Prediction of eye and hair pigmentation phenotypes using the HIrisPlex system in a Brazilian admixed population sample.

Human pigmentation is a complex trait, probably involving more than 100 genes. Predicting phenotypes using SNPs present in those genes is important for forensic purpose. For this, the HIrisPlex tool was developed for eye and hair color prediction, with both models achieving high accuracy among Europeans. Its evaluation in admixed populations is important, since they present a higher frequency of intermediate phenotypes, and HIrisPlex has demonstrated limitations in such predictions; therefore, the performance of this tool may be impaired in such populations. Here, we evaluate the set of 24 markers from the HIrisPlex system in 328 individuals from Ribeirão Preto (SP) region, predicting eye and hair color and comparing the predictions with their real phenotypes. We used the HaloPlex Target Enrichment System and MiSeq Personal Sequencer platform for massively parallel sequencing. The prediction of eye and hair color was accomplished by the HIrisPlex online tool, using the default prediction settings. Ancestry was estimated using the SNPforID 34-plex to observe if and how an individual's ancestry background would affect predictions in this admixed sample. Our sample presented major European ancestry (70.5%), followed by African (21.1%) and Native American/East Asian (8.4%). HIrisPlex presented an overall sensitivity of 0.691 for hair color prediction, with sensitivities ranging from 0.547 to 0.782. The lowest sensitivity was observed for individuals with black hair, who present a reduced European contribution (48.4%). For eye color prediction, the overall sensitivity was 0.741, with sensitivities higher than 0.85 for blue and brown eyes, although it failed in predicting intermediate eye color. Such struggle in predicting this phenotype category is in accordance with what has been seen in previous studies involving HIrisPlex. Individuals with brown eye color are more admixed, with European ancestry decreasing to 62.6%; notwithstanding that, sensitivity for brown eyes was almost 100%. Overall sensitivity increases to 0.791 when a 0.7 threshold is set, though 12.5% of the individuals become undefined. When combining eye and hair prediction, hit rates between 51.3 and 68.9% were achieved. Despite the difficulties with intermediate phenotypes, we have shown that HIrisPlex results can be very helpful when interpreted with caution.

Evaluation of human papillomavirus (HPV) genotyping assays using type-specific HPV L1 reference DNA.

BACKGROUND: Human papillomaviruses (HPV) are known to play a central etiological role in the development of cervical cancer. General HPV genotyping methods consist of PCR with consensus primers combined with various detection methods. OBJECTIVE: The aim was to develop HPV L1 DNA reference materials to evaluate the sensitivity, specificity, and accuracy of genotyping results obtained from the HPV DNA Genotyping Chip (HPV CHIP) and RFMP assays. METHODS: In this study, the Ministry of Food and Drug Safety (MFDS) established reference DNA materials for the L1 gene from 41 subtypes of anogenital HPV to aid in genotyping human papillomavirus (HPV) strains. Of these, 22 subtypes were obtained from cervical scrape samples of Korean women and 19 subtypes were synthesized. These reference materials include 13 high-risk types (HPV-16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68), 3 probable high-risk types (HPV-26, 53, and 66), 16 low-risk types (HPV-6, 10, 11, 27, 34, 40, 42, 43, 44, 54, 55, 61, 70, 72, 73, and 81), and 8 undetermined-risk types (HPV-3, 57, 62, 67, 69, 71, 74, and 84). After confirming the sequences by standard methods, these HPV L1 DNA reference materials were then used to compare results from the HPV DNA Genotyping Chip (HPV CHIP) and restriction fragment mass polymorphism (RFMP) assays. RESULTS: Data collected from the HPV CHIP and RFMP assay showed comparably high sensitivity and accuracy. Both assays could detect 102 or more copies/µl of HPV L1 DNA from 39 types of HPV, with higher accuracy in detecting samples with mixed types of HPV. CONCLUSION: The present study confirms the HPV L1 DNA reference materials developed by MFDS are reliable and useful for the evaluation of HPV genotyping assays.

