An Observational Study of Candida auris Infections with Special Reference to the Clinicomicrobiological Problems Faced in Day-to-Day Practice at a Tertiary Care Hospital in Eastern India

1.Identification, management and prevention of Candida auris infections continue to pose significant problems in global healthcare settings.

2.Awareness amongst healthcare workers and patients is the only way out of this  problem.

3.A high index of suspicion of the clinical microbiologist and treating physician are essential ;  treatment options continue to be limited. 

PATIENTS AND METHODS

Study design: This was a hospital based observational study with cross-sectional design.

Study settings: The study was carried out from January, 2020 to June, 2021at the department of Microbiology at an apex hospital, at a time which paralleled the COVID-19 pandemic.

Study patients: We actively searched for critically ill patients in intensive care units of our hospital and found patients with worsening symptoms, not responding to broad spectrum antibacterial antibiotics. The febrile patients attending outdoor facility or patients with blood stream infection with confirmed bacterial aetiology and those responding to antibacterial antibiotics were excluded from the study population.

Sample size: Taking the prevalence of Candida auris candidemia in intensive care setting as 5.3% [7], our target sample was calculated as sixty patients.

Study procedure: Serial blood culture of these patients was carried out both by conventional and automated procedures. Two sets of aerobic and anaerobic blood culture (with collection of 10-20ml of blood from each patient per 50 ml bottle of Brain Heart infusion broth, prepared inhouse) at a gap of one week were taken, incubated at 35-37°C. We waited for no less than 48 hours for growth signal or appearance of growth followed by identification as per standard guidelines [7,10]. The positive blood culture bottles were sub cultured on 5% Sheep Blood agar plate (SBA), MacConkey Agar (MAC), Sabouraud Dextrose Agar with and without Chloramphenicol (SDA/SDCA) and a selective CHROMagar Candida medium (Difco, Becton Dickinson, Baltimore, MD, USA), as depicted in Figures 1 to 4 [9]. We observed for growth on all the inoculated media after incubation at 35°-37°C maintaining aerobic conditions for a minimum of 48 hours. Gram stain from the growth on culture plates and SDA slope were carried out with the common finding of a Gram positive yeast-like fungus. Germ tube test was done using Candida albicans ATCC 90028 as control, however no germ tube formation was noted in the test isolates. On CHROMagar Candida medium (Difco, Becton Dickinson, Baltimore, MD, USA) we recovered moist, convex, colourless colonies in the initial 24 hours which gradually turned pink purplish on further incubation [7,9,10]. We tested the ability of the yeast of producing pseudo hyphae or other structures by streaking the isolates on cornmeal agar (CMA) plates and incubating the inoculated streak under a sterile coverslip at 30°C for 48 h (Dalmau culture) [9-12]. The capacity to form aggregates was evaluated microscopically by dissolving one C.auris colony in 20mcL of normal saline. The findings from Dalmau culture were non-contributory for all six isolates [7-10]. Following the conventional techniques, we ran the same isolates on automated identification system (VITEK2, bioMérieux, Marcy I’Etoile, France) which corroborated with our findings on conventional system. Antifungal susceptibility test (AFST) was done by disc diffusion method per CLSI guidelines [10,12] using Itraconazole, Voriconazole, Anidulafungin, Caspofungin discs etc. and E-test as per standard guidelines. The disc diffusion results of AFST were confirmed by performing broth microdilution (BMD), which continues to remain the gold standard in carrying out antifungal susceptibility (Figures 5 and 6). We collected the data, enlisted on Microsoft Excel, and analyzed the same as our study continued.

VITEK2 (bioMérieux, Marcy I’Etoile, France) system identified each of the six isolates as Candida auris with 99% probability. As VITEK-2 database was not supportive of Candida auris antifungal sensitivity, further testing was done by disc diffusion method and E-test, in accordance with international guideline and checked by broth microdilution (BMD) [10,11]. The CLSI recommends Mueller-Hinton agar with 2% glucose and 0.5 μg/ml methylene blue dye (GMB) for disc diffusion technique as it enhances reproducibility. The addition of glucose supports fungal growth whereas, methylene blue accentuates the zone edge definition. CLSI dictates similar procedure for inoculum preparation in both disc diffusion and broth dilution based methods with a final stock suspension of 1 × 10⁶ to 5 × 10⁶ cells per ml. A sterile cotton swab was dipped into the isolate suspension, rinsed firmly against the inside wall of the tube to remove excess fluid, and then used to streak the entire agar surface a total of 3 times, with rotation of the plate approximately 60° each time, ensuring even distribution of the inoculum over the agar surface. The agar plates were dried for a minimum of 3 minutes and a maximum of 15 minutes, the antifungal discs were applied maintaining aseptic techniques, maintaining at least 24mm distance between two disk centres. The plates were incubated at 35°C±2°C for no less than 24 hours, the zone diameter for each disk is measured to the nearest millimetre at the point in which there is a prominent reduction of growth. CLSI document M60 provides zone diameter interpretive criteria for Caspofungin, Fluconazole, and Voriconazole. At 100% growth inhibition, the zone diameter endpoints were measured with measuring scales and compared with standard zone diameters, as per CLSI M44, 3^rd Edition [10-11].

