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The outstanding ability of Acinetobacter baumannii to cause outbreaks and acquire multidrug resistance motivated the development of a plethora of typing techniques, which can help infection preventionists and hospital epidemiologists to more efficiently implement intervention controls. Nowadays, the world is witnessing a gradual transition from traditional typing methodology to whole genome sequencing-based approaches. Such approaches are opening new prospects and applications never achieved by existing typing methods. Herein, we provide the reader with an updated review on A. baumannii typing methods recapping the added value of well-established techniques previously applied for A. baumannii and detailing new ones (as clustered regularly interspaced short palindromic repeats-based typing) with a special focus on whole genome sequencing.

Hypervirulent K. pneumoniae (hvKp) is an evolving pathotype that is more virulent than classical K. pneumoniae (cKp). hvKp usually infects individuals from the community, who are often healthy. Infections are more common in the Asian Pacific Rim but are occurring globally. hvKp infection frequently presents at multiple sites or subsequently metastatically spreads, often requiring source control. hvKp has an increased ability to cause central nervous system infection and endophthalmitis, which require rapid recognition and site-specific treatment. The genetic factors that confer hvKp's hypervirulent phenotype are present on a large virulence plasmid and perhaps integrative conjugal elements. Increased capsule production and aerobactin production are established hvKp-specific virulence factors. Similar to cKp, hvKp strains are becoming increasingly resistant to antimicrobials via acquisition of mobile elements carrying resistance determinants, and new hvKp strains emerge when extensively drug-resistant cKp strains acquire hvKp-specific virulence determinants, resulting in nosocomial infection. Presently, clinical laboratories are unable to differentiate cKp from hvKp, but recently, several biomarkers and quantitative siderophore production have been shown to accurately predict hvKp strains, which could lead to the development of a diagnostic test for use by clinical laboratories for optimal patient care and for use in epidemiologic surveillance and research studies., (Copyright © 2019 American Society for Microbiology.)

Microbiological diagnostic procedures have changed rapidly in recent years. This is especially true in the field of molecular diagnostics. Classical culture-based techniques are still the gold standard in many areas; however, they are already complemented by automated and also molecular techniques to guarantee faster and better quality results. The most commonly used techniques include real-time polymerase chain reaction (RT-PCR) based systems and nucleic acid hybridization. These procedures are used most powerfully from direct patient samples or in assays to detect the presence of nonculturable or fastidious organisms. Further techniques such as DNA sequencing are not yet used routinely for urological samples and can be considered experimental. However, in conjunction with dropping prices and further technical developments, these techniques promise to be used much more in the near future. Regarding bacterial identification from culture, mass spectrometry (MALDI-TOF MS) has become the technique of choice in recent years especially in Europe. It has tremendously shortened the time to result. This is now going to be extended to antibiotic susceptibility testing. This is of paramount importance in view of ever rising antimicrobial resistance rates. Techniques described in this review offer a faster and better microbiological diagnosis. Such continuous improvements are critical especially in times of cost pressure and rising antimicrobial resistance rates. It is in our interest to provide the best possible care for patients and in this regard a good and effective communication between the laboratory and the clinician is of vital importance.

Within less than a decade matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has become a gold standard for microbial identification in clinical microbiology laboratories. Besides identification of microorganisms the typing of single strains as well as the antibiotic and antimycotic resistance testing has come into focus in order to speed up the microbiological diagnostic. However, the full potential of MALDI-TOF MS has not been tapped yet and future technological advancements will certainly expedite this method towards novel applications and enhancement of current practice. So, the following chapter shall be rather a brainstorming and forecast of how MALDI-TOF MS will develop to influence clinical diagnostics and microbial research in the future. It shall open up the stage for further discussions and does not claim for overall validity.

