RESUMO
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.
Assuntos
Mecônio , Detecção do Abuso de Substâncias , Humanos , Recém-Nascido , Gravidez , Feminino , Detecção do Abuso de Substâncias/métodos , Mecônio/química , Transtornos Relacionados ao Uso de Substâncias/diagnóstico , Transtornos Relacionados ao Uso de Substâncias/urina , Imunoensaio/métodos , Cordão Umbilical , Exposição Materna/efeitos adversosRESUMO
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.
Assuntos
Antipsicóticos , Clozapina , Esquizofrenia , Antipsicóticos/efeitos adversos , Clozapina/efeitos adversos , Monitoramento de Medicamentos/métodos , Humanos , Esquizofrenia/tratamento farmacológico , FumarRESUMO
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.
Assuntos
Hiperplasia Suprarrenal Congênita , Espectrometria de Massas em Tandem , 17-alfa-Hidroxiprogesterona , Hiperplasia Suprarrenal Congênita/diagnóstico , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Teste em Amostras de Sangue Seco , Humanos , Recém-Nascido , Reprodutibilidade dos Testes , EsteroidesAssuntos
Serviços de Laboratório Clínico/organização & administração , Epidemia de Opioides , Transtornos Relacionados ao Uso de Opioides/diagnóstico , Detecção do Abuso de Substâncias/métodos , Carga de Trabalho , Humanos , Transtornos Relacionados ao Uso de Opioides/tratamento farmacológico , Manejo da Dor/métodosRESUMO
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.
Assuntos
Neoplasias das Glândulas Suprarrenais/epidemiologia , Paraganglioma/epidemiologia , Feocromocitoma/epidemiologia , Saúde da População/estatística & dados numéricos , Vigilância da População/métodos , Adolescente , Adulto , Idoso , Alberta/epidemiologia , Algoritmos , Estudos de Coortes , Feminino , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
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.
Assuntos
Álcoois/sangue , Cromatografia Gasosa/métodos , Etilenoglicol/sangue , Ionização de Chama/métodos , Calibragem , Humanos , Limite de Detecção , Controle de Qualidade , Reprodutibilidade dos TestesRESUMO
BACKGROUND: There is limited information about the effects of instituting CLSI Document C56A recommended workflows for the automated detection of hemolysis, lipemia and icterus (HIL) in different clinical laboratories and patient populations. We describe a process to develop and tailor automated reporting rules that are appropriate for the local laboratory population. METHODS: Automated decision algorithms were generated and applied to 2 high volume labs serving community and hospital populations. Proposed rules were applied to the datasets offline to predict the outcomes, and then were further optimized prior to implementation. RESULTS: Introduction of automated serum indices decreased HIL flagging compared to manual flagging. Hemolysis flagging was the greatest in all 3 patient populations, and was successfully reduced for LD, CK and AST by optimized rules that incorporated both the H-index result and the analyte result. Changes in flagging rates were also patient population specific, particularly for icterus which was a problem in hospitalized populations but not in the community. Overall, concordance between manual and automated flagging methods was very low in both laboratories. CONCLUSIONS: We demonstrate that flagging algorithms may not be universally transferable due to lab specific and population specific factors and demonstrate the benefits of local, a priori testing of algorithms prior to implementation.
