Comparison of two MALDI-TOF MS systems for theidentification of clinically relevant anaerobic bacteriain Argentina

Abstract The aim of this study was to compare the performance of two MALDI-TOF MS systems in the identification of clinically relevant strict anaerobic bacteria. The 16S rRNA gene sequencing was the gold standard method when discrepancies or inconsistencies were observed between platforms. A total of 333 isolates were recovered from clinical samples of different centers in Buenos Aires City between 2016 and 2021. The isolates were identified in duplicate using two MALDI-TOF MS systems, BD Bruker Biotyper (Bruker Daltonics, Bremen, Germany) and Vitek MS (bioMèrieux, Marcy-l'Etoile, France). Using the Vitek MS system, the identification of anaerobic isolates yielded the following percentages: 65.5% (n: 218) at the species or species-complex level, 71.2% (n: 237) at the genus level, 29.4% (n: 98) with no identification and 5.1% (n: 17) with misidentification. Using the Bruker Biotyper system, the identification rates were as follows: 85.3% (n: 284) at the species or species-complex level, 89.7% (n: 299) at the genus level, 14.1% (n: 47) with no identification and 0.6% (n: 2) with misidentification. Differences in the performance of both methods were statistically significant (p-values <0.0001). In conclusion, MALDI-TOF MS systems speed up microbial identification and are particularly effective for slow-growing microorganisms, such as anaerobic bacteria, which are difficult to identify by traditional methods. In this study, the Bruker system showed greater accuracy than the Vitek system. In order to be truly effective, it is essential to update the databases of both systems by increasing the number of each main spectrum profile within the platforms.

Resumen El objetivo de este estudio fue comparar el desempeño de dos sistemas MALDI-TOF MS en la identificación de bacterias anaerobias estrictas de interés clínico. La secuenciación del gen 16S ARNr fue el método de referencia utilizado cuando se observaron discrepancias o inconsistencias entre plataformas. Se recuperaron 333 aislados de muestras clínicas de diferentes centros de la Ciudad Autónoma de Buenos Aires entre 2016 y 2021. Los aislados se identificaron por duplicado mediante dos sistemas MALDI-TOF MS: el BD Bruker Biotyper (Bruker Daltonics, Bremen, Alemania) y el Vitek MS (bioMèrieux, Marcy-l'Etoile, Francia). A través del sistema Vitek MS, los mismos fueron identificados a nivel de especie o complejo de especies en un 65,5% (n: 218) y de género en un 71,2% (n: 237), mientras que no se identificaron en un 29,4% (n: 98) y fue incorrecta en el 5,1% (n: 17). Mediante el sistema Bruker Biotyper, dichos valores fueron del 85,3% (n: 284), del 89,7% (n: 299), del 14,1% (n: 47) y del 0,6% (n: 2), respectivamente. La diferencia entre ambos métodos fue estadísticamente significativa (p<0,0001). En conclusión, los sistemas MALDI-TOF MS aceleran la identificación microbiana. Son especialmente útiles para los microorganismos de crecimiento lento, como las bacterias anaerobias, que son difíciles de identificar con los métodos tradicionales. El sistema Bruker demostró ser más preciso que el Vitek MS. Para que estos métodos sean realmente efectivos es fundamental actualizar las bases de datos de ambos sistemas e incrementar el número de espectros de referencia dentro de las plataformas.

The influence of growth time on the identification of Bartonella henselae strains by MALDI-TOF mass spectrometry

ABSTRACT Bartonella spp. are bacteria responsible for neglected diseases worldwide. Bartonella henselae is the species most associated with human infections. It is associated with a large spectrum of clinical manifestations and is potentially fatal. The identification of Bartonella spp. is considered a challenge in clinical routine. These bacteria are fastidious, and the time required to isolate them varies from one to six weeks. MALDI-TOF mass spectrometry has emerged as an application for research on Bartonella spp. , and has still been little explored. We investigated whether three different B. henselae strains with different growth times—14 and 28 days—could be correctly identified by MALDI-TOF mass spectra fingerprint comparison and matching. We found that the spectra from strains with different growth times do not match each other, leading to misidentification. We suggest creating database entries with multiple spectra from strains with different growth times to increase the chances of accurate identification of Bartonella spp. by MALD-TOF MS.