Microfluidic technologies enable unique studies in the field of microbiology to facilitate our understanding of microorganisms. Using miniaturized and high-throughput experimental capabilities in microfluidics, devices with controlled microenvironments can be created for microbial studies in research fields such as healthcare and green energy. In this research highlight, we describe recently developed tools for diagnostic assays, high-throughput mutant screening, and the study of human disease development as well as a future outlook on microbes for renewable energy.

Purpose of Review: Molecular methods for the diagnosis of Neisseria gonorrhoeae are replacing bacterial culture in many settings. This review focuses on recent progress in the development of molecular tests to detect resistant N. gonorrhoeae both to enhance surveillance and to guide decisions about individual patient management., Recent Findings: Assays to enhance surveillance have been developed to detect determinants of resistance for all antibiotics used as first-line gonorrhoea treatment, or to detect specific 'superbug' strains, but few have been applied in clinical practice. The most advanced strategy relevant to individual case management is to identify ciprofloxacin-sensitive strains so that unnecessary use of ceftriaxone can be avoided. Cross-reactivity with pharyngeal commensal Neisseria species reduces specificity and is a challenge for many assays., Summary: Progress with laboratory-based molecular tests to detect gonococcal resistance is being made but substantial challenges remain. No laboratory-based assay has been subjected to a field evaluation and no assay so far can be used as a point-of-care test. Given the threat of antimicrobial resistance, now is the time to exploit the molecular technologies used for diagnosis and to invest in the development of molecular gonococcal resistance tests that can be implemented for public health good.

By diversifying culture conditions, in a strategy named culturomics, we were able in a short time to grow 124 new bacterial species from human stools, including 39 strict anaerobes. To describe these microorganisms, we use genome sequencing and MALDI-TOF mass spectrometry. Both tools have been major breakthroughs in clinical microbiology over the past decade, have previously been used for taxonomic purposes, and have the advantage over chemotaxonomic methods and DNA-DNA hybridization, to exhibit an excellent intra- and inter-laboratory reproducibility. We developed a polyphasic taxonomic strategy including MALDI-TOF MS and genomic analyses to describe new bacterial species associated with human beings. This strategy, that we have named taxono-genomics, was used to propose the description of 48 new species, the names of 13 of which have officially been validated. In this manuscript, we briefly reviewed the pros and cons of the currently validated taxonomic tools and propose that genomic sequencing and MALDI-TOF mass spectrometry may be incorporated in the taxonomic classification of prokaryotes., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Staphylococcus aureus is a major human pathogen, causing disease in both community and healthcare settings. Over the past two decades, the epidemiology of S. aureus disease has changed dramatically, with the emergence and spread of community-associated methicillin-resistant S. aureus clones. This epidemiological shift, coupled with the association between delayed antimicrobial therapy and increased mortality in S. aureus bacteraemia, has greatly facilitated advances in the rapid molecular diagnosis of S. aureus. Rapid molecular testing for S. aureus can greatly reduce laboratory turnaround time, and in some circumstances, may lead to improved clinical outcomes. In addition, advances in DNA sequencing technology and bioinformatic analysis have shed new lights on the molecular epidemiology and transmission dynamics of S. aureus. In this context, we provide an overview of the key advances in the molecular diagnosis and typing of S. aureus, with a particular focus on the clinical impact and utility of genomic technologies.

