Introduction
The paper that is critical analysed and evaluated further on in this document, is about rapid and accurate molecular identification of the emerging multi-drug resistant pathogen, Candida auris. Candida auris is a type of fungus. It is grow as yeast. Candida auris is one of the species of fungus that cause candidiasis in humans. Candida auris also cause invasive infections in humans who have weak immune system (Satoh et al., 2009). Candida auris could be a recently delinate rare agent of fungemia (infection in blood stream). Candida auris also effect central nervous system and many other organs like kidneys, liver, bones, muscles, joints, spleen and eyes. Candida auris is famous for its multi-drug resistance against antifungal drugs (Richtel and Jacobs, 2019). Candida auris is causing deep untreated health related infections with high mortality rate across the world. Quick identification of Candida auris is of major importance for the implementation of public health measures to control the extent of infection (Ben-Ami et al., 2017) (Lockhart et al., 2017).
They hypothesized that Candida auris can be misdiagnosed by basic or commercially available phenotypic identification methods. It is difficult to identify Candida auris. For proper identification of Candida auris they presented a rapid, trustworthy, easy to use and interpret PCR and real time PCR assays (Kordalewska et al., 2017). In the study it was tested in 140 fungal isolates in which 44 isolates were Candida auris from different clinical setups for both PCR and real time PCR assay taken after by electrophoresis or melting temperature analysis, separately. The recognizable proof comes about from the measures were 100% concordant with DNA sequencing comes about. These molecular measures overcome the lacks of existing phenotypic tests to recognize and related species. The primary objectives of the study was the proper identification of Candida auris by using molecular based diagnostic tools.
In the article it is said that contagions due to the Candida auris have high death rate due to its resistance against popular antifungal drugs (fluconazole, voriconazole and itraconazole) and intrusiveness (Control and Prevention, 2016a). Candida auris needs proper molecular identification tools for exact diagnosis. This refers to an article in which it is described that Candida auris is present almost worldwide. Candida auris was first reported in Japan in 2009 as a new specie. It was isolated and identified from external ear discharge of a 70 year old patient (Satoh et al., 2009) . After 2009, three cases of Candida auris were reported from South Korea in 2011, and two of three patient were died because of complications of persistent fungemia (Lee et al., 2011). Candida auris have being reported from many countries after that such as India, Pakistan, South Africa (Lockhart et al., 2017), Kuwait (Emara et al., 2015), Colombia (Nagata et al., 2012), Venezuela (Li et al., 2005), United Kingdom (Borman et al., 2017) and United States (McCarthy, 2016) (Vallabhaneni et al., 2016).
This study showed that the nearly all Candida auris isolates are extraordinary resistant to fluconazole, with the former azoles presenting mutable antifungal activity and isavuconazole and posaconazole being the foremost active ones. Many Candida auris isolates also showed resistant to amphotericin B and a few were resistant to echinocandins (Control and Prevention, 2016a) (Larkin et al., 2017). It was also found in another study that the Candida auris showed too much resistance against antifungal drugs like fluconazole, voriconazole and itraconazole. If fngal infection persisted in patient, antifungal drugs like fluconazole and amphotericin B therapy also showed no better effect on health of patient. Such drastically multi-resistance will lead to fatal outcomes (Chowdhary et al., 2013) (Lee et al., 2011).
This already gained knowledge serves as a basic background for the current study. The present paper mentions that some clinical isolates of multi drug resistant Candida auris can be interpreted and treated on the basis of their MICs. Candida auris showed zone in MICs to major antifungal drug classes such as asazoles, echinocandins, and polyenes. But this treatment option is also limited to the some extent. For this Candida auris must be properly identified and isolated otherwise their MICs against antifungal drugs will give wrong results (Arendrup et al., 2017). Many times VITEK2 system also misidentify the Candida auris (Kathuria et al., 2015).
