Candidemia in pediatric outpatients receiving home total parenteral nutrition.

This is a cohort study of pediatric outpatients receiving total parenteral nutrition (TPN) and follow-up care in a Tennessee hospital between January and June 1999. The study was conducted following an increase in the incidence of candidemia. Of 13 children receiving home TPN, five had candidemia; three were due to Candida parapsilosis. Case patients were more likely to have an underlying hematologic disease (P = 0.02) as well as previous history of fungemia (P = 0.02). Two case patients had successive candidemia episodes 3 months apart; karyotypes and RAPD profiles of each patient's successive C. parapsilosis isolates were similar. Candida spp. were frequently detected in hand cultures from cohort members (four of 10) and family member caregivers (nine of 11); C parapsilosis was isolated from five caregivers. Our findings underscore the challenges of maintaining stringent infection control practices in the home health care setting and suggest the need for more intensive follow-up and coordination of home TPN therapy among pediatric patients.

Molecular epidemiology of Candida albicans strains isolated from the oropharynx of HIV-positive patients at successive clinic visits.

Candida albicans strain diversity and fluconazole resistance were prospectively analyzed in oral strains from 29 adult human immunodeficiency virus (HIV)-positive patients followed for > 1 year who had five or more culture-positive clinic visits. Molecular typing consisted of genomic blots probed with the Ca3 repetitive element. Sixteen patients had one or more episodes of oropharyngeal candidiasis (OPC), 12 (75%) maintained the original genotype, whereas the remaining four patients had a succession of 2-3 genotypes. The original genotype, either alone or mixed with another strain or with non-C. albicans Candida spp., was recovered from oral lesions in 13 of 15 evaluable (86.7%) patients. C. dubliniensis was the infecting yeast in the remaining two patients. Different patterns of fluconazole resistance occurred in three OPC patients. One patient's infecting strain became less susceptible. A second patient was infected with a resistant genotype and a progressively more susceptible minor genotype variant. C. dubliniensis isolates from the third patient varied in susceptibility. Thirteen colonized patients who never developed OPC harbored a greater variety of C. albicans genotypes (2-6) than their infected counterparts (P = 0.35). OPC patients maintained their original endogenous C. albicans strains for prolonged periods, whether or not they demonstrated decreased in vitro susceptibility to fluconazole. The adaptation and maintenance of an endogenous C. albicans strain within its host may be linked to as yet uncharacterized factors.

Postsurgical Candida albicans infections associated with an extrinsically contaminated intravenous anesthetic agent.

From 16 to 30 April 1990, four of 364 (1%) postsurgical patients at one hospital developed Candida albicans fungemia or endophthalmitis. The case patients' surgeries were clustered on two days. To identify risk factors for C. albicans infections, we conducted a cohort study comparing these 4 patients with 67 control patients who had surgeries on the same days but did not acquire C. albicans infections. The participation of anesthesiologist 9 (relative risk [RR], undefined; P < 0.001) and receipt of intravenous propofol, an anesthetic agent without preservative, which was administered by an infusion pump (RR, 8.8; P = 0.048) were identified as risk factors for C. albicans infections. The anesthetic had been recently introduced in the hospital. Hand cultures of 8 of 14 (57%) anesthesiologists were positive for Candida species; one yielded C. albicans. Anesthesiologist 9 was the only one to use stored syringes of propofol in the infusion pump and to reuse propofol syringes. DNA fingerprinting with a digoxigenin-labeled C. albicans repetitive element 2 probe and electrophoretic karyotyping showed two distinct banding patterns among patient isolates. We hypothesize that extrinsic contamination of propofol by anesthesiologist 9 likely resulted in C. albicans infections. These data suggest that strict aseptic techniques must be used when preparing and administering propofol.

Molecular cloning, characterization, and expression of the M antigen of Histoplasma capsulatum.

