Brent A. Lasker

Analysis of Polymorphic Microsatellite Markers for Typing Penicillium marneffei Isolates

ABSTRACT Penicillium marneffei is an emerging opportunistic dimorphic fungal pathogen that is endemic in Southeast Asia. A typing method based on the analysis of size polymorphisms in microsatellite loci was investigated. Three loci available from the GenBank database were identified to harbor microsatellites. PCR primers flanking the microsatellite repeats were designed with one primer in the set fluorescently labeled. PCR products were then sized by automated capillary electrophoresis. As expected for a haploid fungus, a single band was observed for each microsatellite locus for all isolates. Polymorphic microsatellite marker (PMM) analysis detected a total of 22 different allelic types for 35 isolates of P. marneffei with a high discriminatory power (D = 0.956). Microsatellites I, II, and III detected 14, 10, and 7 alleles, respectively. The reproducibility of length polymorphisms was confirmed by using different DNA preparations from the same isolate or by repeated runs from the same DNA preparation. PMM profiles for eight isolates passaged in vitro for 7 to 8 weeks were identical to the original culture, demonstrating short-term stability and reproducibility. PCR products were not observed for other dimorphic fungi or human DNA. Comparison of allelic frequencies in isolates obtained from China and Thailand identified distinct allele combinations, suggesting the potential geographic isolation of populations. Due to the high discriminatory power, reproducibility, and potential for high throughput, PMM analysis may provide a good typing method for epidemiologic and surveillance investigations of P. marneffei.

Mycobacterium septicum sp. nov., a new rapidly growing species associated with catheter-related bacteraemia.

Rapidly growing mycobacteria are capable of causing several clinical diseases in both immunosuppressed and immunocompetent individuals. A previously unidentified, rapidly growing mycobacterium was determined to be the causative agent of central line sepsis in a child with underlying metastatic hepatoblastoma. Four isolates of this mycobacterium, three from blood and one from the central venous catheter tip, were studied. Phenotypic characterization, HPLC and genetic analysis revealed that while this organism most closely resembled members of the Mycobacterium fortuitum complex and Mycobacterium senegalense, it differed from all previously described species. Phenotypic tests useful in differentiating this species from similar rapidly growing mycobacteria included: growth at 42 degrees C, hydrolysis of acetamide, utilization of citrate, production of arylsulfatase (3-d), acidification of D-mannitol and i-myo-inositol, and susceptibility to erythromycin, vancomycin and tobramycin. The name Mycobacterium septicum is proposed for this new species. The type strain has been deposited in Deutsche Sammlung von Mikroorganismen und Zellkulturen as DSM 44393T and in the American Type Culture Collection as strain ATCC 700731T.

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.

Evaluation of performance of four genotypic methods for studying the genetic epidemiology of Aspergillus fumigatus isolates.

ABSTRACT In the present investigation, 49 Aspergillus fumigatus isolates obtained from four nosocomial outbreaks were typed by Afut1 restriction fragment length polymorphism (RFLP) analysis and three PCR-based molecular typing methods: random amplified polymorphic DNA (RAPD) analysis, sequence-specific DNA primer (SSDP) analysis, and polymorphic microsatellite markers (PMM) analysis. The typing methods were evaluated with respect to discriminatory power (D), reproducibility, typeability, ease of use, and ease of interpretation to determine their performance and utility for outbreak and surveillance investigations. Afut1 RFLP analysis detected 40 types. Thirty types were observed by RAPD analysis. PMM analysis detected 39 allelic types, but SSDP analysis detected only 14 types. All four methods demonstrated 100% typeability. PMM and RFLP analyses had comparable high degrees of discriminatory power (D = 0.989 and 0.988, respectively). The discriminatory power of RAPD analysis was slightly lower (D = 0.971), whereas SSDP analysis had the lowest discriminatory power (D = 0.889). Overall, SSDP analysis was the easiest method to interpret and perform. The profiles obtained by PMM analysis were easier to interpret than those obtained by RFLP or RAPD analysis. Bands that differed in staining intensity or that were of low intensity were observed by RAPD analysis, making interpretation more difficult. The reproducibilities with repeated runs of the same DNA preparation or with different DNA preparations of the same strain were high for all the methods. A high degree of genetic variation was observed in the test population, but isolates were not always similarly divided by each method. Interpretation of band profiles requires understanding of the molecular mechanisms responsible for genetic alternations. PMM analysis and Afut1 RFLP analysis, or their combination, appear to provide the best overall discriminatory power, reproducibility, ease of interpretation, and ease of use. This investigation will aid in planning epidemiologic and surveillance studies of A. fumigatus.

