Strain Typing Methods Research Articles

Use of PCR-BasedMycobacterium tuberculosisGenotyping To Prioritize Tuberculosis Outbreak Control Activities

Genotypic analysis of Mycobacterium tuberculosis isolates is increasingly applied in direct support of tuberculosis outbreak control activities. This is facilitated by PCR-based strain typing methods that enable the genotypic characterization of samples containing small numbers of M. tuberculosis cells. By using DNA extracted directly from primary diagnostic cultures, PCR-based methods were applied to a tuberculosis outbreak investigation and to surveillance in King County, Washington. In the outbreak investigation, five epidemiologically linked M. tuberculosis isolates had a unique pattern at mycobacterial interspersed repeating unit (MIRU) loci 10 and 23 when the pattern was compared to the patterns in a local MIRU locus database. In order to quickly identify new cases involving this strain (termed SBRI10), targeted genotyping at these two loci was performed with cultures from epidemiologically associated tuberculosis cases. Isolates with the characteristic genotypes at loci 10 and 23 were further analyzed by use of a 12-locus MIRU panel and by repetitive-unit-sequence-based PCR (rep-PCR). Between May 2004 and January 2005, 82 cases were screened, of which 14 were identified for further analysis and 13 were confirmed to be infected with SBRI10. Between September 2005 and August 2006, surveillance universal genotyping was performed by using the 12-locus MIRU panel with DNA from primary diagnostic enrichment cultures. A total of 161 samples were submitted for analysis, and 156 were successfully typed. Fifty-one cases formed 18 presumptive clusters by MIRU locus typing. Of these, 30 cases were confirmed to be members of 11 clusters by rep-PCR. Presumptive genotypic data were available rapidly, sometimes within 2 weeks of diagnosis. In this fashion, PCR-based genotyping provided data that can be used to prioritize disease control activities.

Legionella: State of the Art 30 Years After Its Recognition

This book is the result of an international conference on Legionella held in Chicago in 2005. It is a vast compendium of 131 papers divided into four major sections, namely, Clinical and Diagnostic Aspects of Legionnaire's Disease; Epidemiology and Strain Typing Methods; Microbiology, Pathogenesis, Immunology and Genetics; and Environmental Biology, Detection, Prevention and Control.

Epidemiological typing of methicillin-resistant Staphylococcus aureus strains by Fourier transform infrared spectroscopy

A rapid and simple typing system is needed for controlling the spread of epidemic methicillin-resistant Staphylococcus aureus (MRSA), currently one of the most widespread multi-resistant nosocomial pathogens in Canadian hospitals. Fourier transform infrared (FTIR) spectroscopy was used to subtype 85 isolates representing five strains of epidemic Canadian MRSA (CMRSA). Spectral fingerprints of whole cells grown on Que-Bact® Universal Medium No. 2 were transformed to first derivative peak-height normalized files and examined visually and by singular-value decomposition (SVD). Distinguishing spectral regions were processed by principal component analysis (PCA), self-organizing map and K-nearest neighbor supervised cluster analysis. Among the visually identified regions, 1070–1050 and 1155–1137 cm − 1 were found suitable for discrimination of CMRSA-4 and CMRSA-2 respectively, while CMRSA-1, CMRSA-3, and CMRSA-5 each exhibited distinctive spectral profiles in the 1123–1094 cm − 1 region. The combination, 1123–1094, 1174–1154 and 2904–2864 cm − 1 separated the five CMRSA with 84.6% correct classification by PCA. Five clusters were also obtained using the SVD-selected regions 1096–1066, 1118–1090 and 2914–2880 cm − 1 , with 87.8% correct classification based on visual examination of the PCA scores plot and 97% based on supervised cluster analysis. These results demonstrate that FTIR spectroscopy has considerable potential as a rapid (1-hour) and simple method for MRSA strain typing and monitoring in clinical settings.