The major diagnostic antigens of Histoplasma capsulatum are the H and M antigens, pluripotent glycoproteins that elicit both humoral and T-cell-mediated immune responses. These antigens may play a role in the pathogenesis of histoplasmosis. M antigen is considered immunodominant because antibodies against it are the first precipitins to arise in acute histoplasmosis and are commonly present during all phases of infection. The biological activity of monomolecular M antigen and its ability to elicit a protective immune response to H. capsulatum are largely unknown. A molecular approach was used to identify the biological nature of M antigen, including its purification from histoplasmin, partial digestion with proteinases, and reverse-phase high-performance liquid chromatography to separate the released peptides. The amino acid sequences of the purified peptides were obtained by Edman degradation, and using degenerate oligonucleotide primers for PCR, a 321-bp fragment of the gene encoding the M antigen was amplified from genomic H. capsulatum DNA. This fragment was used to screen an H. capsulatum genomic DNA library, leading to the isolation, cloning, and sequencing of the full-length gene. The M gene consists of 2, 187-bp DNA encoding a protein of 80,719 Da, which has significant homology to catalases from Aspergillus fumigatus, Aspergillus niger, and Eimericella nidulans. A cDNA was generated by reverse transcription-PCR and cloned into the expression vector pQE40. The identity of the cloned, expressed protein was confirmed by Western blotting. The recombinant fusion protein was immunoreactive with monoclonal antibodies raised against M antigen, with polyclonal mouse anti-M antiserum, and with a serum sample from a patient with histoplasmosis. The gene encoding the major immunodominant M antigen of H. capsulatum is a presumptive catalase, and the recombinant protein retains serodiagnostic activity.

Rapid identification of Candida species with species-specific DNA probes.

Rapid identification of Candida species has become more important because of an increase in infections caused by species other than Candida albicans, including species innately resistant to azole antifungal drugs. We previously developed a PCR assay with an enzyme immunoassay (EIA) format to detect amplicons from the five most common Candida species by using universal fungal primers and species-specific probes directed to the ITS2 region of the gene for rRNA. We designed probes to detect seven additional Candida species (C. guilliermondii, C. kefyr, C. lambica, C. lusitaniae, C. pelliculosa, C. rugosa, and C. zeylanoides) included in the API 20C sugar assimilation panel, five probes for species not identified by API 20C (C. haemulonii, C. norvegica, C. norvegensis, C. utilis, and C. viswanathii), and a probe for the newly described species C. dubliniensis, creating a panel of 18 Candida species probes. The PCR-EIA correctly identified multiple strains of each species tested, including five identified as C. albicans by the currently available API 20C database but determined to be C. dubliniensis by genotypic and nonroutine phenotypic characteristics. Species identification time was reduced from a mean of 3.5 days by conventional identification methods to 7 h by the PCR-EIA. This method is simple, rapid, and feasible for identifying Candida species in clinical laboratories that utilize molecular identification techniques and provides a novel method to differentiate the new species, C. dubliniensis, from C. albicans.

Sequence analysis of the internal transcribed spacer 2 (ITS2) from yeast species within the genus Candida.

Nucleotide sequences of the internal transcribed spacer 2 (ITS2) regions were determined for 13 species within the genus Candida, representing a collection of those species pathogenic for humans. No two species had identical sequences and the sizes of ITS2 varied fourfold, representing an apparent continuous gradient of nucleotides. When present, sequence homologies were observed in the 5' end of ITS2, and many species exhibited more limited homologies within three known conserved domains found in other yeasts. Cluster analysis of primary sequence revealed a concordance with a known taxonomic subfamily and suggests that certain species within the genus form a similar grouping. A majority of species exhibited similar presumptive RNA secondary structures, consistent with the hypothesis that these spacer regions are essential for correct processing of the 5.8S and 28S subunits.

Comparison of 5.8S and ITS2 rDNA RFLP patterns among isolates of Acremonium obclavatum, A. kiliense, and A. strictum from diverse sources.

Polymerase chain reaction (PCR) products of nuclear 5.8S and internal transcribed spacer regions (ITS2) of rDNA from reference cultures of Acremonium obclavatum (a rarely recognized species first reported from India) were compared with cultures of Acremonium spp. isolated from Georgia, USA. Digestion of amplicons sequentially with Hinfl and Sau3AI divided the isolates into four restriction fragment length polymorphism (RFLP) groups. A representative isolate of primary colonizers of insulation facings from a building in Georgia appeared identical to the type culture of A. obclavatum, whereas other cultures from Indian soils showed variation in the ITS2 region that divided them into further subgroups. Reference cultures of A. kiliense (ATCC 14489) and A. strictum (ATCC 10141) and two additional isolates from metropolitan Atlanta, assigned to this latter species complex on a morphological basis, represented two additional RFLP groups both of which were distinct from the RFLP groups in A. obclavatum. A. kiliense and A. strictum could be placed into similar subgroups on the basis of morphological differences and distinct RFLP patterns.

Candida parapsilosis bloodstream infections in neonatal intensive care unit patients: epidemiologic and laboratory confirmation of a common source outbreak.

