Biological Testing and Interpretation of Laboratory Results Associated with Detecting Newborns with Substance Exposure.

BACKGROUND: Substance use during pregnancy is common, as is biological testing that is intended to help identify prenatal exposures. However, there is no standardized requirement for biological testing with either maternal or newborn specimens, nor is there standardization related to when testing occurs, how frequently testing occurs, what specimen(s) to test, what substances to test for, or how to perform testing. CONTENT: We review common specimen types tested to detect maternal and newborn substance exposure with a focus on urine, meconium, and umbilical cord tissue. We also review common analytical methods used to perform testing, including immunoassay, and mass spectrometry platforms. Considerations regarding the utilization of testing relative to the purpose of testing, the drug analyte(s) of interest, the specific testing employed, and the interpretation of results are emphasized to help guide decisions about clinical utilization of testing. We also highlight specific examples of unexpected results that can be used to guide interpretation and appropriate next steps. SUMMARY: There are strengths and limitations associated with all approaches to detecting substance exposure in pregnant persons as well as biological testing to evaluate a newborn with possible substance exposure. Standardization is needed to better inform decisions surrounding evaluation of substance exposures in pregnant people and newborns. If biological sampling is pursued, testing options and results must be reviewed in clinical context, acknowledging that false-positive and -negative results can and do occur.

Identification of tobacco-specific nitrosamines as disinfection byproducts in chloraminated water.

Tobacco-specific nitrosamines (TSNAs) exist in environmental waters; however, it is unknown whether TSNAs can be produced during water disinfection. Here we report on the investigation and evidence of TSNAs as a new class of disinfection byproducts (DBPs). Using five common TSNAs, including (methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) as the targets, we first developed a solid phase extraction (SPE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) method capable of rapidly determining these TSNAs at levels as low as 0.02 ng/L in treated water. Using this highly sensitive method, we investigated the occurrence and formation potential (FP) (precursor test conducted in the presence of chloramines) of TSNAs in treated water from two wastewater treatment plants (WWTPs) and seven drinking water treatment plants (DWTPs). NNAL was detected in the FP samples, but not in the samples before the FP test, confirming NNAL as a DBP. NNK was detected in the treated wastewater before the FP test, but its concentration increased significantly after chloramination in two of three tests. Thus, NNK could be a DBP and/or a contaminant in wastewater. Moreover, these TSNAs were detected in FP tests of wastewater-impacted DWTP plant influents in 9 of 11 samples. However, TSNAs were not detected at full-scale DWTPs, except for at one DWTP with high ammonia where breakpoint chlorination was not achieved. The concentration of the sum of five TSNAs (0.3 ng/L) was 100-fold lower than NDMA, suggesting that TSNAs have a minor contribution to total nitrosamines in water. We examined several factors in the treatment process and found that chlorine or ozone may destroy TSNA precursors and granular activated carbon (GAC) treatment may remove the precursors. Further research is warranted into the efficiency of these processes at different DWTPs using sources of varying water quality.

Ultra pressure liquid chromatography-negative electrospray ionization mass spectrometry determination of twelve halobenzoquinones at ng/L levels in drinking water.

We report here the characterization of twelve halobenzoquinones (HBQs) using electrospray ionization (ESI) high resolution quadrupole time-of-flight mass spectrometry. The high resolution negative ESI spectra of the twelve HBQs formed two parent ions, [M + H(+) + 2e(-)], and the radical M(-•). The intensities of these two parent ions are dependent on their chemical structures and on instrumental parameters such as the source temperature and flow rate. The characteristic ions of the HBQs were used to develop an ultra pressure liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method. At the UPLC flow rate (400 µL/min) and under the optimized ESI conditions, eleven HBQs showed the stable and abundant transitions [M + H(+) + 2e(-)] → X(-) (X(-) representing Cl(-), Br(-), or I(-)), while dibromo-dimethyl-benzoquinone (DBDMBQ) showed only the transition of M(-•) → Br(-). The UPLC efficiently separates all HBQs including some HBQ isomers, while the MS/MS offers exquisite limits of detection (LODs) at subng/mL levels for all HBQs except DBDMBQ. Combined with solid phase extraction (SPE), the method LOD is down to ng/L. The results from analysis of authentic samples demonstrated that the SPE-UPLC-MS/MS method is reliable, fast, and sensitive for the identification and quantification of the twelve HBQs in drinking water.