Aspergillus strain typing in the genomics era

Multiple reasons may justify a need for strain typing purposes, but the most common reason is to delineate the epidemiological relationships between isolates. The availability of whole genome sequences has greatly influenced our ability to develop highly targeted and efficient strain typing methods fur these purposes. Some strain typing methods may serve dual goals: not only can they be used to discriminate between multiple isolates of a certain species, they can also aid in the recognition, identification, description and validation process of a fungal species.

Transformation of yeast by infectious prion particles

We present methods to prepare infectious Sup35 protein aggregates and use them for genetic transformation of yeast. The protein aggregates are prepared from bacterially expressed recombinant protein, which is converted to amyloid fibers by extended incubation or by nucleated growth using yeast prion particles as seeds. The aggregates are introduced into yeast by a modified spheroplast transformation protocol. The phenotype of the yeast transformants is further characterized by robust prion strain typing methods. The methodology can be used to introduce different [ PSI +] particles to many laboratory yeast genetic backgrounds. It can be adapted for applications in other yeast prion systems as well.

Strain differentiation of Trichophyton rubrum by randomly amplified polymorphic DNA and analysis of rDNA nontranscribed spacer

Trichophyton rubrum is the most common pathogen causing dermatophytosis. Molecular strain-typing methods have recently been developed to tackle epidemiological questions and the problem of relapse following treatment. A total of 67 strains of T. rubrum were screened for genetic variation by randomly amplified polymorphic DNA (RAPD) analysis, with two primers, 5'-d[GGTGCGGGAA]-3' and 5'-d[CCCGTCAGCA]-3', as well as by subrepeat element analysis of the nontranscribed spacer of rDNA, using the repetitive subelements TRS-1 and TRS-2. A total of 12 individual patterns were recognized with the first primer and 11 with the second. Phylogenetic analysis of the RAPD products showed a high degree of similarity (>90 %) among the epidemiologically related clinical isolates, while the other strains possessed 60 % similarity. Specific amplification of TRS-1 produced three strain-characteristic banding patterns (PCR types); simple patterns representing one copy of TRS-1 and two copies of TRS-2 accounted for around 85 % of all isolates. It is concluded that molecular analysis has important implications for epidemiological studies, and RAPD analysis is especially suitable for molecular typing in T. rubrum.

Rapid molecular typing of Burkholderia pseudomallei, isolated in an outbreak of melioidosis in Singapore in 2004, based on variable-number tandem repeats

An increase in the number of reported melioidosis cases was observed in the first 4 months of 2004. These cases were associated with a significant increase in case-fatality rate compared with the past 5 years. In order to exclude the possibility of a single source, including the possibility of intentional release of Burkholderia pseudomallei, we applied a multiplex PCR-based multilocus variable-number tandem repeat (VNTR) assay to determine the clonality of the clinical isolates. Our investigation indicated that a total of 30 different VNTR types could be distinguished in the 32 clinical isolates of B. pseudomallei obtained during this period, thus indicating that infection was unlikely to have occurred from a single source. Our experience underscores the usefulness of a rapid strain typing method in augmenting an epidemiological investigation into an infectious disease outbreak, particularly at a time where the intentional use of biological agents is a potential threat to public health.

Analysis of a Uropathogenic Escherichia coli Clonal Group by Multilocus Sequence Typing

