robust and broadly reflective of incident HD patients in the subcontinent.

This study has several limitations. The sample size is small because we chose to focus exclusively on incident HD patients who were likely to follow-up at our centre for at least 2 weeks in order to assess the effect of colonization on CABSI incidence. Since ours is a tertiary referral center with a majority hailing from other parts of the country, this resulted in a high attrition rate on follow-up. Although MRSA colonization rates do not differ between studies that performed one versus multiple screenings,[15] we cannot rule out that attrition may have affected the observed rate of colonization by gram-negative organisms. Given that the majority of CABSIs were gram negative, swabs from the hand, groin and/or perineum may have been useful to determine if gram-negative bacteremia resulted from endogenous transfer of gram-negative bacteria from these sites to the exit site due to poor patient hand hygiene practice. We did not obtain swabs from the dialysis environment or dialysis unit health care workers to determine if transfer from these reservoirs contributed to gram-negative CABSIs. Lastly, the results of our study may not be generalizable to other countries where the population microbial resistance dynamics, personal and/or dialysis unit hygiene practices may differ.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form the patients have given their consent for their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published, and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Financial support and sponsorship

This study was funded by an Internal Fluid Research Grant from Christian Medical College, Vellore. [IRB Minute number 10683 (OBSERVE) dated 1^st June 2017, Account number 22Z232].

Conflicts of interest

There are no conflicts of interest.