Typing of bacterial isolates has been used for decades to study local outbreaks as well as in national and international surveillances for monitoring newly emerging resistant clones. Despite being recognized as a nosocomial pathogen, the precise modes of transmission of Stenotrophomonas maltophilia in health care settings are unknown. Due to the high genetic diversity observed among S. maltophilia clinical isolates, the typing results might be better interpreted if also environmental strains were included. This could help to identify preventative measures to be designed and implemented for decreasing the possibility of outbreaks and nosocomial infections. In this review, we attempt to provide an overview on the most common typing methods used for clinical epidemiology of S. maltophilia strains, such as PCR-based fingerprinting analyses, pulsed-field gel electrophoresis, multilocus variable number tandem repeat analysis, and multilocus sequence type. Application of the proteomic-based mass spectrometry by matrix-assisted laser desorption ionization-time of flight is also described. Improvements of typing methods already in use have to be achieved to facilitate S. maltophilia infection control at any level. In the near future, when novel Web-based platforms for rapid data processing and analysis will be available, whole genome sequencing technologies will likely become a highly powerful tool for outbreak investigations and surveillance studies in routine clinical practices., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Legionnaires' disease (LD) is an often severe and potentially fatal form of bacterial pneumonia caused by an extensive list of Legionella species. These ubiquitous freshwater and soil inhabitants cause human respiratory disease when amplified in man-made water or cooling systems and their aerosols expose a susceptible population. Treatment of sporadic cases and rapid control of LD outbreaks benefit from swift diagnosis in concert with discriminatory bacterial typing for immediate epidemiological responses. Traditional culture and serology were instrumental in describing disease incidence early in its history; currently, diagnosis of LD relies almost solely on the urinary antigen test, which captures only the dominant species and serogroup, Legionella pneumophila serogroup 1 (Lp1). This has created a diagnostic "blind spot" for LD caused by non-Lp1 strains. This review focuses on historic, current, and emerging technologies that hold promise for increasing LD diagnostic efficiency and detection rates as part of a coherent testing regimen. The importance of cooperation between epidemiologists and laboratorians for a rapid outbreak response is also illustrated in field investigations conducted by the CDC with state and local authorities. Finally, challenges facing health care professionals, building managers, and the public health community in combating LD are highlighted, and potential solutions are discussed., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Bacterial culture was the first method used to describe the human microbiota, but this method is considered outdated by many researchers. Metagenomics studies have since been applied to clinical microbiology; however, a "dark matter" of prokaryotes, which corresponds to a hole in our knowledge and includes minority bacterial populations, is not elucidated by these studies. By replicating the natural environment, environmental microbiologists were the first to reduce the "great plate count anomaly," which corresponds to the difference between microscopic and culture counts. The revolution in bacterial identification also allowed rapid progress. 16S rRNA bacterial identification allowed the accurate identification of new species. Mass spectrometry allowed the high-throughput identification of rare species and the detection of new species. By using these methods and by increasing the number of culture conditions, culturomics allowed the extension of the known human gut repertoire to levels equivalent to those of pyrosequencing. Finally, taxonogenomics strategies became an emerging method for describing new species, associating the genome sequence of the bacteria systematically. We provide a comprehensive review on these topics, demonstrating that both empirical and hypothesis-driven approaches will enable a rapid increase in the identification of the human prokaryote repertoire., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The emergence of Acinetobacter baumannii during recent decades as an important nosocomial pathogen responsible of worldwide, intensively documented, outbreaks has resulted in a need for effective epidemiological typing methods. Throughout the years, many typing methods for A. baumannii epidemiological studies have been proposed from phenotypic to molecular methods. Currently, the use of phenotypic typing methods have declined considerably and been progressively replaced by molecular methods. In this review, we introduce the current molecular methods available for A. baumannii typing. Each method has its own advantages and disadvantages, and the selection of an appropriate genotyping method depends on studied objectives. This review sheds light on questions in different epidemiological settings and most molecular methods used to fit these objectives.