Although many strain typing methods exist for pathogenic Escherichia coli, most have drawbacks in terms of resolving power, interpretability, or scalability. For this reason, multilocus sequence typing (MLST) is an appealing alternative. However, its applicability to different pathogens in specific epidemiologic contexts is not well understood. Here, we applied a previously established MLST method based on housekeeping genes to a well-characterized collection of uropathogenic E. coli isolates to compare the discriminatory ability of this procedure with that of enterobacterial repeat intergenic consensus (ERIC2) PCR, serogrouping, and pulsed-field gel electrophoresis (PFGE). Among 45 E. coli isolates studied, 17 different multilocus sequence types (ST) were identified. One MLST group (designated ST69 complex) was comprised of 22 isolates, all belonging to uropathogenic and bacteremic E. coli strains previously defined as clonal group A (CgA) by ERIC2 PCR. The ST69 strains contained five different serogroups and 14 PFGE types. ERIC2 PCR CgA strains belonging to different MLST groups were also identified. Interestingly, one cow E. coli isolate, previously shown by PFGE to be closely related to a human uropathogenic CgA strain, was found to cluster with the ST69 strains. All of the other animal and environmental CgA isolates had different MLST profiles. The discriminatory power of this MLST method based on housekeeping genes appears to be higher than that of ERIC2 PCR but lower than that of PFGE for epidemiologic study of uropathogenic E. coli.

Multiple-locus variable-number tandem repeat analysis for strain typing of Clostridium perfringens

Clostridium perfringens is ubiquitous in the environment and causes diseases in man and animals, with syndromes ranging from enteritis, enterotoxemia, and sudden death to food poisoning and gas gangrene. Understanding the epidemiology of these infections and of the evolution of virulence in C. perfringens necessitate an efficient, time and cost effective strain typing method. Multiple-locus variable-number tandem repeat analysis (MLVA) has been applied to typing of other pathogens and we describe here the development of a MLVA scheme for C. perfringens. We characterized five variable tandem repeat (VNTR) loci, four of which are contained within protein encoding genes and screened 112 C. perfringens isolates to evaluate typability, reproducibility, and discriminatory power of the scheme. All the isolates were assigned a MLVA genotype and the technique has excellent reproducibility, with a numerical index of discrimination for the five VNTR loci of 0.995. Thus MLVA is an efficient tool for C. perfringens strain typing, and being PCR based makes it rapid, easy, and cost effective. In addition, it can be employed in epidemiological, ecological, and evolutionary investigations of the organism.

Strain Typing of German Transmissible Spongiform Encephalopathies Field Cases in Small Ruminants by Biochemical Methods

Following the implementation of a large scale transmissible spongiform encephalopathies (TSE) surveillance programme of small ruminants, evidence for a natural transmission of bovine spongiform encephalopathy (BSE) to a French goat has been found. During the years 2002-2004, a massive TSE rapid testing programme on >250,000 small ruminants was carried out in Germany. In this national survey, 186 scrapie-affected sheep were found which originated from 78 flocks. The majority of these cases were of the classical TSE type (115 sheep belonging to 14 outbreaks). However, 71 cases coming from 64 flocks were of the novel atypical scrapie type. According to the regulation EU 999/2001, all TSE cases in small ruminants have to be examined by strain typing methods to explore any possibility of the existence of BSE cases in the field sheep population. Here we report on a biochemical typing strategy (termed FLI-test), which includes the determination of molecular masses, antibody binding affinities and glycosylation pattern of the TSE induced abnormal prion protein. Based on this typing approach none of the analysed German classical TSE outbreaks (total number of analysed sheep: 36) displayed biochemical features indicative for a BSE infection. However, in two cases distinct but BSE-unrelated PrP(Sc) types were found, which alludes to the existence of different scrapie strains in the German sheep population.

Microbial DNA Typing by Automated Repetitive-Sequence-Based PCR

Repetitive sequence-based PCR (rep-PCR) has been recognized as an effective method for bacterial strain typing. Recently, rep-PCR has been commercially adapted to an automated format known as the DiversiLab system to provide a reliable PCR-based typing system for clinical laboratories. We describe the adaptations made to automate rep-PCR and explore the performance and reproducibility of the system as a molecular genotyping tool for bacterial strain typing. The modifications for automation included changes in rep-PCR chemistry and thermal cycling parameters, incorporation of microfluidics-based DNA amplicon fractionation and detection, and Internet-based computer-assisted analysis, reporting, and data storage. The performance and reproducibility of the automated rep-PCR were examined by performing DNA typing and replicate testing with multiple laboratories, personnel, instruments, DNA template concentrations, and culture conditions prior to DNA isolation. Finally, we demonstrated the use of automated rep-PCR for clinical laboratory applications by using isolates from an outbreak of Neisseria meningitidis infections. N. meningitidis outbreak-related strains were distinguished from other isolates. The DiversiLab system is a highly integrated, convenient, and rapid testing platform that may allow clinical laboratories to realize the potential of microbial DNA typing.

