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Genetic identification of Candida species in HIV-positive patients using the polymerase chain reaction and restriction fragment polymorphism analysis of its DNA
Abstract
The polymerase chain reaction was used to amplify a targeted region: an internal transcribed spacer region of the ribosomal DNA from 114 Candida isolates and 65 reference strains. Unique product sizes were obtained for Candida glabrata, C. guillermondii and C. inconspicua. Isolates of C. albicans, C. tropicalis, C. dubliniensis and C. krusei could be identified following restriction digestion of the PCR products. The methods proved to be both simple and reproducible and may offer potential advantages over phenotyping methods.
... The PCR/RFLP (restriction fragment length polymorphism) is relatively simple to perform. It only requires standard molecular biology equipment, provides instantly recognizable results, and is easily transferable to most (12)(13)(14). ...
... In the present study, we investigated three of the most important methodologies described for the identification of Candida species by PCR/RFLP (12)(13)(14) in order to evaluate them for the identification of fungemia agents. The results obtained demonstrated that it is possible to use a single RLFP/PCR procedure, on the basis of size and structural differences in the rDNA space regions, for correct identification. ...
... Although PCR/RFLP can be considered to be an "old tool" for PCR product analysis, it is still being used for studies of microorganism characterization because of its simplicity, reliability, easy adaptation for identifying several genera or species, and because it does not require expensive materials or equipment (12)(13)(14). These characteristics are important in cost-effective studies and indicate that the use of this technique will probably become routinely accepted in mycology laboratories before the DNA sequencing or real-time PCR methods are used. ...
Prompt and specific identification of fungemia agents is important in order to define clinical treatment. However, in most cases conventional culture identification can be considered to be time-consuming and not without errors. The aim of the present study was to identify the following fungemia agents: Candida albicans, Candida parapsilosis, Candida tropicalis, Candida glabrata, Cryptococcus neoformans, Cryptococcus gattii, and Histoplasma capsulatum using the polymerase chain reaction and restriction fragment length polymorphism analysis (PCR/RFLP). More specifically: a) to evaluate 3 different amplification regions, b) to investigate 3 different restriction enzymes, and c) to use the best PCR/RFLP procedure to indentify 60 fungemia agents from a culture collection. All 3 pairs of primers (ITS1/ITS4, NL4/ITS5 and Primer1/Primer2) were able to amplify DNA from the reference strains. However, the size of these PCR products did not permit the identification of all the species studied. Three restriction enzymes were used to digest the PCR products: HaeIII, Ddel and Bfal. Among the combinations of pairs of primers and restriction enzymes, only one (primer pair NL4/ITS5 and restriction enzyme Ddel) produced a specific RFLP pattern for each microorganism studied. Sixty cultures of fungemia agents (selected from the culture collection of Fundação de Medicina Tropical do Amazonas--FMTAM) were correctly identified by PCR/RFLP using the prime pair NL4/ITS5 and Ddel. We conclude that the method proved to be both simple and reproducible, and may offer potential advantages over phenotyping methods.
... The variable region V3-V4 of the 16S rRNA gene (~510 bp) was amplified from 2 µL of pre-amplified DNA using primers MSQ-16SV3F (5 -TACGGRAGGCWGCAG-3) and PCR1R-460 (5 -TTACCAGGGTATCTAATCCT-3), as described by Frétin et al. (2018) [6]. To target the ITS2 region of the fungal population, the extracted DNA from the teat suspensions and milk samples were pre-amplified using the primers NL4 (5 -GGTCCGTGTTTCAAGACGG-3 ) and ITS5 (5 -GGAAGTAAAAGTCG TAACAAGG-3 ), as described by Irobi et al. (1999), for 17 amplification cycles [20]. Then 2 µL of pre-amplified DNA were amplified with primers ITS3f (5 -GCATCGATGAAGAACG CAGC-3 ) and ITS4_KYO1 (5 -TCCTCCGCTTWTTGWTWTGC-3 ), as described by Bokulich and Mills (2013), for 30 cycles [21]. ...
... The variable region V3-V4 of the 16S rRNA gene (~510 bp) was amplified from 2 µL of pre-amplified DNA using primers MSQ-16SV3F (5 -TACGGRAGGCWGCAG-3) and PCR1R-460 (5 -TTACCAGGGTATCTAATCCT-3), as described by Frétin et al. (2018) [6]. To target the ITS2 region of the fungal population, the extracted DNA from the teat suspensions and milk samples were pre-amplified using the primers NL4 (5 -GGTCCGTGTTTCAAGACGG-3 ) and ITS5 (5 -GGAAGTAAAAGTCG TAACAAGG-3 ), as described by Irobi et al. (1999), for 17 amplification cycles [20]. Then 2 µL of pre-amplified DNA were amplified with primers ITS3f (5 -GCATCGATGAAGAACG CAGC-3 ) and ITS4_KYO1 (5 -TCCTCCGCTTWTTGWTWTGC-3 ), as described by Bokulich and Mills (2013), for 30 cycles [21]. ...
In dairy cattle, teat disinfection at the end of milking is commonly applied to limit colonization of the milk by pathogenic microorganisms via the teat canal. The post-milking products used can irritate the teat skin and unbalance its microbial population. Our study aimed to assess the impact of different milking products on the balance of the microbial communities on the teat skin of cows and in their milk. For 12 weeks at the end of each milking operation, three groups of seven Holstein dairy cows on pasture received either a chlorhexidine gluconate-based product (G) or a hydrocolloidal water-in-oil emulsion (A), or no post-milking product (C). The composition of the bacterial and fungal communities on the teat skin and in the milk were characterized using a culture-dependent method and by high-throughput sequencing of marker genes to obtain amplicon sequence variants (ASVs). The individual microbiota on the cows’ teat skin was compared for the first time to that of a cow pool. In contrast to the milk, the post-milking treatment influenced the microbiota of the teat skin, which revealed a high microbial diversity. The water-in-oil emulsion appeared to slightly favour lactic acid bacteria and yeasts and to limit the development of undesirable bacteria such as Pseudomonas and Staphylococcus.
... Twenty nanograms of the extracted DNA served as template for PCR amplification. PCR reactions were performed in a total volume of 25 μL containing PCR buffer (final concentration: 1x; 10 mM Tris-HCl (pH 8.3) and 50 mM KCl), 1.5 mM MgCl 2 , 200 nM of the primers ITS5 (5′-GGAAGTAAAAGTCGTAACAAGG-3′) and NL4 (5′-GGTCCGTGTTTCAAGACGG-3′) (both primers were described previously by Irobi et al. [20]), 50 μM dNTPs, and 1 U ampli-Taq DNA polymerase. PCR was performed using a thermocycler (Kyratec SuperCycler, Seoul, South Korea) with the following conditions: initial denaturation for 5 min at 94°C, followed by 40 cycles for 30 s at 94°C (denaturation), 30 s at 50°C (annealing), and 90 s at 72°C (extension), with a final extension for 10 min at 72°C. ...
... The amplicons were digested with 10 U of the restriction enzyme DdeI (Thermo Fisher Scientific, Vilnius, Lithuania) for 3 h at 37°C and subjected to electrophoresis as described above. The sizes of the PCR products and restriction fragments generated from the isolates were compared with the corresponding previously described nucleotide sequences [20]. ...