The routine use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has revolutionized microorganism identification in the clinical microbiology laboratory. Building from these now common microorganism identification strategies, this review explores future clinical applications of MALDI-TOF MS. This includes practical approaches for laboratorians interested in implementing direct identification processing methods for MALDI-TOF detection of microbes in bloodstream infection (BSI) and urinary tract infection (UTI), as well as, post-analytical approaches for classifying MALDI-TOF spectral data to detect characteristics other and species-level identification (e.g. strain-level classification, typing, and resistance mechanisms)., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Objective: To estimate the impact of new tuberculosis diagnostics on tuberculosis transmission given the complex contextual factors that can lead to patient loss before diagnosis or treatment., Methods: An epidemic model of tuberculosis specifying discrete steps along the tuberculosis diagnostic pathway was constructed. The model was calibrated to the epidemiology of tuberculosis and human immunodeficiency virus (HIV) infection in the United Republic of Tanzania and was used to assess the impact of a new diagnostic tool with 70% sensitivity for smear-negative pulmonary tuberculosis. The influence of contextual factors on the projected epidemic impact of the new diagnostic tool over the decade following introduction was explored., Findings: With the use of smear microscopy, the incidence of tuberculosis will decline by an average of 3.94% per year. If the new tool is added, incidence will decline by an annual 4.25%. This represents an absolute change of 0.31 percentage points (95% confidence interval: 0.04-0.42). However, the annual decline in transmission with use of the new tool is less when existing strategies for the diagnosis of smear-negative cases have high sensitivity and when symptomatic individuals delay in seeking care. Other influential contextual factors include access to tuberculosis care, patient loss before diagnosis, initial patient default after diagnosis and treatment success rate., Conclusion: When implementing and scaling up the use of a new diagnostic tool, the operational context in which diagnosis and treatment take place needs to be considered.

Clinical laboratory practices affect patient care and disease surveillance. It is recommended that laboratories routinely use both culture for Escherichia coli O157 and a method that detects Shiga toxins (Stx) to identify all Stx-producing E. coli (STEC) and that labs send broths or isolates to a public health laboratory. In 2007, we surveyed laboratories serving Foodborne Diseases Active Surveillance Network sites that performed on-site enteric disease diagnostic testing to determine their culture and nonculture-based testing practices for STEC identification. Our goals were to measure changes over time in laboratory practices and to compare reported practices with published recommendations. Overall, 89% of laboratories used only culture-based methods, 7% used only Stx enzyme immunoassay (EIA), and 4% used both Stx EIA and culture-based methods. Only 2% of laboratories reported simultaneous culture for O157 STEC and use of Stx EIA. The proportion that ever used Stx EIA increased from 6% in 2003 to 11% in 2007. The proportion that routinely tested all specimens with at least one method was 66% in 2003 versus 71% in 2007. Reference laboratories were less likely than others to test all specimens routinely by one or more of these methods (48% vs. 73%, p=0.03). As of 2007, most laboratories complied with recommendations for O157 STEC testing by culture but not with recommendations for detection of non-O157 STEC. The proportion of laboratories that culture stools for O157 STEC has changed little since 2003, whereas testing for Stx has increased., (© Mary Ann Liebert, Inc.)

Purpose of Review: Faster identification of infecting microorganisms and treatment options is a first-ranking priority in the infectious disease area, in order to prevent inappropriate treatment and overuse of broad-spectrum antibiotics. Standard bacterial identification is intrinsically time-consuming, and very recently there has been a burst in the number of commercially available nonphenotype-based techniques and in the documentation of a possible clinical impact of these techniques., Recent Findings: Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is now a standard diagnostic procedure on cultures and hold promises on spiked blood. Meanwhile, commercial PCR-based techniques have improved with the use of bacterial DNA enrichment methods, the diversity of amplicon analysis techniques (melting curve analysis, microarrays, gel electrophoresis, sequencing and analysis by mass spectrometry) leading to the ability to challenge bacterial culture as the gold standard for providing earlier diagnosis with a better 'clinical' sensitivity and additional prognostic information., Summary: Laboratory practice has already changed with MALDI-TOF MS, but a change in clinical practice, driven by emergent nucleic acid-based techniques, will need the demonstration of real-life applicability as well as robust clinical-impact-oriented studies.

