Cross Platform Standardisation of an Experimental Pipeline for Use in the Identification of Dysregulated Human Circulating MiRNAs.

INTRODUCTION: Micro RNAs (miRNAs) are a class of highly conserved small non-coding RNAs that play an important part in the post-transcriptional regulation of gene expression. A substantial number of miRNAs have been proposed as biomarkers for diseases. While reverse transcriptase Real-time PCR (RT-qPCR) is considered the gold standard for the evaluation and validation of miRNA biomarkers, small RNA sequencing is now routinely being adopted for the identification of dysregulated miRNAs. However, in many cases where putative miRNA biomarkers are identified using small RNA sequencing, they are not substantiated when RT-qPCR is used for validation. To date, there is a lack of consensus regarding optimal methodologies for miRNA detection, quantification and standardisation when different platform technologies are used. MATERIALS AND METHODS: In this study we present an experimental pipeline that takes into consideration sample collection, processing, enrichment, and the subsequent comparative analysis of circulating small ribonucleic acids using small RNA sequencing and RT-qPCR. RESULTS, DISCUSSION, CONCLUSIONS: Initially, a panel of miRNAs dysregulated in circulating blood from breast cancer patients compared to healthy women were identified using small RNA sequencing. MiR-320a was identified as the most dysregulated miRNA between the two female cohorts. Total RNA and enriched small RNA populations (<30 bp) isolated from peripheral blood from the same female cohort samples were then tested for using a miR-320a RT-qPCR assay. When total RNA was analysed with this miR-320a RT-qPCR assay, a 2.3-fold decrease in expression levels was observed between blood samples from healthy controls and breast cancer patients. However, upon enrichment for the small RNA population and subsequent analysis of miR-320a using RT-qPCR, its dysregulation in breast cancer patients was more pronounced with an 8.89-fold decrease in miR-320a expression. We propose that the experimental pipeline outlined could serve as a robust approach for the identification and validation of small RNA biomarkers for disease.

Comparative genome analysis identifies novel nucleic acid diagnostic targets for use in the specific detection of Haemophilus influenzae.

Haemophilus influenzae is recognised as an important human pathogen associated with invasive infections, including bloodstream infection and meningitis. Currently used molecular-based diagnostic assays lack specificity in correctly detecting and identifying H. influenzae. As such, there is a need to develop novel diagnostic assays for the specific identification of H. influenzae. Whole genome comparative analysis was performed to identify putative diagnostic targets, which are unique in nucleotide sequence to H. influenzae. From this analysis, we identified 2H. influenzae putative diagnostic targets, phoB and pstA, for use in real-time PCR diagnostic assays. Real-time PCR diagnostic assays using these targets were designed and optimised to specifically detect and identify all 55H. influenzae strains tested. These novel rapid assays can be applied to the specific detection and identification of H. influenzae for use in epidemiological studies and could also enable improved monitoring of invasive disease caused by these bacteria.

Comparison of Established Diagnostic Methodologies and a Novel Bacterial smpB Real-Time PCR Assay for Specific Detection of Haemophilus influenzae Isolates Associated with Respiratory Tract Infections.

Haemophilus influenzae is a significant causative agent of respiratory tract infections (RTI) worldwide. The development of a rapid H. influenzae diagnostic assay that would allow for the implementation of infection control measures and also improve antimicrobial stewardship for patients is required. A number of nucleic acid diagnostics approaches that detect H. influenzae in RTIs have been described in the literature; however, there are reported specificity and sensitivity limitations for these assays. In this study, a novel real-time PCR diagnostic assay targeting the smpB gene was designed to detect all serogroups of H. influenzae. The assay was validated using a panel of well-characterized Haemophilus spp. Subsequently, 44 Haemophilus clinical isolates were collected, and 36 isolates were identified as H. influenzae using a gold standard methodology that combined the results of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and a fucK diagnostic assay. Using the novel smpB diagnostic assay, 100% concordance was observed with the gold standard, demonstrating a sensitivity of 100% (95% confidence interval [CI], 90.26% to 100.00%) and a specificity of 100% (95% CI, 63.06% to 100.00%) when used on clinical isolates. To demonstrate the clinical utility of the diagnostic assay presented, a panel of lower RTI samples (n = 98) were blindly tested with the gold standard and smpB diagnostic assays. The results generated were concordant for 94/98 samples tested, demonstrating a sensitivity of 90.91% (95% CI, 78.33% to 97.47%) and a specificity of 100% (95% CI, 93.40% to 100.00%) for the novel smpB assay when used directly on respiratory specimens.