Evaluation of the HOOF-Print assay for typing Brucella abortus strains isolated from cattle in the United States: results with four performance criteria

BackgroundA fundamental question that arises during epidemiological investigations of bacterial disease outbreaks is whether the outbreak strain is genetically related to a proposed index strain. Highly discriminating genetic markers for characterizing bacterial strains can help in clarifying the genetic relationships among strains. Under the auspices of the European Society of Clinical Microbiology and Infectious Diseases, the European Study Group for Epidemiological Markers (ESGEM) established guidelines for evaluating the performance of typing systems based of a number of criteria. Recently, HOOF-Print genotype analysis, a new method for typing Brucella abortus strains based on hypervariability at eight tandem repeat loci, was described. This paper evaluates the HOOF-Print assay by four of the criteria set out by the ESGEM: typeability, reproducibility, power of discrimination, and concordance with other typing methods.ResultsThe HOOF-Print Assay was evaluated with a test population composed of 97 unrelated field isolates and 6 common laboratory strains of B. abortus. Both typeability and reproducibility of the assay were excellent. Allele diversity and frequency varied widely among the eight loci, ranging from 1 to 13 alleles. The power of discrimination, measured by the Hunter-Gaston discrimination index (HGDI), varied by locus ranging from 0 to 0.89, where a maximal value of 1.0 indicates discrimination of all strains. The HGDI values calculated for subgroups sorted by biovar were similar to the values determined for the whole population. None of the individual loci achieved the recommended HGDI threshold of 0.95, but the HGDI of the composite profiles was 0.99 (93 unique genotypes from 97 field strains evaluated), well above the recommended threshold. By comparison, the HGDI value for biovar typing was 0.61 in a test population biased with disproportionate numbers of the less common biovars. Cluster analysis based on HOOF-Print genotypes assembled the strains into hierarchical groups with no apparent association with the time or location of strain isolation. Likewise, these hierarchical groups were not homogeneous with regard to biotype. In one extreme case, two field isolates with identical fingerprints were identified as different biovars by conventional methods.ConclusionThe main purpose of this study was to assess the ability of HOOF-Print genotyping to discriminate unrelated field strains of B. abortus, and whether the assay met established requirements for bacterial strain typing methods. The discriminatory power of the assay was remarkable, considering the genetic homogeneity found among species within the genus. The assay met or exceeded all of the recommended levels for the performance criteria of typeability, reproducibility, and power of discrimination, however some inconsistencies with conventional biovar typing were observed. Nevertheless, the results indicate that with cautious interpretation, multilocus genotyping of polymorphic tandem repeats by HOOF-Print analysis could be a valuable complement to routine epidemiological investigations into localized B. abortus outbreaks.

Strain differentiation of Trichophyton rubrum by random amplification of polymorphic DNA (RAPD)

Trichophyton rubrum is an important cause of dermatomycoses. Molecular strain typing methods have recently been developed to address questions about epidemiology and source of relapse following treatment. This report describes the application of RAPD for molecular strain differentiation of this fungus utilizing the primers 1- (5'-d[GGTGCGGGAA]-3') and 6- (5'-d[CCCGTCAGCA]-3'). A total of five RAPD patterns were observed among 10 strains of T. rubrum, with each of the primers used. We conclude that RAPD analysis using primers 1 and 6 can be used in epidemiological studies.

