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 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 35  |  Issue : 2  |  Page : 58-62

Clinicomycological study of Candida isolates in a tertiary care hospital: A pilot study


Department of Microbiology, Sri Ramachandra Institute of Higher Education and Research, Chennai, Tamil Nadu, India

Date of Submission04-Feb-2021
Date of Decision04-Aug-2021
Date of Acceptance16-Aug-2021
Date of Web Publication27-Nov-2021

Correspondence Address:
Anupma Jyoti Kindo
Department of Microbiology, Sri Ramachandra Institute of Higher Studies and Research, Chennai, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jms.jms_28_21

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  Abstract 


Background: Candida infection is on the rise with an increasing number of nonalbicans species. Therefore, the need to speciate Candida rapidly and accurately is of the utmost importance. The purpose of this study was to speciate Candida using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), to analyze the correlation of the isolates with the clinical condition, and to study the outcome of the patient.
Materials and Methods: PCR-RFLP using universal primers ITS1 and ITS4 was done to speciate all isolates of Candida; patient details were collected to analyze the clinical condition and the outcome of the patient.
Results: The most common species of Candida isolated was Candida tropicalis 14 (56%) followed by Candida albicans 5 (20%), Candida auris 3 (14%), Candida parapsilosis 1 (4%), Candida orthopsilosis 1 (4%), and Candida kefyr 1 (4%). Majority of the samples that were collected were urine samples 15 (60%). The average duration of hospital stay was found to be 13.8 days. A number of underlying risk factors were present such as patients with diabetes, sepsis, malignancy, covid19 infection, surgical patients, preterm patients, elderly patients, and patients on long-term steroids.
Conclusion: Candidemia is on the rise nowadays with nonalbicans species responsible for the majority of the infections. Since the outcome of the patient depends on rapid diagnosis and prompt initiation of antifungal agents PCR-RFLP proves to be a rapid and reliable test to identify most of the prevailing species of Candida.

Keywords: Antifungals, Candida, candidemia, polymerase chain reaction, restriction fragment length polymorphism


How to cite this article:
Mishra S, Subramanian A, Kindo AJ. Clinicomycological study of Candida isolates in a tertiary care hospital: A pilot study. J Med Soc 2021;35:58-62

How to cite this URL:
Mishra S, Subramanian A, Kindo AJ. Clinicomycological study of Candida isolates in a tertiary care hospital: A pilot study. J Med Soc [serial online] 2021 [cited 2022 May 27];35:58-62. Available from: https://www.jmedsoc.org/text.asp?2021/35/2/58/331340




  Introduction Top


Candida is an opportunistic fungus, which can be found as a part of normal flora in the body. One hundred and fifty different species of Candida are known, out of which Candida albicans, Candida tropicalis, Candida parapsilosis, Candida krusei, and Candida glabrata are medically important.[1] C. tropicalis is the most common type of Candida in India.[2] Candida usually causes mucocutaneous infections, but if severe, it can cause invasive disease which is known as candidemia. Candida can be found in the oral cavity of at least 75% of the population and it is seen that about 70% of women suffer from vulvovaginal candidiasis.[3] It causes 8% of all infections and is the 4th main cause of bloodstream infection.[4] Due to its property to be able to switch between the yeast and hyphal form, it is able to survive and adapt to different temperatures, form biofilms and cause disseminated disease.[5] Although Candida is a commensal organism and is seen in various parts of a healthy individual, it becomes pathogenic in states of immunosuppression such as acquired immunodeficiency syndrome, pregnancy, patients undergoing surgery, organ transplantation, and patients on chemotherapy.[4] Metabolic conditions such as diabetes and injudicious usage of high-dose antibiotics and antifungals increase the chances of acquiring Candida. It is seen that one of the major causes of an increase in nonalbicans Candida is the indiscriminate use of azoles.[6] Since identifying the species of Candida quickly and effectively is necessary to be able to start the appropriate antifungal, in this study, we use polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) to speciate Candida. The study was also done to analyze the correlation of isolates with the patient's clinical condition and to study the outcome of the patient.


