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Query: EC:3.1.30.2 (endonuclease)
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The availability of an epidemiologic typing system for Candida species that is sensitive, rapid, inexpensive, and easy to perform would clearly be an advantage to the mycologist, microbiologist, and epidemiologist in the ongoing struggle to understand the epidemiology and pathogenesis of candidiasis. This is particularly true given the increasing prominence of organisms such as C. albicans and C. tropicalis which are ubiquitous members of the normal flora yet are also important causes of nosocomial bloodstream infection. Unfortunately, the ideal epidemiologic typing system does not yet exist. Current data suggest that the molecular typing methods of restriction endonuclease digestion of genomic DNA with ethidium bromide staining (DEtBr typing) and electrophoretic karyotyping using pulsed-field electrophoresis offer rapid, simple, and sensitive means of discriminating strains of Candida species. These methods appear at present to be the most practical typing methods for both large- and small-scale epidemiologic studies. Other typing methods using specific DNA probes provide a powerful means of identifying strains and will undoubtedly be applied more broadly in the future. Thus far, studies employing molecular typing methods have documented that (1) most patients are colonized by one strain of Candida species, (2) isolates of Candida species recovered from blood or deep tissue sites are generally identical to those obtained from colonization sites before infection developed, and (3) nosocomial transmission of a single strain of C. albicans may occur, particularly in an intensive care unit setting. Given the limitations of the available typing methods and the complex nature of the patients at risk for candidiasis, both the epidemiologist and laboratory scientist must use these methods with clear epidemiologic objectives in mind. Whenever possible, all organisms to be typed should be typed by the same person on the same day, and typing should always include unrelated as well as epidemiologically related isolates. Additional studies, based upon sound epidemiologic principles, will be necessary to clarify the role of the various molecular typing methods as epidemiologic markers of Candida species and to further our understanding of the epidemiology and pathogenesis of candidiasis.
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PMID:The use of molecular techniques for epidemiologic typing of Candida species. 173 71

An attempt was made to produce DNA probes that could be used as a rapid and efficient means of detecting candidiasis (invasive Candida infection) in immunocompromised patients. Whole DNA from Candida albicans was digested with restriction endonuclease, and the resulting fragments were randomly cloned into a plasmid vector. Several recombinant plasmids were evaluated for cross-hybridization to various other Candida species, other fungal DNAs, and to nonfungal DNAs. Cross reactions were observed between the probes and different yeasts, but none with unrelated DNAs. Some recombinants were genus-specific, and two of these were applied to the analysis of C. albicans growth curves. It became evident that, although both 32P- and biotin-labelled probes could be made quite sensitive, a possible limitation in their diagnostic potential was the poor liberation of Candida DNA from cells. Thus, better methods of treatment of clinical specimens will be required before such probes will be useful in routine diagnosis.
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PMID:Development of DNA probes for Candida albicans. 306 59

During a six-month period, 600 gynecological samples were collected from 585 women with typical herpes lesions, women with non-herpes symptoms (ie, vaginitis, moniliasis, trichomoniasis, etc), and normal women seen at the student health center gynecological clinic and processed for herpes simplex virus (HSV) isolation. From these specimens, 29 samples from 25 of the 585 women (4.3%) were positive for HSV. When these isolates were typed using plaque diameter in chick cells, heat stability of viral thymidine kinase (T.K.), and restriction endonuclease patterns it was found that 18 samples (15 patients or 60%) were HSV-2 and 11 samples (10 patients or 40%) were HSV-1. Inapparent HSV infections constituted 20.0% of the virologically confirmed samples (5 of 25 patients) and represented 0.9% of the total patients studied (5 of 585). The inapparent infections were about equally divided between the two HSV types (2 were HSV-2 and 3 were HSV-1), and 4 of 5 occurred in the presence of clinically diagnosed monilia.
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PMID:Inapparent genital herpes simplex virus infection in college women. 629 59