The aim of this study was to evaluate the diagnostic performance of in-house FISH (fluorescence in situ hybridisation) procedures for the direct identification of invasive fungal infections in blood cultures and cerebrospinal fluid (CSF) samples and to compare these FISH results with those obtained using traditional microbiological techniques and PCR targeting of the ITS1 region of the rRNA gene. In total, 112 CSF samples and 30 positive blood cultures were investigated by microscopic examination, culture, PCR-RFLP and FISH. The sensitivity of FISH for fungal infections in CSF proved to be slightly better than that of conventional microscopy (India ink) under the experimental conditions, detecting 48 (instead of 46) infections in 112 samples. The discriminatory powers of traditional microbiology, PCR-RFLP and FISH for fungal bloodstream infections were equivalent, with the detection of 14 fungal infections in 30 samples. However, the mean times to diagnosis after the detection of microbial growth by automated blood culture systems were 5 hours, 20 hours and 6 days for FISH, PCR-RFLP and traditional microbiology, respectively. The results demonstrate that FISH is a valuable tool for the identification of invasive mycoses that can be implemented in the diagnostic routine of hospital laboratories.
... According to Li (1997), ribosomal regions exhibit a low interspecific polymorphism, and a high interspecific variability. It has been demonstrated that restriction patterns generated from the complex ITS regions (non-coding and variable) and the 18S and 25S rRNA genes encoding regions (rDNA) are helpful in measuring close fungus genealogical relationships, including important Candida species such as between C. albicans and C. dubliniensis (Irobi et al, 1999;McCullough et al, 1999). Irobi et al (1999) used RFLPs generated from ITS of rDNA to differentiate isolates of C. albicans, C. tropicalis, C. dubliniensis and C. krusei from 114 Candida isolates and 65 reference strains. ...
... It has been demonstrated that restriction patterns generated from the complex ITS regions (non-coding and variable) and the 18S and 25S rRNA genes encoding regions (rDNA) are helpful in measuring close fungus genealogical relationships, including important Candida species such as between C. albicans and C. dubliniensis (Irobi et al, 1999;McCullough et al, 1999). Irobi et al (1999) used RFLPs generated from ITS of rDNA to differentiate isolates of C. albicans, C. tropicalis, C. dubliniensis and C. krusei from 114 Candida isolates and 65 reference strains. McCullough et al (1999) used two molecular methods to characterize unusual strains of C. albicans and to compare them with authentic strains of C. dubliniensis and type I C. stellatoidea. ...
Opportunistic fungal pathogens are becoming increasingly important causes of both community-acquired and nosocomial infections. The most important fungal pathogens are yeast species belonging to the genus Candida. These species show differences in levels of resistance to antifungal agents and mortality. Consequently, it is important to correctly identify the causative organism to the species level. Identification of Candida dubliniensis in particular remains problematic because of the high degree of phenotypic similarity between this species and Candida albicans. However, as the differences between both are most pronounced at the genetic level, several studies have been conducted in order to provide a specific and rapid identification fingerprinting molecular test. In most candidal infectious, no single DNA fingerprinting technique has evolved as a dominant method, and each method has its advantages, disadvantages and limitations. Moreover, the current challenge of these techniques is to compile standardized patterns in a database for interlaboratory use and future reference. This review provides an overview of most common molecular fingerprinting techniques currently available for discrimination of C. albicans and C. dubliniensis.
... The variable region V3-V4 of the 16S rRNA gene was amplified from 2 µL of pre-amplified DNA with primers MSQ-16SV3F (5 -TACGGRAGGCWGCAG-3 ) [23] and PCR1R-460 (5 -TTACCAGGGTATCTAATCCT-3 ) as described by Frétin et al. [12]. To target the ITS2 region, the extracted DNA from teat suspensions and milk samples were pre-amplified using the primers NL4 (5 -GGTCCGTGTTTCAAGACGG-3 ) and ITS5 (5 -GGAAGTAAAAGTCGTAACAAGG-3 ) as described by Irobi et al. [24] for 17 amplification cycles. Then 2 µL of pre-amplified DNA were amplified with primers ITS3f (5 -GCATCGATGAAGAACGCAGC-3 ) and ITS4_KYO1 (5 -TCCTCCGCTTWTTGWTWTGC-3 ) as described by Bokulich and Mills [25] for 30 cycles. ...
The supplementation of animal feed with microbial additives remains questioning for the traditional or quality label raw milk cheeses with regard to microbial transfer to milk. We evaluated the effect of dietary administration of live yeast on performance and microbiota of raw milk, teat skin, and bedding material of dairy cows. Two balanced groups of cows (21 primiparous 114 ± 24 DIM, 18 multiparous 115 ± 33 DIM) received either a concentrate supplemented with Saccharomyces cerevisiae CNCM I-1077 (1 × 1010 CFU/d) during four months (LY group) or no live yeast (C group). The microbiota in individual milk samples, teat skins, and bedding material were analysed using culture dependent techniques and high-throughput amplicon sequencing. The live yeast supplementation showed a numerical increase on body weight over the experiment and there was a tendency for higher milk yield for LY group. A sequence with 100% identity to that of the live yeast was sporadically found in fungal amplicon datasets of teat skin and bedding material but never detected in milk samples. The bedding material and teat skin from LY group presented a higher abundance of Pichia kudriavzevii reaching 53% (p < 0.05) and 10% (p < 0.05) respectively. A significant proportion of bacterial and fungal ASVs shared between the teat skin and the milk of the corresponding individual was highlighted.
... albicans). These results were confirmed by others [40][41][42]. ...
... For differentiating between C. albicans and C. dubliniensis by PCR-RFLP, some authors have used three [15] while others have used two enzymes [8,16] utilizing different targets; however use of additional enzymes adds to the cost of the test. Single enzyme has been preferred by most of the researchers who have used different combination of targets and enzymes [9,[17][18][19]. ...
Aims: To retrospectively evaluate 186 stock strains of C. albicans strains isolated from oral cavity of
HIV negative patients with various malignancies for the presence of C. dubliniensis isolates among
them by PCR-RFLP.
Place and Duration of Study: Department of Microbiology, Sher-i-Kashmir Institute of Medical
Sciences, Srinagar, between October 2013 and October 2014.
Methodology: This study included 186 stock strains of C. albicans tentatively identified by
phenotypic methods like germ tube formation in human serum, colony color on chromogeniccandida differential agar, characteristic morphology on corn meal agar and assimilation of sugars
isolated from HIV negative patients with various malignancies. DNA extraction was performed by
chemical method. PCR amplification of ITS1-5.8S-ITS2 rDNA region was achieved using the ITS1
and ITS4 primer pairs which amplify the ITS region of both species, providing a single PCR product
of expected size (540 bp). There is no visible difference between these two species with regard to
their ITS PCR products. Digestion of amplified products was performed by using restriction enzyme
BlnI (AvrII) which cleaves DNA where there is a CCTAGG sequence. The products of digestion
generate one band of 540 bp for C. albicans, and two bands of 200 bp and 340 bp for
C. dubliniensis because BlnI has one recognition site within the ITS region of C. dubliniensis,
whereas none within that of C. albicans.
Results: Of the 186 isolates tested, no C. dubliniensis was found by PCR-RFLP.
Conclusion: Our results of not finding C. dubliniensis in this subset of patients support the need for
further investigations into the prevalence of this species among other clinical samples and other
susceptible patient populations.
... (12). The PCR-RFLP method has been used for the genetic identification of Candida spp. in other studies as well (10,19). Isogai et al. and Williams et al. used the restriction enzymes HaeIII and HaeIII, DdeI, and BfaI, respectively, after amplification of the ITS1-ITS4 regions, for the identification of clinically important Candida spp (7,20). ...
