Acquired activated protein C resistance, thrombophilia and adverse pregnancy outcomes: a study performed in an Irish cohort of pregnant women.

The combination of thrombophilia and pregnancy increases the risk of thrombosis and the potential for adverse outcomes during pregnancy. The most significant common inherited risk factor for thrombophilia is activated protein C resistance (APCR), a poor anticoagulant response of APC in haemostasis, which is mainly caused by an inherited single-nucleotide polymorphism (SNP), factor V G1691A (FV Leiden) (FVL), referred as inherited APCR. Changes in the levels of coagulation factors: FV, FVIII, and FIX, and anticoagulant factors: protein S (PS) and protein C (PC) can alter APC function causing acquired APCR. Prothrombin G20210A and methylenetetrahydrofolate reductase (MTHFR) C677T are prothrombotic SNPs which in association with APCR can also increase the risk of thrombosis amongst Caucasians. In this study, a correlation between an acquired APCR phenotype and increased levels of factors V, VIII, and IX was demonstrated. Thrombophilic mutations amongst our acquired APCR pregnant women cohort are relatively common but do not appear to exert a severe undue adverse effect on pregnancy.

Detection of NASBA amplified bacterial tmRNA molecules on SLICSel designed microarray probes.

BACKGROUND: We present a comprehensive technological solution for bacterial diagnostics using tmRNA as a marker molecule. A robust probe design algorithm for microbial detection microarray is implemented. The probes were evaluated for specificity and, combined with NASBA (Nucleic Acid Sequence Based Amplification) amplification, for sensitivity. RESULTS: We developed a new web-based program SLICSel for the design of hybridization probes, based on nearest-neighbor thermodynamic modeling. A SLICSel minimum binding energy difference criterion of 4 kcal/mol was sufficient to design of Streptococcus pneumoniae tmRNA specific microarray probes. With lower binding energy difference criteria, additional hybridization specificity tests on the microarray were needed to eliminate non-specific probes. Using SLICSel designed microarray probes and NASBA we were able to detect S. pneumoniae tmRNA from a series of total RNA dilutions equivalent to the RNA content of 0.1-10 CFU. CONCLUSIONS: The described technological solution and both its separate components SLICSel and NASBA-microarray technology independently are applicative for many different areas of microbial diagnostics.

Novel multiplex real-time PCR diagnostic assay for identification and differentiation of Mycobacterium tuberculosis, Mycobacterium canettii, and Mycobacterium tuberculosis complex strains.

Tuberculosis (TB) in humans is caused by members of the Mycobacterium tuberculosis complex (MTC). Rapid detection of the MTC is necessary for the timely initiation of antibiotic treatment, while differentiation between members of the complex may be important to guide the appropriate antibiotic treatment and provide epidemiological information. In this study, a multiplex real-time PCR diagnostics assay using novel molecular targets was designed to identify the MTC while simultaneously differentiating between M. tuberculosis and M. canettii. The lepA gene was targeted for the detection of members of the MTC, the wbbl1 gene was used for the differentiation of M. tuberculosis and M. canettii from the remainder of the complex, and a unique region of the M. canettii genome, a possible novel region of difference (RD), was targeted for the specific identification of M. canettii. The multiplex real-time PCR assay was tested using 125 bacterial strains (64 MTC isolates, 44 nontuberculosis mycobacteria [NTM], and 17 other bacteria). The assay was determined to be 100% specific for the mycobacteria tested. Limits of detection of 2.2, 2.17, and 0.73 cell equivalents were determined for M. tuberculosis/M. canettii, the MTC, and M. canettii, respectively, using probit regression analysis. Further validation of this diagnostics assay, using clinical samples, should demonstrate its potential for the rapid, accurate, and sensitive diagnosis of TB caused by M. tuberculosis, M. canettii, and the other members of the MTC.

APCR, factor V gene known and novel SNPs and adverse pregnancy outcomes in an Irish cohort of pregnant women.

