Revealing nitrogenous VX metabolites and the whole-molecule VX metabolism in the urine of guinea pigs.

VX, a well-known organophosphorus nerve agent (OPNA), poses a significant threat to public safety if employed by terrorists. Obtaining complete metabolites is critical to unequivocally confirm its alleged use/exposure and elucidate its whole-molecular metabolism. However, the nitrogenous VX metabolites containing 2-diisopropylaminoethyl moiety from urinary excretion remain unknown. Therefore, this study applied a newly developed untargeted workflow platform to discover and identify them using VX-exposed guinea pigs as animal models. 2-(N,N-diisopropylamino)ethanesulfonic acid (DiPSA) was revealed as a novel nitrogenous VX metabolite in urine, and 2-(Diisopropylaminoethyl) methyl sulfide (DAEMS) was confirmed as another in plasma, indicating that VX metabolism differed between urine and plasma. It is the first report of a nitrogenous VX metabolite in urine and a complete elucidation of the VX metabolic pathway. DiPSA was evaluated as an excellent VX exposure biomarker. The whole-molecule VX metabolism in urine was characterized entirely for the first time via the simultaneous quantification of DiPSA and two known P-based biomarkers. About 52.1% and 32.4% of VX were excreted in urine as P-based and nitrogenous biomarkers within 24 h. These findings provide valuable insights into the unambiguous detection of OPNA exposure/intoxication and human and environmental exposure risk assessment.

Discriminative detection of soman or VX exposure using europium chelated microparticle-based immunofluorescence microfluidic chip.

The retrospective detection of organophosphorus nerve agents (OPNAs) exposure has been achieved by the off-site analysis of OPNA-human serum albumin (HSA) adducts using mass spectrometry-based detection approaches. However, few specific methods are accessible for on-site detection. To address this, a novel immunofluorescence microfluidic chip (IFMC) testing system combining europium chelated microparticle (EuCM) with self-driven microfluidic chip assay has been established to unambiguously determine soman (GD) and VX exposure within 20 min, respectively. The detection system was based on the principle of indirect competitive enzyme-linked immunosorbent assay. The specific monoclonal antibodies that respectively recognized the phosphonylated tyrosine 411 of GD-HSA and VX-HSA adducts were labeled by EuCM to capture corresponding adducts in the exposed samples. The phosphonylated peptides in the test line and goat-anti-rabbit antibody in the control line were utilized to bind the EuCM-labeled antibodies for signal exhibition. The developed IFMC chip could discriminatively detect exposed HSA adducts with high specificity, demonstrating a low limit of detection at exposure concentrations of 0.5 × 10-6 mol/L VX and 1.0 × 10-6 mol/L GD. The exposed serum samples can be qualitatively detected following an additional pretreatment procedure. This is a novel rapid detection system capable of discriminating GD and VX exposure, providing an alternative method for rapidly identifying OPNA exposure.

A protein standard absolute quantification strategy for enhanced absolute quantification of ricin in complex matrices using in vitro synthesized mutant holoprotein as internal standard by ultra-high-performance liquid chromatography-tandem mass spectrometry.