Background: The incidence of opportunistic infections due to Candida albicans and other Candida spp. has been increasing. Rapid identification of candidiasis is important for the clinical management of immunocompromised patients. Polymerase chain reaction: restriction fragment length polymorphism (PCR-RFLP) is a rapid, sensitive, and specific method for detection of clinically important fungi. Objectives: The purpose of this study was to identify Candida spp. isolated from the oral cavities of HIV-infected patients in southeastern Iran (Kerman), by using PCR-based restriction enzyme digestion. Patients and Methods: We identified 96 Candida isolates obtained from 139 Iranian patients infected with the human immunodeficiency virus (HIV), between April 2009 and April 2010, by using PCR-RFLP assay. Universal primers for the internal transcribed spacer (ITS) region (ITS1-ITS4) of the fungal rRNA genes were used for this assay. Results: We successfully identified the different Candida spp. by using the restriction enzyme MspI. C. albicans was the most commonly identified species (82.2%), followed by C. glabrata (7.29%), C. parapsilosis and C. kefyr (both 4.1%), and C. tropicalis (2%). Conclusions: PCR-RFLP is a highly sensitive, specific, and direct method for fungal detection and can be used for fungal epidemiological studies in HIV-positive and other immunocompromised patients. © 2012, AJUMS. Published by Kowsar M.P.Co. All rights reserved.
... The regions ITS1 and ITS2 from 5.8S rRNA gene are appropriate for diagnosis, identification taxonomy and phylogeny of fungi, which are medically important. Irobi & al. amplified the ITS regions (ITS1, 5.8S, ITS2) of several medically important Candida species, including C. krusei (J IROBI & al. [8]). Universal primers are usually used as a beneficial approach for clinical microbiology diagnosis(YC Chen & al. [9]). ...
Candida (C.) albicans is the most frequent cause of fungal infections. Conventional identification tests of pathogenic yeasts are laborious, time-consuming and sometimes ambiguous because of strains variability. The aim of this study was to evaluate the use of one molecular typing method such as PCR (polymerase chain reaction) followed by RFLP (restriction fragment length polymorphism) analysis in some Candida species identification. In this study we used eight clinical Candida strains belonging to two species: C. albicans and C. krusei isolated from patients with pharyngeal candidiasis. The identification of Candida species by PCR and RFLP analysis was based on the size and variation of 5.8 S rDNA gene and of the internal transcribed spacer 1 (ITS1) and 2 (ITS2) regions. For amplification have been used two universal primers ITS1 and ITS4. The amplification products were digested individually with three different restriction enzymes DdeI, CfoI, and MspI. The restriction patterns obtained for seven strains were identical with the restriction pattern of C. albicans ATCC 10231 reference strain and one was identical with the restriction pattern of C. krusei CMGB 94 reference strain. The identify of Candida species with amplified of ITS region and 5.8S rDNA gene followed by RFLP analysis is practical, short and a reliable method, comparing to the conventional Candida species tests based on fermentation and assimilation.
... However, the PCR-RFLP is relatively simple and easy to perform. The obtained patterns are recognizable, reproducible and easy to decipher (5)(6)(7)(8)(9). As moisture is one of the most important disposing factors for fungal growth, and the Guilan province is located in a high humidity area in the north of Iran, it seems that the incidence of fungal infections particularly candidiasis may increases in this province. ...
Due to the epidemiological alteration in distribution of Candida species as well as significant increasing trend of either intrinsic or acquired in resistance of some of these fungi, the precise identification of Candida species is necessary for effective antifungal therapy and also for prevention of nosocomial infections. PCR-RFLP method is indicated to be a reliable, rapid and simple technique which is able to differentiate the Candida species. In the present study, we applied this method to evaluate the distribution of Candida species in patients affected with cutaneous candidiasis in the Guilan province. 896 clinical cutaneous samples were collected from different parts of skin and nail of suspected patients referred to clinical centers all over the Guilan province during 24 months. Samples were examined directly with 15% KOH and cultured on fungal specific media. Genomic DNA was extracted and the restriction enzyme Msp1 was applied for polymorphism analysis. Totally, 47 yeast strains were successfully isolated from different clinical samples and identified by conventional as well as PCR-RFLP methods. The results indicated that Candida albicans (36.17%) was the most frequent species followed by C. parapsilosis (25.53%), C. tropicalis (19.14%), C. guilliermondii (14.89%), C. famata (2.12%) and C. krusei (2.12%). Female finger nails were the most common location to be affected by Candida species. In conclusion, PCR-RFLP method was successfully used for recognition of clinical Candida species within the Guilan province and obtained results revealed C. albicans as the predominant causative agent of cutaneous candidiasis. However, distribution of other Candida species did not completely consist with the reported distribution of Candida species in other parts of Iran with different climate to the Guilan province.
... Ge, Y.P. et al.C. albicans and its closely related C. dubliniensis by these nucleotide differences. Despite that only a limited number of C. albicans and C. dubliniensis isolates were investigated, those restriction sites for ApaI and BsiEI seemed to be well conserved in these two species.Several PCR-RFLP assays have been described so far to discriminate between C. albicans and C. dubliniensis(Table 1).Irobi et al amplified the ITS regions (ITS1, 5.8S, ITS2) of several medically important Candida species, including C.dubliniensis(8). Further RFLP analysis with BfaI, DdeI orHaeIII revealed distinct differences between the two species.McCullough et al amplified the ITS regions and restricted themwith DdeI. ...
Candida dubliniensis is an emerging pathogen capable of causing superficial as well as systemic infections. Due to its close similarity to C. albcians, conventional methods based on phenotypic traits are not always reliable in identification of C. dubliniensis. In this study, we developed a PCR-restriction fragment length polymorphism (RFLP) assay to identify and discriminate between the two closely related species. The D1/D2 region of 28S rDNA was amplified by PCR and enzymatically digested by ApaI and BsiEI respectively. PCR products of both species were digested into two fragments by ApaI, but those of other yeast species were undigested. BsiEI cut the PCR products of C. albicans into two fragments but not those of C. dubliniensis. Thus two species were differentiated. We evaluated 10 reference strains representing 10 yeast species, among which C. albicans and C. dubliniensis were successfully identified. A total of 56 phenotypically characterized clinical isolates (42 C. albicans isolates and 14 C. dubliniensis isolates) were also investigated for intra-species variability. All tested isolates produced identical RFLP patterns to their respective reference strains except one initially misidentified isolate. Our method offers a simple, rapid and reliable molecular method for the identification of C. albicans and C. dubliniensis.
... PCR-RFLP method has advantages over other molecular techniques, such as restriction fragment length polymorphism with genomic DNA and electrophoretic karyotyping, in being simple and quick. [17] Irobi et al, [18] used RFLP, the fragment of which is generated from ITS of rDNA to differentiate C. albicans, C. tropicalis, C. dubliniensis and C. krusei from 114 Candida isolates and 65 reference strains. McCullough et al, [19] used two molecular techniques to identify unusual strains of C. albicans and to compare them with standard strains of C. dubliniensis and type I C. stellatoidea. ...
Opportunistic infections caused by Non- Candida albicans . have been increasing. Traditional methods that are used to identify clinical isolates of Candida species are time-consuming and not appropriate for rapid, accurate and reliable identification. Purpose: To identify Candida spp isolated from cancer patients using PCR-restriction enzyme. Materials and ethods: Using universal primers, ITS1 and ITS4, in this study, we could amplify ITS1-5.8S-ITS2 rDNA regions at both 80 clinical isolates and 3 standard strains. The PCR products were digested with two restriction enzymes M sp I and B ln I separately. Result: We successfully identified all isolated species using two restriction enzymes (M sp I , B ln I). Candida albicans was the most common species (77.5%), followed by C. glabrata (15%), C. tropicalis (5%), C . krusei (2.5%). Although the primers and enzyme had the ability to identify C . parapsilosis , C . guilliermondii , C . dubliniensis , present isolates did not include these among identified ones. Conclusion: RFLP-PCR using ITSI and ITS4 primers and restriction enzyme is a rapid, easy, reliable and also applicable method in clinical laboratory for identification of medically important Candida spp.