Next-generation sequencing technologies: An overview.

Since the days of Sanger sequencing, next-generation sequencing technologies have significantly evolved to provide increased data output, efficiencies, and applications. These next generations of technologies can be categorized based on read length. This review provides an overview of these technologies as two paradigms: short-read, or "second-generation," technologies, and long-read, or "third-generation," technologies. Herein, short-read sequencing approaches are represented by the most prevalent technologies, Illumina and Ion Torrent, and long-read sequencing approaches are represented by Pacific Biosciences and Oxford Nanopore technologies. All technologies are reviewed along with reported advantages and disadvantages. Until recently, short-read sequencing was thought to provide high accuracy limited by read-length, while long-read technologies afforded much longer read-lengths at the expense of accuracy. Emerging developments for third-generation technologies hold promise for the next wave of sequencing evolution, with the co-existence of longer read lengths and high accuracy.

Analytical validation of an RI sample cartridge with the RapidHIT® ID system.

Evaluating the short tandem repeat (STR) in the field is important for the timely identification of a suspect. Several lines showed that the RapidHIT® ID system is reliable for DNA genotyping with buccal swabs and naked DNA. However, the application of this approach with blood samples has been poorly investigated. Because blood samples are among the most common forensic samples in our laboratory, further studies should be conducted. Here, we assessed the analytical performance of 19 STR loci with a newly developed RapidINTEL (RI) Sample Cartridge Kit by using the blood samples with known genotypes. Several commonly used substrates were included in the sensitivity study, and FTA cards proved to be the most promising sample carrier for blood storage and later identification. There was superior sensitivity and specificity with a 100% concordance rate for 0.5 µL of blood or 7 ng of genomic DNA. The performance for blood samples was comparable with that for the standard protocol. High success rate (90.57%) and high-concordance (100%) genotyping were automatically achieved over a wide range of operating conditions except for TH01. No contamination was observed throughout the study. Hematin, indigo, and humic acid had limited influence on the instrument system, while urea and melanin dramatically affected the genotyping results. Generally, the newly developed RI sample cartridge provided an alternative method for the STR genotyping of single-source blood samples over a wide range of operating conditions.

Pharmacogenomics Biomarker Discovery and Validation for Translation in Clinical Practice.

Interindividual variability in drug efficacy and toxicity is a major challenge in clinical practice. Variations in drug pharmacokinetics (PKs) and pharmacodynamics (PDs) can be, in part, explained by polymorphic variants in genes encoding drug metabolizing enzymes and transporters (absorption, distribution, metabolism, and excretion) or in genes encoding drug receptors. Pharmacogenomics (PGx) has allowed the identification of predictive biomarkers of drug PKs and PDs and the current knowledge of genome-disease and genome-drug interactions offers the opportunity to optimize tailored drug therapy. High-throughput PGx genotyping, from targeted to more comprehensive strategies, allows the identification of PK/PD genotypes to be developed as clinical predictive biomarkers. However, a biomarker needs a robust process of validation followed by clinical-grade assay development and must comply to stringent regulatory guidelines. We here discuss the methodological challenges and the emerging technological tools in PGx biomarker discovery and validation, at the crossroad among molecular genetics, bioinformatics, and clinical medicine.

Evaluation of the GenoType NTM-DR assay performance for the identification and molecular detection of antibiotic resistance in Mycobacterium abscessus complex.

The first objective of this study was to determine the GenoType NTM-DR assay performance for subspecies identification in Mycobacterium abscessus complex isolates. The second objective was to evaluate the GenoType NTM-DR assay ability to detect clarithromycin and amikacin resistance in M. abscessus complex isolates compared with drug susceptibility testing (DST) and PCR sequencing of the erm(41), rrl and rrs genes. The concordance between the GenoType NTM-DR and MLST results concerning subspecies identification was 100%. The wild type and mutated alleles of the rrl and rrs genes were detected by the GenoType NTM-DR assay and PCR sequencing with 100% (115/115) agreement. Similarly, 100% concordance between GenoType NTM-DR and DST was observed for clarithromycin and amikacin testing. Sensitivity for the detection of clarithromycin and amikacin resistance was 100%. The GenoType NTM-DR assay provides a robust and complementary tool to the gold standard methods (MLST and broth microdilution) for subspecies identification and drug resistance detection.