Viridans group streptococci (VGS) are gram-positive microorganisms that can form alpha-hemolytic colonies on sheep blood agar. They reside as normal flora in oral cavity, respiratory, gastrointestinal, urogenital tract and on skin. They can cause bacteremia, endocarditis, meningitis and septicemia following dental procedures. The diagnosis of VGS are difficult since the taxonomic classification and species na-mes may change due in time. Viridans group streptococci are classified into 5 groups (Sanguinis, Mitis, Mutans, Salivarius, Anginosus) according to biochemical reactions and 16S rRNA sequencing. Since Streptococcus pneumoniae is a member of the Mitis group, the other important species in this group deserves investigation. Genetic exchange between Streptococcus mitis, Streptococcus oralis and S.pneumoniae by transformation and lysis mechanisms occur continously as they share the same anatomical region. These mechanisms play role in exchanging capsular and antibiotic resistance genes between these species. The cultivation of VGS usually starts with the inoculation of various patient specimens into sheep blood agar and the detection of alpha-hemolytic colonies. Observation of gram-positive cocci microscopically, the detection of optochin-resistant and bile insoluble colonies with few exceptions are the further important steps in laboratory diagnosis. VGS are then identified at species level by using biochemical reactions, automated diagnostic systems and molecular methods. The last step in the laboratory diagnosis of VGS is antibiotic susceptibility testing which is of outmost importance as penicillin and erythromycin resistance are on rise. In this review article, classification of VGS, similarities between S.pneumoniae and Mitis group streptococci and the laboratory diagnosis of VGS have been discussed.

The genus Pseudomonas was described in 1894 and is one of the most diverse and ubiquitous bacterial genera whose species have been isolated worldwide in all kinds of environments, from the Antarctica to the Tropics, present in sediments, clinical samples, plant, fungi and diseased animal specimens, water, soil, plant rhizosphere, sea, deserts, etc. The taxonomy of the genus has been controversial for years since a lot of bacterial taxa initially included in genus Pseudomonas have been reclassified in other genera or species from a different class of Proteobacteria over the years, as techniques for characterization and classification of microorganisms improved, aiming to set a phylogenetic classification of the species. In this review, the historical evolution of the taxonomy of Pseudomonas is described, and the currently valid criteria and future challenges for taxonomy of the genus and techniques used to achieve the necessary characterization for classifying the species are discussed. Finally, all the validly published Pseudomonas species at present are listed with an overview of their diversity and ecology.

The ability to discriminate bacterial isolates is important for a number of areas of research in Medical Microbiology, particularly in defining bacterial taxonomy and monitoring transmission in epidemiological investigations. Molecular techniques capable of typing bacteria at the level of the genome and proteome are now widely used for these investigations. This review considers two electrophoretic methods for typing bacteria on the basis of their proteomes, namely 1-D SDS-PAGE and 2-DE. The application of these two techniques for bacterial typing is described with reference to two publications that appeared in Electrophoresis [Costa, Electrophoresis 1990, 11, 382-391 and Cash et al., Electrophoresis 1997, 18, 1472-1482]. Even though these methods have been used for nearly 20 years to differentiate bacterial isolates they remain key technologies in proteome-based typing methods. The developments that have arisen from the two key papers are described in order to highlight the advantages and disadvantages in typing bacteria at the level of their proteomes. Some of the difficulties associated with electrophoretic typing methods can be overcome by using non-gel proteomic methods based on MS to provide improved sensitivity and specificity. The application of proteomic methods to investigate bacterial taxonomy, epidemiology and pathogenesis in general has significant potential in furthering our understanding of infectious diseases.

Multilocus sequence typing gives us unparalleled insights into the population biology of bacterial species, including those that cause healthcare-associated and/or community-acquired infections. For many of these species, we now recognize the existence of internationally prevalent clones, which are often resistant to multiple antibiotics.