... PCR-RFLP method has advantages over other molecular techniques, such as restriction fragment length polymorphism with genomic DNA and electrophoretic karyotyping, in being simple and quick. [17] Irobi et al, [18] used RFLP, the fragment of which is generated from ITS of rDNA to differentiate C. albicans, C. tropicalis, C. dubliniensis and C. krusei from 114 Candida isolates and 65 reference strains. McCullough et al, [19] used two molecular techniques to identify unusual strains of C. albicans and to compare them with standard strains of C. dubliniensis and type I C. stellatoidea. ...
Opportunistic infections caused by Non-Candida albicans. have been increasing. Traditional methods that are used to identify clinical isolates of Candida species are time-consuming and not appropriate for rapid, accurate and reliable identification.
To identify Candida spp isolated from cancer patients using PCR-restriction enzyme.
Using universal primers, ITS1 and ITS4, in this study, we could amplify ITS1-5.8S-ITS2 rDNA regions at both 80 clinical isolates and 3 standard strains. The PCR products were digested with two restriction enzymes MspI and BlnI separately.
We successfully identified all isolated species using two restriction enzymes (MspI, BlnI). Candida albicans was the most common species (77.5%), followed by C. glabrata (15%), C. tropicalis (5%), C. krusei (2.5%). Although the primers and enzyme had the ability to identify C. parapsilosis, C. guilliermondii, C. dubliniensis, present isolates did not include these among identified ones.
RFLP-PCR using ITSI and ITS4 primers and restriction enzyme is a rapid, easy, reliable and also applicable method in clinical laboratory for identification of medically important Candida spp.
... 51 Less precise but adequate resolution may be achieved by restriction enzyme analysis of the amplicon. 24,30 Repetitive sequence-PCR (REP-PCR), a version of randomly amplified polymorphic DNA (RAPD) in which primers target repetitive sequence elements, have been used for fungal identification. 52,53 However, this re-quires pure cultures as the starting material, which is useful in some applications but is not an acceptable precondition for a clinical fungal diagnostic tool. ...
Fungal infections pose unique challenges to molecular diagnostics; fungal molecular diagnostics consequently lags behind bacterial and viral counterparts. Nevertheless, fungal infections are often life-threatening, and early detection and identification of species is crucial to successful intervention. A high throughput PCR-based method is needed that is independent of culture, is sensitive to the level of one fungal cell per milliliter of blood or other tissue types, and is capable of detecting species and resistance mutations. We introduce the use of high resolution melt analysis, in combination with more sensitive, inclusive, and appropriately positioned panfungal primers, to address these needs. PCR-based amplification of the variable internal transcribed regions of the rDNA genes generates an amplicon whose sequence melts with a shape that is characteristic and therefore diagnostic of the species. Simple analysis of the differences between test and reference melt curves generates a single number that calls the species. Early indications suggest that high resolution melt analysis can distinguish all eight major species of Candida of clinical significance without interference from excess human DNA. Candida species, including mixed and novel species, can be identified directly in vaginal samples. This tool can potentially detect, count, and identify fungi in hundreds of samples per day without further manipulation, costs, or delays, offering a major step forward in fungal molecular diagnostics.
... Several authors have demonstrated that it is possible to use restriction patterns generated from amplified ribosomal DNA sequences to differentiate between closely related species such as C. albicans and C. dubliniensis. Irobi et al. [75] amplified the internal transcribed spacer (ITS) regions of several Candida species, including C. albicans and C. dubliniensis and found that both these species produced a PCR product of approximately 1200 kb. Further analyses of restriction patterns after digestion with BfaI, DdeI or HaeIII revealed that there are distinct differences between these two species, enabling their differentiation. ...
Candida albicans is increasing as an opportunistic pathogen causing candidemia and candidiasis worldwide. In addition, other non-albicans Candida species are now also associated with pertinent infections. These include the closely related C. dubliniensis, which shares many phenotypic similarities with C. albicans. These similarities pose problems in the identification of isolates and have previously led to misidentification of these species. As a result, several identification techniques based on phenotypic and genotypic characteristics have been developed to differentiate between these Candida species. This review will focus on the similarities and differences between these two Candida species highlighting different identification methods and their advantages and disadvantages.
... Invasive procedures or immunosuppression increases the risk of fungemia (24,34), and drug-resistant strains have emerged (9,21). Although Candida albicans is still the predominate agent of nosocomial infection, serious infections caused by other yeasts have increased in frequency (8,11,29,34). For example, species of Cryptococcus other than Cryptococcus neoformans, previously considered to be nonpathogenic saprophytes, have been reported to cause cryptococcosis (28). ...
Species-specific polymorphisms in the noncoding internal transcribed spacer 2 (ITS2) region of the rRNA operon provide accurate identification of clinically significant yeasts. In this study, we tested the hypothesis that ITS1 noncoding regions contain diagnostically useful alleles. The length of ITS1 region PCR products amplified from 40 species (106 clinical strains, 5 reference strains, and 30 type strains) was rapidly determined with single-base precision by automated capillary electrophoresis. Polymorphisms in the PCR product length permitted 19 species to be distinguished by ITS1 alone, compared with 16 species distinguished by using only ITS2. However, combination of both ITS alleles permitted identification of 30 species (98% of clinical isolates). The remaining 10 species with PCR products of similar sizes contained unique ITS alleles distinguishable by restriction enzyme analysis. DNA sequence analysis of amplified ITS1 region DNA from 79 isolates revealed species-specific ITS1 alleles for each of the 40 pathogenic species examined. This provided identification of unusual clinical isolates, and 53 diagnostic ITS1 sequences were deposited in GenBank. Phylogenetic analyses based on ITS sequences showed a similar overall topology to 26S rRNA gene-based trees. However, different species with identical 26S sequences contained distinct ITS alleles that provided species identification with strong statistical support. Together, these data indicate that the analysis of ITS polymorphisms can reliably identify 40 species of clinically significant yeasts and that the capacity for identifying potentially new pathogenic species by using this database holds significant promise.
... The main advantage of using amplification targets from regions of DNA which are conserved among all Candida species is that a PCR product can be obtained from all species using a single set of PCR primers and conditions optimal for that set of primers. Following amplification, species-specific probes can be designed from the more variable DNA regions, located between primer binding sites, for the identification of specific organisms (Elie et al., 1998;Ellepola et al., 2003;Coignard et al., 2004) or amplicons can be subjected to gel electrophoresis, with or without the use of restriction enzyme digestion, followed by ethidium bromide staining to differentiate among the species (Buchman et al., 1990;Crampin and Matthews 1993;Hopfer et al., 1993;Rand et al., 1994;Wildfeuer et al., 1996;Morace et al., 1997;Irobi et al., 1999;Cirak et al., 2003;Graf et al., 2004). Increased sensitivity can be achieved by Southern blotting of such gels onto nylon membranes and by detection with radiolabeled or colorimetric probes (Crampin and Matthews, 1993;Miyakawa et al., 1993;Burgener-Kairuz et al., 1994;Holmes et al., 1994;Jordan, 1994;van Deventer et al., 1995;Loeffler et al., 2000a). ...
Invasive candidiasis is associated with high morbidity and mortality. Clinical diagnosis is complicated by a lack of specific clinical signs and symptoms of disease. Laboratory diagnosis is also complex because circulating antibodies to Candida species may occur in normal individuals as the result of commensal colonization of mucosal surfaces thereby reducing the usefulness of antibody detection for the diagnosis of this disease. In addition, Candida species antigens are often rapidly cleared from the circulation so that antigen detection tests often lack the desired level of sensitivity. Microbiological confirmation is difficult because blood cultures can be negative in up to 50% of autopsy-proven cases of deep-seated candidiasis or may only become positive late in the infection. Positive cultures from urine or mucosal surfaces do not necessarily indicate invasive disease although can occur during systemic infection. Furthermore, differences in the virulence and in the susceptibility of the various Candida species to antifungal drugs make identification to the species level important for clinical management. Newer molecular biological tests have generated interest but are not yet standardized or readily available in most clinical laboratory settings nor have they been validated in large clinical trials. Laboratory surveillance of at-risk patients could result in earlier initiation of antifungal therapy if sensitive and specific diagnostic tests, which are also cost effective, become available. This review will compare diagnostic tests currently in use as well as those under development by describing their assets and limitations for the diagnosis of invasive candidiasis.