Halobenzoquinones in swimming pool waters and their formation from personal care products.

Halobenzoquinones (HBQs) are a class of disinfection byproducts (DBPs) of health relevance. In this study, we aimed to uncover which HBQs are present in swimming pools. To achieve this goal, we developed a new method capable of determining eight HBQs while overcoming matrix effects to achieve reliable quantification. The method provided reproducible and quantitative recovery (67-102%) and detection limits of 0.03-1.2 ng/L for all eight HBQs. Using this new method, we investigated water samples from 10 swimming pools and found 2,6-dichloro-1,4-benzoquinone (2,6-DCBQ) in all the pools at concentrations of 19-299 ng/L, which was as much as 100 times higher than its concentration in the input tap water (1-6 ng/L). We also identified 2,3,6-trichloro-(1,4)benzoquinone (TriCBQ), 2,3-dibromo-5,6-dimethyl-(1,4)benzoquinone (DMDBBQ), and 2,6-dibromo-(1,4)benzoquinone (2,6-DBBQ) in some swimming pools at concentrations of <0.1-11.3, <0.05-0.7, and <0.05-3.9 ng/L, respectively, but not in the input tap water. We examined several factors to determine why HBQ concentrations in pools were much higher than in the input tap water. Higher dissolved organic carbon (DOC), higher doses of chlorine and higher temperatures enhanced the formation of HBQs in the pools. In addition, we conducted laboratory disinfection experiments and discovered that personal care products (PCPs) such as lotions and sunscreens can serve as precursors to form additional HBQs, such as TriCBQ, 2,6-dichloro-3-methyl-(1,4)benzoquinone (DCMBQ), and 2,3,5,6-tetrabromo-(1,4)benzoquinone (TetraB-1,4-BQ). These results explained why some HBQs existed in swimming pools but not in the input water. This study presents the first set of occurrence data, identification of new HBQ DBPs, and the factors for their enhanced formation in the swimming pools.

UV-induced transformation of four halobenzoquinones in drinking water.

Halobenzoquinones (HBQs) are a group of emerging disinfection byproducts (DBPs) found in treated drinking water. Because the use of UV treatment for disinfection is becoming more widespread, it is important to understand how the HBQs may be removed or changed due to UV irradiation. Water samples containing four HBQs, 2,6-dichloro-1,4-benzoquinone (DCBQ), 2,3,6-trichloro-1,4-benzoquinone (TCBQ), 2,6-dichloro-3-methyl-1,4-benzoquinone (DCMBQ), and 2,6-dichloro-1,4-benzoquinone (DBBQ), were treated using a modified bench scale collimated beam device, mimicking UV treatment. Water samples before and after UV irradiation were analyzed for the parent compounds and products using a high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method. As much as 90% of HBQs (0.25 nmol L(-1)) in both pure water and tap water were transformed to other products after UV254 irradiation at 1000 mJ cm(-2). The major products of the four HBQs were identified as 3-hydroxyl-2,6-dichloro-1,4-benzoquinone (OH-DCBQ) from DCBQ, 5-hydroxyl-2,6-dichloro-3-methyl-1,4-benzoquinone (OH-DCMBQ) from DCMBQ, 5-hydroxyl-2,3,6-trichloro-1,4-benzoquinone (OH-TCBQ) from TCBQ, and 3-hydroxyl-2,6-dibromo-1,4-benzoquinone (OH-DBBQ) from DBBQ. These four OH-HBQs were further modified to monohalogenated benzoquinones when the UV dose was higher than 200 mJ cm(-2). These results suggested possible pathways of UV-induced transformation of HBQs to other compounds. Under the UV dose commonly used in water treatment plants, it is likely that HBQs are partially converted to other halo-DBPs. The occurrence and toxicity of these mixed DBPs warrant further investigation to understand whether they pose a health risk.