Ricin is a highly toxic protein toxin that poses a potential bioterrorism threat due to its potency and widespread availability. However, the accurate quantification of ricin through absolute mass spectrometry (MS) using a protein standard absolute quantification (PSAQ) strategy is not widely practiced. This limitation primarily arises from the presence of interchain disulfide bonds, which hinder the production of full-length isotope-labeled ricin as an internal standard (IS) in vitro. In this study, we have developed a novel approach for the absolute quantification of ricin in complex matrices using recombinant single-chain and full-length mutant ricin as the protein IS, instead of isotope-labeled ricin, in conjunction with ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The amino acid sequence of the ricin mutant internal standard (RMIS) was designed by introducing site mutations in specific amino acids of trypsin/Glu-C enzymatic digestion marker peptides of ricin. To simplify protein expression, the A-chain and B-chain of RMIS were directly linked to replace the original interchain disulfide bonds. The RMISs were synthesized using an Escherichia coli expression system. An appropriate RMIS was selected as the protein IS based on consistent digestion efficiency, UHPLC-MS/MS behavior, antibody recognition function, lectin activity, and proper depurination activity with intact ricin. The RMIS was utilized to simultaneously quantify A- and B-chain marker peptides of ricin through UHPLC-MS/MS. This method was thoroughly validated using a milk matrix. By employing internal protein standards, this quantitative strategy overcomes the challenges posed by variations in extraction recoveries, matrix effects, and digestion efficiency encountered when working with different matrices. Consequently, calibration curves generated from milk matrix-spiked samples were utilized to accurately and precisely quantify ricin in river water and plasma samples. Moreover, the established method successfully detected intact ricin in samples obtained from the sixth Organization for the Prohibition of Chemical Weapons (OPCW) exercise on biotoxin analysis. This study presents a novel PSAQ strategy that enables the accurate quantification of ricin in complex matrices.

Simultaneous solvent extraction and quantification of eleven amine compounds related to Chemical Weapon Convention in soils via hydrophilic interaction liquid chromatography-tandem mass spectrometry.

The detection of Chemical Weapon Convention (CWC)-related amine compounds including the precursors or degradation products of V-type organophosphorus nerve agent, nitrogen mustard and 3-quinuclidinyl benzilate is an important aspect for verifying their intact chemical warfare agents. This work focuses on the development of a novel formulation for the simultaneous solvent extraction of eleven CWC-related amine compounds, from the four-type soil matrices including environmental standard soil, sand, clay, and loam. Extracts were well separated on the hydrophilic interaction liquid chromatography (HILIC) and then detected by MS/MS multiple reaction monitoring mode. The type and component of solvent mixtures were optimized to cover a wide range of polarity over all eleven amine compounds with high extraction efficiencies. Extraction parameters, such as the proportion of methanol, water and NH4OH, the times and the period of extraction, and volumes of extraction solution were optimized. The results indicated that a mixed solvent of methanol/water (44:53, v/v) in 3.0% NH4OH was the optimal formulation for extraction of all 11 analytes with high mean extraction recoveries (64.4-96.1%). Specificity and sensitivity were well improved by the good separation of 11 analytes from four-type soil matrices using these optimized HILIC parameters. This method was fully validated for each analyte in four soil matrices. The linear range of 11 analytes was 0.50/0.75-500 ng·g-1 with correlation coefficient (R2) ≥0.990, and intra/inter-day accuracies were 70.3-125% with relative standard deviation (RSD) ≤19.3%. Limit of detection (LOD) of 11 analytes ranged from 0.01 to 0.5 ng·g-1, which was far lower than those reported in previous studies. The built method accomplishes simultaneously quantitative and trace measurement of all eleven CWC-related amine compounds within a single solvent extraction and detection. It only takes a small amount of soil samples and possesses the highest sensitivity over all previous methods. This study provides an optional recommended operating procedure for determination of CWC-related amine compounds in four typical types of complex soils during chemical weapons verification.

Screening of monoclonal antibodies against specific phosphonylation sites and analysis of serum samples exposed to soman and VX using an indirect competitive enzyme-linked immunosorbent assay.