... Investigation of the sequence polymorphisms in the internal transcribed spacer region (ITS) of the rRNA genes (Chen et al., 2000;Borst et al., 2003) is a common way to discriminate between these fungal species. Precise solutions are restriction fragment length polymorphism (PCR-RFLP) (Graf et al., 2004;Irobi et al., 1999), multiple PCR with speciesspecific primers (Kanbe et al., 2002), or blot hybridization (Posteraro et al., 2000), but PCR fingerprinting (Bartie et al., 2001) and randomly amplified polymorphic DNA analysis methods (Bautista-Munoz et al., 2003) have also been described. These methods are time consuming and labor intensive. ...
Several phenotypic methods have been used for the differentiation of Candida albicans and Candida dubliniensis, but molecular investigations are considered most reliable in their diagnostic value. Here, we suggest a rapid real-time polymerase chain reaction assay where the discrimination was achieved through melting point analysis with the help of the nonspecific fluorescent dye SybrGreen.
Purpose:
Bloodstream infections are major causes of morbidity and mortality that lead to prolonged hospital stays and higher medical costs. In this study, we aimed to evaluate the MinION nanopore sequencer for the identification of the most dominant pathogens in positive blood culture bottles.
Methodology:
16S and ITS1-5.8S-ITS2 rRNA genes were amplified by PCR reactions with barcoded primers using nine clinical isolates obtained from positive blood bottles and 11 type strains, including five types of Candida species. Barcoded amplicons were mixed, and multiplex sequencing with the MinION sequencer was performed. In addition, barcoded PCR amplicons were sequenced by Sanger sequencing to validate the performance of the MinION.
Results:
The bacterial and Candida spp. identified by MinION sequencing, based on the highest homology of reference sequences from the NCBI gene databases, agreed with the matrix-assisted laser desorption ionization time of flight mass spectrometry results, excepting the closely related species Streptococcusand Escherichia coli. The 'pass' reads obtained within about 10 min of sequencing were sufficient to identify the pathogens. The average values of sequence identities with 1D2 chemistry and the R9.5 flow cell were around 99 %; thus, frequent sequence errors did not affect species identification based on amplicon sequencing.
Conclusion:
We have established a rapid, portable and economical technique for the identification of pathogens in positive blood culture bottles through a novel MinION nanopore sequencer amplicon sequencing scheme, which replaces traditional Sanger sequencing.
Background/aim: The identification of Candida species isolated from clinical specimens provides information about antifungal
susceptibility and sheds light on the choice of empirical treatment. In the present study, restriction enzyme analysis of C. albicans and
non-albicans Candida species previously identified by conventional methods was done to evaluate the utility of restriction enzyme
analysis for more rapid and reliable identification of Candida species.
Materials and methods: A total of 146 Candida strains isolated from various clinical specimens and ATCC strains were included. PCR
products were digested with MwoI for all species and with BslI for C. parapsilosis and C. tropicalis strains.
Results: The strains were identified by conventional methods as 40 C. albicans, 27 C. parapsilosis, 26 C. tropicalis, 25 C. glabrata, 11 C.
kefyr, 10 C. krusei, and 7 C. guilliermondii strains. Restriction digestion with MwoI was able to distinguish between five different species
(C. albicans, C. krusei, C. guilliermondii, C. kefyr, and C. glabrata), while BslI digestion could distinguish between C. tropicalis and C.
parapsilosis.
Conclusion: Restriction enzyme analysis with MwoI and BslI can be used for the identification of Candida species in situations where
rapid identification is necessary or conventional methods are problematic.
Background: Candidiasis is a widespread fungal disease caused by different candida spp. Traditional methods for these yeasts, are time-consuming and in some cases are not successful. New molecular techniques based on differences in DNA targets are faster and more useful. The aim of this study was identification of the most medically common Candida species by PCR-RFLP analysis and their prevalence in Isfahan, central, Iran. Methods: Yeast genomic DNA was extracted from living cultures using FTA-filters and ITS1-ITS2 region was amplified by PCR method and was digested by restriction enzyme MspI. RFLP products were loaded on agarose gel and yeast species were identified acorging to differences in their band patterns. Finding: One hundered and eighty two isolates were evaluated from different body locations comprising nail, vagina, groin, blood, wound etc, from which 86 isolates (47.2%) were identified as C. albicans, 31 (17%) as C. parapsilosis, 19 (10.4%) as C. kefyr, 15 (8.2%) as C. tropicalis, 14 (7.7%) as C. glabrata, 14 isolates (7.7%) as C. krusei, 3 isolates (1.6%) as C. guilliermondii. Totally 47.2% isolates were C. albicans and 52.8% isolates were non- albicans spp. Conclusion: Increasing of non-albicans species and their more resistance to antifungal drugs than C. albicans is an important topic of fungal infections that needed precise epidemiological surveys. This study can be a leader for more applicable studies of researchers.
Background: The incidence of invasive mycoses is increasing worldwide. PCR-RFLP was applied to the identification of 10 reference strains and 90 cultures of agents of invasive mycoses. In addition, the new approach was applied to detect fungal agents in 120 biological samples (blood, cerebrospinal fluid and bone marrow). PCR-RFLP results were compared with the ones obtained with conventional methods (culture, microscopy, and biochemical testing).
Results: The assays carried out with the reference strains (Candida albicans, Candida parapsilosis, Candida tropicalis, Candida krusei, Candida guilliermondii, Cryptococcus neoformans, Cryptococcus gattii and Histoplasma capsulatum), demonstrated that the RFLP profiles were correctly predicted by the in silico investigation and allowed unequivocal identification of all chosen reference strains. The PCR-RFLP also identified 90 cultures of agents of invasive mycoses correctly, 2.5 times faster than the conventional assays. Evaluating PCR-RFLP with biological samples it was observed that the PCR was found to be 100% accurate and the RFLP profiles allowed the identification of the etiological agents: C. neoformans (n = 3) and C. gattii (n = 1) in CSF samples, H. capsulatum (n = 1) in bone marrow and C. albicans (n = 2) in blood cultures. The detection and identification by PCR-RFLP were found to be between two to ten times faster than the conventional assays.
Conclusion: The results showed that PCR-RFLP is a valuable tool for the identification of invasive mycoses that can be implemented in hospital laboratories, allowing for a high number of clinical analyses per day.
Fluorescence in situ hybridization (FISH) has been shown to be useful for the detection of Candida and Cryptococcus species in blood culture materials. FISH procedures for the detection of Histoplasma capsulatum var. capsulatum have not been reported so far. This study describes the development and evaluation of fluorescently labeled rRNA-targeting FISH probes to detect and identify H. capsulatum in blood cultures. All three analyzed H. capsulatum reference strains and clinical isolates showed positive signals with the newly designed specific oligonucleotide probes for H. capsulatum, whereas negative reactions were observed for all three nontarget yeast species and the two nontarget bacteria. The assay was also successfully applied for detections of H. capsulatum cells in pre-incubated blood culture samples of patients with clinical suspicion of histoplasmosis (n = 33). The described FISH-based assay was shown to be easy to apply, sensitive, and specific (compared to polymerase chain reaction) for the detection and identification of H. capsulatum in this proof-of-principle analysis. Larger multicentric assessments are recommended for a thorough diagnostic evaluation of the procedure.