Assuntos
Serviços de Laboratório Clínico , Hemólise , Hiperlipidemias/sangue , Icterícia/sangue , Algoritmos , Automação , HumanosRESUMO
Acesulfame (ACE) and sucralose (SUC) have become recognized as ideal domestic wastewater contamination indicators. Liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) analysis is commonly used; however, the sensitivity of SUC is more than two orders of magnitude lower than that of ACE, limiting the routine monitoring of SUC. To address this issue, we examined the ESI behavior of both ACE and SUC under various conditions. ACE is ionic in aqueous solution and efficiently produces simple [M-H](-) ions, but SUC produces multiple adduct ions, limiting its sensitivity. The formic acid (FA) adducts of SUC [M+HCOO](-) are sensitively and reproducibly generated under the LC-MS conditions. When [M+HCOO](-) is used as the precursor ion for SUC detection, the sensitivity increases approximately 20-fold compared to when [M-H](-) is the precursor ion. To further improve the limit of detection (LOD), we integrated the large volume injection approach (500µL injection) with ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), which reduced the method detection limit (MDL) to 0.2ng/L for ACE and 5ng/L for SUC. To demonstrate the applicability of this method, we analyzed 100 well water samples collected in Alberta. ACE was detected in 24 wells at concentrations of 1-1534ng/L and SUC in 8 wells at concentrations of 65-541ng/L. These results suggest that wastewater is the most likely source of ACE and SUC impacts in these wells, suggesting the need for monitoring the quality of domestic well water.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Sacarose/análogos & derivados , Edulcorantes/análise , Espectrometria de Massas em Tandem/métodos , Tiazinas/análise , Poluentes Químicos da Água/análise , Água Doce/química , Limite de Detecção , Espectrometria de Massas , Espectrometria de Massas por Ionização por Electrospray/métodos , Sacarose/análiseRESUMO
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.
Assuntos
Cromatografia Líquida/métodos , Nitrosaminas/análise , Piridinas/análise , Extração em Fase Sólida/métodos , Espectrometria de Massas em Tandem/métodos , Purificação da Água , Cloraminas , Cloro , Desinfecção , Espectrometria de Massas , Ozônio , Nicotiana , Água/análise , Qualidade da ÁguaRESUMO
We report liquid chromatographic separation with tandem mass spectrometry determination of 12 endogenous estrogens and their intact conjugates in blood and urine and its application to study effects of exemestane treatment on estrogen generation and metabolism in postmenopausal women with estrogen-dependent breast cancer. A 0.5 mL aliquot of each urine or serum sample is fractionated with solid phase extraction to a fraction of free estrogen and another fraction of their conjugates. The reversed phase LC/MS/MS determines dansylated estrogens with positive ionization and intact conjugates with negative ionization. The method provides reproducible separation and limit of detection as low as 1 pg mL(-1) for free estrogens and 0.03 ng mg(-1) creatinine for the conjugates in serum and urine samples. The method enabled us to acquire unique concentration profiles of 12 endogenous estrogens and their intact conjugates in 30 breast cancer patients before and after one month of exemestane treatment. Exemestane suppressed total serum and urinary estrogens by 11-97% (P<0.0001) and 8.7-91% (P<0.0001), respectively. Specifically, these data show that exemestane preferentially suppressed E1, E1-3S, E1-3G, and E2-17G more than other estrogens. Linear regression analysis of estrogen concentrations before and after treatment showed correlation coefficients of 0.8385 (n=289, P<0.0001) and 0.8863 (n=360, P<0.0001). This study provides urinary and blood estrogen concentration profiles in breast cancer patients to demonstrate the effect of exemestane on estrogen generation, supporting inhibition of aromatase activity.
Assuntos
Androstadienos/uso terapêutico , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Cromatografia Líquida de Alta Pressão , Congêneres do Estradiol/análise , Estrogênios/análise , Espectrometria de Massas em Tandem , Congêneres do Estradiol/sangue , Congêneres do Estradiol/urina , Estrogênios/sangue , Estrogênios/urina , Feminino , Humanos , Pós-Menopausa , Extração em Fase SólidaRESUMO
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.
Assuntos
Benzoquinonas/química , Água Potável/química , Halogênios/química , Raios Ultravioleta , Cromatografia Líquida de Alta Pressão , Espectrofotometria Ultravioleta , Espectrometria de Massas em TandemRESUMO
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(-â¢). 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(-)] â X(-) (X(-) representing Cl(-), Br(-), or I(-)), while dibromo-dimethyl-benzoquinone (DBDMBQ) showed only the transition of M(-â¢) â 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.