BACKGROUND: Activated Protein C Resistance (APCR), a poor anticoagulant response of APC in haemostasis, is the commonest heritable thrombophilia. Adverse outcomes during pregnancy have been linked to APCR. This study determined the frequency of APCR, factor V gene known and novel SNPs and adverse outcomes in a group of pregnant women. METHODS: Blood samples collected from 907 pregnant women were tested using the Coatest Classic and Modified functional haematological tests to establish the frequency of APCR. PCR-Restriction Enzyme Analysis (PCR-REA), PCR-DNA probe hybridisation analysis and DNA sequencing were used for molecular screening of known mutations in the factor V gene in subjects determined to have APCR based on the Coatest Classic and/or Modified functional haematological tests. Glycosylase Mediated Polymorphism Detection (GMPD), a SNP screening technique and DNA sequencing, were used to identify SNPs in the factor V gene of 5 APCR subjects. RESULTS: Sixteen percent of the study group had an APCR phenotype. Factor V Leiden (FVL), FV Cambridge, and haplotype (H) R2 alleles were identified in this group. Thirty-three SNPs; 9 silent SNPs and 24 missense SNPs, of which 20 SNPs were novel, were identified in the 5 APCR subjects. Adverse pregnancy outcomes were found at a frequency of 35% in the group with APCR based on Classic Coatest test only and at 45% in the group with APCR based on the Modified Coatest test. Forty-eight percent of subjects with FVL had adverse outcomes while in the group of subjects with no FVL, adverse outcomes occurred at a frequency of 37%. CONCLUSIONS: Known mutations and novel SNPs in the factor V gene were identified in the study cohort determined to have APCR in pregnancy. Further studies are required to investigate the contribution of these novel SNPs to the APCR phenotype. Adverse outcomes including early pregnancy loss (EPL), preeclampsia (PET) and intrauterine growth restriction (IGUR) were not significantly more frequent in subjects with APCR compared to normal pregnant women however Pregnancy induced hypertension (PIH) was found to be associated with FVL in our study group.

Co-occurrence of the West European (Gr.III) and North American (Gr.I) ribotypes of Alexandrium tamarense (Dinophyceae) in Shetland, Scotland.

An investigation into the diversity of the dinoflagellate Alexandrium was carried out during August 2007 within two fjordic sea lochs in the Shetland Isles, Scotland. The co-occurrence in the water column of the non-toxic West European (W.E. or Gr.III) and the neurotoxic North American (N.A. or Gr.I) ribotypes of A. tamarense was demonstrated using fluorescent in situ hybridisation. A patch of A. tamarense (W.E.) localised at approximately 10 m depth and extending over 6 km was detected in 'Clift Sound' with concentrations locally reaching approximately 1 x 10(4) cells l(-1). A. tamarense (N.A.) was also observed there but despite the presence of toxins in net haul samples collected locally, concentrations were low and near limits of detection. Alexandrium concentrations were approximately 1.5 x 10(3) cells l(-1) in 'Vaila Sound', where both W.E. and N.A. ribotypes were detected with equal relative abundances in some samples. Given the patchiness of A. tamarense populations and their possible organisation in thin layer structures, better vertical resolution through fine-scale sampling will be necessary for population dynamic studies. Implications for the shellfish industry are substantial since harmful microalgae patches may not be detected during routine monitoring. Moreover, the co-occurrence of morphologically indistinct toxic and non-toxic ribotypes will necessitate implementing molecular methods for their discrimination.

Real-time PCR detection of Dinophysis species in Irish coastal waters.

Diarrhetic shellfish toxin-producing Dinophysis species occur in Irish coastal waters throughout the year. Dinophysis acuta and Dinophysis acuminata are the most commonly occurring species and are responsible for the majority of closures of Irish mussel farms. This study describes the development of a qualitative real-time polymerase chain reaction (PCR) assay for identification of D. acuta and D. acuminata in Irish coastal waters. DNA sequence information for the D1-D2 region of the large ribosomal sub-unit (LSU) was obtained, following single-cell PCR of D. acuta and D. acuminata cells isolated from Irish coastal locations. PCR primers and hybridization probes, specific for the detection of D. acuta, were designed for real-time PCR on the LightCycler™. The LightCycler™ software melt curve analysis programme determined that D. acuta was identified by a melt-peak at 61°C, while D. acuminata cells produced a melt peak at 48°C. The limit of detection of the real-time PCR assay was determined to be one to ten plasmid copies of the LSU D1-D2 target region for both species and one to five D. acuminata cells. Lugol's preserved water samples were also tested with the assay. The real-time PCR assay identified Dinophysis species in 100% of samples found to contain Dinophysis species by light microscopy and had a greater than 90% correlation with light microscopy for identification of D. acuta and D. acuminata in the samples. The assay can identify and discriminate D. acuta and D. acuminata at low numbers in Irish waters and has the potential to add value to the Irish phytoplankton monitoring programme.

tmRNA--a novel high-copy-number RNA diagnostic target--its application for Staphylococcus aureus detection using real-time NASBA.