Detection and differentiation of Plum pox virus using real-time multiplex PCR with SYBR Green and melting curve analysis: a rapid method for strain typing

A real-time multiplex PCR procedure with melting curve analysis, using the green fluorescence dye SYBR Green I, was developed for rapid and reliable identification of Plum pox virus (PPV) isolates of strains D and M. Members of the different strains were identified by their distinctive melting temperatures ( T ms); 84.3–84.43 °C for D isolates, and 85.34–86.11 °C for M isolates. The associated amplicon sizes were 114 and 380 bp, respectively. The procedure was used for detection and identification of PPV in both herbaceous and woody hosts. The T m for members of a particular strain was very similar, with a host effect that did not hinder strain identification. Universal primers included in the study detected all isolates of PPV tested, amplifying a 74 bp fragment. The T m of this fragment varied from 80.12 to 81.52 °C and may have supplementary value for PPV identification. SYBR Green-based detection was compared to detection using a hybridization LUX fluorogenic primer. Better resolution of the melting peaks was observed with SYBR Green I, than with the LUX primers, hence strain identification with SYBR Green I was more reliable. This is a simple approach to PPV strain identification with the relatively inexpensive dye SYBR Green I, and eliminates any need for electrophoretic analysis of amplicons or RFLP patterns using ethidium bromide.

Methicillin-ResistantStaphylococcus aureus1

Methicillin-resistant Staphylococcus aureus (MRSA) is first and foremost a pathogen of healthcare settings. It is the most common pathogen associated with nosocomial infections in the United States, particularly nosocomial pneumonia and surgical site infections. It is also a frequent cause of bloodstream and skin and soft tissue infections. The percentage of S. aureus isolates resistant to oxacillin/methicillin in U.S. intensive care units increased from 30% to 40% in the mid-1990s to 57% in 2002. Data from a recent Duke Infection Control Outreach Network survey indicate that of patients with healthcare-associated MRSA infections, 39% were from nursing homes, 37% had been hospitalized in the previous 90 days, 10% had received home health care, and 10% received dialysis. Data suggest that MRSA bacteremia is associated with an increased likelihood of death, longer hospital stays, and increased cost of hospitalization, when compared with bacteremia levels caused by methicillin-susceptible strains. Increasing resistance to vancomycin among MRSA also complicates therapy, which is already difficult because of multidrug resistance among healthcare-associated MRSA. Because spread of MRSA in healthcare settings is often clonal, hand hygiene and barrier precautions are often effective in interrupting spread. Targeted surveillance for MRSA is also a useful aid for infection control. Data from the Duke network indicate that the spread of MRSA can be curtailed in healthcare settings, given vigilance and adequate funding of infection control activities. MRSA is now spreading in community settings. Reports from the early 1980s indicate that patients in the community without established risk factors for MRSA (i.e., recent hospitalization, residence in a long-term care facility, or dialysis) sought medical care with MRSA infections. In the late 1990s, four children in Minnesota and North Dakota died from community-associated MRSA infections. The isolates were susceptible to most non-β-lactam drugs, had pulsed-field gel electrophoresis (PFGE) profiles that differed from typical healthcare-associated MRSA, and contained the Panton-Valentine leukocidin toxin. Prospective surveillance for MRSA in Minnesota at 12 sentinel hospitals (6 in metropolitan areas and 6 in rural areas) indicated that community-associated MRSA patients were significantly younger than healthcare-associated MRSA patients and more likely to have skin and soft tissue infections than respiratory or urinary tract infections. A study in Texas showed that incision and drainage of abscesses due to community-associated MRSA was more effective management than administering antimicrobial agents alone, particularly since many patients were given ineffective antimicrobial agents (i.e., β-lactam agents). Molecular analysis of the community-associated MRSA strains showed that the methicillin resistance gene mecA is typically carried on a much smaller genetic element than is seen in healthcare-associated MRSA. Four distinct elements, called staphylococcal chromosome cassette mec (or SCCmec), have been described. In the United States, SCCmec type II, which is approximately 60 kb in size and also carries an erythromycin resistance determinant, predominates among healthcare-associated MRSA, while SCCmec type IV, which is only ≈23 kb in length and carries no other resistance determinants, is typically associated with community-associated MRSA. Three major strain typing methods, PFGE, multi-locus sequence typing (MLST), and staphylococcal protein A typing (spa typing), are used to study the spread of MRSA. MLST identified a series of five major lineages (also called clonal complexes) of MRSA globally, while spa typing and PFGE subdivide this group into approximately a dozen epidemic clones. Virulence determinates for MRSA include a series of enterotoxins, toxic shock toxin, and