Skin pigmentation and genetic variants in an admixed Brazilian population of primarily European ancestry.

Although many genes have been shown to be associated with human pigmentary traits and forensic prediction assays exist (e.g. HIrisPlex-S), the genetic knowledge about skin colour remains incomplete. The highly admixed Brazilian population is an interesting study population for investigation of the complex genotype-phenotype architecture of human skin colour because of its large variation. Here, we compared variants in 22 pigmentary genes with quantitative skin pigmentation levels on the buttock, arm, and forehead areas of 266 genetically admixed Brazilian individuals. The genetic ancestry of each individual was estimated by typing 46 AIM-InDels. The mean proportion of genetic ancestry was 68.8% European, 20.8% Sub-Saharan African, and 10.4% Native American. A high correlation (adjusted R2 = 0.65, p < 0.05) was observed between nine SNPs and quantitative skin pigmentation using multiple linear regression analysis. The correlations were notably smaller between skin pigmentation and biogeographic ancestry (adjusted R2 = 0.45, p < 0.05), or markers in the leading forensic skin colour prediction system, the HIrisPlex-S (adjusted R2 = 0.54, p < 0.05). Four of the nine SNPs, OCA2 rs1448484 (rank 2), APBA2 rs4424881 (rank 4), MFSD12 rs10424065 (rank 8), and TYRP1 1408799 (rank 9) were not investigated as part of the HIrisPlex-S selection process, and therefore not included in the HIrisPlex-S model. Our results indicate that these SNPs account for a substantial part of the skin colour variation in individuals of admixed ancestry. Hence, we suggest that these SNPs are considered when developing future skin colour prediction models.

DNA methylation age estimation in blood samples of living and deceased individuals using a multiplex SNaPshot assay.

Many studies in the forensic field have reported that analysis of DNA methylation is the most reliable method of predicting age. In a previous study, 5 CpG sites located in ELOVL2, FHL2, KLF14, C1orf132 and TRIM59 genes were tested for age prediction purposes in blood, saliva and buccal swab samples from Korean individuals using a multiplex methylation SNaPshot assay. The main goals of the present study were i) to replicate the same multiplex SNaPshot assay in blood samples from Portuguese individuals, ii) to compare DNA methylation status between two different populations and iii) to address putative differences in the methylation status between blood from living and deceased individuals. Blood samples from 59 living individuals (37 females, 22 males; aged 1-94 years-old) and from 62 deceased individuals (13 females, 49 males; aged 28-86 years-old) were evaluated. The specific primers were those previously described. Linear regression models were used to analyse relationships between methylation levels and chronological age using IBM SPSS software v.24. Our results allowed to build a final age prediction model (APM) for blood samples of living individuals with 3 CpG sites, at ELOVL2, FHL2 and C1orf132 genes, explaining 96.3% of age variation, with a mean absolute deviation (MAD) from chronological age of 4.25 years. Some differences were found in the extent of the age association in the targeted loci comparing Portuguese with Korean individuals. The final APM built for deceased individuals included 4 CpG sites, at ELOVL2, FHL2, C1orf132 and TRIM59 genes, explaining 79.3% of age variation, with a MAD of 5.36 years. Combining both sets of samples from living and deceased individuals, the most accurate APM with 4 CpGs, at ELOVL2, FHL2, C1orf132 and TRIM59 genes, explained 92.5% of variation in age, with a MAD of 4.97 years. In conclusion, our study replicated in blood samples of Portuguese living individuals a previous SNaPshot assay for age estimation. The possibility that age markers might be population specific and that postmortem changes can alter the methylation status among specific loci was suggested by our data. Our study showed the usefulness of the multiplex methylation SNaPshot assay for forensic analysis in blood samples of living and deceased individuals.