  Materials and Methods Top


A prospective cross-sectional study was carried out for 2 months at the Department of Microbiology, Sri Ramachandra Institute of Higher Education and Research, Chennai, a tertiary care center.

All clinical isolates of Candida were taken up for the study. Totally 25 strains were taken. ATCC strain of C. albicans 90028 was taken as standard strain.

All Candida isolates were subcultured on Sabouraud's Dextrose Agar.

Molecular identification

DNA extraction

DNA was extracted from all clinical isolates by phenol–chloroform method. 500 μL of lysis buffer (10 mM TRIS, pH-8), 1 mM ethylenediaminetetraacetic acid (pH-8), 3% SDS and 100 mM NaCl) was taken in an Eppendorf. Candida was suspended in lysis buffer, and it was kept in the water bath at 100°C for 2 min. Equal volume of Phenol:Chloroform (500 μL) was added to it and mixed well. It was centrifuged at 10,000 rpm for 5 min. The aqueous layer was then transferred to a fresh Eppendorf; 500 μL of chloroform was added and was centrifuged at 10,000 rpm for 5 min. DNA precipitation was done by adding an equal amount of Isopropyl Alcohol, which was centrifuged and then washed with 300 μL of 70% ethanol. Then, 50 μl of TE buffer was added and stored at −20°C.

Polymerase chain reaction assay

The master mix was prepared containing 10 μL of PCR mix (Sigma), 0.5 μL of forward (ITS-1) and reverse primer (ITS-4) (Sigma), 2 μL of template DNA and 7 μL of sterile nuclease-free water, total volume of the mixture being 20 μL. This was then subjected to PCR which was performed as initial denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 30 s, and final extension at 72°C for 5 min. PCR products were electrophoresed 1.5% agarose and visualized under trans-ultraviolet light.

Restriction fragment length polymorphism

Two μL of enzyme buffer, 0.5 μL of Msp I (GeNei, Bangalore) enzyme, 7.5 μL of nuclease-free sterile water and 10 μL of PCR product were added in a 200-μL PCR tube, and it was incubated at 37°C for 2 h. RFLP products were electrophoresed at 2% agarose gel.

Patient details such as demographic details, diagnosis on admission, duration of stay in the hospital, course in the hospital, medications (antibiotics/antifungal usage), culture reports, COVID19 status, and underlying illnesses were collected from the hospital discharge summary, and the laboratory reports and the patients were followed up until discharge from the hospital.


  Results Top


Among the 25 isolates that were collected over 2 months (August and September 2020), 6 species of Candida were identified using PCR-RFLP [Figure 1] and [Figure 2], they were - C. tropicalis 14 (56%), C. albicans 5 (20%), Candida auris 3 (14%), C. parapsilosis 2 (8%), Candida orthopsilosis 1 (4%), Candida kefyr 1 (4%).
Figure 1: BandingpatternofrestrictionfragmentlengthpolymorphismproductsofdifferentCandida species

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Figure 2: PolymerasechainreactionsubjectedtovariousCandida species. Lane1:DNAladder,Lane2:Candida albicans, Lane 3: Candida tropicalis, Lane 4: Candida parapsilosis, Lane 5: Candida kefyr, Lane 6: Candida orthopsilosis, Lane 7: Candida auris,Lane8:C.ATCCCandida albicans

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Seventeen (68%) of them males and 8 (32%) of them were female. Out of the 25 samples collected - 15 (60%) of them were urine samples, 8 (32%) were blood samples, 1 (4%) was pus sample, and 1 (4%) was tissue sample [Figure 3].
Figure 3: Breakup of the Candidaisolatesfromthevariousclinicalspecimens