Biological variation in clozapine and metabolite reporting during therapeutic drug monitoring.

BACKGROUND: Clozapine (CLO) is an atypical antipsychotic used in management of treatment-resistant schizophrenia. Adverse drug reactions are caused by both CLO and its primary metabolite, norclozapine (NCLO). We defined the biological variability of CLO, NCLO, and the CLO to NCLO ratio (CNR) as well as assess the impact of reporting CLO and NCLO routinely. METHODS: The CVi and CVg were calculated from 1904 results from 247 patients by CV-ANOVA, and ANOVA, respectively, for CLO, NCLO, and the CNR. Association between each were also analyzed against a number of parameters including age and gender, complete blood count (CBC), kidney and liver function tests, lipids, and glucose within 24 h of CLO measurement. RESULTS: For CLO, NCLO and CNR, the CVi was calculated as 19.3%, 19.2%, and 14.7%, and the CVg was 46.9%, 51.4%, and 36.3%, respectively. A total of 87 patients (19.7%) demonstrated higher NCLO results than CLO, with a ratio as low as 0.47. Kidney function was also found to have a statistically significant relationship to CLO and NCLO levels. CONCLUSIONS: We provide data for biological variability of CLO metabolism as well as while providing some evidence for reporting NCLO values clinically.

Measuring steroids from dried blood spots using tandem mass spectrometry to diagnose congenital adrenal hyperplasia.

BACKGROUND: Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders that occur due to defects in the steroidogenesis pathway. Approximately 90% of CAH cases can be diagnosed by the measurement of serum 17-hydroxyprogesterone alone. However, the quantification of six additional steroids could significantly improve CAH laboratory diagnosis. Using dried blood spot (DBS) as specimen of choice can further improve patient care due to the small sample volume required for CAH diagnosis in neonates. METHODS: An optimized DBS sample preparation method was employed for steroids quantification without the need of derivatization. A LC-MS/MS assay was developed and optimized using a reverse phase-ultra high-pressure liquid chromatography (RP-UHPLC) system combined with triple quadrupole mass spectrometry using positive electrospray ionization mode. RESULTS: The assay was validated according to CLSI analytical guidelines, including lower limit of quantification (LLOQ), linearity, precision, accuracy, carryover, and method comparison. The analytical measuring range of the method for all steroids was 2.5, 5, or 10 ng/ml to 250 ng/ml in DBS, r2 ≥ 0.995. The LLOQ, intra-day and inter-day precision were 0.11-1.8 ng/ml, 1.2-6.4 ng/ml, 1.8-11.5%, and 5.3-13.8%, respectively. CONCLUSIONS: Our LC-MS/MS assay simultaneously detects 7 steroids for the diagnosis of CAH and can be readily implemented in clinical laboratories to provide superior analytical performance over traditional immunoassays.

Epidemiology of pheochromocytoma and paraganglioma: population-based cohort study.