Organophosphorus nerve agents (OPNAs) covalently bind to tyrosine 411 of human serum albumin (HSA) and the formed adducts are stable biomarkers of OPNA exposure. The detection of these adducts has been limited to mass spectrometry techniques combined with protein digestion. Here, we developed indirect competitive ELISA (icELISA) methods to verify OPNA exposure by the detection of OPNA-phosphonylated adducts at tyrosine 411 residue (OPNA-HSA adducts), in which monoclonal antibodies (mAbs) against phosphonylation sites at tyrosine 411 were introduced. The two mAbs were prepared by the fourth generation of rabbit mAb technology using the phosphonylated peptides of LVRY(GD or VX)TKKVPQC as the haptens. These mAbs were screened using our developed competitive ELISA method and then selected based on their individual affinity and selectivity. As a result, we obtained two mAbs that recognized the HSA Tyr 411 adduct of GD (mAb-5G2) or VX (mAb-12B9), respectively. They shared the highest affinity exhibiting a Kd value of about 10-6 mol/L of the OPNA exposure concentration. They also had remarkable selectivity, which could especially recognize their individual OPNA-HSA adducts in a native state but did not recognize other OPNA-HSAs and unadducted HSAs. Especially for mAb-12B9, it could clearly distinguish VX-HSA and GB-HSA between which there was only one alkyl difference in their phosphonyl portion of the adducted sites. The two mAbs were then used to build the icELISA method for analysis of the serum samples exposed to OPNA. It was found that the detectable lowest GD- and VX-exposed concentrations in serum samples were respectively 1.0 × 10-6 mol/L and 10.0 × 10-6 mol/L. This study provides one novel approach and strategy for the retrospective detection of OPNA exposure, and the two mAbs have great potential to be extended for point-of-care testing of OPNA intoxication.

Rapid Differential Detection of Abrin Isoforms by an Acetonitrile- and Ultrasound-Assisted On-Bead Trypsin Digestion Coupled with LC-MS/MS Analysis.

The high toxic abrin from the plant Abrus precatorius is a type II ribosome-inactivating protein toxin with a human lethal dose of 0.1-1.0 µg/kg body weight. Due to its high toxicity and the potential misuse as a biothreat agent, it is of great importance to developing fast and reliable methods for the identification and quantification of abrin in complex matrices. Here, we report rapid and efficient acetonitrile (ACN)- and ultrasound-assisted on-bead trypsin digestion method combined with HPLC-MS/MS for the quantification of abrin isoforms in complex matrices. Specific peptides of abrin isoforms were generated by direct ACN-assisted trypsin digestion and analyzed by HPLC-HRMS. Combined with in silico digestion and BLASTp database search, fifteen marker peptides were selected for differential detection of abrin isoforms. The abrin in milk and plasma was enriched by immunomagnetic beads prepared by biotinylated anti-abrin polyclonal antibodies conjugated to streptavidin magnetic beads. The ultrasound-assisted on-bead trypsin digestion method was carried out under the condition of 10% ACN as denaturant solvent, the entire digestion time was further shortened from 90 min to 30 min. The four peptides of T3Aa,b,c,d, T12Aa, T15Ab, and T9Ac,d were chosen as quantification for total abrin, abrin-a, abrin-b, and abrin-c/d, respectively. The absolute quantification of abrin and its isoforms was accomplished by isotope dilution with labeled AQUA peptides and analyzed by HPLC-MS/MS (MRM). The developed method was fully validated in milk and plasma matrices with quantification limits in the range of 1.0-9.4 ng/mL for the isoforms of abrin. Furthermore, the developed approach was applied for the characterization of abrin isoforms from various fractions from gel filtration separation of the seeds, and measurement of abrin in the samples of biotoxin exercises organized by the Organization for the Prohibition of Chemical Weapons (OPCW). This study provided a recommended method for the differential identification of abrin isoforms, which are easily applied in international laboratories to improve the capabilities for the analysis of biotoxin samples.

Direct Acetonitrile-Assisted Trypsin Digestion Method Combined with LC-MS/MS-Targeted Peptide Analysis for Unambiguous Identification of Intact Ricin.