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Sophisticated technologies to prolong the lives of severely debilitated patients have become more commonplace. Ironically,
as a result of these medical advances, a population of patients more vulnerable to fungal infections has been produced. Individuals
who would previously have died from major illnesses now survive in a debilitated state, often requiring intensive care for
prolonged periods of time. As a consequence, the incidence of invasive fungal infections among critically-ill patients has
increased significantly over the past decade. Most fungal infections in the intensive care unit (ICU) are caused by opportunistic
pathogens, principallyCandidaspecies, which are commonly found as normal commensals of the human gastrointestinal tract, skin, and/or mucosa. Less commonly,
infections in critically-ill patients are caused by moulds, such asAspergillusspecies, which are ubiquitous in the environment. These fungi do not ordinarily cause invasive infection unless the host is
immunocompromised or otherwise debilitated; therefore, the inherently weakened condition of most critically-ill patients,
along with the multiplicity of invasive procedures and immunosuppressive drugs used to treat underlying illnesses in this
patient population, make them particularly susceptible to invasive fungal diseases.
Background/Aim: Causative agents most frequently encountered in systemic infections are bacteria, although fungi that cause invasive infections have also emerged, mostly in immune-compromised patients. The early detection and adequate treatment of bloodstream infections are critical for successful treatment. The aim of this study was to develop a rapid and efficient method for the detection and differentiation of the most common fungal pathogens.
Real-time Polymerase chain reaction (PCR) and consecutive high-resolution melting analysis was used for the detection and differentiation of fungal pathogens.
The developed analysis procedure proved appropriate for discrimination of the ten most relevant Candida species, four Aspergillus species, and Cryptococcus neoformans. The sensitivity of the PCR reaction was 5, which is suitable for the detection of these fungi in blood.
This technique is not adaptable as a general identification method, but it is highly useful when certain fungal species are to be expected in clinical samples.
As there are four major molecular types of Cryptococcus neoformans (VNI, VNII, VNIII and VNIV) and four molecular types of Cryptococcus gattii (VGI, VGII, VGIII and VGIV), it is important to identify the specific groups causing cryptococcosis in different geographical regions. Here, we investigated the molecular types of 57 cryptococcal isolates from patients in a tertiary care hospital in the state of Amazonas, Brazil, between 2006 and 2010. The isolates were characterised by PCR fingerprinting using the M13 minisatellite and confirmed by URA5-RFLP analysis, and the presence of specific genes from the mating type locus (MATα and MATa) of these species was analysed by PCR. Most of the patients were male (66.7%), between 16 and 30 years of age (51.7%), and HIV-positive (75.0%). Most isolates were collected from cerebrospinal fluid samples (71.7%). Most of the C. neoformans isolates (n=40) were characterised as members of the VNI molecular group (n=39), a unique isolate was characterised as VNII whereas all isolates of C. gattii (n=17) were members of the VGII molecular group. With regard to mating types, 55 isolates were type 'α', and only two were type 'a'. This study revealed the prevalence of the VNI molecular group and provides the first reported observation of the VNII molecular group in the northern region of Brazil.
There is a high interest in Candida species other than Candida albicans because of the rise and the epidemiological shifts in candidiasis. These emerging Candida species are favored by the increase of immunocompromised patients and the use of new medical practices, and m. Most oropharyngeal candidiasis can be foundare observed in those HIV-infected patients infected with human immunodeficiency virus (HIV). Candida dubliniensis is a recently described opportunistic pathogen that is closely related to C. albicans but differs from it with respect to epidemiology, certain virulence characteristics, and the ability to develop fluconazole resistance in vitro. C. dubliniensis has been linked to oral candidiasis in AIDS patients, although it has recently been associated to invasive disease. C. dubliniensis shares diagnostic characteristics with C. albicans, as germ tube- and chlamydospore-production, and it is generally misclassified as C. albicans by standard diagnostic procedures. Several recent studies have attempted to elucidate useful phenotypic and genotypic characteristics for separating both species. A large variety of methods have been developed with the aim of facilitating rapid and, accurate identification of this species. These have included differential chromogenic isolation platesculture media, direct immunological tests, and enhanced manual and automated biochemical and enzymatic panels. Chromogenic isolation media, as CHROMagar Candida, demonstrate better detection rates than traditional media, and allow the presumptive identification of C. dubliniensis by means of colony color (dark-green colonies). API 20 C AUX system is considered a reference method, but ID 32 C strip, the VITEK Yeast Biochemical Card and the VITEK 2 ID-YST system correctly identify most C. dubliniensis isolates, being the latter the most accurate. Spectroscopic methods, such as Fourier transformed-infrared spectroscopy, offer potential advantages. However, many authors consider that standard methods for differentiation of Candida species are time-consuming, often insensitive and can fail to distinguish C. dubliniensis. To overcome these low sensitivity, poor specificity and intolerable delay,drawbacks, molecular tools have been developed to discriminate C. dubliniensis, and particularly those based on the polymerase chain reaction. But, molecular tools prove difficult and too complex for routine use in the clinical laboratory setting and new developments are necessary. Moreover, an increased resistance to antifungal drugs has been described. Although preliminary studies indicate that most strains of C. dubliniensis are susceptible to antifungal agents, fluconazole-resistant strains have been detected. Furthermore, fluconazole-resistant strains are easily derived in vitro, showing an increased expression of multidrug resistance transporters, as MDR1.
Restriction fragment length polymorphism analysis of the 5.8S rRNA gene and the internal transcribed spacers (ITS1 and ITS2) was used for examination of 66 isolates belonging to 19 species. Intraspecies variability was found in the examined region of 11 species (Candida albicans, C. catenulata, C. colliculosa, C. glabrata, C. kefyr, C. melinii, C. parapsilosis, C. guillermondii, C. solanii, C. tropicalis, Saccharomyces cerevisiae). Region of ITS-5.8S rDNA was amplified using the primers ITS1 and ITS4. The amplicons were digested by HaeIII, HinfI and CfoI. The recognized intraspecies variability was confirmed in the second step, in which the shorter fragments of this region were amplified using primers ITS1 and ITS2 and analyzed by capillary electrophoresis. Considerable intraspecific variability renders this method unsuitable for species identification, whereas it can be useful for epidemiological tracing of isolates.
Most strains of Candida albicans are capable of switching frequently and reversibly between a number of phenotypes distinguishable by colony morphology. A number of different switching systems have been defined according to the limited set of phenotypes in each switching repertoire, and each strain appears to possess a single system. Switching can affect many aspects of cellular physiology and morphology and appears to be a second level of phenotypic variability superimposed upon the bud-hypha transition. The most dramatic switching system so far identified is the "white-opaque transition." This system dramatizes the extraordinary effects switching can have on the budding cell phenotype, including the synthesis of opaque-specific antigens, the expression of white-specific and opaque-specific genes, and the genesis of unique cell wall structures. Switching has been demonstrated to occur at sites of infection and between episodes of recurrent vaginitis, and it may function to generate variability in commensal and infecting populations for adaptive reasons. Although the molecular mechanisms involved in the switch event are not understood, recent approaches to its elucidation are discussed and an epigenetic mechanism is proposed.
The PCR was used to amplify a targeted region of the ribosomal DNA from 84 Candida isolates. Unique product sizes were obtained for Candida guilliermondii, Candida (Torulopsis) glabrata, and Candida pseudotropicalis. Isolates of Candida albicans, Candida tropicalis, Candida stellatoidea, Candida parapsilosis, and Candida krusei could be identified following restriction digestion of the PCR products.