Molecular biology-based techniques based on microbial genotype or DNA sequence have emerged as a basic tool in biological research and in the establishment of large databases of characterized organisms. Genotyping methods have the potential to provide information on subtypes of the organism and their source and/or origin of infection, and to recognize particularly virulent strains of the organism and monitor vaccination programs. Pulsed-field gel electrophoresis, ribotyping, CTX typing, amplified fragment length polymorphism, enterobacterial intergenic consensus sequence-PCR, multilocus sequence typing and microarray methods are more often used for the determination of genetic changes of toxigenic and nontoxigenic Vibrio cholerae strains, origin of infection and relationship between clinical and environmental strains, with the simultaneous detection of the number of copies and types of CTX prophages and genes required for persistence in diverse aquatic environments. This review will discuss DNA-based techniques for the molecular analysis of V. cholerae, its application and future directions.

A questionnaire-based survey was performed to accumulate data on methodologies used in microbiology laboratories involved in epidemiological typing. Genotyping by PFGE and MLST are currently clearly preferred over phenotyping. The overall wish is to increase the activities by over 20% and additional resources would be used to invest in real-time typing.

Infections caused by foodborne bacterial pathogens continue to be a major public health issue around the world. During the past decade, pulsed-field gel electrophoresis (PFGE) has become the gold standard for molecular subtyping and source tracking of most foodborne bacteria. Owing to problems inherent in PFGE technology, new methods have been developed focusing on DNA sequence-based subtyping. This review discusses the feasibility of using multilocus sequence typing, multiple-locus variable-number tandem repeat analysis, single nucleotide polymorphisms, microarrays, whole genome sequencing and mass spectrometry for subtyping foodborne bacterial pathogens.

The W-Beijing strain of tuberculosis has been identified in many molecular epidemiological studies as being particularly prevalent. This identification has been made possible through the development of a number of genotyping technologies including spoligotyping. Highly prevalent genotypes associated with outbreaks, such as the W-Beijing strain, are implicitly regarded as fast spreading. Here we present a quantitative method to identify "emerging" strains, those that are spreading faster than the background rate inferred from spoligotype data. The approach uses information about the mutation process specific to spoligotypes, combined with a model of both transmission and mutation. The core principle is that if two comparable strains have the same number of isolates, then the strain with fewer inferred mutation events must have spread faster if the mutation process is common. Applying this method to four different data sets, we find not only the W-Beijing strain, but also a number of other strains, to be emerging in this sense. Importantly, the strains that are identified as emerging are not simply those with the largest number of cases. The use of this method should facilitate the targeting of individual genotypes in intervention programs.

Background: Recent advances in nucleic acid-based methods to detect bacteria offer increased sensitivity and specificity over traditional microbiological techniques. The potential benefit of nucleic acid-based testing to the clinical laboratory is reduced time to diagnosis, high throughput, and accurate and reliable results., Methods: Several PCR and hybridization tests are commercially available for specific organism detection. Furthermore, hundreds of nucleic acid-based bacterial detection tests have been published in the literature and could be adapted for use in the clinical setting. Contamination potential, lack of standardization or validation for some assays, complex interpretation of results, and increased cost are possible limitations of these tests, however, and must be carefully considered before implementing them in the clinical laboratory., Conclusions: A major area of advancement in nucleic acid-based assay development has been for specific and broad-range detection of bacterial pathogens.

The importance of the intraspecific typing of salmonellae for effective epidemiological analysis is shown. The methods (phenotypical and genotypical), used for the epidemiological marking of salmonellae, are characterized, their advantages and drawbacks, as well as prospects of their further use, are given. The importance of the standardization of molecular-genetic methods to ensure data exchange between different laboratories and the investigation of international outbreaks of enteric infections is pointed out. The international projects PulseNet and Salmgene of the surveillance of Salmonella infection, based on genotyping of Salmonella isolates, are presented.