Simple Protocol for Generating and Genotyping Genome-Edited Mice With CRISPR-Cas9 Reagents.

The simple protocol described in this article aims to provide all required information, as a comprehensive, easy-to-follow step-by-step method, to ensure the generation of the expected genome-edited mice. Here, we provide protocols for the preparation of CRISPR-Cas9 reagents for microinjection and electroporation into one-cell mouse embryos to create knockout or knock-in mouse models, and for genotyping the resulting offspring with the latest innovative next-generation sequencing methods. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Designing the best RNA guide for your gene disruption/editing strategy Basic Protocol 2: Preparing and validating CRISPR-Cas9 reagents Basic Protocol 3: Preparing and injecting CRISPR-Cas9 compounds into fertilized mouse oocytes Basic Protocol 4: Genotyping genome-edited mice Support Protocol: Genotyping for CRISPR-generated "indel" mutations.

Evaluation of KRAS, NRAS and BRAF mutations detection in plasma using an automated system for patients with metastatic colorectal cancer.

BACKGROUND: Cell-free DNA detection is becoming a surrogate assay for tumor genotyping. Biological fluids often content a very low amount of cell-free tumor DNA and assays able to detect very low allele frequency mutant with a few quantities of DNA are required. We evaluated the ability of the fully-automated molecular diagnostics platform Idylla for the detection of KRAS, NRAS and BRAF hotspot mutations in plasma from patients with metastatic colorectal cancer (mCRC). MATERIALS AND METHODS: First, we evaluated the limit of detection of the system using two set of laboratory made samples that mimic mCRC patient plasma, then plasma samples from patients with mCRC were assessed using Idylla system and BEAMing digital PCR technology. RESULTS: Limits of detection of 0.1%, 0.4% and 0.01% for KRAS, NRAS and BRAF respectively have been reached. With our laboratory made samples, sensitivity up to 0.008% has been reached. Among 15 patients' samples tested for KRAS mutation, 2 discrepant results were found between Idylla and BEAMing dPCR. A 100% concordance between the two assays has been found for the detection of NRAS and BRAF mutations in plasma samples. CONCLUSIONS: The Idylla system does not reach as high sensitivity as assays like ddPCR but has an equivalent sensitivity to modified NGS technics with a lower cost and a lower time to results. These data allowed to consider the Idylla system in a routine laboratory workflow for KRAS, NRAS and BRAF mutations detection in plasma.

Application of MIRU-VNTR on smear slides: a shortcut for detection of polyclonal infections in tuberculosis patients.

Mixed (polyclonal) infections are one of the main problems in tuberculosis (TB) management. The best available method for detecting polyclonal infections in TB is mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR). According to multiple studies, MIRU-VNTR method can be applied to detect TB-related polyclonal infections in sputum samples or cultures. Setup of MIRU-VNTR on smear slides can be an efficient approach, regardless of the limitations of cultures and sputum samples in many laboratories. The present study aimed at investigating the diagnostic potential of MIRU-VNTR on smear slides in detecting mixed infections. Ziehl-Neelsen-stained microscopic slides were prepared from 14 clinical specimens. For amplifying 24 MIRU-VNTR loci, PCR assay was performed on the smear slides, clinical specimens, and cultures. Based on the 24-locus MIRU-VNTR analysis, polyclonal infections were reported in 42.85% of smear slides, while the corresponding rate was estimated at 57.1% (8/14) in the clinical samples. In the corresponding cultures, the rate of mixed infection was 7.14% (1/14). Use of smear slides can be a safe option for transferring clinical specimens between environmental and reference laboratories. Considering their significant impact on TB treatment, it is essential to diagnose mixed infections in low-resource countries with a high prevalence of mixed infections. The present findings show that direct MIRU-VNTR on smear slides can be conveniently used for the detection of mixed infections.