Structure and diversity among rhizobial strains, populations and communities–a review

Published studies of rhizobial populations, communities and other strain collections were analysed to identify trends in strain richness, strain dominance and genetic diversity within and between locations. For individual populations and communities, strain richness indices were calculated by dividing the number of strain types identified by the number of isolates recovered. Where possible, strain dominance (the proportion of rhizobial isolates represented by each strain type) was also calculated. Analysis of the genetic diversity of populations, communities and rhizobial strain collections originating from diverse legume hosts and locations, was confined to studies using multilocus enzyme electrophoresis (MLEE) so that diversity could be compared on the basis of published H values. Strain richness indices were highly variable (0.02–0.94) and were influenced by both the discriminatory power of the strain typing method and the type and number of legume species used to recover rhizobia from soil. The strain richness of populations recovered either directly from soil, or from the nodules of trap hosts inoculated with the same soil, was similar. Because the arithmetic relationship between the number of strain types and number of isolates varies between different populations and communities, strain richness curves are proposed as the most appropriate method for reporting rhizobial structural diversity. Comparison of over 50 rhizobial populations and communities from published studies showed that strain dominance patterns in nodules were often similar. Typically, a single strain type occupies more than 30% of nodules with the majority of strain types being recovered at low frequency (∼75% of published populations and communities). Rhizobial populations and communities characterised by MLEE varied in their genetic diversity, with H values ranging from 0.06 to 0.78. In several studies, most of the genetic diversity within a site could be recovered from the nodules of a single plant. When hierarchical analyses were performed on populations within and between sites, the genetic diversity within sites was similar to the genetic diversity between sites. Similarly, the genetic diversity of strain collections originating from multiple hosts and locations was no more diverse than some individual populations and communities. Strain richness and genetic diversity measures were not always correlated for rhizobial populations. Populations with low strain richness were sometimes genetically diverse, and the relationship between the diversity measures varied for different legume species at the same location. We suggest that both strain richness and genetic diversity measures are required to fully describe rhizobial population and community diversity.

Effects of Different Experimental Conditions on the PrPSc Core Generated by Protease Digestion: IMPLICATIONS FOR STRAIN TYPING AND MOLECULAR CLASSIFICATION OF CJD

The discovery of molecular subtypes of the pathological prion protein PrPSc has provided the basis for a novel classification of human transmissible spongiform encephalopathies (TSEs) and a potentially powerful method for strain typing. However, there is still a significant disparity regarding the understanding and nomenclature of PrPSc types. In addition, it is still unknown whether a specific PrPSc type is associated with each TSE phenotypic variant. In sporadic Creutzfeldt-Jakob disease (sCJD), five disease phenotypes are known, but only two major types of PrPSc, types 1 and 2, have been consistently reproduced. We further analyzed PrPSc properties in sCJD and variant CJD using a high resolution gel electrophoresis system and varying experimental conditions. We found that pH varies among CJD brain homogenates in standard buffers, thereby influencing the characteristics of protease-treated PrPSc. We also show that PrPSc type 1 and type 2 are heterogeneous species which can be further distinguished into five molecular subtypes that fit the current histopathological classification of sCJD variants. Our results shed light on previous disparities in PrPSc typing, provide a refined classification of human PrPSc types, and support the notion that the pathological TSE phenotype is related to PrPSc structure.