OBJECTIVE: Despite the significant morbidity and mortality associated with pheochromocytoma and paraganglioma, little is known about their epidemiology. The primary objective was to determine the incidence of pheochromocytoma and paraganglioma in an ethnically diverse population. A secondary objective was to develop and validate algorithms for case detection using laboratory and administrative data. DESIGN: Population-based cohort study in Alberta, Canada from 2012 to 2019. METHODS: Patients with pheochromocytoma or paraganglioma were identified using linked administrative databases and clinical records. Annual incidence rates per 100 000 people were calculated and stratified according to age and sex. Algorithms to identify pheochromocytoma and paraganglioma, based on laboratory and administrative data, were evaluated. RESULTS: A total of 239 patients with pheochromocytoma or paraganglioma (collectively with 251 tumors) were identified from a population of 5 196 368 people over a period of 7 years. The overall incidence of pheochromocytoma or paraganglioma was 0.66 cases per 100 000 people per year. The frequency of pheochromocytoma and paraganglioma increased with age and was highest in individuals aged 60-79 years (8.85 and 14.68 cases per 100 000 people per year for males and females, respectively). An algorithm based on laboratory data (metanephrine >two-fold or normetanephrine >three-fold higher than the upper limit of normal) closely approximated the true frequency of pheochromocytoma and paraganglioma with an estimated incidence of 0.54 cases per 100 000 people per year. CONSLUSION: The incidence of pheochromocytoma and paraganglioma in an unselected population of western Canada was unexpectedly higher than rates reported from other areas of the world.

Characterization and determination of chloro- and bromo-benzoquinones as new chlorination disinfection byproducts in drinking water.

We report the characterization and determination of 2,6-dichloro-1,4-benzoquinone and three new disinfection byproducts (DBPs): 2,6-dichloro-3-methyl-1,4-benzoquinone, 2,3,6-trichloro-1,4-benzoquinone, and 2,6-dibromo-1,4-benzoquinone. These haloquinones are suspected bladder carcinogens and are likely produced during drinking water disinfection treatment. However, detection of these haloquinones is challenging, and consequently, they have not been characterized as DBPs until recently. We have developed an electrospray ionization tandem mass spectrometry technique based on our observation of unique ionization processes. These chloro- and bromo-quinones were ionized through a reduction step to form [M + H](-) under negative electrospray ionization. Tandem mass spectra and accurate mass measurements of these compounds showed major product ions, [M + H - HX](-), [M + H - HX - CO](-), [M + H - CO](-), and/or X(-) (where X represents Cl or Br). The addition of 0.25% formic acid to water samples was found to effectively stabilize the haloquinones in water and to improve the ionization for analysis. These improvements were rationalized from the estimates of pK(a) values (5.8-6.3) of these haloquinones. The method of tandem mass spectrometry detection, combined with sample preservation, solid phase extraction, and liquid chromatography separation, enabled the detection of haloquinones in chlorinated water samples collected from a drinking water treatment plant. The four haloquinones were detected only in drinking water after chlorination treatment, with concentrations ranging from 0.5 to 165 ng/L, but were not detectable in the untreated water. This method will be useful for future studies of occurrence, formation pathways, toxicity, and control of these new halogenated DBPs.

Aeromonas salmonicida Type I pilus system contributes to host colonization but not invasion.

The host-adherence strategies employed by Aeromonas salmonicida subsp, salmonicida, the etiological agent of an infectious bacteremia of salmonids, are poorly understood. In addition to the outer protein coat or S-layer, A. salmonicida has both Type I and Type IV pili loci. The A. salmonicida Type I or Fim pilus is encoded by an operon with genes for a chaperone, an usher, and 3 pilus subunits and is predicted to be similar to the Pap fimbriae of uropathogenic Escherichia coli, which are considered significant virulence factors. A Fim-deficient strain of A. salmonicida strain A449, delta fim, was created by deleting this operon. Virulence of delta fim was unchanged in direct live challenges of Atlantic salmon Salmo salar L., a natural host for A. salmonicida. A measure of clinically inapparent (covert) infections suggested Fim was required to establish or maintain a covert infection. This was confirmed by an ex vivo adherence and invasion assay using freshly excised salmon gastrointestinal (GI) tract, which showed that, compared to the parental strain, the ability of the isogenic delta fim mutant strain to adhere to the salmon GI tract was reduced but, once adhered, its ability to invade was unchanged. Thus the Fim pilus functions as an adhesin in A. salmonicida and the presence of a functional Fim improved the efficiency of A. salmonicida infection of Atlantic salmon.