We developed a microtitration plate enzyme immunoassay to detect PCR-amplified DNA from Candida species. Nucleotide sequences derived from the internal transcribed spacer (ITS) region of fungal rDNA were used to develop species-specific oligonucleotide probes for Candida albicans, C. tropicalis, C. parapsilosis, and C. krusei. No cross-hybridization was detected with any other fungal, bacterial, or human DNAs tested. In contrast, a C. (Torulopsis) glabrata probe cross-reacted with Saccharomyces cerevisiae DNA but with no other DNAs tested. Genomic DNA purified from C. albicans blastoconidia suspended in blood was amplified by PCR with fungus-specific universal primers ITS3 and ITS4. With the C. albicans-specific probe labeled with digoxigenin, a biotinylated capture probe, and streptavidin-coated microtitration plates, amplified DNA from a few as two C. albicans cells per 0.2 ml of blood could be detected by enzyme immunoassay.
PCR of a Candida albicans cytochrome P-450 lanosterol-alpha-demethylase (P450-L1A1) gene segment is a rapid and sensitive method of detection in clinical specimens. This enzyme is a target for azole antifungal action. In order to directly detect and identify the clinically most important species of Candida, we cloned and sequenced 1.3-kbp fragments of the cytochrome P450-L1A1 genes from Torulopsis (Candida) glabrata and from Candida krusei. These segments were compared with the published sequences from C. albicans and Candida tropicalis. Amplimers for gene sequences highly conserved throughout the fungal kingdom were first used; positive PCR results were obtained for C. albicans, T. glabrata, C. krusei, Candida parapsilosis, C. tropicalis, Cryptococcus neoformans, and Trichosporon beigelii DNA extracts. Primers were then selected for a highly variable region of the gene, allowing the species-specific detection from purified DNA of C. albicans, T. glabrata, C. krusei, and C. tropicalis. The assay sensitivity as tested for C. albicans in seeded clinical specimens such as blood, peritoneal fluid, or urine was 10 to 20 cells per 0.1 ml. Compared with results obtained by culture, the sensitivity, specificity, and efficiency of the species-specific nested PCR tested with 80 clinical specimens were 71, 95, and 83% for C. albicans and 100, 97, and 98% for T. glabrata, respectively.
Reference strains from 48 selected serovars representing eight species of Leptospira were examined by two polymerase chain reaction (PCR)-based strategies. First, mapped restriction site polymorphisms (MRSP) were examined in PCR products from portions of rrs (16S rRNA gene) and rrl (23S rRNA gene). Twenty MRSP and 2 length polymorphisms were used to group reference strains into 16 MRSP profiles. Species assignments were consistent with those obtained by a second method, genomic fingerprinting with arbitrarily primed PCR, in which strains within a species were characterized by many shared arbitrarily primed PCR products. The results of both of these methods were in general agreement with those of previous studies that used DNA-DNA relatedness and confirmed the high level of divergence among the recognized species of Leptospira. However, Leptospira meyeri serovar ranarum and evansi strains were indistinguishable from some strains of Leptospira interrogans sensu stricto. Intervening sequences of about 485 to 740 bp were located near base 1230 in rrl of some strains.
Primers complementary to the region of genes coding for rRNA in Candida albicans were used in PCRs to detect yeast DNA extracted from blood samples containing various Candida species. One fragment (105 bp) was amplified from all yeasts tested, whereas a second (684 bp) was only amplified when C. albicans DNA was present. The level of sensitivity was 15 +/- 5 (mean +/- standard error) CFU of C. albicans per ml of blood.
The reported incidence of fungal infections associated with non-albicans species from the Candida genus is increasing. Most of these infections occur in immunocompromised patients, particularly those infected with HIV. The role of molecular genetic techniques alongside the existing techniques for the identification and typing of these organisms is discussed. Species-specific genomic DNA fragments cloned from C. tropicalis and C. krusei have been developed for identification and strain typing. Analysis of tRNA profiles has been shown to be effective for the identification of C. glabrata, C. guilliermondii, C. parapsilosis and C. tropicalis. A PCR method employing primers complimentary to large ribosomal subunit genes and the lanosterol-alpha-demethylase gene has been applied for several species, including C. glabrata, C. krusei and C. tropicalis. Strain typing by comparison of genomic DNA fingerprints has been demonstrated for C. tropicalis and C. krusei following hybridisation analysis with species-specific probes. Synthetic oligonucleotide probes--which do not have to be species-specific and which can detect minor polymorphisms--have also been used for strain typing of isolates of several non-albicans species. Random amplification of polymorphic DNA (RAPD) has also been used for analysis of C. glabrata, C. lusitaniae and C. tropicalis isolates. The potential for the application of these and other techniques to Candida spp. taxonomy--and the example of a recently discovered novel species, C. dubliniensis--is discussed.
Candida albicans is among the most common fungal pathogens. Infections caused by C. albicans and other Candida species can be life threatening in individuals with impaired immune function. Genetic analysis of C. albicans pathogenesis is complicated by the diploid nature of the species and the absence of a known sexual cycle. Through a combination of parasexual techniques and molecular approaches, an effective genetic system has been developed. The close relationship of C. albicans to the more extensively studied Saccharomyces cerevisiae has been of great utility in the isolation of Candida genes and development of the C. albicans DNA transformation system. Molecular methods have been used for clarification of taxonomic relationships and more precise epidemiologic investigations. Analysis of the physical and genetic maps of C. albicans and the closely related Candida stellatoidea has provided much information on the highly fluid nature of the Candida genome. The genetic system is seeing increased application to biological questions such as drug resistance, virulence determinants, and the phenomenon of phenotypic variation. Although most molecular analysis to data has been with C. albicans, the same methodologies are proving highly effective with other Candida species.
Fungemias were reviewed in 110 immunocompromised patients hospitalized between November 1, 1974, and December 31, 1977, at Memorial Sloan-Kettering Cancer Center (MSKCC). The incidence of Candida tropicalis fungemia increased each year. Seventy-six percent of the patients with C. tropicalis fungemia and 32.5 percent of those with C. albicans fungemia had either leukemia or lymphoma. Seventy-seven percent of the C. parapsilosis fungemias were related to total parenteral nutrition. Thirty-seven percent of the patients with C. albicans fungemia were receiving oral prophylactic nystatin therapy. The source of fungemia was often difficult to determine: in 60 percent of the patients, only blood cultures were positive for C. tropicalis or Torulopsis glabrata; no cultures were positive for the fungus from any other site before the episode occurred. Serologic tests, including a highly sensitive passive hemagglutination test, showed fourfold increases in titer only inconsistently. A passive hemagglutination-inhibition test for circulating antigen was positive in 50.9 percent of 57 patients with fungemia who were tested and may be a valid indication for treatment. Fungemia usually represented a severe and often fatal disease. The over-all mortality of the 110 patients with fungemia was 79 percent whereas only 23 percent of the patients with C. parapsilosis fungemia died. Among the patients who received more than 200 mg of amphotericin B, 71 percent died despite treatment.
Fungal infections may account for 5-50% of serious infections in intravenous drug users, and for 5-50 per 100,000 hospital admissions. The fungi most commonly encountered are Candida and Aspergillus spp. Candidosis may be disseminated, with lesions in superficial structures, the eye and the skeletal system, or limited to the eye, the heart (as endocarditis) or the central nervous system. Aspergillosis usually presents as endophthalmitis or as central nervous system infection. Mucormycosis is also met with occasionally, and various fungi may cause endophthalmitis or endocarditis. Antifungal therapy for intravenous drug use-related infections is no different from that for similar mycoses in other patients, but the management of intravenous drug users requires considerable clinical skill.
Candida albicans is among the most common fungal pathogens. Infections caused by C. albicans and other Candida species can be life threatening in individuals with impaired immune function. Genetic analysis of C. albicans pathogenesis is complicated by the diploid nature of the species and the absence of a known sexual cycle. Through a combination of parasexual techniques and molecular approaches, an effective genetic system has been developed. The close relationship of C. albicans to the more extensively studied Saccharomyces cerevisiae has been of great utility in the isolation of Candida genes and development of the C. albicans DNA transformation system. Molecular methods have been used for clarification of taxonomic relationships and more precise epidemiologic investigations. Analysis of the physical and genetic maps of C. albicans and the closely related Candida stellatoidea has provided much information on the highly fluid nature of the Candida genome. The genetic system is seeing increased application to biological questions such as drug resistance, virulence determinants, and the phenomenon of phenotypic variation. Although most molecular analysis to data has been with C. albicans, the same methodologies are proving highly effective with other Candida species.