A real-time nucleic acid sequence-based amplification assay, targeting tmRNA, was designed for the rapid identification of Staphylococcus aureus. The selectivity of the assay was confirmed against a panel of 76 Staphylococcus strains and species and 22 other bacterial species. A detection limit of 1 cell equivalent was determined for the assay. A chimeric in vitro transcribed internal amplification control was developed and included in the assay. Application of the assay in natural and artificially contaminated unpasteurized (raw) milk enabled detection of 1-10 CFUS. aureus mL(-1) in 3-4 h, without the need for culture enrichment. Staphylococcus aureus was detected in all artificially contaminated milk samples (n=20) and none of the natural milk samples (n=20). Microbiological analysis of the natural milk samples was performed in parallel according to ISO 6888-3 and confirmed the absence of S. aureus. The method developed in this study has the potential to enable the specific detection of S. aureus in raw milk in a significantly shorter time frame than current standard methods. The assay further demonstrates the usefulness of tmRNA/ssrA as a nucleic acid diagnostic target.

Evaluation of a novel real-time PCR test based on the ssrA gene for the identification of group B streptococci in vaginal swabs.

BACKGROUND: Despite the implementation of prevention guidelines, early-onset group B streptococci (GBS) disease remains a cause of neonatal morbidity and mortality worldwide. Strategies to identify women who are at risk of transmitting GBS to their infant and the administration of intrapartum antibiotics have greatly reduced the incidence of neonatal GBS disease. However, there is a requirement for a rapid diagnostic test for GBS that can be carried out in a labour ward setting especially for women whose GBS colonisation status is unknown at the time of delivery. We report the design and evaluation of a real-time PCR test (RiboSEQ GBS test) for the identification of GBS in vaginal swabs from pregnant women. METHODS: The qualitative real-time PCR RiboSEQ GBS test was designed based on the bacterial ssrA gene and incorporates a competitive internal standard control. The analytical sensitivity of the test was established using crude lysate extracted from serial dilutions of overnight GBS culture using the IDI Lysis kit. Specificity studies were performed using DNA prepared from a panel of GBS strains, related streptococci and other species found in the genital tract environment. The RiboSEQ GBS test was evaluated on 159 vaginal swabs from pregnant women and compared with the GeneOhm StrepB Assay and culture for the identification of GBS. RESULTS: The RiboSEQ GBS test is specific and has an analytical sensitivity of 1-10 cell equivalents. The RiboSEQ GBS test was 96.4% sensitive and 95.8% specific compared to "gold standard" culture for the identification of GBS in vaginal swabs from pregnant women. In this study, the RiboSEQ GBS test performed slightly better than the commercial BD GeneOhm StrepB Assay which gave a sensitivity of 94.6% and a specificity of 89.6% compared to culture. CONCLUSION: The RiboSEQ GBS test is a valuable method for the rapid, sensitive and specific detection of GBS in pregnant women. This study also validates the ssrA gene as a suitable and versatile target for nucleic acid-based diagnostic tests for bacterial pathogens.

Fluorescent labeling of NASBA amplified tmRNA molecules for microarray applications.

BACKGROUND: Here we present a novel promising microbial diagnostic method that combines the sensitivity of Nucleic Acid Sequence Based Amplification (NASBA) with the high information content of microarray technology for the detection of bacterial tmRNA molecules. The NASBA protocol was modified to include aminoallyl-UTP (aaUTP) molecules that were incorporated into nascent RNA during the NASBA reaction. Post-amplification labeling with fluorescent dye was carried out subsequently and tmRNA hybridization signal intensities were measured using microarray technology. Significant optimization of the labeled NASBA protocol was required to maintain the required sensitivity of the reactions. RESULTS: Two different aaUTP salts were evaluated and optimum final concentrations were identified for both. The final 2 mM concentration of aaUTP Li-salt in NASBA reaction resulted in highest microarray signals overall, being twice as high as the strongest signals with 1 mM aaUTP Na-salt. CONCLUSION: We have successfully demonstrated efficient combination of NASBA amplification technology with microarray based hybridization detection. The method is applicative for many different areas of microbial diagnostics including environmental monitoring, bio threat detection, industrial process monitoring and clinical microbiology.

Evaluation of taxa-specific real-time PCR, whole-cell FISH and morphotaxonomy analyses for the detection and quantification of the toxic microalgae Alexandrium minutum (Dinophyceae), Global Clade ribotype.