Mutations in the Aeromonas salmonicida subsp. salmonicida type III secretion system affect Atlantic salmon leucocyte activation and downstream immune responses.

Deletion mutants of Aeromonas salmonicida subsp. salmonicida were used to determine the effect of the type three secretion system (TTSS) on Atlantic salmon anterior head kidney leucocytes (AHKL). One strain had a deletion in the outer membrane pore gene, ascC; and the other in three effector genes: aopO, aopH and aexT (we call this strain Deltaaop3). Host cell invasion success and 24h survival were depressed in DeltaascC, as was 24h survival of Deltaaop3, when compared to the wild type strain. Challenge of AHKLs with A449 or TTSS mutants stimulated expression of the inflammatory mediators IL-8, IL-1 and TNFalpha at two bacterial concentrations (A(600) 0.1, 0.01). Expression of IL-12 was not stimulated in DeltaascC challenged cells, whereas A449 and Deltaaop3 challenge resulted in an up-regulation of IL-12 in AHKLs, 2- and 4-fold higher than PBS, respectively. Only the wild type strain elicited a significant increase in IL-10 expression (5.5x at A(600) 0.1). Inducible nitric oxide synthetase (iNOS) and arginase (I+II) genes were also significantly up-regulated upon exposure to all strains. However, iNOS:arginase ratio was elevated in the effector mutant challenge. These results suggest that A. salmonicida subsp. salmonicida may enhance survival within the host cell through polarization of macrophages/leucocytes to an alternative, rather than classical, activation state. Furthermore, the short-term survival and lack of T-cell signalling cytokine stimulation in DeltaascC, may help explain its inefficiency at providing protection to subsequent wild type challenge.

Identification of N-nitrosamines in treated drinking water using nanoelectrospray ionization high-field asymmetric waveform ion mobility spectrometry with quadrupole time-of-flight mass spectrometry.

We report a nanoelectrospray ionization (nESI) with high-field asymmetric waveform ion mobility spectrometry (FAIMS) and tandem mass spectrometry (MS-MS) method for determination of small molecules of m/z 50 to 200 and its potential application in environmental analysis. Integration of nESI with FAIMS and MS-MS combines the advantages of these three techniques into one method. The nESI provides efficient sample introduction and ionization and allows for collection of multiple data from only microliters of samples. The FAIMS provides rapid separation, reduces or eliminates background interference, and improves the signal-to-noise ratio as much as 10-fold over nESI-MS-MS. The tandem quadrupole time-of-flight MS detection provides accurate mass and mass spectral measurements for structural identification. Characteristics of FAIMS compensation voltage (CV) spectra of seven nitrosamines, N-nitrosodimethylamine (NDMA), N-nitrosomethylethylamine (NMEA), N-nitrosodiethylamine (NDEA), N-nitrosodi-n-propylamine (NDPA), N-nitrosodi-n-butylamine (NDBA), N-nitrosopiperidine (NPip), and N-nitrosopyrrolidine (NPyr), were analyzed. The optimal CV of the nitrosamines (at DV -4000 V) were: -1.6 V, NDBA; 2.6 V, NDPA; 6.6 V, NPip; 8.8 V, NDEA; 13.2 V, NPyr; 14.4 V, NMEA; and 19.4 V, NDMA. Fragmentation patterns of the seven nitrosamines in the nESI-FAIMS-MS-MS were also obtained. The specific CV and MS-MS spectra resulted in positive identification of NPyr and NPip in a treated water sample, demonstrating the potential application of this technique in environmental analysis.

Contribution of type IV pili to the virulence of Aeromonas salmonicida subsp. salmonicida in Atlantic salmon (Salmo salar L.).