Genetic characterization of 19 X-STRs in Sierra Leone population from Freetown.

A total of 550 individuals (265 males and 285 females) from Sierra Leone, a west-African coastal country, were genotyped using the Microreader™ 19X ID System kit. No significant deviations from the Hardy-Weinberg equilibrium were observed. A total of 250 alleles were identified with corresponding allele frequencies spanning from 0.0012 to 0.6762. PIC of the loci ranged from 0.4615 to 0.9481. The CPE, CPDF, and CPDM were 0.9999997856, 0.999999999999999999995774, and 0.999999999998997, respectively. The highly combined MECKruger, MECKishida, MECDesmarais, and MECDesmarais Duo were achieved as 0.99999992508, 0.999999999990802, 0.999999999990836, and 0.99999998412, respectively. Genetic comparisons revealed that genetic homogeneity existed in similar ethno origin or geographic origin populations. This is a pioneering genetic investigation using the Microreader™ 19X ID System kit in the population of Sierra Leone.

QuantStudio™ 12K Flex OpenArray® System as a Tool for High-Throughput Genotyping and Gene Expression Analysis.

Real time technology provides great advancements over PCR-based methods for a broad range of applications. With the increased availability of sequencing information, there is a need for the development and application of high-throughput real time PCR genotyping and gene expression methods that significantly broaden the current screening capabilities. Thermo Fisher Scientific (USA) has released a platform (QuantStudio™ 12K Flex system coupled with OpenArray® technology) with key elements required for high-throughput SNP genotyping and gene expression analysis. This allows for a rapid screening of large numbers of TaqMan® assays (up to 256) in many samples (up to 480) per run. This advanced real-time method involves the use of an array composed of 3,000 through-holes running on the QuantStudio™ 12K with OpenArray® block. The aim of this chapter is to outline the OpenArray® approach while providing a comprehensive in-depth review of the scientific literature on this topic. In agreement with a large number of independent studies, we conclude that the use of OpenArray® technology is a rapid and accurate method for high-throughput and large-scale systems biology studies with high specificity and sensitivity.

Validation of the Spartan RXCYP2C19 Genotyping Assay Utilizing Blood Samples.

The antiplatelet agent clopidogrel, a prodrug that requires bioactivation through the cytochrome P450 2C19 (CYP2C19) enzyme, is commonly prescribed post-percutaneous coronary intervention (PCI). Genetic variation in CYP2C19 contributes to individual variability in clopidogrel response, and can lead to adverse cardiovascular events. Incorporating CYP2C19 testing during routine clinical care helps identify high-risk patients, and provides the opportunity for pharmacotherapeutic interventions in the early post-PCI period. The Spartan RX CYP2C19 System has emerged as an optimal genotyping assay for use in clinical care due to ease of use, utilization of buccal swabs, and rapid turnaround time. However, workflow constraints related to sample collection and processing, storage, time, and personnel were encountered when integrating testing into clinical care. To improve clinical workflow and successfully implement CYP2C19 genotyping at our institution, we validated the Spartan RX System to return genotype utilizing blood samples. Our Molecular Diagnostic Laboratory tested 26 known reference materials and both blood and buccal swab samples from 23 patients and volunteers using the Spartan RX Assay. Genotype results were 100% concordant between DNA from blood and buccal swabs for all patients or volunteers, and consistent with expected results for the 26 reference materials. For reproducibility, three samples were tested in at least four separate runs, with all resulting genotypes in agreement between runs. Post-validation, the laboratory began offering CYP2C19 testing during clinical care. DNA extracted from blood can serve as a genomic DNA source for the Spartan RX Assay. Alteration of the methodology allowed for clinical implementation to support genotype-guided therapy.

Novel use of a Clinical Laboratory Improvements Amendments (CLIA)-certified Cyclin-Dependent Kinase N2C (CDKN2C) loss assay in sporadic medullary thyroid carcinoma.