Analysis on Quality of Sojae Semen Praeparatum Based on Traditional Quality Evaluation / 中国实验方剂学杂志

ObjectiveBased on the quality evaluation experience of "it is better to have a fragrant and strong aroma" summarized by materia medica of past dynasties， the chemical components of Sojae Semen Nigrum（SSN） and Sojae Semen Praeparatum（SSP） were systematically compared and analyzed， and the main fermentation products in different fermentation time were quantitatively analyzed， so as to clarify the transformation law of internal components in the processing process and provide scientific basis for the modern quality control of SSP. MethodUltra performance liquid chromatography-quadrupole tandem time-of-flight mass spectrometry（UPLC-Q-TOF-MS） was used for the structural identification of the chemical constituents of SSN and SSP， and with the aid of Progenesis QI v2.3 software， the negative ion mode was employed for principal component analysis（PCA） pattern recognition， and the data were analyzed with the aid of orthogonal partial least squares-discriminant analysis（OPLS-DA） for two-dimensional data to obtain S-plot， and components with |P|>0.1 were selected as the differential constituents. The contents of isoflavonoids in SSP during fermentation was determined by UPLC， and the samples were taken every 8 h in the pre-fermentation period and every 2 d in the post-fermentation period， and the dynamic changes of isoflavonoid contents in different fermentation stages were analyzed. The contents of amino acids and nucleosides in SSP and SSN from different fermentation stages were quantitatively analyzed by phenyl isothiocyanate（PITC） pre-column derivatization and high performance liquid chromatography（HPLC） gradient elution， and the contribution of flavor substances to the "delicious" taste of SSP was discussed by taste intensity value（TAV）. ResultA total of 19 kinds of differential components were screened out， mainly soybean saponins and isoflavones， and their contents decreased significantly or even disappeared after fermentation. In the pre-fermentation process of SSP， glycoside bond hydrolysis mainly occurred， and isoflavone glycosides in SSN were degraded and converted into the corresponding aglycones， the content of flavor substances such as amino acids increased gradually. In the post-fermentation process， protein degradation mainly occurred， after 8 d of post-fermentation， the content of isoflavones was basically stable， while the total content of amino acids increased by 8-40 times on average. Different amino acids form the special flavor of SSP， such as the TAV of glutamate is always ahead of other flavor substances， and sweet substances such as alanine and valine have made relatively great contributions to SSP. ConclusionBased on the law of constituent transformation， combined with the traditional evaluation index of "fragrant and strong"， it is difficult to control the fermentation degree of SSP by the existing standards in the 2020 edition of Chinese Pharmacopoeia. It is suggested that description of the characteristics of SSP be refined and changed to "fragrant， delicious and slightly sweet"， and at the same time， the post-fermentation index compounds such as glutamic acid， alanine and valine should be added as the quality control indicators of SSP， so as to standardize the production process and improve the quality of SSP.

Qualitative and Quantitative Analysis of Chemical Constituents in Liu Junzitang by UPLC-Q-TOF-MS/MS and UPLC / 中国实验方剂学杂志

ObjectiveTo establish a qualitative and quantitative analysis method for chemical constituents in Liu Junzitang（LJZT）， and to clarify its material basis. MethodThe chemical constituents in LJZT were analyzed by ultra performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS/MS）， and the resulting compounds were identified by using databases， such as MassBank， PubChem， ChemSpider， Traditional Chinese Medicine Systems Pharmacology Database and Analytical Platform（TCMSP）， and by combining with relevant literature. UPLC was used to establish a quantitative method for analysis of 9 compounds in LJZT， including liquiritin， hesperidin， lobetyolin， liquiritigenin， glycyrrhizic acid， nobiletin， tangeretin， atractylenolide Ⅱ and Ⅰ. ResultBy combining the relevant literature， database and MS information， a total of 79 compounds were identified from LJZT， including 31 flavonoids， 15 terpenoids， 14 nitrogen-containing compounds， 6 phenylpropanoids， 6 organic acids and 7 other compounds. The established quantitative analytical method for the nine representative components showed good linearity within their respective linear ranges， and the precision， stability， reproducibility and recovery were in accordance with the requirements. The quantitative results showed that the contents of liquiritin， hesperidin， lobetyolin， liquiritigenin， glycyrrhizic acid， nobiletin， tangeretin， atractylenolide Ⅱ and Ⅰ in LJZT were 0.376 5， 2.602 1， 0.082 6， 0.128 1， 1.778 6， 0.015 7， 0.006 7， 0.030 4， 0.003 2 mg·g-1， respectively. ConclusionThe established method can quickly， sensitively and accurately analyze the chemical constituents in LJZT， clarify that the material basis of LJZT is mainly flavonoids， terpenoids and nitrogen-containing compounds， and simultaneously determine the contents of the 9 components， which can lay a foundation for the research on quality control， mechanism and clinical application of LJZT.

