Update on the epidemiological typing methods for Acinetobacter baumannii.

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 Klebsiella pneumoniae.

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.

MALDI-TOF MS in the Microbiology Laboratory: Current Trends.

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.

[New microbiological techniques]. / Neue mikrobiologische Techniken.

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.

Microfluidics in microbiology: putting a magnifying glass on microbes.

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.

Molecular tests for the detection of antimicrobial resistant Neisseria gonorrhoeae: when, where, and how to use?

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.

From culturomics to taxonomogenomics: A need to change the taxonomy of prokaryotes in clinical microbiology.

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.

Contemporary genomic approaches in the diagnosis and typing of Staphylococcus aureus.

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.

Current and emerging Legionella diagnostics for laboratory and outbreak investigations.

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.

The rebirth of culture in microbiology through the example of culturomics to study human gut microbiota.

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.

An overview of various typing methods for clinical epidemiology of the emerging pathogen Stenotrophomonas maltophilia.

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.

Current molecular methods in epidemiological typing of Acinetobacter baumannii.

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.

Beyond identification: emerging and future uses for MALDI-TOF mass spectrometry in the clinical microbiology laboratory.

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).

The impact of new tuberculosis diagnostics on transmission: why context matters.

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.

Laboratory practices for the identification of Shiga toxin-producing Escherichia coli in the United States, FoodNet sites, 2007.

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.

[Classical and new approaches in laboratory diagnosis of viridans streptococci]. / Viridans streptokoklarin laboratuvar tanisinda klasik ve yeni yaklasimlar.

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.

Molecular diagnosis of bloodstream infections: planning to (physically) reach the bedside.

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.