BACKGROUND: The cyclin-dependent-kinase inhibitor/retinoblastoma pathway has been implicated in sporadic medullary thyroid carcinoma tumorigenesis. Somatic CDKN2C loss has been associated with decreased overall survival in medullary thyroid carcinoma patients. We evaluated CDKN2C loss in a prospective clinical environment using a novel Clinical Laboratory Improvement Amendments-certified assay to confirm its association with aggressive disease and to interrogate response to targeted therapy. METHODS: Patients with advanced sporadic medullary thyroid carcinoma underwent tumor genotyping for the purpose of management of targeted therapy and prognostication. RESULTS: Of patients with informative CDKN2C assay results, 30 (51.8%) were haploinsufficient/1N and 28 (48.3%) were 2N. Forty patients (69.0%) had a somatic RET mutation, and 36.9% had alterations of both genes. Thirty patients (51.7%) were treated with systemic therapy. Presence of genetic alterations in CDKN2C or RET did not predict treatment response. Patients with 1N CDKN2C loss had significantly shorter time-to-distant-metastasis than patients with normal copy number (P = .03). CONCLUSION: This is the first evaluation in the clinical setting of CDKN2C haploinsufficiency in sporadic medullary thyroid carcinoma. Although a larger cohort and longer follow-up will be required, loss seems to be associated with more aggressive disease and may indicate patients that might receive benefit from treatment with a CDK inhibitor.

Human short tandem repeat identification using a nanopore-based DNA sequencer: a pilot study.

Short tandem repeats (STRs) are repetitive DNA sequences that are highly polymorphic and widely used for personal identification in the field of forensic medicine. The standard method for determining the repeat number of STRs is capillary electrophoresis of PCR products; however, the use of DNA sequencing has increased because it can identify same-sized alleles with nucleotide substitutions (iso-alleles). In this study, we performed human STR genotyping using a portable nanopore-based DNA sequencer, the MinION, and evaluated its performance. Because the sequence quality obtained by MinION is considerably lower than those obtained with other DNA sequencers, we developed an original scoring scheme for judging the genotypes from MinION reads. Analysis of seven human samples for 21-45 STR loci yielded an average of 857 thousand reads per sample, and the accuracy of genotyping and iso-allele identification reached 75.7% and 82%, respectively. Although the accuracy is higher than that reported previously, further improvements are required before this method can be practically applied.

Comparison of an in-house 'home-brew' and commercial ViroSeq integrase genotyping assays on HIV-1 subtype C samples.

BACKGROUND: Roll-out of Integrase Strand Transfer Inhibitors (INSTIs) such as dolutegravir for HIV combination antiretroviral therapy (cART) in sub-Saharan Africa necessitates the development of affordable HIV drug resistance (HIVDR) assays targeting the Integrase gene. We optimised and evaluated an in-house integrase HIV-1 drug resistance assay (IH-Int) and compared it to a commercially available assay, ViroSeq™ Integrase Genotyping kit (VS-Int) amongst HIV-1 clade C infected individuals. METHODS: We used 54 plasma samples from treatment naïve participants and one plasma sample from a patient failing INSTI based cART. Specimens were genotyped using both the VS-Int and IH-Int assays. Stanford HIV drug resistance database were used for integrase resistance interpretation. We compared the major and minor resistance mutations, pairwise nucleotide and amino-acid identity, costs and assay time. RESULTS: Among 55 specimens tested with IH-Int, 53 (96.4%) successfully amplified compared to 45/55 (81.8%) for the VS-Int assay. The mean nucleotide and amino acid similarity from 33 paired sequences was 99.8% (SD ± 0.30) and 99.8% (SD ± 0.39) for the IH-Int and VS-Int assay respectively. The reagent cost/sample were 32 USD and 147 USD for IH-Int and VS-Int assay, respectively. All sequenced samples were confirmed as HIV-1 subtype C. CONCLUSIONS: The IH-Int assay had a high amplification success rate and high concordance with the commercial assay. It is significantly cheaper compared to the commercial assay. Our assay has the needed specifications for routine monitoring of participants on Dolutegravir based regimens in Botswana.

