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Comparison of Methods for DNA Extraction from Candida Albicans

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Comparison of Methods for DNA Extraction from Candida Albicans Department of Medical Biochemistry and Microbiology Uppsala University Comparison of methods for DNA extraction from Candida albicans Ashraf Dadgar 2006 Department of Clinical Microbiology, Uppsala University Hospital, SE- 751 85 Uppsala Sweden Supervisor: Åsa Innings and Björn Herrmann ABSTRACT Invasive Candida infection is an increasing cause of morbidity and mortality in the immunocompromised patient. Molecular diagnosis based on genomic amplification methods, such as real time PCR, has been reported as an alternative to conventional culture for early detection of invasive candidiasis. The template DNA extraction step has been the major limitation in most reported nucleic acid based assays, due to problems in breaking fungal cell walls and incomplete purification in PCR inhibitor substances. The aim of this study was to compare enzymatic cell wall disruption using recombinant lyticase with mechanical disruption using glass beads. The QIAamp tissue kit was compared with two automated DNA extraction robots, the BioRobot M48 and NucliSens easyMAG, to determine their sensitivity, reliability and duration for DNA release of C. albicans. Mechanical cell wall disruption shortened and facilitated the extraction procedure, but the quantity of released DNA was significantly lower than when enzymatic cell wall disruption was used. Use of robots did not significantly shorten the DNA extraction time, compared with manual DNA extraction. However the NucliSens easyMAG resulted in a higher yield of target DNA compared to the BioRobot M48 and the manual QIAamp tissue kit. KEYWORDS: Candida albicans, candidemia, DNA extraction, cell wall disruption, real time PCR 2 SAMMANFATTNING Invasiva svampinfektioner är ett stort problem hos patienter med dåligt immunförsvar. Förekomst av invasiva svampinfektioner har ökat under senare år och medför hög dödlighet. En svampinfektion som inte snabbt diagnostiseras och behandlas kan bli livshotande om patientens kondition är dålig. Candida albicans är den vanligaste orsaken till invasiva svampinfektioner. Med traditionell svampidentifiering kan det ta dagar till veckor att isolera och artbestämma svampen. En snabbare metod att detektera Candida är att använda sig av molekylärbiologiska metoder som påvisar svampens arvsmassa, DNA. Svampar har en cellvägg som är svår att bryta ner och därför är DNA extraktionssteget ett av de mest rapporterade problemen vid DNA svampdiagnostik. Syftet med denna studie var att jämföra enzymatisk och mekanisk cellväggsnedbrytning av C. albicans med hjälp av enzymet lyticase respektive glaskulor. Vi jämförde också en manuell metod med två automatiska robotar för att bestämma deras känslighet, tillförlitlighet och tidsåtgång för DNA-extraktion från C. albicans. De slutsatser som nåtts är att den enzymatiska cellväggsnedbrytningen var känsligare men betydligt mer tidskrävande än den mekaniska cellväggsnedbrytningen. Denna studie visade även att en av de automatiska systemen extraherade signifikant mer DNA än den manuella metoden. 3 INTRODUCTION Invasive fungal infection has become a major cause of morbidity and mortality in immunocompromised patients, for example, neuropenic patients with hematological malignancies and recipients of allogeneic bone marrow transplants [1]. The most common fungi causing disease are Candida species [2]. The genus Candida is comprised of more than 200 species, of which approximately a dozen have been associated with human infection. Of these the most important cause of disease is Candida albicans [3]. There is an increasing incidence of bloodstream infections caused by Candida species and this genus now ranks as the fourth most common cause of nosocomial bloodstream infections [4]. A prospective epidemiological survey of candidaemia has been performed in central Sweden from January 1998 to December 1999. Out of a total of 191 reported cases, C. albicans was identified in 128 cases (67%), followed by C. glabrata in 30 (15,7%) and C. parapsilosis in 14 (7,3%) [5]. C. albicans is an opportunistic and a commensal organism that is carried by a large proportion of the population on the mucosal surfaces of the gastrointestinal and urogenital tract without clinical symptoms. C. albicans causes both superficial infections and life threatening systemic candidiasis in immunocompromised hosts, such as AIDS patients, cancer patients and other immunosupressed individuals [6]. Candida virulence factors include the ability to adhere to host tissues, production of tissue damaging secreted enzymes, and morphological changes that may enhance tissue penetration and avoidance of immune surveillance. There is evidence of an important role of mononuclear phagocytes, as well as protective antibodies and T-helper cells, in primary and acquired resistance to systemic and disseminated candidiasis. Neutrophils are still considered to be the most important effector cells. Therefore, sustained neutropenia results in a predisposition to disseminated fungal infection [6]. Candida infections are treated with antifungal agents such as azole drugs, mainly fluconazole, expensive drugs with high incidences of side effects. Treatment of candidiasis patients is further hampered by limited choice of antifungal agents and the appearance of clinical isolates resistant to azole drugs [7]. 