Candida albicans is an important human yeast pathogen that accounts for the majority of superficial and systemic infections caused by the Candida genus (Fan et al., 2008; Sobel, 2015). Candidemia, the most common form of systemic candidiasis, is the fourth most frequent cause of nosocomial bloodstream infections with a mortality rate of about 50% (Gudlaugsson et al., 2003; Kibbler et al., 2003). The morbidity of invasive candidiasis has been increasing in recent years due to the widespread use of broad-spectrum antibiotics, hormone drugs, and immunosuppressants, while invasive treatment such as endotracheal intubation and mechanically ventilation is also an important risk factor (Kim and Sudbery, 2011; Tang et al., 2016). Because Candida spp. differ in their patterns of resistance to common antifungals, differentiation of C. albicans from other Candida species is required for appropriate preventative and antimicrobial therapy (Kim and Brehm-Stecher, 2015). Moreover, the number of clinical C. albicans isolates resistant to antifungal agents is on the rise (Liu et al., 2014). Therefore, early and accurate identification of C. albicans is critical, especially in patients with suspected symptoms of invasive candidiasis.

Conventional C. albicans detection methods based on phenotype include blood culture, microscopic examination, and biochemical identification (Bouchara et al., 1996; Alam et al., 2014). However, they are time-consuming, labor-intensive with low sensitivity. The long period of waiting time required to diagnose C. albicans infection often leads to a delay in the start of treatment with antifungal drugs. Additionally, several molecular biological methods have been applied to the detection of C. albicans, such as polimerase chain reaction (PCR; Holmes et al., 1994; Vahidnia et al., 2015), real-time PCR (Guiver et al., 2001; Maaroufi et al., 2003), mass spectrometry (Zehm et al., 2012), and immunoassay (Gunasekera et al., 2015). Nevertheless, these techniques are relatively complex and require expertise and expensive instruments. Thus, a simpler, more cost-effective method is needed.

Polymerase spiral reaction (PSR), a novel isothermal nucleic acid testing (INAT) method based on auto cycling strand displacement activity using Bst DNA polymerase, is characterized by rapidity, high specificity and high sensitivity (Liu et al., 2015). Different from PCR, which needs a strictly temperature-controlled and sophisticated instrument, PSR is performed under isothermal conditions. Thus, a thermostat water bath or metal bath is sufficient to initiate the PSR reaction. Compared to other established INAT techniques, PSR does not require an initial incubation at high temperature or the utilization of a DNA helicase in the reaction mix to attain the denaturation of DNA double helix (Walker et al., 1992; Notomi et al., 2000). PSR is being widely applied to the detection of bacteria, antibiotic resistance genes, and viruses (Dong et al., 2015).

Internal transcribed spacer 2 (ITS2), a region between 5.8S and 28S fungal ribosomal DNA (rDNA), is frequently used for identification of fungal species (Turenne et al., 1999). In this research, we designed primers targeting the ITS2 sequence and determined the specificity and sensitivity of PSR detection for C. albicans. Subsequently, the clinical applicability of the PSR method for identifying C. albicans was evaluated in intensive care unit (ICU) and hematological patients with proven or suspected candidiasis infections.

Normally living as a harmless commensal, C. albicans commonly resides in mucosal surfaces of the gastrointestinal and genitourinary tracts (Zucchi et al., 2010). However, overgrowth of C. albicans will result in mucosal and superficial diseases such as oral candidiasis and vulvo vaginal candidiasis (VVC). Moreover, C. albicans can enter the systemic circulation through epithelial tissue, causing critical invasive candidiasis such as bloodstream infections, bronchopneumonia, and meningitis. Unfortunately, early diagnosis of invasive candidiasis remains a challenge because of unapparent symptoms (e.g., a prolonged low-grade fever) and time-consuming fungal culture methods (Tang et al., 2016). Thus, early and accurate identification of C. albicans is very important.

The development of INAT methods has greatly facilitated clinical screening and on-site diagnosis (Li and Macdonald, 2015). In this study, the PSR method, a recently developed INAT technique, was used to detect C. albicans targeting the ITS2 gene. Additionally, two auxiliary primers were designed to improve the reaction velocity. When the Bst DNA polymerase incorporates a deoxynucleoside triphosphate into the nascent DNA, the by-products include a hydrogen ion (Pourmand et al., 2006). The accumulation of hydrogen ions results in the decrease of pH value, which enabled the use of pH indicator. Both continuous monitoring with a real-time turbidimeter and visual inspection with the aid of cresol red and phenol red were used to assess the PSR amplification of the ITS2 gene, with an identical sensitivity of 6.9 pg/μl within 60 min, which was 10 times more sensitive than traditional PCR (69.0 pg/μl). Moreover, no false-positive amplification was observed when testing non-C. albicans species.

The PSR method requires a constant-temperature environment, so a thermostatic water bath or even a vacuum cup is sufficient to perform the PSR reaction. In contrast, PCR involves temperature cycling. It is also worth mentioning that covering the reaction mixture with a wax seal was essential for minimizing cross contamination, without disturbing the PSR reaction.

The clinical applicability of PSR was confirmed through a surveillance of C. albicans in blood samples. The test results indicated that 34/122 of samples tested positive, which was in coincidence with the culture method. However, the latter requires at least 2–3 days, while it takes less than 60 min for PSR-based detection. There were two false-negative results by PCR assay (sensitivity: 94.1%), indicating PSR is more sensitive than PCR for detection of samples with low C. albicans loads. In addition, the PSR method is easy to operate and obviates the need for complicated instruments. It is fast and convenient to identify the result as positive or negative through color change. Thus, we think PSR has a great potential for clinical screening. The antifungal susceptibility testing of C. albicans isolates revealed that amphotericin B may be beneficial when azole antifungal agents are ineffective for VVC treatment.

We established a novel PSR detection method for C. albicans, which meets the guidelines proposed by the World Health Organization for developing diagnostic techniques—namely, ASSURED (affordable, sensitive, specific, user-friendly, robust and rapid, equipment-free, and deliverable; Mabey et al., 2004). We anticipate its routine use for on-site testing in clinical settings, especially in situations where resources are limited.

DD, DZ, XH, and DA did the experiment. XJ and LB provided the clinical samples. ZY and SH designed the experiments. NL analyzed the data. WL and LH managed the project and wrote the article.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.