Optimizing PCR for Mouse Genotyping: Recommendations for Reliable, Rapid, Cost Effective, Robust and Adaptable to High-Throughput Genotyping Protocol for Any Type of Mutation.

Genotyping consists of searching for a DNA sequence variation localized at a well-defined locus in the genome. It is an essential step in animal research because it allows the identification of animals that will be bred to generate and maintain a colony, euthanized to control the available space in the animal facility, or used in experiment protocols. Here we describe polymerase chain reaction (PCR) genotyping protocols for fast, sensitive, easy, and cost-effective characterization of mouse genotype. We discuss optimization of parameters to improve the reliability of each assay and propose recommendations for enhancing reproducibility and reducing the occurrence of inconclusive genotyping. All steps required for efficient genotyping are presented: tissue collection; sample verification and direct DNA lysis; establishment of a robust genotyping strategy with reliable, rapid, and cost-effective assays; and finally, transition to high-throughput automatized PCR, including mix miniaturization and automation. © 2019 The Authors. Basic Protocol 1: Tissue sampling methods and procedure Basic Protocol 2: Sample verification and DNA lysis Basic Protocol 3: Design of a genotyping strategy Basic Protocol 4: Moving to high-throughput genotyping.

Interplay between Rapid Diagnostic Tests and Antimicrobial Stewardship Programs among Patients with Bloodstream and Other Severe Infections.

BACKGROUND: Antimicrobial stewardship programs (ASPs) aim to provide optimal antimicrobial therapy to patients quickly to improve the likelihood of overcoming infection while reducing the risk of adverse effects. Rapid diagnostic tests (RDTs) for infectious diseases have become an integral tool for ASPs to achieve these aims. CONTENT: This review explored the demonstrated clinical value of longer-standing technologies and implications of newer RDTs from an antimicrobial stewardship perspective. Based on available literature, the focus was on the use of RDTs in bloodstream infections (BSIs), particularly those that perform organism identification and genotypic resistance detection, phenotypic susceptibility testing, and direct specimen testing. Clinical implications of rapid testing among respiratory, central nervous system, and gastrointestinal infections are also reviewed. SUMMARY: Coupling RDTs with ASPs facilitates the appropriate and timely use of test results, translating into improved patient outcomes through optimization of antimicrobial use. These benefits are best demonstrated in the use of RDT in BSIs. Rapid phenotypic susceptibility testing offers the potential for early pharmacokinetic/pharmacodynamic optimization, and direct specimen testing on blood may allow ASPs to initiate appropriate therapy and/or tailor empiric therapy even sooner than other RDTs. RDTs for respiratory, central nervous system, and gastrointestinal illnesses have also shown significant promise, although more outcome studies are needed to evaluate their full impact.

Advances in Rapid Molecular Blood Culture Diagnostics: Healthcare Impact, Laboratory Implications, and Multiplex Technologies.

BACKGROUND: For far too long, the diagnosis of bloodstream infections has relied on time-consuming blood cultures coupled with traditional organism identification and susceptibility testing. Technologies to define the culprit in bloodstream infections have gained sophistication in recent years, notably by application of molecular methods. CONTENT: In this review, we summarize the tests available to clinical laboratories for molecular rapid identification and resistance marker detection in blood culture bottles that have flagged positive. We explore the cost-benefit ratio of such assays, covering aspects that include performance characteristics, effect on patient care, and relevance to antibiotic stewardship initiatives. SUMMARY: Rapid blood culture diagnostics represent an advance in the care of patients with bloodstream infections, particularly those infected with resistant organisms. These diagnostics are relatively easy to implement and appear to have a positive cost-benefit balance, particularly when fully incorporated into a hospital's antimicrobial stewardship program.