Ricin is a type II ribosome-inactivating protein toxin consisting of A and B chains linked by one interchain disulfide bond. Because of its high toxicity depending on both chains together, confirming the presence of both A and B chains of intact ricin is required during the investigation of the illegal production and application. Here, we report a novel and sensitive acetonitrile (ACN)-assisted trypsin digestion method for unambiguous identification of intact ricin by simultaneous detection of its marker peptides from A and B chains. Marker peptides were generated with a simple procedure by direct cleaving the native ricin at 45 °C for 4 h using Promega modified sequencing grade trypsin under the assistance of 10% ACN, and then directly analyzed by ultrahigh performance liquid chromatography tandem mass spectrometry. The type of trypsin was found to be one critical factor for cleavage of intact ricin based on a significant difference in the yields of specific peptides generated while using various types of trypsin. A low content of ACN in enzymatic buffer significantly reduced the digestion time from overnight to 4 h. There was commonly a better MS response of marker peptides when using the developed ACN-assisted trypsin digestion method than methanol-assisted trypsin digestion within the same 4 h. Totally, seven specific peptides with high sensitivity and specificity including three in the A-chain (TA7, TA11, and TA10) and four in the B-chain (TB6, TB14-ss-TB16, TB20, and TB18) were obtained as good marker peptides for unambiguous identification of intact ricin. The lowest concentration of native ricin for unambiguous identification was 20 ng/mL, in which three marker peptides from both the A-chain and B-chain could be measured with a minimum of three ion transitions. Combined with affinity enrichment, the developed approach was successfully applied for the measurement of intact ricin from the complicated matrix samples of the second, third, and fourth biotoxin exercises organized by the Organisation for the Prohibition of Chemical Weapons (OPCW). This study has provided a recommended detection method combined with one novel ACN-assisted trypsin digestion with MS for forensic unambiguous confirmation of trace ricin intact with high confidence.

Quantitative detection of ricin in beverages using trypsin/Glu-C tandem digestion coupled with ultra-high-pressure liquid chromatography-tandem mass spectrometry.

The toxic protein of ricin has drawn wide attention in recent years as a potential bioterrorism agent due to its high toxicity and wide availability. For the verification of the potential anti-terrorism activities, it is urgent for the quantification of ricin in food-related matrices. Here, a novel strategy of trypsin/Glu-C tandem digestion was introduced for quantitative detection of ricin marker peptides in several beverage matrices using isotope-labeled internal standard (IS)-mass spectrometry. The ricin in beverages was captured and enriched by biotinylated anti-ricin polyclonal antibodies conjugated to streptavidin magnetic beads. The purified ricin was cleaved using the developed trypsin/Glu-C tandem digestion method and then quantitatively detected by ultra-high-pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) with isotope-labeled T7A and TG11B selected as IS. The use of trypsin/Glu-C digestion allows shorter peptides, which are more suitable for MS detection, to be obtained than the use of single trypsin digestion. Under the optimized tandem digestion condition, except for T7A in the A-chain, two resulting specific peptides of TG13A, TG28A from the A-chain and two of TG11B, TG33B from the B-chain were chosen as novel marker peptides with high MS response. The uniqueness of the selected marker peptides allows for unambiguous identification of ricin among its homologous proteins in a single run. The MS response of the four novel marker peptides is increased by more than 10 times compared with that of individual corresponding tryptic peptides. Both the marker peptides of A-chain T7A and B-chain TG11B were selected as quantitative peptides based on the highest MS response among the marker peptides from their individual chains. The limit of detection (LOD) of ricin is 0.1 ng/mL in PBS and 0.5 ng/mL in either milk or orange juice. The linear range of calibration curves for ricin were 0.5-300 ng/mL in PBS, 1.0-400 ng/mL in milk, and 1.0-250 ng/mL in orange juice. The method accuracy ranged between 82.6 and 101.8% for PBS, 88.9-105.2% for milk, and 95.3-118.7% for orange juice. The intra-day and inter-day precision had relative standard deviations (%RSD) of 0.3-9.4%, 0.7-8.9%, and 0.2-6.9% in the three matrices respectively. Furthermore, whether T7A or TG11B is used as a quantitative peptide, the quantitative results of ricin are consistent. This study provides not only a practical method for the absolute quantification of ricin in beverage matrices but also a new strategy for the investigation of illegal use of ricin in chemical weapon verification tasks such as OPCW biotoxin sample analysis exercises.

LC-HRMS Screening and Identification of Novel Peptide Markers of Ricin Based on Multiple Protease Digestion Strategies.