Statistical analysis

The collected data were collected and analyzed using Microsoft Excel as the study continued.

RESULTS

We reached at a diagnosis of Candida auris as the etiological agent of febrile illness in six out of sixty cases under investigation. The yeast was isolated repeatedly and conclusively by both automated and conventional blood culture systems which correlated with poor and deteriorating clinical condition of the patients. The mean hospital stay of the patients in our study was 14 days. All the patients received initial treatment outside our institution and were admitted to our intensive care units. We identified sixty suspected cases of C. auris candidemia, out of which we carried out thorough investigations on six patients with successful isolation, identification, and susceptibility pattern of the fungus (study work flow demonstrated in Figure 7)

Fever was the common symptom. Two of our patients had history of road traffic accident. All of them were on mechanical ventilator support and received initial care outside our institution. 1 of the 6 patient was on Total parenteral nutrition (TPN) and 1 required renal replacement therapy (RRT). Laboratory parameters of the patients showed a normal leucocyte count, with mild neutrophilia, raised CRP and Procalcitonin, normal serum electrolytes, mild transaminitis, raised serum globulin, which remained unchanged during the whole course of their stay. We could not reveal any bacteriological, parasitological or viral etiology of fever, even after intensive work up (Table 3.1)

The antifungal susceptibility testing (AFST) of the isolates revealed; all resistant to Nystatin, Fluconazole, Voriconazole; 4 out of 6 isolates were found to be sensitive to Anidulafungin (Table 3.2).All six patients were on intravenous broad spectrum antibacterial antibiotics before we could reach at a diagnosis of Candida auris candidemia following which they were started on intravenous echinocandin monotherapy like Anidulafungin 200 mg loading dose as slow infusion with normal saline and then 100 mg once per day for 14 days or  in combination with ketoconazole [12-14]. After initial two days of antifungal administration the patients showed marked clinical improvement. Fever started subsiding and subsequent blood cultures taken after 7 days and 14 days, [15] were sterile and patient was clinically stable. There was no marked change in their laboratory parameters during the whole course of stay. Environmental and samples from other patients in near vicinity were requested from the treating units, as detection of this fungus as a coloniser has great significance in assessment of clinical burden of infection [16-20].

Through this study of ours we tried to conduct a microbiological review of suspected Candida auris infections and actively searched for confirmed cases at our hospital over a considerable amount of time and hammer on the difficulties faced in day to day practice. We progressed with the premeditated knowledge of this yeast being a nosocomial colonizer often isolated from surfaces, most often multi-drug resistant, difficult to treat, patients required strict isolation facilities and a dedicated team-based treatment often involving multiple disciplines and merits for tracing the contacts and environmental sampling [21-24]. While selecting our patients we followed studies conducted by Rudramurthy et al and Chowdhary et al. which revealed that C. auris accounted for >5% Candidemia in Indian intensive care units and almost 30% of infections in individual hospitals [25,28] C. auris Candidemia cases have been reported across 5 continents. The risk factors for developing C. auris infections were almost similar in most centers, which included; abdominal surgeries, prolonged hospital stay, ICU admission, diabetic state, presence of indwelling central venous catheter etc. [24-26]. Significance of C. auris isolation depended only on clinical correlation. Our study progressed in parallels with the COVID-19 pandemic. All sixty suspected cases were from various intensive care units and were having risk factors for developing C. auris infections, however we could thoroughly investigate six confirmed cases as we faced logistical and practical difficulties in recruiting and investigating all suspected cases. The kin or next of kin of most suspected patients were unwilling to undergo further investigations.

This fungus is known to pose difficulty in identification as a ‘pathogen’ with automated systems and require clinical correlation and close surveillance of the patients and environment before reaching at a diagnosis. During our study we carried out serial blood culture of patients with the targets of ruling out bacterial etiology and isolation of yeast, either by conventional and/or automated blood culture system and followed by identification by conventional methods including CHROMagar Candida medium and VITEK-2 followed by antifungal susceptibility tests (AFST) by disc diffusion method and E-test, using Itraconazole, Voriconazole, Anidulafungin, Caspofungin discs etc followed by confirmation with broth microdilution (BMD). Misidentification of Candida auris has been a known hindrance in initiation of prompt and specific therapy, since discovery. In an exhaustive Indian study by Kathuria et al [4], the investigators used Internal transcriber spacer(ITS)sequencing for confirmation of  88.2% of 102 yeast isolates as Candida auris which were previously misidentified as C. haemulonii or C. famata with the VITEK system. Several other studies impress upon the fact that C. auris continues to remain elusive in most laboratories as most commercially available identification systems do not have an updated yeast database. The high-end laboratories can utilise Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF-MS) which is considered a rapid and a dedicated system for diagnosis of Candida auris, however availability is still restricted in centres like ours. We found instances when researchers have tried to find the reason behind this tendency of Candida auris to be misidentified as something else.  Chatterjee et al. and Sharma et al [17,18] in their studies involving Whole Genome Sequencing (WGS) have showed close phylogenetic resemblance of Candida auris and Candida lusitaniae, both being intrinsically resistant to most antifungals. C. auris has been demonstrated to have pathogenicity resembling C.albicans by Borman et al. In other studies, both C.auris and C.albicans demonstrated biofilm forming ability, adherence to polymers, making the pathogen more persistent, more drug resistant in a healthcare setting [23,24,26].