A rapid culture independent methodology to quantitatively detect and identify common human bacterial pathogens associated with contaminated high purity water.

BACKGROUND: Water and High Purity Water (HPW) distribution systems can be contaminated with human pathogenic microorganisms. This biocontamination may pose a risk to human health as HPW is commonly used in the industrial, pharmaceutical and clinical sectors. Currently, routine microbiological testing of HPW is performed using slow and labour intensive traditional microbiological based techniques. There is a need to develop a rapid culture independent methodology to quantitatively detect and identify biocontamination associated with HPW. RESULTS: A novel internally controlled 5-plex real-time PCR Nucleic Acid Diagnostics assay (NAD), was designed and optimised in accordance with Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines, to rapidly detect, identify and quantify the human pathogenic bacteria Stenotrophomonas maltophilia, Burkholderia species, Pseudomonas aeruginosa and Serratia marcescens which are commonly associated with the biocontamination of water and water distribution systems. The specificity of the 5-plex assay was tested against genomic DNA isolated from a panel of 95 microorganisms with no cross reactivity observed. The analytical sensitivities of the S. maltophilia, B. cepacia, P. aeruginosa and the S. marcescens assays are 8.5, 5.7, 3.2 and 7.4 genome equivalents respectively. Subsequently, an analysis of HPW supplied by a Millipore Elix 35 water purification unit performed using standard microbiological methods revealed high levels of naturally occurring microbiological contamination. Five litre water samples from this HPW delivery system were also filtered and genomic DNA was purified directly from these filters. These DNA samples were then tested using the developed multiplex real-time PCR NAD assay and despite the high background microbiological contamination observed, both S. maltophilia and Burkholderia species were quantitatively detected and identified. At both sampling points the levels of both S. maltophilia and Burkholderia species present was above the threshold of 10 cfu/100 ml recommended by both EU and US guidelines. CONCLUSIONS: The novel culture independent methodology described in this study allows for rapid (<5 h), quantitative detection and identification of these four human pathogens from biocontaminated water and HPW distribution systems. We propose that the described NAD assay and associated methodology could be applied to routine testing of water and HPW distribution systems to assure microbiological safety and high water quality standards.

Rapid nucleic acid diagnostics for the detection of antimicrobial resistance in Gram-negative bacteria: is it time for a paradigm shift?

A key component for tackling the ever more serious antimicrobial resistance problem in Gram-negative bacteria is the introduction of rapid nucleic acid diagnostics. Successful incorporation of new diagnostic technologies has the potential benefit of improving not only patient treatment but also infection control and antimicrobial stewardship. However, there are still many hurdles to overcome, such as the complexity of resistance mechanisms in Gram-negative bacteria, the discrepancy between phenotype and genotype and the difficulty in distinguishing pathogens from background commensals. A small number of manufacturers have introduced tests to the market that concentrate partly or specifically on resistance determinants in Gram-negative bacteria. These are currently predominantly based on different types of PCR technology. The development of new technologies, such as whole-genome sequencing and the combination of MALDI-TOF with PCR, holds much promise for the introduction of improved diagnostics for the future.

A current overview of commercially available nucleic acid diagnostics approaches to detect and identify human gastroenteritis pathogens.

