Sequence-identification of Candida species isolated from candidemia.

BACKGROUND: Candida species are the most prevalent cause of invasive fungal infections such as candidemia. Candidemia is a lethal fungal infection among immunocompromised patients worldwide. Main pathogen is Candida albicans but a global shift in epidemiology toward non-albicans species have reported. Species identification is imperative for good management of candidemia as a fatal infection. The aim of the study is to identify Candida spp. obtained from candidemia and determination of mortality rate among this population.
MATERIALS AND METHODS: The study was performed during February 2014 to March 2015 in Tehran, Iran. Two-hundred and four blood cultures were evaluated for fungal bloodstream infection. Identification of isolates was carried out using phenotypic tests and polymerase chain reaction sequencing technique.
RESULTS: Twenty-two out of 204 patients (10.8%) had candidemia. Candida parapsilosis was the most prevalent species (45.4%), followed by C. albicans (31.8%) and Candida glabrata (22.7%). Male to female sex ratio was 8/14.
CONCLUSIONS: The emergence of resistant strains of Candida species should be considered by physicians to decrease the mortality of this fatal fungal infection by appropriate treatment.

INTRODUCTION

Candidemia is a significant public health problem among immunocompromised patients worldwide. During the last two decades, epidemiologic studies show that Candida species are the fourth most prevalent cause of nosocomial bloodstream infection (BSI) and are connected to high morbidity and mortality.[123] Risk factors contain exposure to broad-spectrum antibiotics, cytotoxic chemotherapy, corticosteroids, prolonged use of intravascular catheters, and dialysis.[45] Invasive candidiasis (IC) involving BSI continues to increase worldwide and approach 50%.[67] Main pathogen is Candida albicans but a global shift toward non-albicans species such as Candida tropicalis, Candida krusei, and Candida glabrata have detected.[891011] This epidemiologic changing is of concern due to the varying susceptibility to antifungal agents with some of these emerging non-albicans species.[1213] Due to the varying antifungal susceptibility of clinical isolates, Candida spp. identification is necessary for good management of candidemia as a fatal infection. The goal of the present investigation is to identify Candida spp. obtained from candidemia and determination of mortality rate among this population.

MATERIALS AND METHODS

Strains

Between February 2014 and March 2015, 204 blood cultures were evaluated for fungal infection. The samples were collected from Imam Khomeini Hospital Complex, Pediatrics Center, Baghiatallah, Hazrat Rasool, Imam Hossein, and Shariati Hospitals, Tehran, Iran. Direct microscopy, chlamydoconidia, and germ-tube production, and subcultured onto CHROMagar Candida (Paris, France) were used for phenotypic identification.

Molecular identification

Polymerase chain reaction

DNA extraction was performed using of Whatman FTA filter matrix technology as delineated formerly.[1415] Briefly, a loopful of a single colony was suspended in 80–100 μl of distilled water and 5 μl of the suspension was transferred to a disc of FTA card (4 mm in diameter) and incubated at 25°C for at least 5 h. The dried papers were eluted in 400 μl sterile water for 10 s, then the paper was transferred to a new microtube containing 40 μl distilled water and incubated at 95°C for 15 min. The paper discs were removed, and the water including DNA was used for polymerase chain reaction (PCR) and stored at −20°C. PCR amplification and DNA sequencing of the ITS1-5.8SrDNA-ITS2 region was used for the identification of all Candida strains. The universal fungal primers ITS1 (5′-TCC GTA GGT GAA CCT GCG G-3′) and ITS4 (5′-TCC TCC GCT TAT TGA TAT GC-3′) were used to amplify the entire ITS rDNA region.[16] PCR mixture contained 5 μl of 10X reaction buffer, 0.4 mM dNTPs, 1.5 mM MgCl2, 2.5 U of Taq polymerase, 30 pmol of each ITS1 and ITS4 primers, and 2 μl of extracted DNA in a final volume of 50 μl.