Aeromonas salmonicida subsp. salmonicida, a bacterial pathogen of Atlantic salmon, has no visible pili, yet its genome contains genes for three type IV pilus systems. One system, Tap, is similar to the Pseudomonas aeruginosa Pil system, and a second, Flp, resembles the Actinobacillus actinomycetemcomitans Flp pilus, while the third has homology to the mannose-sensitive hemagglutinin pilus of Vibrio cholerae. The latter system is likely nonfunctional since eight genes, including the gene encoding the main pilin subunit, are deleted compared with the orthologous V. cholerae locus. The first two systems were characterized to investigate their expression and role in pathogenesis. The pili of A. salmonicida subsp. salmonicida were imaged using atomic force microscopy and Tap- and Flp-overexpressing strains. The Tap pili appeared to be polar, while the Flp pili appeared to be peritrichous. Strains deficient in tap and/or flp were used in live bacterial challenges of Atlantic salmon, which showed that the Tap pilus made a moderate contribution to virulence, while the Flp pilus made little or no contribution. Delivery of the tap mutant by immersion resulted in reduced cumulative morbidity compared with the cumulative morbidity observed with the wild-type strain; however, delivery by intraperitoneal injection resulted in cumulative morbidity similar to that of the wild type. Unlike the pili of other piliated bacterial pathogens, A. salmonicida subsp. salmonicida type IV pili are not absolutely required for virulence in Atlantic salmon. Significant differences in the behavior of the two mutant strains indicated that the two pilus systems are not redundant.

The genome of Aeromonas salmonicida subsp. salmonicida A449: insights into the evolution of a fish pathogen.

BACKGROUND: Aeromonas salmonicida subsp. salmonicida is a Gram-negative bacterium that is the causative agent of furunculosis, a bacterial septicaemia of salmonid fish. While other species of Aeromonas are opportunistic pathogens or are found in commensal or symbiotic relationships with animal hosts, A. salmonicida subsp. salmonicida causes disease in healthy fish. The genome sequence of A. salmonicida was determined to provide a better understanding of the virulence factors used by this pathogen to infect fish. RESULTS: The nucleotide sequences of the A. salmonicida subsp. salmonicida A449 chromosome and two large plasmids are characterized. The chromosome is 4,702,402 bp and encodes 4388 genes, while the two large plasmids are 166,749 and 155,098 bp with 178 and 164 genes, respectively. Notable features are a large inversion in the chromosome and, in one of the large plasmids, the presence of a Tn21 composite transposon containing mercury resistance genes and an In2 integron encoding genes for resistance to streptomycin/spectinomycin, quaternary ammonia compounds, sulphonamides and chloramphenicol. A large number of genes encoding potential virulence factors were identified; however, many appear to be pseudogenes since they contain insertion sequences, frameshifts or in-frame stop codons. A total of 170 pseudogenes and 88 insertion sequences (of ten different types) are found in the A. salmonicida genome. Comparison with the A. hydrophila ATCC 7966T genome reveals multiple large inversions in the chromosome as well as an approximately 9% difference in gene content indicating instances of single gene or operon loss or gain.A limited number of the pseudogenes found in A. salmonicida A449 were investigated in other Aeromonas strains and species. While nearly all the pseudogenes tested are present in A. salmonicida subsp. salmonicida strains, only about 25% were found in other A. salmonicida subspecies and none were detected in other Aeromonas species. CONCLUSION: Relative to the A. hydrophila ATCC 7966T genome, the A. salmonicida subsp. salmonicida genome has acquired multiple mobile genetic elements, undergone substantial rearrangement and developed a significant number of pseudogenes. These changes appear to be a consequence of adaptation to a specific host, salmonid fish, and provide insights into the mechanisms used by the bacterium for infection and avoidance of host defence systems.

Cell-electronic sensing of particle-induced cellular responses.