Background: Polymorphic tandem repeat typing is a new generic technology which has been proved to be very efficient for bacterial pathogens such as B. anthracis, M. tuberculosis, P. aeruginosa, L. pneumophila, Y. pestis. The previously developed tandem repeats database takes advantage of the release of genome sequence data for a growing number of bacteria to facilitate the identification of tandem repeats. The development of an assay then requires the evaluation of tandem repeat polymorphism on well-selected sets of isolates. In the case of major human pathogens, such as S. aureus, more than one strain is being sequenced, so that tandem repeats most likely to be polymorphic can now be selected in silico based on genome sequence comparison., Results: In addition to the previously described general Tandem Repeats Database, we have developed a tool to automatically identify tandem repeats of a different length in the genome sequence of two (or more) closely related bacterial strains. Genome comparisons are pre-computed. The results of the comparisons are parsed in a database, which can be conveniently queried over the internet according to criteria of practical value, including repeat unit length, predicted size difference, etc. Comparisons are available for 16 bacterial species, and the orthopox viruses, including the variola virus and three of its close neighbors., Conclusions: We are presenting an internet-based resource to help develop and perform tandem repeats based bacterial strain typing. The tools accessible at http://minisatellites.u-psud.fr now comprise four parts. The Tandem Repeats Database enables the identification of tandem repeats across entire genomes. The Strain Comparison Page identifies tandem repeats differing between different genome sequences from the same species. The "Blast in the Tandem Repeats Database" facilitates the search for a known tandem repeat and the prediction of amplification product sizes. The "Bacterial Genotyping Page" is a service for strain identification at the subspecies level.

Microbiology has entered the postgenomic era and it is clear that bacterial typing should aim to be based on analysis of complete genomes. Although complete genome sequencing for epidemiological typing remains unrealistic for the present, microarrays that provide information on gene content are now becoming available. Microarrays comprised of several thousand probes on glass slides can now be manufactured in the laboratory using robotic arrayers. The gene probes are either PCR products or synthetic oligonucleotides that can be irreversibly attached to a reactive glass surface. The target nucleic acids to be hybridized to the probe array are tagged with fluorescent dyes. Relative probe hybridization signals can be measured when two or more different preparations are labeled with distinguishable fluorophores. Microarrays that include probes for every gene within a genome provide excellent comparative data, although a focus on variable genes may be more useful for typing purposes. Composite arrays of variable genes are under development.

Rapid and accurate diagnosis of symptomatic patients is a cornerstone of global tuberculosis control strategies. Remarkable progress has recently been made, upgrading the speed and quality of mycobacteriology diagnostic services in industrialized countries, but for most of the world where TB is a large public health burden those gains are still unrealized. Deficiencies in current case-finding tools in disease endemic countries have made it difficult to ensure access to good diagnostics at all health service levels, leaving many patients undiagnosed. Additionally, in well-established TB control programs where diagnostic access has been ensured, efforts to interrupt disease transmission have been hampered by the insensitivity and late detection of smear microscopy. Fortunately, technical progress in diagnostics is resulting in a number of improved tools, including some appropriate for low-income settings. Important work remains, however, before new diagnostic tools can be meaningfully integrated into national TB control programs of high-burden countries and before TB control strategies can take them into account. The design and quality of clinical trials evaluating new diagnostics must be improved, clinical and laboratory services that would allow rapid response to test results need to be enhanced, and basic and operational research to appraise the impact and cost-effectiveness of new diagnostic technologies must be carried out. This paper describes some of the recent advances in TB diagnostic technologies and puts them into perspective for global tuberculosis control.

The basic premise in any bacterial typing scheme is that epidemiologically related isolates are derived from the clonal expansion of a single precursor. In simple terms this means that a certain characteristic is more useful than others on the basis that it is conserved within a strain but diverse with a species. Such a definition is appropriate when considering the spread of a species within a ward hospital or even a community. However, as we have developed more and more refined molecular techniques for analysis of the bacterial genome, such a definition has become stressed when considering the evolution that is occurring especially amongst drug-resistant bacteria whose genetic make-up is changing due to the selective pressure induced by drug usage in the hospital or community. Increased sophistication does not necessarily bring improved typing of bacterial strains which might only be variants of the same clonal type. Molecular epidemiology is perhaps too exacting to be practical in certain circumstances and one should be cautious in its interpretation and extrapolation. It is open to debate whether we shall see in the next century unanimity in the choice of pheno- or genotypic tests to be used for typing of bacterial strains.