BACKGROUND: Candida parapsilosis is a common cause of sporadic and epidemic infections in neonatal intensive care units (NICUs). When a cluster of C. parapsilosis bloodstream infections occurred in NICU patients in a hospital in Louisiana, it provided us with the opportunity to conduct an epidemiologic investigation and to apply newly developed molecular typing techniques. METHODS: A case-patient was defined as any NICU patient at Louisiana State University Medical Center, University Hospital, with a blood culture positive for C. parapsilosis during July 20 to 27, 1991. To identify risk factors for C. parapsilosis bloodstream infection, a cohort study of all NICU infants admitted during July 17 to 27, 1991, was performed. Electrophoretic karyotyping was used to assess the relatedness of C. parapsilosis isolates. RESULTS: The receipt of liquid glycerin given as a suppository was identified as a risk factor (relative risk, 31.2; 95% confidence intervals, 4.3 to 226.8). Glycerin was supplied to the NICU in a 16-oz multidose bottle. Bottles used at the time of the outbreak were not available for culture. All six available isolates from four case-patients had identical chromosomal banding patterns; six University Hospital non-outbreak isolates had different banding patterns. CONCLUSIONS: This study demonstrates the utility of combined epidemiologic and laboratory techniques in identifying a novel common source for a C. parapsilosis bloodstream infection outbreak and illustrates that extreme caution should be exercised when using multidose medications in more than one patient.

Microtitration plate enzyme immunoassay to detect PCR-amplified DNA from Candida species in blood.

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.

Nucleotide sequence analysis of the 5.8S rDNA and adjacent ITS2 region of Candida albicans and related species.

We have determined the nucleotide sequence for the DNA encoding the 5.8S RNAs and downstream internal transcribed spacer (ITS2) regions for Candida albicans and the taxonomically related species C. parapsilosis, C. tropicalis, C. glabrata and C. krusei. Phylogenetic analysis of all known fungal 5.8S RNA sequences revealed a close relationship between C. tropicalis and C. parapsilosis, and to a lesser extent C. albicans within the yeast-like fungi. This group can itself be delineated from predominantly filamentous species. The more distal relationships between Candida (Torulopsis) glabrata and C. krusei support previous findings based on small (18S) ribosomal RNA sequence analysis, suggesting a greater degree of evolutionary divergence of these species from the C. albicans group. Among strains of C. albicans we observed conservation of the ITS2 region at the nucleotide level. Conservation was also observed for a more limited number of C. parapsilosis strains. Although the 3' region of the ITS spacer was species specific, sequence homology was observed in the 5' end within the albicans/parapsilosis/tropicalis group. Our findings suggest a rapid approach to species identification through the use of non-conserved regions flanked by highly conserved, functional domains.

Genomic heterogeneity in the yeast Candida parapsilosis.

Candida parapsilosis shows a wide intraspecies variation in chromosome/homolog size distribution. As a prerequisite for delineating modes of transmission, we have undertaken an analysis of genetic variation at different levels. In the present study we have observed that a majority of isolates display similar electrophoretic karyotype patterns consistent for the species, with variations in the smaller group of chromosomes. In two strains we observed phenotypic "switching"; one of these also exhibited a mixed karyotypic subpopulation. In contrast, a few isolates displayed a greater degree of chromosome/homolog size variation. We also observed, through randomly amplified polymorphic DNA (RAPD) analysis, results consistent with those of pulsed-field electrophoresis. Isolates displaying a high degree of chromosome/homolog variation also displayed a high degree of variation in genomic "fingerprints". Polymorphisms, although present, were much reduced in the majority of isolates. These parallel observations suggest a common underlying mechanism. Our results are consistent with the hypothesis that chromosome-sized variations in C. parapsilosis are due to random genetic events. A similar mechanism has been hypothesized for the taxonomically related yeast Candida albicans.

Persistence of the same Candida albicans strain despite fluconazole therapy. Documentation by pulsed-field gel electrophoresis.

Candida albicans and other Candida species have emerged as major nosocomial pathogens associated with a high mortality. Therapeutic options for fungal infections are limited. Amphotericin B has been the mainstay of treatment for serious systemic candidal infections, but it is relatively toxic and associated with a variety of side effects. Fluconazole has been proposed as alternative therapy for the treatment of systemic candidiasis including candidemia. We report the case of a patient with fungemia in whom fluconazole failed to eradicate C. albicans and C. tropicalis. These pathogens were recovered from sputum and urine cultures, respectively, on day 12 of intravenous fluconazole therapy. Molecular epidemiologic techniques employing pulsed-field gel electrophoresis confirmed the persistence of the same C. albicans strain. Susceptibility studies showed a marked change in MICs of fluconazole between 24 and 48 hr, with an increase from less than or equal to 1.25 to greater than 80 micrograms/ml. Controlled trials will be needed to delineate the role of fluconazole in the treatment of disseminated candidiasis and its efficacy in comparison with amphotericin B. Amphotericin B should remain the drug of choice for such infections until data from controlled trials are available.