Both ricin and R. communisagglutinin (RCA120), belonging to the type II ribosome-inactivating proteins (RIPs-Ⅱ), are derived from the seeds of the castor bean plant. They share very similar amino acid sequences, but ricin is much more toxic than RCA120. It is urgently necessary to distinguish ricin and RCA120 in response to public safety. Currently, mass spectrometric assays are well established for unambiguous identification of ricin by accurate analysis of differentiated amino acid residues after trypsin digestion. However, diagnostic peptides are relatively limited for unambiguous identification of trace ricin, especially in complex matrices. Here, we demonstrate a digestion strategy of multiple proteinases to produce novel peptide markers for unambiguous identification of ricin. Liquid chromatography-high resolution MS (LC-HRMS) was used to verify the resulting peptides, among which only the peptides with uniqueness and good MS response were selected as peptide markers. Seven novel peptide markers were obtained from tandem digestion of trypsin and endoproteinase Glu-C in PBS buffer. From the chymotrypsin digestion under reduction and non-reduction conditions, eight and seven novel peptides were selected respectively. Using pepsin under pH 1~2 and proteinase K digestion, six and five peptides were selected as novel peptide markers. In conclusion, the obtained novel peptides from the established digestion methods can be recommended for the unambiguous identification of ricin during the investigation of illegal use of the toxin.

A sensitive quantification approach for detection of HETE-CP adduct after benzyl chloroformate derivatization using ultra-high-pressure liquid chromatography tandem mass spectrometry.

Sulfur mustard (HD) reacts with human serum albumin (HSA) at Cys34 and produces a long-term biomarker of HD exposure. Here, we present a novel, sensitive, and convenient method for quantification of HD exposure by detection of HD-HSA adducts using pronase digestion, benzyl chloroformate (Cbz-Cl) derivatization, and ultra-high-pressure liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). The HSA in HD-exposed plasma in vitro was precipitated with acetone and digested (2 h, 50 °C) with pronase to form the alkylated dipeptide, S-hydroxyethylthioethyl-CysPro (HETE-CP). The HETE-CP adduct was derivatized with Cbz-Cl to generate N-carbobenzoxy HETE-CP (HETE-C(Cbz)P). The derivatized product was analyzed by UHPLC-MS/MS. HD surrogate, 2-chloroethyl ethyl sulfide (2-CEES), was introduced as a non-isotope internal standard (ISTD) instead of traditional d8-HD for quantification. The method was found to be linear between 1.00 and 200 ng/mL HD exposure (R2 > 0.998) with precision of ≤ 9.0% relative standard deviation (RSD) and accuracy ranged between 97.1 and 111%. The limit of detection (LOD) is 0.500 ng/mL (S/N~5), over 15 times lower than that of the previous method (7.95 ng/mL). Time-consuming affinity purification or solid phase extraction (SPE) is not needed in the experiment and the operation takes less than 5 h. This study provides a new strategy and useful tool for retrospective analysis of HD exposure by HETE-CP biomarker detection. Graphical abstract Flow diagram for quantification of sulfur mustard exposure by detection of HETE-CP dipeptide adduct after benzyl chloroformate derivatization using ultra-high-pressure liquid chromatography tandem mass spectrometry.

Simultaneous quantification of four metabolites of sulfur mustard in urine samples by ultra-high performance liquid chromatography-tandem mass spectrometry after solid phase extraction.