Furthermore, the present text refers that the exact mechanism of multi-drug resistance of Candida auris is still unclear. But it’s too much spread is an alarming situation for public health (Mizusawa et al., 2017). Candida auris is of extraordinary concern to open wellbeing organizations, due to the plausibility that biological and epidemiologic variables mat trigger an indeed more broad around the world development of Candida auris diseases (Clancy and Nguyen, 2017). Many microbiological laboratories misdiagnosed Candida auris because it is morphologically close to Candida haemulonii and many other fungal species (C. famata, C. guilliermondii, C. lusitaniae, C. parapsilosis, C. sake, Rhodotorula glutinis, and Saccharomyces cerevisiae) (Mizusawa et al., 2017) (Kathuria et al., 2015). Microbiological laboratories were using basic or commercially available methods to identify candida species that’s why the Candida auris was not properly identified and isolated. Few clinical microbiological laboratories do not able to recognize all candida at the species level so they reported all the candida as “candida species” (Control and Prevention, 2016b). It is concluded that misidentification of Candida auris is a major cause of its spreading. Subsequently, it is imperative for clinical microbiologically and other health laboratories to quickly and precisely distinguish Candida auris to assist anticipate health care associated episode and make strides survival among infected patients by enabling appropriate early antifungal treatment execution.
Treatment options against this multi-drug resistant fungus is bigger problem because it is not identified by basic or commercially available methods. It is also resistant toward three major antifungal classes (azoles, echinocandins, and polyenes). Given the current demonstrative direness encompassing this pathogen, the point of this work was to create molecular based strategies that can rapidly and precisely identify Candida auris .The performance of the proposed technique was assessed employing a comprehensive panel of clinical confines with a wide range of variable fungal species.
Methodology
This study is about the fast molecular detection of multi-drug resistant Candida auris. Before alert of CDC in 2016, deep infections in organs and multi drug resistant infections caused by Candida auris are not identifiable because of basic or commercially available diagnostic techniques adopted by clinical and other health related laboratories (Control and Prevention, 2016b). Candida auris isolated after followed proper identification molecular based methods. This is very important to identify the Candida auris for proper antifungal treatment (Chowdhary et al., 2016) and also for performance of control measures to inhibit its further dispersion in public (Schelenz et al., 2016) .
The work was performed on 140 different isolates of fungus. These 140 isolates were incuded: 44 Candida auris, 9 C. Albicans, 3 C. Dubliniensis, 1 C. famata, 10 C. glabrata, 4 C. guilliermondii, 6 C. Duobushaemulonii, 7 C. haemulonii, 10 C. krusei, 6 C. lusitaniae, 3 C. Orthopsilosis, 4 C. Metapsilosis, 2 C. kefyr, 1 C. sake, 10 C. Parapsilosis, 11 C. tropicalis, 2 Saccharomyces cerevisiae, 3 Rhodotorula mucilaginosa, 1 Aspergillus fumigatus, 1 A. Niger, 1 A. Flavus, and 1 Fusarium solani and only 1 genomic DNA sample of human. They collected all samples from different clinical setups. All 140 isolates were primarily cultured on yeast-pepton-dextrose agar before testing. C. Duobushaemulonii, C. Haemulonii and C. Sake isolates requires 24°C temperature to grow on culture media but all other isolates requires 37°C temperature. Identification was confirmed by performing rDNA sequencing (whole sequences of transcribed internal spacer 1, 5.8S ribosomal RNA gene, partial regions of the 28S and 18S rRNA genomic sequence and centeral transcribed spacer 2) and BLAST analysis were performed after amplification with “Fun-rDNAF” for forward primer (5′-GGTCATTTAGAGGAAGTAAAAGTCG-3′) and “Fun-rDNAR” for reverse primer (5′-YGATATGCTTAAGTTCAGCGGGTA-3′).
Phenotypically based identification techniques such as biochemical test, chrome agar, automated instruments, API and VITEK system are not useful for proper identification of C.auris (Kathuria et al., 2015). C.auris are proper identified by using molecular techniques. In this study both real-time and conventional PCR assays are present were for proper identification of Candida auris. These days, legitimate recognizable proof of “Candida species” involves the application of particular strategies like MALDI-TOF (matrix-assisted laser desorption ionization–time of flight). Molecular distinguishing proof that is based on the sequenced regions of D1-D2 sequences of the 28S rDNA (ribosomal DNA) are also used. Be that as it may, because of the lack of Candida auris section within the FDA approved library, it remains undetected by Vitek-MS and Bruker Biotyper as it were, when a further research practice only RUO library comprising Candida auris is consolidated can correct the identification of Candida auris be found by both “MALDI systems”.