4 Non-albicans Candida (NAC) species cause 35-65% of fall candidaemia. NAC species are emerging as both colonizers and pathogens causing nosocomial fungal bloodstream infections. The most common species are C. tropicalis, C. glabrata, C. krusei and C. parapsilosis, which as a group represent about one-half of all Candida spp. isolated from blood cultures. Two general problems are associated with the occurrence of NAC. The virulence and pathogenicity of some NAC species, mainly in the immunocompromised host, results in significant mortality. Another issue of concern is the occurrence of resistance to currently available antifungal drugs [8]. Candida glabrata infections can be mucosal or systemic and are common in immunocompromised persons or those with diabetes mellitus. In contrast to other Candida species, C. glabrata is not dimorphic. As a consequence, it is found as blastoconidia, both as commensal and as a pathogen. Treatment of C. glabrata infections is difficult due to frequent resistant to many azole antifungal agents, especially fluconazole [9]. Candida krusei is often seen in leukaemic patients and bone marrow transplant recipients. It is rare in surgical and intensive care patients and neonates. In general, C. krusei is primarily resistant to fluconazole but sensitive to itraconazole, ketoconazole and amphotericin [8]. Candida tropicalis is the four most commonly isolated NAC species after C. glabrata , C. parapsilosis and C. krusei. The incidence of infection due to C. tropicalis does not seem to be increasing. C. tropicalis is seen more frequently in cancer patients. Animal models indicate that the virulence and pathogenicity of C. tropicalis is at least as virulent as C. albicans. C. tropicalis was early found to have lower sensitivity to ketokonazole and moconazole. Lately also fluconazole and amfotericin B resistance have been reported [8]. 5 In 1995, a new Candida species, C. dubliniensis, closely related to C. albicans, was identified in cases of oral candidiasis in HIV-infected individuals. Despite the very close phylogenetic relationship between C. albicans and C. dubliniensis and the fact that they share a large number of phenotypic traits, epidemiological and virulence model data indicate that the former is a far more successful pathogen. Resistance to fluconazole has been reported with C. dubliniensis [3]. C. parapsilosis causes 17-50% of fungaemia in children compared with 2,5-12% in adult surgical or intensive care unit populations. C. parapsilosis has the lowest mortality of all NAC, 8%, compared to 21% for C. albicans, 40% for C. glabrata and 30% C. krusei. C. parapsilosis is sensitive to most available anti mycotic drugs on the market [8]. Candida gulliermondi rarely causes infection in human. No specific risk factors have been described for C. gulliermondi. There are no data on virulence and pathogenicity in comparison with C. albicans or other NAC species, but C. gulliermondi can undoubtedly cause invasive infections in man. C. gulliermondi is sensitive to fluconazole [8]. As candidiasis incidence continue to rise, quick laboratory identification of Candida is becoming increasingly important for a growing population of patients at-risk. Early initiation of antifungal therapy is critical in reducing the high mortality rate in these patients. The traditional reference method for detection of blood infections is blood culture. Significant improvements have been made over the last decades with this method. For example optimization of culture media attempting to shorten the turnaround time to detection of negative or positive results, and to increase the strength and yield for the assay. Culture detection is often delayed because of slow or absent growth of fungal isolate from clinical specimens. Blood cultures are positive for fewer than 50% of patients with hepatosplenic candidiasis [10]. C. albicans can be identified by germ tube information tests. C. albicans, C. krusei and C. tropicalis can presumptively be identified by growth on CHROMagar medium, and 6 other species of Candida can be identified by rapid (in 4 h) enzymatic tests. However each of these procedures requires the organism to be grown on solid medium for at least 24 h, and more often 48 h, before such tests
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