Conventional and molecular epidemiology of trimethoprim-sulfamethoxazole resistance among urinary Escherichia coli isolates

Background Antibiotic resistance is increasing in Escherichia coli, the most common cause of urinary tract infections, but its epidemiology has not been well described. We evaluated the epidemiology of trimethoprim-sulfamethoxazole–resistant E. coli in a large, public health care system in Denver, Colorado. Methods Outpatients with E. coli urinary tract infections during the first 6 months of 1998 were evaluated retrospectively. A prospective study was then performed to confirm the rate of trimethoprim-sulfamethoxazole resistance. We used several strain-typing methods (pulsed-field gel electrophoresis, ribotyping, serotyping) to evaluate the molecular epidemiology of the resistance. Results The rate of trimethoprim-sulfamethoxazole resistance was similar in the retrospective (24% [161/681]) and prospective (23% [30/130]) phases of the study ( P = 0.89). Almost all trimethoprim-sulfamethoxazole–resistant strains (98%) were resistant to at least one other antibiotic. Risk factors for infection with a resistant strain included age ≤3 years, Hispanic ethnicity, recent travel outside the United States, and a prior urinary tract infection. However, rates of resistance were >15% among nearly all of the subgroups. Most strains had high-level resistance (>1000 μg/mL) to trimethoprim-sulfamethoxazole. Of the 23 resistant isolates evaluated, 10 (43%) belonged to the clone A group. There was no correlation between conventional epidemiologic characteristics and the molecular mechanism of resistance or strain type. Conclusion Resistance to trimethoprim-sulfamethoxazole among E. coli isolates among patients in a Denver public health care system is common, with high rates of resistance even among patients without risk factors.

The role of molecular biology and nucleic Acid technology in the study of human infection and epidemiology.

In this review, we will focus on recent molecular typing methods that can be applied to different pathogens and assess their values and limitations. Resistant subgroups within a species of pathogenic organisms often emerge as dominant strains under the selective pressures of antibiotics, vaccinations, and new health care practices. The emergence of resistant organisms has added to the burden and cost of health care-related infections. Pathogens derived from a common ancestry are often difficult to distinguish by conventional methods, and the practice of clinical microbiology and infectious disease epidemiology must adapt to this problem. Conventional strain typing methods provide a limited means of distinguishing epidemic from endemic or sporadic isolates of pathogens. Nucleic acid-based methods complement conventional and serologic methods of organism isolation and typing. Often, these genomic methods offer more discrimination and details than the phenotype-based conventional methods. Highly sensitive molecular techniques are capable of detecting single base pair substitutions and resolving the mechanism of underlying complex variation.

Reflections on the question: What does molecular mycology have to do with the clinician treating the patient?

After a talk on regulation of gene transcription in Candida albicans, a clinical mycologist was heard to ask: "What difference does all that make to the lady dying of disseminated Candida infection in her hospital bed?" The rapid expansion of research in fungal diseases is widening the communication gap between individuals with responsibility for patient care and those who study pathogenic fungi at the level of molecular biology. DNA-based technologies have produced real advances for patient care by delivering superior methods for fungus identification and strain typing that will soon find a routine place in patient management. Molecular research into the fine detail of the host-pathogen interplay in fungal disease has also made great advances, though the spin-offs to benefit the clinician are not yet obvious. Detection of fungal DNA as a non-culture diagnostic method still requires considerable refinement before it can be regarded as a routinely useful approach, and genomic-based strategies for discovery of novel antifungal drugs from molecular targets have so far produced no agents that have entered development. It is inevitable that, in time, several aspects of molecular mycology research will become important basic knowledge for the clinician who is treating the patient. Therefore, clinicians and bench scientists with specialist interest in mycoses need to retain a reasonable level of mutual comprehension and respect of each other's work rather than assuming their professional paths are divergent.