Gastroenteritis is caused by a wide range of viral, bacterial and parasitic pathogens and causes millions of deaths worldwide each year, particularly in infant populations in developing countries. Traditional microbiological culture and immunological based tests are time consuming, laborious and often lack diagnostic specificity and sensitivity. As a result patients can receive suboptimal and/or inappropriate antimicrobial treatment. In recent years, rapid nucleic acid diagnostics (NAD) technologies have become available to complement or even bypass and replace these traditional microbiological culture and immunological based tests. The main purpose of this review is to describe a number of recently available multiparametric commercial tests, to support the rapid and accurate clinical diagnosis of human gastroenteritis. These state of the art technologies have the ability to identify a wide range of microorganisms associated with enteric gastroenteritis. Following further technological innovation and more comprehensive clinical validation studies, these NAD tests have the potential to impact on the economic burden of health care systems. These rapid NAD tests can also be used to guide improved patient therapy in a timely manner which will reduce the extent of morbidity and mortality associated with these infections globally.

Diagnostics method for the rapid quantitative detection and identification of low-level contamination of high-purity water with pathogenic bacteria.

High-purity water (HPW) can be contaminated with pathogenic microorganisms, which may result in human infection. Current culture-based techniques for the detection of microorganisms from HPW can be slow and laborious. The aim of this study was to develop a rapid method for the quantitative detection and identification of pathogenic bacteria causing low-level contamination of HPW. A novel internally controlled multiplex real-time PCR diagnostics assay was designed and optimized to specifically detect and identify Pseudomonas aeruginosa and the Burkholderia genus. Sterile HPW, spiked with a bacterial load ranging from 10 to 10(3) cfu/100 ml, was filtered and the bacterial cells were removed from the filters by sonication. Total genomic DNA was then purified from these bacteria and subjected to testing with the developed novel multiplex real-time PCR diagnostics assay. The specific P. aeruginosa and Burkholderia genus assays have an analytical sensitivity of 3.5 genome equivalents (GE) and 3.7 GE, respectively. This analysis demonstrated that it was possible to detect a spiked bacterial load of 1.06 × 10(2) cfu/100 ml for P. aeruginosa and 2.66 × 10(2) cfu/100 ml for B. cepacia from a 200-ml filtered HPW sample. The rapid diagnostics method described can reliably detect, identify, and quantify low-level contamination of HPW with P. aeruginosa and the Burkholderia genus in <4 h. We propose that this rapid diagnostics method could be applied to the pharmaceutical and clinical sectors to assure the safety and quality of HPW, medical devices, and patient-care equipment.

Advances in multiparametric molecular diagnostics technologies for respiratory tract infections.

PURPOSE OF REVIEW: Respiratory tract infections (RTIs) are caused by a variety of bacterial, viral, fungal, and other pathogens and cause millions of deaths each year. Current standard microbiological culture-based tests are laborious and time consuming. Thus, patients are initially treated empirically, leading to inappropriate use of antibiotics. The purpose of this article is to provide clinicians and scientists with a review of recently available commercial multiparametric molecular diagnostics tests for the detection of RTIs so that they can be considered for use instead of, or in combination with, traditional culture techniques. RECENT FINDINGS: Several technologies have become commercially available for the multiparametric molecular detection of RTIs in the past decade including tests based on PCR-array, PCR-mass spectrometry, and multiplex qPCR technologies. The majority of these tests are for the detection of viruses, but more recently companies have begun to focus on detection of viruses, bacteria, and associated drug resistances in a single product to maximize the information provided to the clinician by a single test. SUMMARY: We describe the recent advances in commercial multiparametric molecular diagnostics technologies for the diagnosis of RTIs. Combining the specific and sensitive molecular detection of bacteria, viruses, fungi, and drug resistances is key if molecular methods are to replace traditional culture. The reliability of the molecular drug-resistance markers chosen, the need for the quantitative detection of some organisms, and throughput are also important considerations for new technology developers.

Overview of invasive fungal infections.