Sequencing

The amplicons were purified using the ethanol purification method, and cycle sequencing reactions in forward direction were performed (Bioneer, Korea). The sequencing products were analyzed with Chromas 2.3 (http://chromas.software.informer.com/2.4/). Resulting sequences of isolates were evaluated using NCBI BLAST searches against fungal sequences existing in DNA databases (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

RESULTS

Twenty-two out of 204 patients (10.8%) had candidemia. Age range of patients was between 16 days and 89 years (mean age; 28.8). Predisposing factors included catheter (31.8%), cancer (22.7%), pneumonia (9.1%), diabetes (9.1%), dialysis (4.5%), hypoparathyroidism (4.5%), cerebral infarction (4.5%), candiduria (4.5%), severe burn and inflammation of the esophagus due to ingestion of chemical materials (4.5%), and congenital heart defect (4.5%). Eight patients (36.4%) were males and 14 patients (63.6%) were females. Mortality rate was 4.5% (n = 2). Candida parapsilosis was the most prevalent species (45.4%), followed by C. albicans (31.8%) and C. glabrata (22.7%). Colony features on CHROMagar Candida confirmed our findings. C. albicans, C. parapsilosis, and C. glabrata caused green, white, and pink colonies, respectively. Seventeen patients (77.2%) were taking antibiotics and nine patients (40.9%) were taking Cortone (Cortisone Acetate). Nine patients (40.9%) were hospitalized in Pediatric Intensive Care Unit (ICU), eight patients (36.3%) in ICU, two patients (9.1%) in Neonatal ICU, one patient (4.5%) in heart-lung transplant ward, one patient (4.5%) in liver transplantation unit, and one patient (4.5%) in the bone marrow transplant unit. Table 1 summarizes the details of patients entered in this study.

Table 1

Characteristics of patients with candidemia in this study

DISCUSSION

Candida species are the most prevalent cause of invasive fungal infections. During 1995–2002, the frequency of Candida species in blood cultures in the United States rose from 8% to 12%.[14] Because of C. albicans remains the most common Candida spp. causing IC worldwide; however, the incidence of BSIs due to C. albicans was found to have decreased,[15] rather than C. glabrata has emerged as a prominent and potentially multidrug-resistant Candida species.[16] For example, the incidence of candidemia in consequence of C. glabrata in Atlanta was shown to have increased from 1 case/100,000 people/year in 1992–1993 to 4.5 cases/100,000/year in 2008–2009.[17] C. glabrata was isolated from 22.7% of patients in the present investigation, too. Pfaller et al.[15] showed that 5.7% patients with candidemia were infected with 2 or more species of Candida, whereas mixed fungal infection was not found in this study. They reported 51.2% of patients had a concurrent bacterial infection as a result of immunocompromised nature of these patients. Bacterial coinfection was seen in 18.2% of cases in this study [Table 1]. Cleveland et al.[18] revealed 61% of patients were in an ICU within the 14 days before or after candidemia. Many studies reported that men are infected to Candida BSI more frequently than women;[2318] however, 63.6% of all patients were female in this study. Many studies[3192021] revealed C. albicans as the most common Candida species of candidemia; nevertheless, C. parapsilosis was predominant species in the present investigation. Matsumoto et al.[21] reported 20% mortality rate among candidemia patients, but in this study, crude mortality was 4.5% (n = 2). The proportion of C. parapsilosis isolates varied considerably among the participating hospitals (the majority of isolates were obtained from pediatrics center). Burning of tissue is generally one of the first steps of systemic fungal infection. Candida infection in burn patients has been connected to prolonged hospitalization and high mortality. A 3-year-old female with candidemia had esophagus chemical burn as a predisposing factor in this study; however, she was healed due to the appropriate antifungal therapy. Lotfi et al.[22] identified C. parapsilosis as the most prevalent species from candidemia (38%) in accordance with the present investigation (40.1%). Similar to the present survey, Ghahri et al.[23] by PCR-restriction fragment length polymorphism technique revealed C. parapsilosis as the most common yeast pathogen isolated from candidemia patients (34.4%). Mortality rate was 12.5% in their study. Limitations of this study include limited follow-up data, lack of antifungal susceptibility testing results, and restricted information on antifungal dosing practices.

CONCLUSIONS

The findings of this investigation confirm the high rate of candidemia in Tehran, Iran, and appear changes in epidemiological data such as increased proportion of C. parapsilosis and C. glabrata infections. Periodic surveillance studies are recommended to monitor alterations in the epidemiology of bloodstream Candida infections among high-risk population, management of serious conditions of disease, and early hospital discharge policy to control this fatal disease.

Financial support and sponsorship

Isfahan University of Medical Sciences, Isfahan, Iran (No. 394310).

Conflicts of interest

There are no conflicts of interest.