Investigation of an epidemic of invasive aspergillosis: utility of molecular typing with the use of random amplified polymorphic Dna probes

When seven immunocompromised patients developed invasive aspergillosis during construction at a hospital, new methods were performed to compare fungal isolates and a case-control study was conducted to determine risks for infection. Typing of Aspergillus flavus with the use of restriction endonuclease analysis and restriction fragment length polymorphism using random amplified polymorphic DNA reactions to generate DNA probes revealed different patterns between isolates from two patients and a similar pattern among those from one patient, a health care worker, and an environmental source. Case patients were more likely than controls to have longer periods of hospitalization (median, 83 vs. 24 days; P < 0.01), neutropenia (median, 33 vs. 6 days; P < 0.05), and exposure to broad spectrum antimicrobials (median, 56 vs. 15 days; P = 0.08). No patients restricted to protected areas developed aspergillosis. Risk of exposure of immunocompromised patients to opportunistic organisms stirred up by construction activity may be decreased by admitting these patients to protected areas away from construction activity and by restricting traffic from construction sites to these areas. Although typing of A. flavus isolates did not reveal a single type or source of organism responsible for infection, this method may facilitate epidemiologic investigation of possible nosocomial sources and transmission in similar settings.

Characterization of a novel gene for strain typing reveals substructuring of Aspergillus fumigatus across North America.

ABSTRACT Fifty-five epidemiologically linked Aspergillus fumigatus isolates obtained from six nosocomial outbreaks of invasive aspergillosis were subtyped by sequencing the polymorphic region of the gene encoding a putative cell surface protein, Afu3g08990 (denoted as CSP). Comparative sequence analysis showed that genetic diversity was generated in the coding region of this gene by both tandem repeats and point mutations. Each unique sequence in an outbreak cluster was assigned an arbitrary number or CSP sequence type. The CSP typing method was able to identify “clonal” and genotypically distinct A. fumigatus isolates, and the results of this method were concordant with those of another discriminatory genotyping technique, the Afut1 restriction fragment length polymorphism typing method. The novel single-locus sequence typing (CSP typing) strategy appears to be a simple, rapid, discriminatory tool that can be readily shared across laboratories. In addition, we found that A. fumigatus isolates substructured into multiple clades; interestingly, one clade consisted of isolates predominantly representing invasive clinical isolates recovered from cardiac transplant patients from two different outbreak situations. We also found that the A. fumigatus isolate Af293, whose genome has been sequenced, possesses a CSP gene structure that is substantially different from those of the other A. fumigatus strains studied here, highlighting the need for further taxonomic study.

Outbreak of invasive aspergillosis among renal transplant recipients.

Invasive aspergillosis (IA) is rare among renal transplant recipients (RTRs). We investigated a cluster of IA among RTRs at a California hospital from January to February 2001, when construction was ongoing. We conducted a cohort study among RTRs who were hospitalized between January 1 and February 5, 2001, to determine risk factors for IA. IA was defined using established guidelines. Four IA cases occurred among 40 RTRs hospitalized during the study period. Factors associated with an increased risk of IA included prolonged hemodialysis, lengthy corticosteroid treatment posttransplant, and use of sirolimus alone or with mycophenolate (P<0.05). After the study period, three additional RTRs developed IA; two Aspergillus isolates recovered from these patients had indistinguishable profiles by DNA fingerprinting, suggesting common-source exposure. This study suggests that immunosuppressed RTRs can be at an increased risk for IA. Measures to prevent IA in these patients should be taken during hospital construction.

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.

Cluster of Cases of Invasive Aspergillosis in a Transplant Intensive Care Unit: Evidence of Person-to-Person Airborne Transmission

In October 1998, a patient developed deep surgical-site and organ-space infection with Aspergillus fumigatus 11 days after undergoing liver retransplantation; subsequently, 2 additional patients in the transplant intensive care unit had invasive pulmonary infection with A. fumigatus diagnosed. It was determined that debriding and dressing wounds infected with Aspergillus species may result in aerosolization of spores and airborne person-to-person transmission.

Interlaboratory reproducibility of a microsatellite-based typing assay for Aspergillus fumigatus through the use of allelic ladders: proof of concept

An interlaboratory study was performed with the aim of investigating the reproducibility of a multiplex microbial microsatellite-based typing assay for Aspergillus fumigatus in different settings using a variety of experimental and analytical conditions and with teams having variable prior microsatellite typing experience. In order to circumvent problems with exchange of sizing data, allelic ladders are introduced as a straightforward and universally applicable concept for standardization of such typing assays. Allelic ladders consist of mixtures of well-characterized reference fragments to act as reference points for the position in an electrophoretic trace of fragments with established repeat numbers. Five laboratories independently analysed six microsatellite markers in 18 samples that were provided either as DNA or as A. fumigatus conidia. Allelic data were reported as repeat numbers and as sizes in nucleotides. Without the use of allelic ladders, size differences of up to 6.7 nucleotides were observed, resulting in interpretation errors of up to two repeat units. Difficulties in interpretation were related to non-specific amplification products (which were resolved with explanation) and bleed-through of the different fluorescent labels. In contrast, after resolution of technical or interpretive problems, standardization of sizing data by using allelic ladders enabled all participants to produce identical typing data. The use of allelic ladders as a routine part of molecular typing using microsatellite markers provides robust results suitable for interlaboratory comparisons and for deposition in a global typing database.