The dinoflagellate genus Alexandrium contains neurotoxin-producing species that have adversely affected the aquaculture industry in many countries. The morphological similarity between Alexandrium species has led to the development of molecular methods for the discrimination, enumeration and monitoring of toxic and nontoxic species. A quantitative real-time PCR assay (qRT-PCR) targeting the internal transcribed spacer 1-5.8S rRNA gene using hybridization probe technology was developed for the potentially toxic species Alexandrium minutum (Global Clade) (GC). The assay was specific with a detection limit of less than one cell equivalent. The assay was used to detect and quantify A. minutum (GC) in seawater samples collected during summer 2007 in Cork Harbour, Ireland. The results were compared with those obtained using whole-cell FISH (WC-FISH) and morphotaxonomy analyses. Alexandrium minutum did not reach high bloom concentrations over the sampling period (maximum of c. 6 x 10(4) cells L(-1)), and the average concentrations determined using qRT-PCR, WC-FISH and morphotaxonomy did not significantly differ in eight of nine comparisons. Regression curves showed positive relationships between the methods; WC-FISH and qRT-PCR slightly under- and overestimated, respectively, the A. minutum concentrations compared with the morphotaxonomy method. The qRT-PCR assay for A. minutum (GC) offers high-throughput sample analysis and may prove suitable for implementation in microalgae monitoring programmes and assist in population dynamics studies of the species.

Rapid detection of Listeria monocytogenes in food using culture enrichment combined with real-time PCR.

A rapid method for the detection of Listeria monocytogenes in foods combining culture enrichment and real-time PCR was compared to the ISO 11290-1 standard method. The culture enrichment component of the rapid method is based on the ISO standard and includes 24h incubation in half-Fraser broth, 4h incubation in Fraser broth followed by DNA extraction and real-time PCR detection of the ssrA gene of L. monocytogenes. An internal amplification control, which is co-amplified with the same primers as the L. monocytogenes DNA, was also included in the assay. The method has a limit of detection of 1-5CFU/25g food sample and can be performed in 2 working days compared to up to 7days for the ISO standard. A variety of food samples from retail outlets and food processing plants (n=175) and controls (n=31) were tested using rapid and conventional methods. The rapid method was 99.44% specific, 96.15% sensitive and 99.03% accurate when compared to the standard method. This method has the potential to be used as an alternative to the standard method for food quality assurance providing rapid detection of L. monocytogenes in food.

Integrated microfluidic tmRNA purification and real-time NASBA device for molecular diagnostics.

We demonstrate the first integrated microfluidic tmRNA purification and nucleic acid sequence-based amplification (NASBA) device incorporating real-time detection. The real-time amplification and detection step produces pathogen-specific response in < 3 min from the chip-purified RNA from 100 lysed bacteria. On-chip RNA purification uses a new silica bead immobilization method. On-chip amplification uses custom-designed high-selectivity primers and real-time detection uses molecular beacon fluorescent probe technology; both are integrated on-chip with NASBA. Present in all bacteria, tmRNA (10Sa RNA) includes organism-specific identification sequences, exhibits unusually high stability relative to mRNA, and has high copy number per organism; the latter two factors improve the limit of detection, accelerate time-to-positive response, and suit this approach ideally to the detection of small numbers of bacteria. Device efficacy was demonstrated by integrated on-chip purification, amplification, and real-time detection of 100 E. coli bacteria in 100 microL of crude lysate in under 30 min for the entire process.

Rapid real-time PCR detection of Listeria monocytogenes in enriched food samples based on the ssrA gene, a novel diagnostic target.

A real-time PCR assay was designed to detect a 162-bp fragment of the ssrA gene in Listeria monocytogenes. The specificity of the assay for L. monocytogenes was confirmed against a panel of 6 Listeria species and 26 other bacterial species. A detection limit of 1-10 genome equivalents was determined for the assay. Application of the assay in natural and artificially contaminated culture enriched foods, including soft cheese, meat, milk, vegetables and fish, enabled detection of 1-5 CFU L. monocytogenes per 25g/ml of food sample in 30h. The performance of the assay was compared with the Roche Diagnostics 'LightCycler foodproof Listeria monocytogenes Detection Kit'. Both methods detected L. monocytogenes in all artificially contaminated retail samples (n=27) and L. monocytogenes was not detected by either system in 27 natural retail food samples. The method developed in this study has the potential to enable the specific detection of L. monocytogenes in a variety of food types in a time-frame considerably faster than current standard methods. The potential of the ssrA gene as a nucleic acid diagnostic (NAD) target has been demonstrated in L. monocytogenes. We are currently developing NAD tests based on the ssrA gene for a range of common foodborne and clinically relevant bacterial pathogens.