Improved diagnostic procedures should be an effective way to control infectious diseases and the spread of antibiotic resistance. To do so, diagnostics will need to be obtained within 1 hour of sampling and will require nucleic acid-based amplification tests directly on the clinical specimen.

A number of high-resolution molecular typing systems have been developed in recent years. Their availability raises the new issues of selecting the method (s) best suited for a particular purpose and interpreting and communicating typing results. Most of the currently available methods are comparative only: they allow testing of a sample of isolates for delineation of those closely related from those markedly different in genomic backgrounds. This approach is adequate for outbreak investigation, allowing determination of clonal spread in a microenvironment and identification of the source of infection. Comparative methods with sufficient resolution for most pathogens include restriction fragment-length polymorphism (RFLP), pulsed-field gel electrophoresis (PFGE), and arbitrarily primed and randomly amplified polymorphic DNA-polymerase chain reaction (PCR) analysis. For surveillance systems, monitoring clonal spread and prevalence in populations over extended periods of time requires library typing systems. These must be standardized, must have a high throughput, and must use a uniform nomenclature. Promising or validated methods include serotyping, insertion sequence fingerprinting, ribotyping, PFGE, amplified fragment-length polymorphism (AFLP), infrequent-restriction-site amplification PCR, interrepetitive element PCR typing (rep-PCR) and PCR-RFLP of polymorphic loci. PCR methods generating arrays of size-specific amplicons (AFLP, rep-PCR) can be more reproducibly analyzed by using denaturing polyacrylamide gel or capillary electrophoresis with automated laser detection. Binary probe typing systems appear optimal and should be enhanced further through use of DNA chip technology. In these systems, amplification of polymorphic regions is followed by solid-phase hybridization with a reference panel of sequence-variant specific probes. The resulting binary type results allow determination of reproducible, numeric profiles. However, interpretation and nomenclature of typing results for large-scale surveillance purposes still require a better understanding of population structure and microevolution of most microbial pathogens.

For many years, isolation in tissue culture (TC) was considered the test of choice for diagnosis of Chlamydia trachomatis infection. Non-culture tests, such as direct fluorescent antibody (DFA) and enzyme immunoassay (EIA) which detected chlamydial antigens in clinical specimens, made chlamydia diagnostic tests more widely available. DFA and EIA were less sensitive than TC and had some false positive results which compromised our ability to use these tests in low prevalence settings. Direct nucleic acid probes are available, but do not appear to be more sensitive than EIA. It was only with the introduction of amplified DNA tests [polymerase chain reaction (PCR) and ligase chain reaction (LCR)] that non-culture tests became available that were actually more sensitive than TC. Unfortunately these tests are also more expensive than the antigen detection methods. Until there is a fairly sophisticated cost benefit analysis or a change in the pricing of these tests, it seems obvious that TC will remain, the best choice where medical/legal implications are important, DFA will probably remain a widely used tests for laboratories that process relatively small numbers of specimens and EIAs will play a role where cost is major factor and large numbers of specimens require bulk processing. Where they are affordable, the amplified DNA tests are to be preferred as they are far more sensitive than these other non-culture tests.

After years of decline, infections due to Mycobacterium tuberculosis appear to be increasing in the developed world. This has led to a search for improved methods of typing strains in order to answer new questions concerning the epidemiology of tuberculosis. Owing to the phenotypic and genotypic homogeneity of the species, classical fingerprinting methods, including phage typing, serotyping and multilocus enzyme electrophoresis, have proven ineffective. However, new methods based on the analysis of repetitive DNA sequences have been introduced and are now being used to elucidate the emerging pattern of disease.