Timothy J. Lott

Epidemiologic and Molecular Characterization of an Outbreak of Candida parapsilosis Bloodstream Infections in a Community Hospital

ABSTRACT Candida parapsilosis is an important cause of bloodstream infections in the health care setting. We investigated a large C. parapsilosis outbreak occurring in a community hospital and conducted a case-control study to determine the risk factors for infection. We identified 22 cases of bloodstream infection with C. parapsilosis: 15 confirmed and 7 possible. The factors associated with an increased risk of infection included hospitalization in the intensive care unit (adjusted odds ratio, 16.4; 95% confidence interval, 1.8 to 148.1) and receipt of total parenteral nutrition (adjusted odds ratio, 9.2; 95% confidence interval, 0.9 to 98.1). Samples for surveillance cultures were obtained from health care worker hands, central venous catheter insertion sites, and medical devices. Twenty-six percent of the health care workers surveyed demonstrated hand colonization with C. parapsilosis, and one hand isolate was highly related to all case-patient isolates by tests with the DNA probe Cp3-13. Outbreak strain isolates also demonstrated reduced susceptibilities to fluconazole and voriconazole. This largest known reported outbreak of C. parapsilosis bloodstream infections in adults resulted from an interplay of host, environment, and pathogen factors. Recommendations for control measures focused on improving hand hygiene compliance.

Assessment of Ribosomal Large-Subunit D1-D2, Internal Transcribed Spacer 1, and Internal Transcribed Spacer 2 Regions as Targets for Molecular Identification of Medically Important Aspergillus Species

ABSTRACT Molecular approaches are now being developed to provide a more rapid and objective identification of fungi compared to traditional phenotypic methods. Ribosomal targets, especially the large-subunit RNA gene (D1-D2 region) and internal transcribed spacers 1 and 2 (ITS1 and ITS2 regions), have shown particular promise for the molecular identification of some fungi. We therefore conducted an assessment of these regions for the identification of 13 medically important Aspergillus species: Aspergillus candidus, Aspergillus (Eurotium) chevalieri, Aspergillus (Fennellia) flavipes, Aspergillus flavus, Aspergillus fumigatus, Aspergillus granulosus, Aspergillus (Emericella) nidulans, Aspergillus niger, Aspergillus restrictus, Aspergillus sydowii, Aspergillus terreus, Aspergillus ustus, and Aspergillus versicolor. The length of ribosomal regions could not be reliably used to differentiate among all Aspergillus species examined. DNA alignment and pairwise nucleotide comparisons demonstrated 91.9 to 99.6% interspecies sequence identities in the D1-D2 region, 57.4 to 98.1% in the ITS1 region, and 75.6 to 98.3% in the ITS2 region. Comparative analysis using GenBank reference data showed that 10 of the 13 species examined exhibited a ≤1-nucleotide divergence in the D1-D2 region from closely related but different species. In contrast, only 5 of the species examined exhibited a ≤1-nucleotide divergence from sibling species in their ITS1 or ITS2 sequences. Although the GenBank database currently lacks ITS sequence entries for some species, and major improvement in the quality and accuracy of GenBank entries is needed, current identification of medically important Aspergillus species using GenBank reference data seems more reliable using ITS query sequences than D1-D2 sequences, especially for the identification of closely related species.

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.

Towards understanding the evolution of the human commensal yeast Candida albicans

Allelic frequencies and relationships for one dimorphic locus and three unlinked polymorphic loci have been determined for 114 unrelated isolates of Candida albicans, including 14 laboratory reference strains and 50 strains from each of two geographic regions. Although there was no indication of geographical partitioning, there were significant correlations for specific allelic pairs among loci and little evidence that any alleles were in Hardy-Weinberg equilibrium. This gives additional support for the concept that the primary mode of genetic inheritance in this species is clonal, with other intracellular genetic events playing a lesser role in the creation of genomic diversity. Through inference of this and other known attributes of closely related Candida species, such as sequence analysis of IS1 and the ITS2 (internal transcribed spacer 2) region of the rDNA cistron, the deduced phylogeny suggests an evolutionarily recent origin for many frequently isolated strains. This finding will be of interest in the context of understanding pathogenicity and drug resistance in this human commensal yeast.

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.

Molecular genotyping of Candida parapsilosis group I clinical isolates by analysis of polymorphic microsatellite markers.