We report a new technique for the continuous and real-time measurement of microparticle-induced cellular responses using a real-time cell-electronic sensing (RT-CES) technology. The method involves the use of microelectrode-embedded microwells seeded with one of two lung cancer carcinoma cell lines (A549 and SK-MES-1), allowing for continuous measurements of impedance. The change in impedance that is automatically converted to the cell index is linearly correlated with the numbers of the seeding cells during the log phase, providing quantitative measurement of cytotoxicity. After 24 h of initial incubation in 96 microwells, the cultures are treated with microparticles, and changes in the cell index are monitored in real time. Multiple data, including dose response curves, IC(50) (a concentration inhibiting 50% cell growth), and cell-specific and particulate-specific cell responses, are obtained from a single set of experiments. SK-MES-1 cells consistently showed more severe effects and lower IC(50) values than A549 cells when they were treated with quartz particle suspensions. The different effects detected using the RT-CES technique were related to morphological change and apoptosis, supported by the scanning electronic microscopy and flow cytometry results. The method is further used to test the cytotoxicity of two PM(10) standard reference materials of urban air dust and diesel particulates, demonstrating the potential application of this new technique for biomonitoring of air particulates.

Comparative genomics profiling of clinical isolates of Aeromonas salmonicida using DNA microarrays.

BACKGROUND: Aeromonas salmonicida has been isolated from numerous fish species and shows wide variation in virulence and pathogenicity. As part of a larger research program to identify virulence genes and candidates for vaccine development, a DNA microarray was constructed using a subset of 2024 genes from the draft genome sequence of A. salmonicida subsp. salmonicida strain A449. The microarray included genes encoding known virulence-associated factors in A. salmonicida and homologs of virulence genes of other pathogens. We used microarray-based comparative genomic hybridizations (M-CGH) to compare selected A. salmonicida sub-species and other Aeromonas species from different hosts and geographic locations. RESULTS: Results showed variable carriage of virulence-associated genes and generally increased variation in gene content across sub-species and species boundaries. The greatest variation was observed among genes associated with plasmids and transposons. There was little correlation between geographic region and degree of variation for all isolates tested. CONCLUSION: We have used the M-CGH technique to identify subsets of conserved genes from amongst this set of A. salmonicida virulence genes for further investigation as potential vaccine candidates. Unlike other bacterial characterization methods that use a small number of gene or DNA-based functions, M-CGH examines thousands of genes and/or whole genomes and thus is a more comprehensive analytical tool for veterinary or even human health research.

One-step extraction and quantitation of toxic alcohols and ethylene glycol in plasma by capillary gas chromatography (GC) with flame ionization detection (FID).

OBJECTIVES: Clinical analysis of volatile alcohols (i.e. methanol, ethanol, isopropanol, and metabolite acetone) and ethylene glycol (EG) generally employs separate gas chromatography (GC) methods for analysis. Here, a method for combined analysis of volatile alcohols and EG is described. DESIGN AND METHODS: Volatile alcohols and EG were extracted with 2:1 (v:v) acetonitrile containing internal standards (IS) 1,2 butanediol (for EG) and n-propanol (for alcohols). Samples were analyzed on an Agilent 6890 GC FID. The method was evaluated for precision, accuracy, reproducibility, linearity, selectivity and limit of quantitation (LOQ), followed by correlation to existing GC methods using patient samples, Bio-Rad QC, and in-house prepared QC material. RESULTS: Inter-day precision was from 6.5-11.3% CV, and linearity was verified from down to 0.6mmol/L up to 150mmol/L for each analyte. The method showed good recovery (~100%) and the LOQ was calculated to be between 0.25 and 0.44mmol/L. Patient correlation against current GC methods showed good agreement (slopes from 1.03-1.12, and y-intercepts from 0 to 0.85mmol/L; R(2)>0.98; N=35). Carryover was negligible for volatile alcohols in the measuring range, and of the potential interferences tested, only toluene and 1,3 propanediol interfered. The method was able to resolve 2,3 butanediol, diethylene glycol, and propylene glycol in addition to the peaks quantified. CONCLUSIONS: Here we describe a simple procedure for simultaneous analysis of EG and volatile alcohols that comes at low cost and with a simple liquid-liquid extraction requiring no derivitization to obtain adequate sensitivity for clinical specimens.