Sixty-two Candida albicans isolates from the oral cavities of 28 patients with AIDS who were receiving fluconazole therapy were typed by restriction endonuclease analysis followed by pulsed-field gel electrophoresis; these isolates were then tested for fluconazole susceptibility by a standard broth dilution method. Sequential isolates (range, 2-4) were evaluated for 22 patients; only one isolate was evaluated for six patients. DNA subtyping revealed a total of 37 different DNA subtypes. Twelve (54.5%) of 22 patients with multiple episodes of oropharyngeal candidiasis were infected with a single DNA subtype throughout the observation period. Ten (45.5%) of 22 patients with multiple episodes of oropharyngeal candidiasis were infected with two or three DNA subtypes during the observation period. In vitro susceptibility tests revealed that MICs of fluconazole ranged from < or = 0.125 microgram/mL to 64 micrograms/mL, with an MIC50 of 0.5 microgram/mL and an MIC90 of 4 micrograms/mL. A significant increase in the MICs (fourfold or greater) of fluconazole for sequential C. albicans isolates was found for 66.6% of the patients infected with a single DNA subtype and for 50% of the patients infected with multiple DNA subtypes. Despite a limited number of patients and isolates, our data suggest that C. albicans isolates that are susceptible to fluconazole at MICs of > or = 8 micrograms/mL in vitro will be less susceptible in vivo to standard doses (100-200 mg/d) of this drug.
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PMID:DNA subtypes and fluconazole susceptibilities of Candida albicans isolates from the oral cavities of patients with AIDS. 775 88

Electrophoretic karyotype and restriction endonuclease analysis of genomic DNA were used for the typing of nine isolates of Candida albicans from the oral cavities of two patients with AIDS--a husband and wife--whose infections became resistant to treatment with fluconazole (400 mg/d). The in vitro susceptibilities of sequential isolates to fluconazole and two other triazoles, itraconazole and the investigational drug D0870, were also evaluated. DNA analysis showed that the isolates responsible for fluconazole-resistant episodes of oropharyngeal candidiasis in the two patients were genetically related. In vitro susceptibility to fluconazole correlated well with clinical outcome. Although the minimal inhibitory concentrations of itraconazole and D0870 for fluconazole-resistant isolates were higher than those for fluconazole-susceptible isolates, both of the former triazoles exhibited good in vitro activity against the isolates tested.
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PMID:Transmission of fluconazole-resistant Candida albicans between patients with AIDS and oropharyngeal candidiasis documented by pulsed-field gel electrophoresis. 852 44

Candida tropicalis has been known to be a major cause of invasive Candida infection. Numerous reports have documented C. tropicalis as the most common species of Candida other than C. albicans. The epidemiology and antifungal susceptibility of C. tropicalis are poorly defined. A series of 89 clinical isolates of C. tropicalis from 56 patients hospitalized at seven different U.S. medical centers were analyzed by restriction endonuclease analysis of genomic DNA (REAG) using the restriction enzymes Sfil and BssHII followed by pulsed-field gel electrophoresis (PFGE). The MICs of the isolates for amphotericin B, 5-fluorocytosine (5FC), fluconazole, itraconazole, and D0870 were determined by microbroth dilution testing. A total of 49 different DNA types were identified among the 89 isolates. Generally, each DNA type represented an individual patient, and serial isolates from the same patient were the same DNA type. Small clusters of patients infected with the same DNA type of C. tropicalis suggested possible nosocomial transmission. The MICs of the various antifungal agents were amphotericin B 0.5 to 2.0 micrograms/ml (MIC90 = 2.0 micrograms/ml), 5FC 0.25 to 1.0 microgram/ml (MIC90 = 0.5 microgram/ml), fluconazole 0.25 to 8.0 micrograms/ml (MIC90 = 1.0 microgram/ml), itraconazole 0.03 to 1.0 microgram/ml (MIC90 = 0.5 microgram/ml), and D0870 0.007 to 0.12 microgram/ml (MIC90 = 0.03 microgram/ml). These data support previous observations that infections caused by C. tropicalis frequently originate from the patient's own endogenous flora. Clusters of a single strain in individual hospitals also suggests that limited nosocomial transmission may occur.
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PMID:Variations in DNA subtype and antifungal susceptibility among clinical isolates of Candida tropicalis. 914 6

Candida krusei is inherently resistant to fluconazole and is an important pathogen responsible for nosocomial candidiasis especially in patients with hematological malignancy. Despite the growing clinical importance of C. krusei infections, little is known of its genetic diversity and molecular epidemiology. Therefore, differentiating between C. krusei isolates is of importance for a better understanding of the epidemiology, mode of transmission and pathogenesis of the organism. We investigated the use of two different methods (restriction endonuclease analysis of genomic DNA (REAG) with Hinfl and polymerase chain reaction by using Arno1 and Arno2 primers) for molecular typing of 56 C. krusei isolates from 56 patients. Ten different types (A-J) were determined by REAG. Depending on the patterns of isolates, the number of the bands varied from 12 to 15 and the size of the fragments varied from 2.0 kb to 6.2 kb. Of the isolates 71.4% were gathered under three major patterns (D, F, H). In the second method, PCR amplified different sizes of fragments varied approximately from 1 kb to 2 kb, which yielded 13 types (a-m) from 56 patients. Four major patterns (d, f, h, k) were observed for 58.9% of the isolates. The genotypes detected by REAG and PCR methods were found to be same in 43 isolates out of 56. As the banding patterns of the isolates were found to be similar in this study, it was thought that an exogenous origin could be the source of infections caused by C. krusei isolates. Both REA of genomic DNA and PCR analysis seem to be useful for the typing of C. krusei, however PCR assay can be preferred as it is a simple and rapid method. As a result, further studies are required for the validation of reproducibility and discriminatory power of these methods.
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PMID:[Molecular typing of clinical Candida krusei isolates by using restriction endonuclease analysis of genomic DNA and polymerase chain reaction]. 1914 85

