Introduction
Candida species is the most frequent emerging cause of fungal infections globally.1 It is also the fourth most significant cause of blood stream infection (BSI) in the United States. Due to various multi-centric studies, the picture is not clear in India. It has been also seen that Candidemia is also linked with an elevated mortality rate, which ranges from 10-49%.2 The most general cause of worldwide Candidemia is Candida albicans, but it has been observed that non Candida albicans (NCA) species like C. tropicalis, C. glabrata, C. parapsilosis, C. krusei and C. guilleirmondii are additionally involved. 2
The most frequent species of Candida has been isolated from blood in India is C.tropicalis. 3 It has been observed from fewer studies that, fluconazole and other triazoles are resistant to Candida species are mostly C. glabrata and C. krusei whereas C. parapsilosis have been found susceptible to azole group of drugs. 4 A few studies have also shown resistance to Amphotericin B and echinocandins. 5, 6
Phenotypic methods used for the isolation of Candida species are sugar assimilation, sugar fermentation, Dalmou plate technique, culture characteristics on SDA or CHROM agar etc, but these methods require 48-72 hrs or even more for the identification. Quick identification and detection of Candida species is necessary for the suitable treatment and protection of patients who suffer from Candidemia. 7
We used molecular methods like PCR-RFLP which are more sensitive, quick, smooth and price- effective for the diagnosis of different species of Candida from blood isolates of neonates.
Materials and Methods
This was a prospective cross-sectional study for which ethical clearance was taken.
Single Candida isolate per patient’s specimen was comprised in the study to avoid duplication of isolates. Mixed cultures were excluded from the study. In Table1, six standard strains used in the study with the sizes of ITS1-ITS4 PCR products for Candida species before and after digestion with MspI restriction enzyme are listed, which were provided by PGI Chandigarh.
Clinical isolates
Entirely 27 successive blood isolates were collected of neonates from the Neonatal Intensive Care Unit (NICU) for two years period at the Microbiology laboratory, Subharti University, Meerut.
All the clinical specimens were cultivated for the isolation of Candida species using standard mycological techniques. The isolates were cultured on Sabouraud’s Dextrose Agar (SDA) and further, they were used for molecular examination.
DNA isolation
By using a True-prep DNA extraction kit (Molbio Diagnostics Pvt Ltd) all the isolates was extracted for DNA. The eluted DNA was stored at -20°C until use. To confirm the presence of DNA, the gel electrophoresis was done.
PCR assay
Amplification for PCR was perform in a final volume of 100 µl. Every reaction consists of 1 µl of template DNA, each forward (ITS1, 5'-TCC GTA GGT GAA CCT GCG G-3'and reverse (ITS4, 5'TCC TCC GCT TAT TGA TAT GC-3') primer at 0.2 µM, each deoxynucleoside triphosphate (dNTP) at 0.1 mM, 10 µl of 10X PCR buffer, and 2.5 U of Taq DNA polymerase. The amplification was pursued by 25 cycles of denaturation at 94°C for 30 s, annealing at 56°C for 45 s, extension at 72°C for 1 min, with a final extension step of 72°C for 7 minutes. 1.5 % (w/v) agarose gel electrophoresis in TAE buffer were used to visualize the amplified products and then they stain with ethidium bromide under UV light and photographed.
RFLP analysis and gel electrophoresis
To perform the digestion, a 20µl aliquot of PCR product was incubated with 10 U of MspI in a final reaction volume of 25µl for 2hr at 37°C. By 1.8% agarose gel electrophoresis in TAE buffer for approximately 45 min at 100V restriction fragments were separated and visualized by staining with ethidium bromide.
Results
By using universal primers ITS-1 and ITS-4, PCR assay successfully amplified the ITS-1 and ITS-2 region of 27 isolates and the amplicon size of approximately 510-870 bp (Figure 1) was obtained from medically important Candida species (C. albicans, C. tropicalis, C. glabrata, C. parapsilosis, C.krusei and C.guilliermondii). The restriction enzyme Msp I was used for the RFLP method in one study which was performed in Iran. 8 (Figure 2).
