-By Kranti Karande
Yeasts have always been a part of human microbiota. Some of the species belonging to the yeast family are opportunistic pathogens leading to infections of cutaneous, mucosal, bloodstream or deep-seated organs known as Candidiasis. These infections have become a major threat to humans. Different species of the Candida genus lead to different medical conditions and demonstrate varied sensitivity towards anti fungal agents used in practice. Failure to commence accurate anti-Candida therapy at an appropriate time has led to an increase in fatal cases of Candidiasis. Hence, it is important to correctly identify the Candida species to start appropriate and timely treatment. There are very few studies of yeast infections in Indian context. Thus this study is important as it is a study of a large number of Indian clinical samples for yeast infection reporting opportunistic and emerging pathogens. The study conducted on 176 clinical samples collected from Bharati Hospitals, Pune, Maharashtra was aimed at identifying the pathogenic species of yeast, understanding their anti fungal susceptibility and cell invasion capabilities.
Existing techniques for species identification of Candida species may be time consuming and even resulting in non-authentic identification if carried out by classical usage of blood culture technique. Recently developed molecular tests provide real time PCR assays and rapid results. Although these methods are specific, they do not improve the diagnostic sensitivity of Candidaemia and Candidiasis. Nonetheless DNA sequencing of amplified PCR products continues to be the most reliable method for authentic fungal species identification. A comparison of three different techniques for yeasts species identification was carried out along with characterization of these species for antifungal susceptibility and cellular invasion capabilities.
The clinical samples obtained were chemically treated as per standard protocol before being microscopically examined to find out the presence of unicellular yeast cells and other yeast organs. Samples with probable yeast were further treated to obtain isolated colonies and consequently obtain pure cultures which were deposited at NCMR- NCCS Pune, India. The colonies were further characterized biochemically using different identification tests.
PCR amplification, DNA sequencing and phylogenetic analysis:
Genomic DNA from each isolate was extracted using lithium acetate. The precipitated DNA was used for PCR amplification (increasing the copies of DNA) using suitable primers. After confirmation of amplification, the PCR product was sequenced to identify different species present in the samples. A phylogenetic tree was constructed individually for each strain to confirm its species identification using known sequences of different Candida species available in GenBank at NCBI.
MALDI-TOF/MS biotyper and chromogenic media method:
Actively growing pure cultures were used for protein extraction for MALDI-TOF MS analysis. The extracted protein samples were analyzed using a 60 Hz Nitrogen Laser and Flex Control software. Chromogenic media screening of yeasts was also performed and identification of colonies was done using colony color and appearance. Chromogenic media is used commonly in clinical laboratories for rapid identification of Candida strains. It is a fast method but not entirely reliable. Test results showed that chromogenic media successfully identified only 64 out of the 75 strains identified as C. albicans by sequencing. Seven strains were misidentified and four remained unidentified. Hence, it is safe to say that chromogenic agar technique is not a reliable method for yeast species identification.
A total of 176 isolates were analyzed using MALDI-TOF/MS and the results were compared with results of DNA sequencing. MALDI-TOF MS could identify 157 of yeast isolates correctly. Overall, the correct identification rates of the 176 yeast isolates to species levels by the Bruker MALDI Biotyper systems was 89.2. This study confirms that MALDI-TOF MS presents an effective alternative to the sequencing for the correct identification of the emerging yeast pathogens however, there is a need to improve the database by regularly adding newer yeasts species from clinical and environmental scenarios.
Invasive fungal infections are becoming common and its rapidity of invasiveness by pathogens demands early arrest of infection by antifungal agents. There are immense changes in the host factors, infecting fungi, and antifungal agents and hence there is a need to accomplish antifungal susceptibility tests. The study conducted involved characterization of yeast species in terms of their antifungal susceptibility. A total of 157 isolates were tested for their susceptibility to antifungal drugs. Clinical yeasts showed increased resistance to fluconazole (55%) which is the most common antifungal used for treatment of candidiasis. Some emerging pathogens were found to be sensitive to most of the tested antifungal agents. It has been observed that strains of C. glabrata have highest resistance among various species of Candida. Many researchers have also isolated multi-drug resistant strains of C. glabrata.
In vitro cell invasion assay involved investigation of the ability of various yeasts isolates to invade epithelial cells (HeLa Cells were used for this study). Since the invasion of host cells by pathogenic yeast cells is one of the important virulence factors in invasive candidiasis, this study is significant for the purpose of understanding potential threats from each Candida species. A total of 88 yeast isolates tested were isolated from invasive Candidiasis patients and only few could show invasion in vitro. Although the results of invasion capabilities study for different species like C. albicans, C. glabrata, C. parapsilosis showed varied results, it was observed that yeast cells invade host cells with the help of pseudo-hyphae or hyphal structures. Some studies have also shown that the transformation of C. albicans into hyphal form increases its interaction with the tissue cells, thus increasing its ability to adhere to human cells and potent invasion.
MALDI-TOF MS is an important tool in clinical set up for microbial identification as it is fast, low-cost, simple to use and wide spectrum applications in the identification of bacteria, archaea, and fungi. However, there is an urgent need to identify the species directly from the sample since it will greatly save on time of treatment which is crucial in serious cases like invasive Candidiasis. At present, both the methods which are considered reliable require pure culture, i.e. DNA sequencing and MALDI-TOF MS. The antifungal susceptibility tests showed that a large number of isolates showed resistance to all antifungals tested. A molecular analysis of all these strains is essential to substantiate the cause and mechanism of resistance. In the present study, nystatin was found to be the most effective antifungal agent. Many new types of yeast were reported in the study as probable human pathogens which should be included in diagnostic protocols.