For extraction Single colony was mixed with bicarbonate, potassium chloride, Tris at 95oC with the addition of some anti-inhibition buffer. PCR analysis was performed by using a solution of extracted DNA. They designed the primers for Candida auris and related species by Integrated DNA Technologies. PCR mixtures were prepared in a total volume of 30 μl for C.auris specific PCR, consisting of 15 μl of 2× EmeraldAmp MAX PCR master mix (TaKaRa Bio Inc.), 1 μl of each primer (CauF and CauR) at 10 μM, and 2 μl of DNA. PCR was performed in a T100 thermal cycler (Bio-Rad Laboratories, Inc.). The thermal profile included an initial denaturation for 3 min at 95°C followed by 30 cycles of 20 s at 95°C, 20 s at 68°C, and 20 s at 72°C. Electropheropherosis was performed on 2% agarose gel and amplicons presence were detected.
Real-time PCR mixtures for both Related-species-specific and Species-specific were up to 30 μl/reaction, 2× One-Step containing 15 μl of “SYBR RT-PCR buffer IV”, each primer 1ul(CauR and CauF, CauRelR, and CauRelF respectively) at 10 μMol, PrimeScript 1 µl of enzyme mix II (TaKaRa Bio, Inc.), and 2 μl of DNA.
Real-time PCR (RT-PCR) was performed on qPCR system of Mx3005P (Stratagene). The Candida auris highly specific assays consisted of total 3-mins incubation period at 95°C, 30 cycles of 15 seconds at 95°C, 20 s at temperature 68°C, and 72°C for 20 s, and finally 5 min at72°C. This assay was consisting of 3 min of incubation at 95 oC, followed by total 30 cycles of 15s, 30s at 66 oC, 72 oC, and then for 5 mins. At the end melting curve was performed for short time.
Analytical sensitivity was checked by making serial dilution from 1-106 CFU per reaction in three copies. Two isolates of Candida auris were primarily tested for specie specificity i.e for Candida auris assay, and only 1 Candida auris, Candida lusitaniae C. Duobushaemulonii and C. Haemulonii were selected for the evaluation of species-specific assay based sensitivity related to Candida auris. They performed the proficiency test as well. Real time assay was performed on 46 isolates and then proficiency testing was performed. To encourage decrease the time to determination, they presented a colony PCR technique. A sterilized tooth pick was used to pick up single colony and it was subjected to the PCR reaction blend rather than the utilizing DNA extract. After that Candida auris related highly specific Polymerase Chain Reaction was performed as mentioned over. 46 isolates confines drawn from the 140 introductory isolates. Those were tested to check the proficiency of the colony PCR. “MxPro software (version 4.1)” (Stratagene) is used for determination of melting temperature of each species. Values of Tm (melting temperature) for each specie was determined by analysis of melting curves by means of the “MxPro software” (version 4.1) (Stratagene). The melting temperature Conveyance was studied by “GraphPad Prism 6.05 software”. The precisions were estimated of Candida auris from the other species by computing both specificity and sensitivity for each assay. Great outcomes were achieved with both tests amid the improvement stage, as well as amid the proficiency validation.
Results and Discussion
Candida auris is identified as a multi-drug resistant pathogen that cause mild to severe diseases in humans. It showed resistant against major antifungal drugs. It is also misidentified by many clinical laboratories because of usage of phenotypically based methods of identification. Candida auris is hard to identify and isolate as it resembles with other species of fungus. This study proposed molecular based method to identify Candida auris by using PCR and real-time PCR assay.
Specific primers were used for identification of Candida auris and other isolates. Two primers (CauF and CauR) were specially used for 163 base pairs long PCR product of Candida auris. In Candida auris most specific Polymerase Chain Reaction and real-time PCR assays, all 44 isolates of Candida auris were showed 163-bp-long PCR product which means 100% sensitivity and 100% specificity. Real time PCR assay also showed similar results. The LOD for this was set up at the level of nearly 10 CFU/reaction. Proficiency test confirmed the accuracy of test. In real time PCR technique that is specific to the Candida auris related species, PCR products were studied for 7 C. haemulonii isolates, 44 C. auris isolates, 6 C. duobushaemulonii isolates, and 6 C. lusitaniae isolates. This assay also showed 100% sensitivity and 100% specificity. The LOD for this assay was recognized at the level of about 1,000 CFU/reaction. Proficiency test panel of 46 isolates were confirmed the accuracy of test.