Four HD urinary metabolites including hydrolysis metabolite thiodiglycol (TDG), glutathione-derived metabolite 1,1'-sulfonylbis[2-S-(N-acetylcysteinyl)ethane] (SBSNAE), as well as the ß-lyase metabolites 1,1'-sulfonylbis[2-(methylsulfinyl)ethane] (SBMSE) and 1-methylsulfinyl-2-[2-(methylthio) ethylsulfonyl]ethane (MSMTESE) are considered as important biomarkers for short-term retrospective detection of HD exposure. In this study, a single method for simultaneous quantification of the four HD metabolites in urine samples was developed using ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The four urinary metabolites were simultaneously extracted from urinary samples using a solid phase extraction (SPE) method with high extraction recoveries for all four metabolites varied in the range of 71.1-103% followed by UHPLC-MS/MS analysis. The SPE is simple and high effective only requiring 0.1mL of urinary samples and 0.5h time consuming. The problem of previous co-elution of TDG and SBSNAE in UHPLC was well solved, and complete separation of TDG, SBSNAE, SBMSE and MSMTESE from SPE-processed urine matrix was obtained to increase specificity and sensitivity. A full method validation was performed for each analyte in urine matrix. The linear range of calibration curves for the four analytes were respectively from 0.50-500ngmL-1 for TDG and SBSNAE, 0.05-500ngmL-1 for SBMSE and MSMTESE with coefficient of determination value (R2) ≥0.990. The limit of detection was 0.25ngmL-1 for TDG and SBSNAE, 0.01ngmL-1 for SBMSE and MSMTESE spiked in normal urine. The intra/inter-day precision for each analyte at three QC levels had relative standard deviation (%RSD) of ≤10.3%, and the intra/inter-day accuracy ranged between 88.0-108%. This developed method allows for simultaneous and trace measurement of four HD urinary metabolites within one single determination with the lowest usage amount of urine samples over all previous methods This study provides a useful tool for early diagnosis and monitoring of HD poisoning for medical treatment with high confidence, avoiding the need for application of several analysis methods.

Simultaneous quantification of soman and VX adducts to butyrylcholinesterase, their aged methylphosphonic acid adduct and butyrylcholinesterase in plasma using an off-column procainamide-gel separation method combined with UHPLC-MS/MS.

This work describes a novel and sensitive non-isotope dilution method for simultaneous quantification of organophosphorus nerve agents (OPNAs) soman (GD) and VX adducts to butyrylcholinesterase (BChE), their aged methylphosphonic acid (MeP) adduct and unadducted BChE in plasma exposed to OPNA. OPNA-BChE adducts were isolated with an off-column procainamide-gel separation (PGS) from plasma, and then digested with pepsin into specific adducted FGES*AGAAS nonapeptide (NP) biomarkers. The resulting NPs were detected by UHPLC-MS/MS MRM. The off-column PGS method can capture over 90% of BChE, MeP-BChE, VX-BChE and GD-BChE from their respective plasma materials. One newly designed and easily synthesized phosphorylated BChE nonapeptide with one Gly-to-Ala mutation was successfully reported to serve as internal standard instead of traditional isotopically labeled BChE nonapeptide. The linear range of calibration curves were from 1.00-200ngmL-1 for VX-NP, 2.00-200ngmL-1 for GD-NP and MeP-NP (R2≥0.995), and 3.00-200ngmL-1 for BChE NP (R2≥0.990). The inter-day precision had relative standard deviation (%RSD) of <8.89%, and the accuracy ranged between 88.9-120%. The limit of detection was calculated to be 0.411, 0.750, 0.800 and 1.43ngmL-1 for VX-NP, GD-NP, MeP-NP and BChE NP, respectively. OPNA-exposed quality control plasma samples were characterized as part of method validation. Investigation of plasma samples unexposed to OPNA revealed no baseline values or interferences. Using the off-column PGS method combined with UHPLC-MS/MS, VX-NP and GD-NP adducts can be unambiguously detected with high confidence in 0.10ngmL-1 and 0.50ngmL-1 of exposed human plasma respectively, only requiring 0.1mL of plasma sample and taking about four hours without special sample preparation equipment. These improvements make it a simple, sensitive and robust PGS-UHPLC-MS/MS method, and this method will become an attractive alternative to immunomagnetic separation (IMS) method and a useful diagnostic tool for retrospective detection of OPNA exposure with high confidence. Furthermore, using the developed method, the adducted BChE levels from VX and GD-exposed (0.10-100ngmL-1) plasma samples were completely characterized, and the fact that VX being more active and specific to BChE than GD was re-confirmed.

Characterization of class 1 integron gene cassettes among clinical bacteria isolated from one large hospital in northern China.