Evaluation of A Liver Yin Deficiency Mouse Model Based on Untargeted Metabolomics / 中国实验方剂学杂志

ObjectiveBased on ultra performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry（UPLC-Q-TOF-MS）， to evaluate the establishment of a mouse model of liver Yin deficiency by thyroid tablet suspension combined with 10% carbon tetrachloride（CCl4） from the perspective of non-targeted metabolomics， in order to lay the foundation for the establishment of a traditional Chinese medicine（TCM） syndrome model. MethodA total of 24 mice were randomly divided into blank group and model group. The model group was given thyroid tablet suspension（0.003 2 g·kg-1） by gavage for 14 consecutive days， and 10% CCl4（5 mL·kg-1） was intraperitoneally injected once a week to establish a liver Yin deficiency model， while the blank group was injected with an equal amount of olive oil intraperitoneally and gavaged with an equal amount of distilled water， and was fed with normal feed. After the modeling was completed， 6 mice in each group were randomly selected， the levels of alanine aminotransferase（ALT）， aspartate aminotransferase（AST）， cyclic adenosine monophosphate（cAMP）， cyclic guanosine monophosphate（cGMP）， interleukin（IL）-6， IL-10， tumor necrosis factor-α（TNF-α）were measured in the mice serum， and malondialdehyde（MDA）， superoxide dismutase（SOD）， total protein（TP）， hydroxyproline（HYP） and other indicators were measured in the mice liver. Liver tissue sections were taken for hematoxylin-eosin（HE） staining and observing pathological changes. The remaining 6 mice in each group were subjected to UPLC-Q-TOF-MS combined with principal component analysis（PCA） and orthogonal partial least squares-discriminant analysis（OPLS-DA） were used to screen differential metabolites in the liver Yin deficiency mouse model， Kyoto Encyclopedia of Genes and Genomes（KEGG） database was used to analyze the corresponding metabolic pathways of differential metabolites. ResultCompared with the blank group， mice in the model group showed liver Yin deficiency manifestations such as reduced body weight， fatigue and sleepiness， disheveled and lusterless hair， irritability. The levels of ALT， cAMP/cGMP， IL-6， AST， MDA， cAMP， TNF-α significantly increased（P<0.05， P<0.01）， while the levels of SOD， IL-10 and cGMP significantly decreased（P<0.05， P<0.01）， and the changes of HYP and TP were not statistically significant. Hepatic steatosis and distortion of the radial arrangement of the liver plate cells were seen in the section images of the model group， endogenous substances were clearly separated， and 252 differential metabolites were identified in the serum samples， which were mainly involved in the metabolic pathways of purine metabolism， steroid hormone biosynthesis and pyrimidine metabolism. A total of 229 differential metabolites were identified in the liver samples， mainly involving nucleotide metabolism， purine metabolism， steroid hormone biosynthesis， pyrimidine metabolism， antifolate resistance， insulin resistance， primary bile acid biosynthesis， prostate cancer， sulfur relay system， arachidonic acid metabolism and other metabolic pathways. ConclusionThe successful establishment of liver Yin deficiency model in mice by CCl4 combined with thyroid hormone is evaluated through the investigation of serum and liver metabolomics， combined with biochemical indicators， which provides a biological basis and experimental foundation for the Yin deficiency syndrome model of TCM.