The PCR, like recombinant DNA technology, has had an enormous impact in both basic and diagnostic aspects of molecular biology because it can produce large amounts of a specific DNA fragment from small amounts of a complex template. Recombinant DNA techniques create molecular clones by conferring on a specific sequence the ability to replicate by inserting it into a vector and introducing the vector into a host cell. PCR represents a form of "in vitro cloning" that can generate, as well as modify, DNA fragments of defined length and sequence in a simple automated reaction. In addition to its many applications in basic molecular biological research, PCR promises to play a critical role in the identification of medically important sequences as well as an important diagnostic one in their detection.
Restriction fragment length polymorphisms in the ribosomal DNA (rDNA) have been shown to be a useful criterion for distinguishing among various isolates of Candida albicans. In a sample of 12 clinical isolates, we found six different classes based on variations in the fragments produced from genomic DNA by EcoRI and visualized after Southern transfer by being probed with a plasmid containing Saccharomyces cerevisiae rDNA. Some of the classes appeared to be heterozygous at the rDNA locus. Similar digestion of other Candida species showed that each could be identified on the basis of its restriction patterns. Since these are highly reiterated genes, the differences were apparent on ethidium bromide-stained gels; Southern transfers were not necessary. EcoRI restriction maps of the rDNA of C. albicans, C. stellatoidea, C. tropicalis, and C. guilliermondii were determined.
Methods are described for extraction of DNA from the yeast form of Candida spp., followed by digestion and electrophoresis of DNA fragments. The resulting gel patterns (greater than 100 bands) were used to type Candida isolates. Four intense bands identified, three of which are present in each isolate (6 to 7, 3.7 or 4.2, and 2.5 to 3 kilobases), appear to be DNA encoding the rRNA. The methods proved to be both simple and reproducible. The patterns were shown to be stable through several hundred doublings from multiple single colonies. A survey of isolates showed that, on the basis of similarity of gel patterns, several Candida species could be sorted into mutually exclusive groups, and subgroups could be created. Analyses of this survey suggested the possible epidemiologic and taxonomic applications of these methods. DNA typing methods appear to offer important potential advantages over phenotyping methods. The methods provide a base for further epidemiologic studies and for further development of techniques, such as the use of cloned probes for studies of DNA homology.
Fungemias were reviewed in 110 immunocompromised patients hospitalized between November 1, 1974, and December 31, 1977, a Memorial Sloan-Kettering Cancer Center (MSKCC). The incidence of Candida tropicalis fungemia increased each year. Seventy-six percent of the patients with C. tropicalis fungemia and 32.5 percent of those with C. albicans fungemia had either leukemia or lymphoma. Seventy-seven percent of the C. parapsilosis fungemias were related to total parenteral nutrition. Thirty-seven percent of the patients with C. albicans fungemia were receiving oral prophylactic nystatin therapy. The source of fungemia was often difficult to determine: in 60 percent of the patients, only blood cultures were positive for C. tropicalis or Torulopsis glabrata; no cultures were positive for the fungus from any other site before the episode occurred. Serologic tests, including a highly sensitive passive hemagglutination test, showed fourfold increases in titer only inconsistently. A passive hemagglutination-inhibition test for circulating antigen was positive in 50.9 percent of 57 patients with fungemia who were tested and may be a valid indication for treatment. Fungemia usually represented a severe and often fatal disease. The over-all mortality of the 110 patients with fungemia was 79 percent whereas only 23 percent of the patients with C. parapsilosis fungemia died. Among the patients who received more than 200 mg of amphotericin B, 71 percent died despite treatment.
CHROMagar Candida is a novel, differential culture medium that is claimed to facilitate the isolation and presumptive identification of some clinically important yeast species. We evaluated the use of this medium with 726 yeast isolates, including 82 isolated directly on the medium from clinical material. After 2 days of incubation at 37 degrees C, 285 C. albicans isolates gave distinctive green colonies that were not seen with any of 441 other yeast isolates representing 21 different species. A total of 54 C. tropicalis isolates also developed distinctive dark blue-gray colonies with a halo of dark brownish purple in the surrounding agar. C. krusei isolates (n = 43) also formed highly characteristic rough, spreading colonies with pale pink centers and a white edge that was otherwise encountered only rarely with isolates of C. norvegensis. Trichosporon spp. (n = 34) formed small, pale colonies that became larger and characteristically rough with prolonged incubation. Most of the other 310 yeasts studied formed colonies with a color that ranged from white to pink to purple with a brownish tint. The only exceptions were found among isolates identified as Geotrichum sp. or Pichia sp., some of which formed colonies with a gray to blue color and which in two instances formed a green pigment or a dark halo in the agar. The specificity and sensitivity of the new medium for the presumptive identification of C. albicans, C. krusei, and C. tropicalis exceeded 99% for all three species. A blinded reading test involving four personnel and 57 yeast isolates representing nine clinically important species confirmed that colonial appearance after 48 h of incubation on CHROMagar Candida afforded the correct presumptive recognition of C. albicans, C. tropicalis, C, krusei, and Trichosporon spp. None of nine bacterial isolates grew on CHROMagar Candida within 72 h, and bacteria (Escherichia coli) grew from only 4 of 104 vaginal, 100 oral, and 99 anorectal swabs. The new medium supported the growth of 19 of 23 dermatophyte fungi tested and 41 of 43 other molds representing a broad range of fungal pathogens and contaminants. In parallel cultures of 348 clinical specimens set up on Sabourand agar and CHROMagar Candida, both media grew yeasts in the same 78 instances. CHROMagar Candida is recommended as a useful isolation medium capable of the presumptive identification of the yeast species most commonly isolated from clinical material and facilitating recognition of mixed yeast cultures.
Two spacer regions outside the ribosomal DNA (rDNA) transcriptional unit in three species of Saccharomyces, S. cerevisiae, S. carlsbergensis and S. pastorianus, were amplified using the polymerase chain reaction. These regions were composed of the 3' external transcribed spacer (ETS) and the intergenic spacer (IGS). Primers were developed from sequence alignments and by taking the reverse complement of a previously described sequence. The region amplified spanned base position 3110 on the 26S rRNA to base position 27 on the 5S rRNA of S. cerevisiae. Nine of the twelve strains used in this study exhibited different restriction profiles, showing that the spacers are highly variable between species. The results suggest that PCR fingerprinting of the non-coding spacer regions can be used to distinguish between closely related Saccharomyces species and may have potential in DNA profiling of other yeasts.
During a study of oral rinses of 130 HIV-infected individuals, both typical and atypical Candida albicans colonies were isolated from ten patients on a yeast differential medium. Typical Candida albicans colonies were light green; atypical colonies were dark green. Both types of colonies were germ tube-positive and produced chlamydospores. However, DNA fingerprinting of the atypical isolates with the Ca3 Candida albicans-specific probe showed that they belonged to the recently described species Candida dubliniensis. Candida dubliniensis colonies could also be differentiated from Candida albicans colonies on isolation plates by the absence of fluorescence of colonies on methyl blue-Sabouraud agar under Wood's light. Among other phenotypic characteristics, only the absence of intracellular beta-glucosidase activity reliably distinguished Candida albicans from Candida dubliniensis. Candida dubliniensis may be underreported in clinical samples because most currently used isolation and identification methods fail to recognize this yeast.
Descriptions of the species, arranged alphabetically
- Barnett