The class 1 integron and complex gene cassettes among different species of clinical isolates in northern China were characterized in this study. 383 clinical isolates were obtained from northern China, and class 1 integrons containing gene cassettes widely distributed among gram negative clinical isolates was observed. We find that the class 1 integron showed positive correlation with multidrug resistance phenotype of gram negative bacteria. In addition, we find that isolates belonged to one species harbored different types of gene cassette arrays, while same types of gene cassette arrays were observed in different species of isolates. The diversity of gene cassette arrays among the isolates indicated the complexity of multidrug resistance in clinical isolates in northern China.

Ehrlichiosis and zoonotic anaplasmosis in suburban areas of Beijing, China.

In 2006, an unusual nosocomial outbreak of anaplasmosis occurred in Anhui Province, China. To follow these emerging tickborne-rickettsioses, a larger survey of Ehrlichia chaffeensis and Anaplasma phagocytophilum seroprevalence among farm worker populations, and the divergence of the partial 16S rRNA gene sequences of A. phagocytophilum among domestic animals, were conducted in Yanqing, Miyun, and Tongzhou Counties in Beijing from March to April, 2009. Blood samples from 562 farmers, 90 goats, 73 cattle, and 2 dogs were collected. IgG antibodies against E. chaffeensis and A. phagocytophilum were assayed by micro-indirect immunofluorescence assay (IFA). Partial fragments of 16S rRNA genes of A. phagocytophilum were amplified from blood DNA from domestic animals and their sequences analyzed. The total E. chaffeensis and A. phagocytophilum seroprevalence among the farm worker population was 16.4% and 14.1%, respectively. For domestic animals, the seropositive rates of A. phagocytophilum for goats, cattle, and dogs, were 2.3%, 0%, and 0%, respectively. The PCR-positive rates for A. phagocytophilum in goats and cattle were 48.9% and 23.9%, respectively. Three dominant genetic groups of Chinese A. phagocytophilum isolates were determined for goats and cattle, and these isolate varieties were broadly identified in China, Japan, and Korea. The prevalence of E. chaffeensis and A. phagocytophilum among farmers and domestic animals in Beijing rural areas was also demonstrated. The diagnoses and differential diagnoses of these emerging infectious diseases should be emphasized in clinics, and further ecological investigation of E. chaffeensis and A. phagocytophilum vectors and hosts is needed.

[Investigation on Ehrlichia chaffeensis and Anaplasma phagocytophilum infection among farmers and domestic animals in rural areas of Beijing, China].

OBJECTIVE: To investigate the status of Ehrlichia (E.) chaffeensis and Anaplasma (A.) phagocytophilum infection among farming populations and domestic animals in the rural area of Beijing, China. METHODS: Blood samples from 562 farmers and 163 blood samples including 90 goats, 71 ox and 2 dogs, were collected. Specificity of IgG antibodies against E. chaffeensis and A. phagocytophilum were tested by micro-indirect immunofluorescent assay (mIFA). 16S rRNA genes of A. phagocytophilum were amplified from the domestic animal blood samples and products were sequenced and analyzed by nested PCR. RESULTS: The positive rates of E. chaffeensis and A. phagocytophilum antibody were 16.5% and 14.0% respectively for farmers. The total positive rates of A. phagocytophilum were 2.3% and 0 for both goats and oxen respectively. No antibody was found for the 2 tested dogs. The PCR positive rates were 48.9% and 23.9% for goats and oxen respectively. Three dominant varieties of A. phagocytophilum were demonstrated in goats and oxen. CONCLUSION: The prevalence rates of E. chaffeensis and A. phagocytophilum were identified in the rural areas of Beijing.

Spotted fever group Rickettsia in Yunnan Province, China.