Structural identification of the related substances in phloroglucinol injection by two-dimensional liquid chromatography-quadrupole time-of-flight mass spectrometry / 药学学报

This study aimed to identify the related substances of phloroglucinol injection by two-dimensional liquid chromatography quadrupole time-of-flight mass spectrometry (2D-LC-Q-TOF/MS). The first-dimensional separation was carried out on an HSS T3 (250 mm × 4.6 mm, 5 μm) column by gradient elution using 1.36 g·L-1 potassium dihydrogen phosphate buffer solution (pH adjusted to 3.0 with diluted phosphoric acid) and acetonitrile as the mobile phases. The separated components were then trapped in switch valve tube lines respectively and delivered to the second-dimensional desalting gradient elution which was performed with a BDS C18 (100 mm × 4.6 mm, 2.4 μm) column using 0.1% formic acid and methanol as the mobile phases. After rapid desalting, electrospray-ionization quadrupole time-of-flight high resolution mass spectrometry was used for determining the accurate masses and elemental compositions of the parents and their product ions for both phloroglucinol and its related substance. Structures of the related substances were then figured out by mass spectrometry elucidation, organic reaction mechanism analysis, and/or comparison with reference substances. Under the established analytical conditions, phloroglucinol and its related substances were adequately separated, 17 main related substances were detected and identified in the injection and its stressed samples for the first time. The identification results can provide reference for the quality control of phloroglucinol injection.

Toxicity Attenuation Mechanism on Processing Method for Aconiti Lateralis Radix Praeparata in Guilingji Based on Urine Metabolomics / 中国实验方剂学杂志

ObjectiveMetabolomics was used to reveal the mechanism of Aconiti Lateralis Radix Praeparata（ALRP） in attenuating toxicity by processing from the aspects of amino acid metabolism， oxidative stress and energy metabolism by analyzing multiple metabolic pathways. MethodTwenty-four rats were randomly divided into control group， raw group and processed group， 8 rats in each group. The raw and processed group were given with 0.64 g·kg-1 of raw ALRP and processed ALRP respectively every day， the control group was given with an equal amount of normal saline once a day. After continuous administration for 7 days， the urine， serum and heart tissue of rats were collected. Pathological examination of the heart was carried out using hematoxylin-eosin（HE） staining， and the activities of lactate dehydrogenase（LDH） and creatine kinase-MB（CK-MB） in serum and cardiac tissues were detected by microplate assay and immunoinhibition assay. The effects of ALRP on rat heart before and after processing were compared and analyzed. Ultra performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS） was used to perform urine metabolomics analysis， and multivariate statistical analysis was used to screen for differential metabolites related to ALRP in attenuating toxicity by processing， and pathway enrichment analysis was carried out to explore the processing mechanism. ResultHE staining showed that no obvious pathological changes were observed in the heart tissue of the control group， while obvious infiltration of inflammatory cells such as plasma cells and granulocytes was observed in the heart tissue of the raw group， indicating that the raw ALRP had strong cardiotoxicity. There was no significant difference in HE staining of heart tissue between the processed group and the control group， indicating that the toxicity of ALRP was significantly reduced after processing. Compared with the control group， the activities of LDH and CK-MB were significantly increased in serum and heart tissue of the raw group， and those were significantly decreased in serum and heart tissue of the processed group， suggesting that the myocardial toxicity of processed ALRP was reduced. A total of 108 endogenous differential metabolites associated with the raw ALRP were screened using multivariate statistical analysis in positive and negative modes， of which 51 differential metabolites were back-regulated by the processed ALRP. Biological analysis of the key regulatory pathways and associated network changes showed that the pathways related to toxicity of ALRP mainly included tryptophan metabolism， arginine and proline metabolism， phenylalanine metabolism， aminoacyl-tRNA biosynthesis， alanine， aspartate and glutamate metabolism， etc. The metabolic pathways related to the attenuation of processed ALRP mainly included aminoacyl-tRNA biosynthesis， tryptophan metabolism， phenylalanine， tyrosine and tryptophan biosynthesis， phenylalanine metabolism and caffeine metabolism. ConclusionThe processing technology of ALRP in Guilingji can significantly attenuate the cardiotoxicity of raw products， the mechanism mainly involves amino acid metabolism， oxidative stress and energy metabolism， which can provide experimental bases for the research related to the mechanism of toxicity reduction of ALRP by processing and its clinical safety applications.