The incidence of invasive fungal infections (IFIs) has seen a marked increase in the last two decades. This is especially evident among transplant recipients, patients suffering from AIDS, in addition to those in receipt of immunosuppressive therapy. Worryingly, this increased incidence includes infections caused by opportunistic fungi and emerging fungal infections which are resistant to or certainly less susceptible than others to standard antifungal agents. As a direct response to this phenomenon, there has been a resolute effort over the past several decades to improve early and accurate diagnosis and provide reliable screening protocols thereby promoting the administration of appropriate antifungal therapy for fungal infections. Early diagnosis and treatment with antifungal therapy are vital if a patient is to survive an IFI. Substantial advancements have been made with regard to both the diagnosis and subsequent treatment of an IFI. In parallel, stark changes in the epidemiological profile of these IFIs have similarly occurred, often in direct response the type of antifungal agent being administered. The effects of an IFI can be far reaching, ranging from increased morbidity and mortality to increased length hospital stays and economic burden.

Global effects of homocysteine on transcription in Escherichia coli: induction of the gene for the major cold-shock protein, CspA.

Homocysteine (Hcy) is a thiol-containing amino acid that is considered to be medically important because it is linked to the development of several life-threatening diseases in humans, including cardiovascular disease and stroke. It inhibits the growth of Escherichia coli when supplied in the growth medium. Growth inhibition is believed to arise as a result of partial starvation for isoleucine, which occurs because Hcy perturbs the biosynthesis of this amino acid. This study attempted to further elucidate the inhibitory mode of action of Hcy by examining the impact of exogenously supplied Hcy on the transcriptome. Using gene macroarrays the transcript levels corresponding to 68 genes were found to be reproducibly altered in the presence of 0.5 mM Hcy. Of these genes, the biggest functional groups affected were those involved in translation (25 genes) and in amino acid metabolism (19 genes). Genes involved in protection against oxidative stress were repressed in Hcy-treated cells and this correlated with a decrease in catalase activity. The gene showing the strongest induction by Hcy was cspA, which encodes the major cold-shock protein CspA. RT-PCR and reporter fusion experiments confirmed that cspA was induced by Hcy. Induction of cspA by Hcy was not caused by nutritional upshift, a stimulus known to induce CspA expression, nor was it dependent on the presence of a functional CspA protein. The induction of cspA by Hcy was suppressed when isoleucine was included in the growth medium. These data suggest that the induction of CspA expression in the presence of Hcy occurs because of a limitation for isoleucine. The possibility that Hcy-induced cspA expression is triggered by translational stalling that occurs when the cells are limited for isoleucine is discussed.

Homocysteine toxicity in Escherichia coli is caused by a perturbation of branched-chain amino acid biosynthesis.

In Escherichia coli the sulfur-containing amino acid homocysteine (Hcy) is the last intermediate on the methionine biosynthetic pathway. Supplementation of a glucose-based minimal medium with Hcy at concentrations greater than 0.2 mM causes the growth of E. coli Frag1 to be inhibited. Supplementation of Hcy-treated cultures with combinations of branched-chain amino acids containing isoleucine or with isoleucine alone reversed the inhibitory effects of Hcy on growth. The last intermediate of the isoleucine biosynthetic pathway, alpha-keto-beta-methylvalerate, could also alleviate the growth inhibition caused by Hcy. Analysis of amino acid pools in Hcy-treated cells revealed that alanine, valine, and glutamate levels are depleted. Isoleucine could reverse the effects of Hcy on the cytoplasmic pools of valine and alanine. Supplementation of the culture medium with alanine gave partial relief from the inhibitory effects of Hcy. Enzyme assays revealed that the first step of the isoleucine biosynthetic pathway, catalyzed by threonine deaminase, was sensitive to inhibition by Hcy. The gene encoding threonine deaminase, ilvA, was found to be transcribed at higher levels in the presence of Hcy. Overexpression of the ilvA gene from a plasmid could overcome Hcy-mediated growth inhibition. Together, these data indicate that in E. coli Hcy toxicity is caused by a perturbation of branched-chain amino acid biosynthesis that is caused, at least in part, by the inhibition of threonine deaminase.