Information about spotted fever group (SFG) rickettsiae in southern China remains sparse. A specific and sensitive real-time PCR assay for detection of SFG rickettsiae was established and used to detect the prevalence rate of SFG rickettsiae in Yunnan Province, China. The limit of detection (LOD) of our real-time PCR was 200 copies per reaction, which is more sensitive than the previously developed nested PCR assays for Rickettsia. We tested 265 blood samples (127 goats, 78 dogs, and 60 cattle) collected from Yunnan Province using the real-time PCR assay and revealed that the prevalence of SFG rickettsiae among dogs, cattle, and goats were 14.10%, 23.33%, and 24.41%, respectively. The SFG rickettsiae detected in animals in Yunnan Province were classified into two genotypes: a unique group that is different from all known SFG rickettsiae species, and R. heilongjiangensis.

[Project to enhance accuracy of nursing assessments performed by medical and surgical ICU nursing staffs].

BACKGROUND: Some nurses do not have the skills necessary to evaluate general ICU patient condition and the meaning of laboratory data adequately. This can influence nursing care quality and patient safety. Between October 16th and 29th, 2009, this project employed a checklist to evaluate nursing assessment accuracy in medical and surgical ICUs. The unexpectedly low accuracy rate of two-thirds (63.4 percent) was caused by factors including poor nursing assessment cognizance, lack of experience performing nursing assessments, nursing assessment skills taught only to experienced nursing staff, and a lack of nursing assessment guidelines. PURPOSE: This project was designed to improve nursing assessment cognizance and accuracy among nursing staff. RESOLUTION: The authors: 1. Established formal nursing assessment guidelines; 2. made a CD-ROM to introduce nursing assessment basics; and 3. employed lectures and simulation exercises to teach nursing assessment skills. RESULTS: Nursing assessment accuracy improved significantly to 85.8 percent and actual nursing assessment cognizance scores ranged between 68.7 and 83.1. CONCLUSIONS: This project effectively improved nursing assessment accuracy and may be considered and referenced by relevant medical organizations.

[Sero-epidemiological investigation on Rickettsia typhi, Bartonella henselae and Orientia tsutsugamushi in farmers from rural areas of Tianjin, 2007 - 2009].

OBJECTIVE: To study the sero-epidemiological status regarding Rickettsia (R.) typhi, Bartonella (B.) henselae and Orientia (O.) tsutsugamushi in farmers from rural areas of Tianjin. METHODS: Field epidemiological surveys were performed in 8 districts (county) of Tianjin city from 2007 to 2009. 886 farmers were randomly recruited and their serum samples collected to detect the specific antibodies of R. typhi, B. henselae and O. tsutsugamushi by micro-indirect immunofluorescence (IFA). RESULTS: The total antibody positive rates of R. typhi increased from 5.0% to 58.2% while B. henselae had an increase from 2.6% to 14.5% and O. tsutsugamushi increased from 1.8% to 39.8%. Geographic distribution showed that farmers living in the central and southeast areas were higher than that in other areas. CONCLUSION: Infections of both R. typhi, B. henselae and O. tsutsugamushi in farmers from Tianjin areas were popular and the antibody positive rates of R. typhi, B. henselae and O. tsutsugamushi had an annual increase.

[Study on effect of Salvia injection in treating primary nephrotic syndrome and on endothelin and serum interleukin-2 receptor in children].

OBJECTIVE: To explore the effect and mechanism of Salvia Injection (SI) in treating primary nephrotic syndrome (PNS) in children. METHODS: Forty-four PNS children were randomly divided into conventional steroid treated group (20 cases) and conventional plus SI intervention treated group (24 cases), the levels of serum endothelin (ET), soluable interleukin-2 receptor (sIL-2R) were observed. RESULTS: Before treatment, plasma ET and sIL-2R in PNS children were higher than those in healthy children significantly (P < 0.01). After treatment, the ET and sIL-2R levels were all obviously improved in both treated groups (P < 0.05) and the improvement in the conventional plus SI intervention treated group was more obvious, the difference between the two treated groups after treatment was significant (P < 0.05). CONCLUSION: Conventional treatment supplemented with SI could more effectively improve the levels of plasma ET and SIL-2R in treating PNS children, and hence alleviate the damage of renal tissue.