Assuntos
Benzoquinonas/análise , Água Potável/química , Cromatografia Líquida de Alta Pressão , Estrutura Molecular , Pressão , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
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.
Assuntos
Benzoquinonas/análise , Produtos Domésticos/análise , Hidrocarbonetos Halogenados/análise , Piscinas , Poluentes Químicos da Água/análise , Adolescente , Adulto , Benzoquinonas/química , Criança , Cromatografia Líquida , Desinfecção , Feminino , Halogenação , Humanos , Hidrocarbonetos Halogenados/química , Íons , Masculino , Espectrometria de Massas , Extração em Fase Sólida , Termodinâmica , Poluentes Químicos da Água/química , Abastecimento de Água , Adulto JovemRESUMO
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.
Assuntos
Benzoquinonas/análise , Benzoquinonas/química , Desinfecção , Ingestão de Líquidos , Halogenação , Espectrometria de Massas/métodos , Água/química , Benzoquinonas/isolamento & purificação , Benzoquinonas/toxicidade , Cromatografia Líquida , Saúde Pública , Extração em Fase Sólida , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem , Neoplasias da Bexiga Urinária/induzido quimicamente , Poluentes Químicos da Água/análise , Poluentes Químicos da Água/química , Poluentes Químicos da Água/isolamento & purificação , Poluentes Químicos da Água/toxicidadeRESUMO
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.
Assuntos
Aeromonas salmonicida/fisiologia , Fímbrias Bacterianas/fisiologia , Doenças dos Peixes/microbiologia , Infecções por Bactérias Gram-Negativas/veterinária , Animais , Infecções por Bactérias Gram-Negativas/microbiologia , Salmo salarRESUMO
N-nitrosodiphenylamine (NDPhA) is a disinfection byproduct (DBP) in drinking water. However, it is not known what governs the formation of NDPhA and which precursor(s) in the raw water is responsible for its formation. We report here diphenylamine (DPhA) as a key precursor of NDPhA, and we describe the effect of water pH and chloramination conditions on the formation of NDPhA. To identify precursors of NDPhA, raw water samples were collected from the same drinking water system in which NDPhA was previously detected. Analysis of the raw water samples showed the presence of 1.3 ng/L of DPhA and no detectable NDPhA. Seven hours after the treatment of the raw water with chloramines, the concentration of DPhA decreased to 0.4 ng/L with corresponding formation of NDPhA (0.4 ng/L). Controlled experiments involving chloramination of DPhA in water showed that chloramines were essential to the formation of NDPhA, and that increasing the pH from 4 to 10 resulted in 64-fold enhancement in NDPhA formation. Removal of DPhA and formation of NDPhA was found by mass imbalance, which led to the identification of two new DBPs, phenazine (MW 180 Da) and a chlorinated phenazine derivative (MW 216 Da), using liquid chromatography tandem mass spectrometry and gas chromatography mass spectrometry. Both new DBPs were detected only in the treated water and not in the raw water. Phenazine and N-chlorophenazine have never been reported as DBPs and neither their occurrence in drinking water nor their health effects are known.
Assuntos
Cloraminas/química , Difenilamina/química , Desinfecção , Nitrosaminas/síntese química , Fenazinas/química , Água/química , Cromatografia Líquida , Concentração de Íons de Hidrogênio , Espectrometria de Massas , Nitrosaminas/química , Padrões de Referência , Soluções , Fatores de TempoRESUMO
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.
Assuntos
Aeromonas salmonicida/imunologia , Doenças dos Peixes/microbiologia , Furunculose/veterinária , Salmo salar , Fatores de Virulência/imunologia , Aeromonas salmonicida/genética , Animais , Citocinas/genética , Citocinas/imunologia , Doenças dos Peixes/genética , Doenças dos Peixes/imunologia , Furunculose/genética , Furunculose/imunologia , Furunculose/microbiologia , Leucócitos/imunologia , Leucócitos/microbiologia , Mutagênese Sítio-Dirigida , Mutação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Fatores de Virulência/genéticaRESUMO
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.