ABSTRACT Candida parapsilosis, a pathogenic yeast, is composed of three newly designated genomic species that are physiologically and morphologically indistinguishable. Nosocomial infections caused by group I C. parapsilosis are often associated with the breakdown of infection control practices and the contamination of medical devices, solutions, and indwelling catheters. Due to the low levels of nucleotide sequence variation that are observed, an investigation of the size polymorphisms in loci harboring microsatellite repeat sequences was applied for the typing of C. parapsilosis group I isolates. PCR primer sets that flank the microsatellite repeats for seven loci were designed. Following amplification by PCR, the size of each amplification product was determined automatically by capillary electrophoresis. A total of 42 C. parapsilosis group I isolates were typed by microsatellite analysis, and their profiles were compared to the hybridization profiles obtained by use of the Cp3-13 DNA probe. A high degree of discrimination (discriminatory power = 0.971) was observed by microsatellite analysis. The number of different alleles per locus ranged from 14 for locus B to 5 for locus C. Microsatellite analysis detected 30 different microsatellite genotypes, with 24 genotypes represented by a single isolate. Comparison of the genotypes obtained by microsatellite analysis and those obtained by analysis of the Cp3-13 hybridization profiles showed that they were similar, and these methods were able to identify related and unrelated isolates. Some discrepancies were observed between the methods and may be due to higher mutation rates and/or homoplasy by microsatellite markers. Identical results were observed between microsatellite analysis and Cp3-13 DNA hybridization profile analysis for C. parapsilosis isolates obtained from two patients, demonstrating the reproducibilities of the methods in vivo. Identical microsatellite profiles were observed for isolates displaying different phenotypic switching morphologies. Indistinguishable Cp3-13 DNA hybridization profiles were observed for six epidemiologically related isolates; however, only three of six primary isolates had identical microsatellite profiles. Size variation at a single locus was observed for three of six isolates obtained either after the outbreak period or from a different body site, suggesting the potential of the method to detect microevolutionary events. Interestingly, for most loci a single allele per strain was observed; in contrast, two alleles per locus were observed for some strains, and consistent with the findings for natural isolates, some isolates may be aneuploid. Due to the potential for high throughput, reproducibility, and discrimination, microsatellite analysis may provide a robust and efficient method for the genotyping of large numbers of C. parapsilosis group I isolates.

Population structure of Candida albicans, a member of the human flora, as determined by microsatellite loci

This study examines the macrogeographic population structure of Candida albicans, a yeast commensal of humans, through a population genetic analysis of 5 microsatellite loci in 13 cities. The populations were predominantly clonal with some recombination. About 5% of the genetic variation is between populations and the overall pattern is one of intermediate differentiation. We did not find a single widespread genotype but instead found high, macrogeographic gene flow in these clinical populations; the most common genotype was limited to Atlanta and San Francisco. Homogeneity is evident within large geographic regions, such as Europe, Asia, and the USA, and isolation by distance accounted for 39% of the variation observed. Overall gene flow for a member of the human flora is variable but can be extensive, with an average of 4.5 migrants per generation (N(m)). Eastern hemisphere populations were less divergent than those of the Americas and Caribbean, consistent with the expansion of humans out of the eastern hemisphere.

The molecular genetics of Candida albicans

Candida albicans, the major fungal pathogen of humans, is a diploid with no known sexual cycle. Recent genetic studies in C. albicans include the physical characterization of the genome, the development of systems for parasexual genetics, the cloning of Candida genes, and the development of methods for integrative and ARS-mediated transformation as well as gene disruption.

Evidence for a Pseudo-Outbreak of Candida guilliermondii Fungemia in a University Hospital in Brazil

ABSTRACT Fungal infections due to Candida species represent an important cause of nosocomial bloodstream infections. We report a large pseudo-outbreak of Candida guilliermondii fungemia that occurred in a university hospital in Brazil. C. guilliermondii was identified in 64 (43%) of the 149 blood samples drawn between June 2003 and July 2004. The samples were from patients in different wards of the hospital but concentrated in pediatric units. None of the patients had clinical signs of fungemia, and observational analysis revealed errors in the collection of blood samples. During the investigation of the pseudo-outbreak, C. guilliermondii was isolated from environmental surfaces and from the skin and nails of members of the nursing team. Through a subtyping analysis it was found that some of the nonpatient isolates were highly related to the patient isolates, and all the patient isolates were highly related. This is consistent with the hypothesis that the pseudo-outbreak was from a limited number of common sources. The adoption of intervention measures was effective in resolving the outbreak, supporting the hypothesis that the outbreak was due to poor techniques of drawing blood samples for culture.

Multilocus sequence type analysis reveals both clonality and recombination in populations of Candida glabrata bloodstream isolates from U.S. surveillance studies.

ABSTRACT The human commensal yeast Candida glabrata is becoming increasingly important as an agent of nosocomial bloodstream infection. However, relatively little is known concerning the genetics and population structure of this species. We have analyzed 230 incident bloodstream isolates from previous and current population-based surveillance studies by using multilocus sequence typing (MLST). Our results show that in the U.S. cities of Atlanta, GA; Baltimore, MD; and San Francisco, CA during three time periods spanning 1992 to 2009, five populations of C. glabrata bloodstream isolates are defined by a relatively small number of sequence types. There is little genetic differentiation in the different C. glabrata populations. We also show that there has been a significant temporal shift in the prevalence of one major subtype in Atlanta. Our results support the concept that both recombination and clonality play a role in the population structure of this species.