Digestion of ITS region by Msp I give rise to two bands in each species except for C. parapsilosis as it does not have any specific restriction site for Msp I. The pattern for bands was different for each Candida species and hence it was simple to differentiate them. All strains were compared with the control strains as shown in the table (Table 1). Dispersal of Candida species isolated from blood of NICU patients by PCR-RFLP are mentioned in (Table 2).
Figure 1
PCR products from Candida species. Lane 1- NC (Negative Control), Lanes 2- ATCC C.albicans, Lane 3-ATCC C.glabrata, Lane 4- ATCC C.krusei, Lane 5-ATCC C.tropicalis, Lane 6-ATCC C.guilliermondii, Lane 7: C.parapsilosis. Lane M: 100bp Molecular size marker
Figure 2
Candida strains with the enzyme MspI. Lanes 1 and7: C.krusei, Lane 2: C. tropicalis, Lane 3: C.albicans Lane 4: C.parapsilosis, Lane 5: C guilliermondii, Lane 6: C.glabrata, Lane M: 100bp Molecular size marker
Table 1
Sizes of ITS1-ITS4 PCR products for Candida species before and after digestion with MspI restriction enzyme.
Discussion
For the handling of Candidemia and to lower the death rate related with it, early diagnosis of Candida from blood stream infections are very important. Currently to diagnose Candida up to species level by using phenotypic methods will take 48 to 72 hrs. Because of restrictions of conventional techniques, genotypic method mainly PCR is being operated for untimely detection of Candida from blood.
For the speedy detection of Candida species, many molecular methods such as DNA sequencing, RAPD (Random Amplified Polymorphic DNA), real time PCR etc. have been developed, but these methods require skilled workers and are more expensive. 9
In our study, PCR-RFLP was performed for 27 Candida samples from the blood of neonatal intensive care unit. Universal primers, ITS1 and ITS2 were able to amplify the ITS region of genomic DNA by PCR. RFLP was done for the amplified products with the help of MSP I restriction enzyme and all the isolates were correctly recognized up to the species level. Six Candida species were detected from NICU patient’s blood samples (C. tropicalis, C.parapsilosis, C.albicans, C.glabrata, C.krusei and C.gullermondii).
In our study, non Candida albicans (NCA) spp. were more commonly isolated than Candida albicans. The same finding also noticed in other studies done by Vaibhav Misra et.al.10 and Asifa Nazir et.al. 11 In epidemiological studies indicated that Candida tropicalis is involved in about 67-90% cases of candidemia. 12 In our study also C.tropicalis was the prominent one among other NCA species.
It has been found that PCR-RFLP is a quick and authentic technique to speciate Candida isolates in several studies also. In one research, MSP I enzyme with PCR-RFLP assay used for detection of medically essential fungi. 13 Similar study from Iran, by Mirhendi et.al. also identified the six Candida isolates by PCR-RFLP. 8
In other study of Iran, clinical isolates of Candida were identified by Real time PCR by High Resolution Melting Analysis (HRMA), and in one study of Vienna, various molecular methods are described for the diagnosis of Candida species. 14, 15
In our study, we found PCR-RFLP to be an easy, speedy and affordable technique for the isolation of Candida species from blood isolates.
The whole procedure of PCR-RFLP will finish in approximately seven hours whereas conventional methods require 48-72 hours to speciate the isolates. This will help to start the actual therapy of Candidemia by clinicians before antifungal sensitivity reports are obtainable. For example C.glabrata is usually resistant to fluconazole, but it is safe and effective in invasive candidiasis treatment in infants. 16 In one study, randomized controlled trials were done and based on that data the recommended antifungal were echinocandins as first line agent in older children and adults for Candidemia and disseminated candidiasis. 17, 18 Therefore, Candidemia sufferers can be treated by Amphotericin B and echinocandins.
Conclusion
PCR-RFLP has an advantage of high discrimination power, potential for wide range of speciation of Candida species and can be used for quality control of our phenotypic methods when these phenotypic methods fail to identify the species. It is also helpful for clinicians to start actual therapy in Candidemia patients before antifungal sensitivity reports are obtainable.
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