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The average duration of hospital stay was 13.8 days (1–34 days). The age of the patients ranged from preterm of 32 weeks to 80 years. The underlying risk factors that were present are shown in [Figure 4]. Apart from these, there were other underlying comorbidities such as hypertension in 5 (11%) patients, coronary artery disease in 4 (9%) patients, Chronic Kidney Disease in 2 (4%) patients; acute kidney injury in 1 (2%) patient, chronic liver disease was in 1 (2%) patient, cerebrovascular disease in 1 (2%) patient, and tuberculosis in 1 (2%) patient. Among 25 patients 6 (13%) patients were COVID19 positive. [Table 1] shows the sample and species of Candida isolated from them. Among 25 patients, 18 (72%) of them were discharged, 4 (16%) of them refused treatment and left against medical advice, and 3 (12%) of them succumbed to infection.
Table 1: Underlying illness in patients with COVID-19 and their sample and isolates

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Figure 4: Underlyingriskfactorspresentinpatients

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  Discussion Top


Candidemia is one of the leading causes of mortality in today's world, and the need to be able to speciate Candida accurately and rapidly has become all the more necessary. In this study, PCR-RFLP was done to speciate Candida since it is a rapid and relatively cheaper method of correctly identifying the species.

Out of the 25 isolates, C. tropicalis was the most prevalent 14 (56%) followed by, C. albicans 5 (20%), C. auris 3 (14%), C. parapsilosis 2 (8%), C. orthopsilosis 1 (4%), and C. kefyr 1 (4%) The pattern shows an emergence of nonalbicans species. C. tropicalis has become the most common species, according to the Indian hospital statistics. We had 10 isolates from urine and 3/8 isolates from blood. C. albicans has become very infrequently isolated species in the present era, one isolate was from appendicular abscess and second one from a tissue sample of a case of necrotizing fascitis of the leg and thigh. Three isolates of C. albicans were from urine and none from blood. Among the urinary isolates, one was from an advanced malignancy of the rectosigmoid which grew C. albicans, the other case was from a patient who had bladder injury due to a fall. The third isolate was from decompensated liver disease with uncontrolled diabetes. All of these patients recovered after treatment. In our study, we had some unusual nonalbicans Candida isolated which were identified as C. auris, C. orthopsilosis, and C. parapsilosis, and C. kefyr. Molecular identification made it possible to identify the rare isolates which are a limitation of phenotypic methods. C. auris has become a pandemic ever since its isolation in Japan.[7] It is being frequently isolated from patients admitted to hospitals, especially in intensive care units (ICUs) in India.[8] Among the 3 C. auris isolates in our study, two were from blood samples and 1 was from urine sample. The first patient was extremely preterm 27 weeks with C. auris sepsis, refractory shock, necrotizing enterocolitis. Blood sample grew C. auris, the treatment was given with micafungin and voriconazole. The treatment should be started after doing an antifungal susceptibility testing, especially for C. auris since it is now a known fact that C. auris is resistant to the commonly used antifungal agents such as azoles and amphotericin B.[9] Some of the isolates may even be resistant to the echinocandins such as caspofungin, micafungin, and anidulafungin.[10] The baby recovered after treatment and was discharged after 3 weeks.

The other patient, a 6-month-old baby diagnosed with metastatic neuroblastoma also from pediatric ICU had C. auris isolated once and C. parapsilosis in another occasion during the same admission. The baby was in the hospital for a month due to the comorbid conditions. Another isolate of C. parapsilosis was from a late preterm 34 weeks' baby. Both these newborns recovered after treatment.

The third isolate of C. auris was a urine from a 54-year-old patient with Gram-negative septicemia caused by Pseudomonas, septic encephalopathy, Type 2 diabetes mellitus, with right knee effusion, erosive gastropathy, and coronary artery disease. The patient left against medical advice.