Lockhart et al in their collaborative study under the aegis of Centers for Disease Control and Prevention (CDC), Atlanta, Georgia, with isolates from 54 patients with C. auris infection from Pakistan, India, South Africa, and Venezuela which showed that 93% resistance to Fluconazole, 35% to Amphotericin B, 7% to Echinocandins; near about 41% were resistant to 2 antifungal classes, and 4% were resistant to 3 classes [27,28]. Rudramurthy et al and some other investigators have shown Posaconazole and Isavuconazole of having promising in-vitro activity against C.auris. However, over the years Echinocandins became the first-line therapy, with limited activity against C.auris biofilm [24,25]. Pioneering studies by Kim et al. and Lee et al on multi-drug resistance of C.auris on 15 and 3 isolates, respectively from South Korea, were instrumental in making the scientific community aware of an emerging drug resistant fungus [2,3]. Both studies revealed resistance of the agent to conventionally used antifungals like Amphotericin B (AMB) and all azoles. In path breaking research conducted by Chowdhary et al in 2013 and 2014, 3 hospitals in New Delhi were identified to be housing C.auris infection demonstrating significant resistance to Fluconazole (FLU) and 5-flucytosine (FC) and showed elevated minimum inhibitory concentrations (MICs) of voriconazole (VRC) and Caspofungin (CFG)[19-20].

Our understanding of Candida auris is evolving. As we continue to learn about the fungus, few possibilities become more probable: high likelihood of the MDR pathogen spreading to unaffected parts of our planet which would eventually tax our laboratories, pharmaceuticals and healthcare delivery systems with greater economic burden, as we would require multifaceted diagnostic tools and more expensive antifungal treatment. Global preparedness to face a future fungal pandemic is a telling requirement.Management, control and prevention of Candida auris leaves ample space for improvement in strategies for containment of outbreaks, multidisciplinary approach and early initiation of treatment, improvement in hospital infection control, including disinfection, hand washing, strict adherence to bio-medical waste disposal guidelines, patient isolation, barrier nursing, restriction of footfalls in hospital environment. High index of suspicion is the key to a fruitful diagnosis of Candida auris. Prevention can be done by contact investigation to identify and detect transmission by collecting swab sample from body sites mainly the skin folds like axilla, groin as well as from patient’s environment. All Candida auris patient should be managed by placing in a single room with contact precaution. When more than one patient with C. auris are identified, infection control measures should be performed. Centers for Disease Control and Prevention (CDC) has laid down guidelines which prescribe daily disinfection of room surfaces with high level disinfectant (HLD), medical equipments by autoclaving or ethylene oxide or gas plasma sterilization. Regular dedicated oral decolonization with 0.2% chlorhexidine mouthwash, 1% chlorhexidine dental gel in ventilated patient have shown to reduce colonization.  Chlorhexidine impregnated protective disk for central vascular catheter exit sites may be used to reduce central line associated blood stream infection. For terminal cleaning, fogging by hydrogen peroxide vapors been used to disinfect patients’ room. Several newer antifungals are in various stages of development against this fungus and are showing promising results e.g. Ibrexafungerp (IBX; SCY-078, SCY078 and MK-3118), Rezafungin (RZF; CD101 and SP3025), Manogepix (MGX; APX001A and E1210), Fosmanogepix (FGX; APX001 and E1211), Olorofim (F901318), Opelconazole (OPC; PC945), Quilseconazole (VT-1129), Oteseconazole (VT-1161), VT-1598, ATI-2307, MGCD290 and VL-2397 [29-31]. For all practical purposes, we can carry out a multicentric study analysing past microbiological records for a minimum one-year duration of patients with high-risk attributes along with enhanced surveillance of laboratory settings for identification of C.auris , which can play vital role in preparing strategies for future prevention of infections.

ACKNOWLEDGMENT: None

Funding: None

Conflict of interest: None

Ethical consideration;

Permission and official approval to carry out the study was obtained from the Institutional Ethics Committee (IEC), IPGMER and SSKM Hospital, Kolkata, West Bengal, India before carrying out our study. All patients’ kin signed a written informed consent