We describe a case of congenital acquired candidiasis in a preterm female delivered through Caesarean section due to the premature rupture of the amniotic membrane. The neonate presented with suspected chorioamnionitis and erythematous desquamative skin. Candida albicans was isolated from the placenta, mouth, groin, and periumbilical lesions. The infant developed candidemia due to Candida albicans and the same yeast was also isolated from a catheter. Culture inoculated with swabs from the mouth and vagina of the mother yielded C. albicans and C. krusei. All C. albicans isolates from the mother and the neonate were visually indistinguishable by molecular typing techniques which included chromosomal karyotyping and restriction endonuclease analysis followed by pulsed-field gel electrophoresis. These findings allowed the clinical condition to be confirmed as congenital acquisition of candidiasis in this case.
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PMID:Congenital candidiasis: confirmation of mother-neonate transmission using molecular analysis techniques. 1930 15

Nosocomial invasive candidiasis (IC) has emerged as a major problem in neonatal intensive care units (NICUs). We investigated herein the temporal clustering of six cases of neonatal IC due to Candida albicans in an NICU. Eighteen isolates obtained from the six neonates and two isolates from two health care workers (HCWs) working at the same unit and suffering from fingers' onychomycosis were genotyped by electrophoretic karyotyping (EK) and restriction endonuclease analysis of genomic DNA by using Sfi I (PFGE-Sfi I). PFGE-Sfi I was more effective in discriminating between temporally related isolates. It showed that (i) both HCWs had specific strains excluding them as a source of infections in neonates. (ii) Isolates collected from three neonates were identical providing evidence of their clonal origin and the occurrence of a horizontal transmission of C. albicans in the unit. (iii) The three remaining neonates had specific strains confirming that the IC cases were coincidental. (iv) Microevolution occurred in one catheter-related candidemia case. Our results illustrate the relevance of the molecular approach to investigate suspected outbreaks in hospital surveys and the effectiveness of PFGE-Sfi I for typing of epidemiologically related C. albicans isolates.
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PMID:Investigation of a cluster of Candida albicans invasive Candidiasis in a neonatal intensive care unit by pulsed-field gel electrophoresis. 2254 75

Type 1 diabetes mellitus (DM) and Graves' disease are autoimmune diseases, and a number of genetic factors, including HLA and CTLA-4 genes, have been reported to contribute to their etiology. The gene responsible for autoimmune polyendocrinopathy- candidiasis-ectodermal dystrophy (APECED) has been cloned and named the autoimmune regulator-1 (AIRE-1) gene. AIRE-1 protein is thought to be a transcription regulatory protein and to have a role in the maintenance of immunological tolerance. The aim of this study was to determine whether heterozygous AIRE-1 gene mutations are associated with childhood-onset type 1 diabetes and Graves' disease in the Japanese population. We investigated 46 children with type 1 DM (29 females and 17 males; age at the time of diagnosis, 0.5-16 yr) and 44 children with Graves' disease (34 females and 10 males; age at the time of diagnosis, 3-16 yr) for the presence of the K83E mutation in exon 2 and the R257X mutation in exon 6 of the AIRE-1 gene. The alleles were identified by polymerase chain reaction of genomic DNA and restriction fragment-length polymorphism analysis (PCR-RFLP) with endonuclease TaqI. Since no patients with type 1 DM or Graves' disease were found to carry the K83E or the R257X heterozygous mutation, we concluded that neither the K83E nor the R257X heterozygous mutation in the AIRE-1 gene seem to be the cause of the more common isolated endocrinopathies, i.e., type 1 diabetes mellitus and Graves' disease, in Japanese children.
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PMID:Absence of Heterozygous K83E and R257X Mutations of the AIRE-1 Gene in 46 Children with Type 1 Diabetes and 44 Children with Graves' Disease. 2479 Mar 5


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