Quantification of ALS1 gene expression in Candida albicans biofilms by RT-PCR using hybridisation probes on the LightCycler.

The fungal pathogen Candida albicans has the ability to grow as a biofilm on synthetic materials. This presents a significant problem in the clinical situation when the organism grows as a biofilm on medical devices resulting in infections which are resistant to antifungal agents. Determining the extent to which certain genes are involved in biofilm formation is an important aspect for the development of strategies to control pathogenic biofilms. ALS1 is a member of the ALS (agglutinin-like sequence) family, the protein products of which are implicated in attachment to endothelial cells and biofilm formation. The expression of ALS1 in biofilms grown on silicone elastomer, a material used in the manufacture of medical devices, and planktonically grown cells was investigated using a novel real-time quantitative reverse transcriptase PCR (q-RT PCR) on the LightCycler. This study demonstrates quantitatively that ALS1 is clearly up-regulated during biofilm growth. The real-time q-RT PCR assay described here has the potential to be used as an indicator of biofilm formation on medical devices.

Evaluation of culture methods and a DNA probe-based PCR assay for detection of Campylobacter species in clinical specimens of feces.

Campylobacter species are the leading agents of bacterial gastroenteritis in developed countries. In this study 320 specimens of feces from patients with symptoms of acute gastroenteritis were cultured for Campylobacter species by direct plating on modified charcoal cefoperazone deoxycholate agar and by enrichment in modified Preston broth, with or without blood added, for 48 h at 37 degrees C prior to plating. A 16S/23S PCR/DNA probe membrane-based colorimetric detection assay was evaluated on a subset of the feces (n = 127), including 18 culture-positive and 109 culture-negative specimens. DNA was extracted directly from the fecal specimens by using the QIAamp DNA stool Minikit for the DNA probe-based PCR assay (PCR/DNA probe assay). A second PCR/DNA probe assay based on the 16S rRNA gene in Campylobacter spp. was applied to all specimens that were culture negative, PCR/DNA positive on initial analysis. Campylobacter species were cultured in 20 of the 320 specimens. The 16S/23S PCR/DNA probe assay detected campylobacter DNA in 17 of 18 (94% sensitivity) culture-positive specimens and in 41 (38%) culture-negative specimens. The presence of campylobacter DNA in 35 of 41 culture-negative specimens was confirmed by the 16S PCR/DNA probe assay. DNA sequence analysis of seven 16S/23S PCR products and five 16S PCR products amplified from a selection of these specimens confirmed the presence of campylobacter DNA and more specifically Campylobacter jejuni, C. concisus, C. curvus, and C. gracilis DNA in these specimens. The molecular assays described in this study are rapid methods for the detection and identification of Campylobacter species in fecal specimens. The finding of Campylobacter spp. DNA in a large number of specimens of feces from patients with no other identified cause of diarrhea may suggest that Campylobacter spp. other than C. jejuni and C. coli may account for a proportion of cases of acute gastroenteritis in which no etiological agent is currently identified.

Extended-spectrum beta-lactamases in Ireland, including a novel enzyme, TEM-102.

Organisms producing extended-spectrum beta-lactamases (ESBLs) have been reported in many countries, but there is no information on the prevalence of ESBL-producing members of the family Enterobacteriaceae in Ireland. A total of 925 isolates of ampicillin-resistant members of the Enterobacteriaceae were received from six hospitals in Ireland over a 3-year period from September 1996 to September 1999. Isolates were screened for ESBL production by the double-disk diffusion (DDD) method. DDD-positive isolates that were (i) confirmed as ESBL producers by National Committee for Clinical Laboratory Standards (NCCLS) confirmatory testing and (ii) susceptible to cefoxitin by disk diffusion were considered ESBL producers. By these criteria, 27 (3%) of the ampicillin-resistant members of the Enterobacteriaceae studied were categorized as ESBL producers. Molecular typing suggested that some intra- and interhospital spread of ESBL-producing isolates had occurred. DNA sequencing of amplified bla(TEM) and bla(SHV) genes resulted in the detection of a novel bla(TEM) ESBL gene, bla(TEM-102) in two isolates (Klebsiella pneumoniae and Enterobacter cloacae) received from the same hospital but isolated from different patients. The study suggests dissemination of ESBL-producing bacteria within the health care system in Ireland and emphasizes the need for measures to control such spread.