Isolation, characterization, and sequencing of Candida albicans repetitive element 2.

A 1059-bp Sau3A fragment, designated Candida albicans repetitive element 2 (CARE-2), was isolated from the genome of the pathogenic yeast, C. albicans. CARE-2 DNA was detected on several C. albicans chromosomes separated by transverse alternating-field electrophoresis. A high degree of interstrain variation in the pattern of hybridizing bands were observed by Southern blot analysis, with a minimum of 10-14 copies of CARE-2 per strain. A low frequency of new CARE-2 polymorphisms was observed over time for three strains grown at 25 degrees C or 37 degrees C. No new CARE-2 polymorphisms were observed from two naturally occurring switch phenotypes. To localize repeated DNA, oligodeoxyribonucleotide probes, each representing a different region of CARE-2, were hybridized to genomic blots. A lower number of copies were observed 5' and 3' to a 600-bp region of CARE-2. Nucleotide (nt) sequence analysis of CARE-2 DNA shows the element is characterized by six perfect direct repeats 6 bp in length and shows no significant DNA similarity with any known nt sequence.

Characterization of CARE-1: Candida albicans repetitive element-1.

A middle repetitive DNA element, Candida albicans repetitive element-1 (CARE-1) has been isolated from the pathogenic yeast C. albicans. CARE-1 appears to be species-specific and constitutes approx. 0.045% of total C. albicans DNA, or a reiteration frequency of about two to twelve copies per haploid genome. The CARE-1 element has been detected on several C. albicans chromosomes separated by field-inversion gel electrophoresis, suggesting that the element is dispersed. Interstrain variation was observed in the number and distribution of hybridizing bands. The element is well conserved, since no nucleotide (nt) heterogeneity was observed when the sequences of two CARE-1 family members isolated from two different chromosomes (A and B) of C. albicans were compared. CARE-1 possesses 467 bp and is characterized by several stretches of A's and T's, short direct repeats and shows no significant homology to any known nt sequence.

Electrophoretic karyotyping of typical and atypical Candida albicans.

Electrophoretic karyotypes of atypical isolates of Candida albicans, e.g., strains that were germ tube negative, failed to express proteinase activity, demonstrated low virulence for mice, formed hyperchlamydoconidia, produced hyperhyphae, or were sucrose negative (including the type strain of Candida stellatoidea), were compared with those of typical C. albicans. Karyotypes of whole-cell DNA of classical C. albicans examined with transverse alternating-field electrophoresis under specific conditions were composed of seven DNA bands with a specific migration pattern. Certain atypical strains and representatives of the three serotypes of C. stellatoidea produced discrete karyotypes with 5 to 10 bands. All isolates demonstrated a significant degree of DNA relatedness, suggesting their conspecificity. Densitometric tracings of DNA bands provided an objective and standardized method for comparing bands within the gels.

An electrophoretic karyotype for Candida albicans reveals large chromosomes in multiples.

Using field-inversion gel electrophoresis we defined an electrophoretic karyotype for the yeast, Candida albicans. The karyotype is distinct from other species of Candida and is species specific. A total of five distinct chromosomal mobility groups were observed, at least four of which are composed of a minimum of two fragments each. From the apparent sizes of these fragments relative to the large chromosomes of the morphologically related yeast Saccharomyces cerevisiae, together with the known genome size of this organism, we conclude that the karyotype is the result of the migration of intact chromosomes.

Three-dimensional similarity mapping reveals nucleotide sequence relationships among human immunodeficiency virus (HIV) isolates.

A method has been developed to compare and graphically display comparisons between many related nucleotide sequences. Principal coordinate analysis, a multidimensional scaling technique, was used to display DNA homology data in three dimensions. Using these methods, an analysis of the env, gag, and gag/pol overlapping regions of human immunodeficiency virus (AIDS virus) clearly demonstrates a clustering of North American isolates relative to African isolates. Elements of the latter group are dissimilar to one another and to the North America group. For the env region, the North American isolates can be resolved into two distinct groups.

The use of microcomputers for the quantitation of light intensity patterns using digitized video signals.

We have developed an inexpensive yet versatile microcomputer-based system for quantitating light intensity levels in autoradiographs. This system employs a standard video camera interfaced to an analog-to-digital convertor. A program has been written for this system which can measure intensities within a defined region of an autoradiograph, permitting an easy and accurate quantitation of spots or bands of irregular shape.