C. kefyr is another very rare species of Candida. C. kefyr usually causes disseminated candidiasis particularly in patients who have hemato-oncological malignancies.[11] The patient in this study had acute pancreatitis with Eschericia coli sepsis and the patient was in the ICU for 11 days grew C. kefyr, the patient succumbed to the illness due to the underlying conditions. High morbidity and mortality rate is seen in patients who are immunocompromised and hematological malignant associated with this species.[12],[13]

Candida orthopsilosis was isolated from a 68-year-old patient with uncontrolled diabetes and diabetic foot ulcer. This patient was started on anidulafungin for 2 weeks, became culture negative after treatment and was discharged from hospital after he recovered from his other comorbidities. During the corona spread, six patients had COVID19 infection, and all of them had raised sugar levels. The urine samples sent from these six patients, it could not be ascertained if candida were due to the infection per se, none of the patients were started on any antifungal drugs. Since repeat samples did not grow a significant count of the yeast. We could not establish them being disseminated as none of the blood isolates from these patients grew Candida. The urine isolates were considered as colonizers.

Multiple risk factors were present in our patients who included an increased hospital stay, long, abdominal surgeries, term antibiotic usage, multiple catheterizations, malignancy, sepsis, and preterm. Our study showed that the average duration of hospital stay was 14 days as compared to another study conducted in India by Chakrabarti et al. in which the duration of hospital stay was 9 days.[8]

Seven of our patients had undergone various surgical procedures, including major surgeries such as appendicular abscess drainage, debulking surgery for carcinoma rectosigmoid, open abdominal surgery for necrotizing enterocolitis and pancreatitis, debulking surgery for pseudomyxoma peritonei, and repair of the urinary bladder and sigmoid colon rupture due to traumatic injury. They developed candidemia after surgery. Many studies show that abdominal surgeries carry the highest risk among different surgeries for causing disseminated Candida infection.[8] A multicentric study on incidence, characteristics, and outcome of ICU-acquired candidemia in India[8] showed that around 37.3% of patients had undergone one or more surgical procedures, nearly 48.4% of these were gastrointestinal, hepatobiliary, and pancreatic surgeries.

Regarding antibiotic therapy as a risk factor in our study, 16 patients were on long-term antibiotic treatment such as meropenem, piperacillin, and tazobactam. A number of studies show that this is a common and independent risk factor associated with candidemia, this is consistent with a study conducted by Xess et al. in 2007,[14] who showed in her study that 196 patients out of 439 grew Candida (71.2%), the most important predisposing factor in candidemia patients being prior use of antibiotic and similarly, Giri et al. in 2013[15] showed that 64% of the patients who developed candidemia were on long-term antibiotics.

Preterm birth is also a common risk factor for candidemia which has been reported very often from tertiary care centers. The reason being that they are immunocompromised and require invasive therapies such as central catheters, endotracheal tubes, and exposed to broad-spectrum antibiotics and steroids, making them highly prone to fungal infection. Another factor is gastric acidity, the use of antacids cause fungal overgrowth.[16]

Among our study, patient group two were preterm and had candidemia with C. auris and C. parapsilosis, however had good prognosis because of prompt diagnosis and treatment.


  Conclusion Top


Even though the sample size was small, being a 2 months short study for speciation of Candida, using molecular technique, we were able to identify some rare species of Candida, which otherwise would not have been possible. Since phenotypic methods are not able to identify all species of Candida, especially the emerging ones. The outcome of the patient depends on rapid diagnosis and prompt initiation of appropriate antifungal agents, PCR-RFLP proves to be a rapid and reliable test to identify most of the prevailing species of Candida.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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Safavieh M, Coarsey C, Esiobu N, Memic A, Vyas JM, Shafiee H, et al. Advances in Candida detection platforms for clinical and point-of-care applications. Crit Rev Biotechnol 2017;37:441-58.  Back to cited text no. 1
    
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Ahmad S, Khan Z, Al-Sweih N, Alfouzan W, Joseph L, Asadzadeh M. Candida kefyr in Kuwait: Prevalence, antifungal drug susceptibility and genotypic heterogeneity. PLoS One 2020;15:e0240426.  Back to cited text no. 11
    
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Sipsas NV, Lewis RE, Tarrand J, Hachem R, Rolston KV, Raad II, et al. Candidemia in patients with hematologic malignancies in the era of new antifungal agents (2001-2007) Stable incidence but changing epidemiology of a still frequently lethal infection. Cancer 2009;115:4745-52.  Back to cited text no. 12
    
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