Analysis of Antidepressant Quality Markers of Bupleuri Radix Before and After Vinegar-processing Based on UPLC-Q-TOF-MS and Network Pharmacology / 中国实验方剂学杂志

ObjectiveTo analyze the antidepressant quality markers（Q-Marker） of Bupleuri Radix（BP） before and after vinegar-processing by ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS）， multivariate statistical analysis and network pharmacology. MethodUPLC-Q-TOF-MS was used to analyze the chemical basis of raw and vinegar-processed products of BP， and principal component analysis（PCA） orthogonal partial least squares-discriminant analysis（OPLS-DA） were used to identify the differential components in BP that changed significantly before and after vinegar-processing， which were regarded as candidate quality markers（Q-Marker）. Then the disease-drug-component-target network related to antidepressant effect of BP was constructed by network pharmacology， and the antidepressant Q-Marker of raw and vinegar-processed products of BP was determined. Rats were randomly divided into blank group， model group， fluoxetine group（2.67 mg·kg-1） and total saponin group（0.72 mg·kg-1）， except the blank group， rats in the other groups were subjected to chronic unpredictable mild stress（CUMS）. Three weeks after the start of modeling， rats in each administration group were given the corresponding dose of drugs once a day for 4 weeks， and rats in the blank and model groups were given normal saline with dose of 10 mL·kg-1. At 1 day before modeling， 21 days and 28 days after administration， body mass weighing， sucrose preference test and open field test were performed on each group . After 28 days of administration， real-time fluorescence quantitative polymerase chain reaction（Real-time PCR） was used to detect the mRNA expression levels of phosphatidylinositol 3-kinase（PI3K）， protein kinase B（Akt）， mammalian target of rapamycin（mTOR）， glycogen synthase kinase-3β（GSK-3β）， forkhead box transcription factor O3a（FoxO3a） and β-catenin in hippocampal tissues of rats in each group， while protein expression levels of PI3K， Akt， mTOR and FoxO3a in hippocampal tissues of rats in each group were detected by Western blot. ResultThere were 19 components in BP showed significant changes before and after vinegar-processing， and 9 components such as saikosaponin A， saikosaponin B1， saikosaponin B2， saikosaponin C and saikosaponin D were identified as potential Q-Marker through S-plot differential marker screening. Combined with the disease-drug-component-target network， saikosaponin A， saikosaponin B1， saikosaponin B2 and saikosaponin D were identified as antidepressant Q-Marker of raw and vinegar-processed products of BP. According to the results of pharmacodynamic tests， after 28 d of administration， compared with the blank group， the body mass， sucrose preference index and open field total score of rats in model group， fluoxetine group and total saponin group decreased significantly（P<0.01）. Compared with the model group， the body mass， sucrose preference index and open field total score in total saponin group increased significantly（P<0.01）. Compared with the blank group， mRNA expression levels of PI3K， Akt， mTOR and β-catenin in hippocampus of rats in the model group decreased significantly（P<0.05）， while mRNA expression levels of GSK-3β and FoxO3a increased significantly（P<0.05）. Compared with the model group， mRNA expression levels of PI3K， Akt， mTOR and β-catenin in hippocampus of rats in the total saponin group were increased significantly（P<0.05）， while mRNA expression levels of GSK-3β and FoxO3a decreased significantly（P<0.05）. Compared with the blank group， the protein expression levels of Akt and mTOR in hippocampus of the model group decreased significantly（P<0.01）， while the protein expression levels of PI3K and FoxO3a increased significantly（P<0.01）. Compared with the model group， the expression level of Akt in hippocampus of the total saponin group increased significantly（P<0.01）， the mTOR expression level was increased but not statistically significant， while the protein expression levels of PI3K and FoxO3a decreased significantly（P<0.01）. ConclusionThe chemical constituents of BP changed greatly after vinegar-processing， and the antidepressant Q-Marker of raw and vinegar-processed products of BP was determined by chemical basis， pharmacodynamics， network pharmacology and signaling pathway， which provided a reference for further research on quality control， pharmacodynamic substance basis and processing mechanism of BP.

Exploration of potential active ingredients and mechanism of action of Xihuang pill-medicated serum against glioma based on HPLC-Q-TOF-MS/MS, network pharmacology and experimental verification / 药学学报

Qualitative analysis of the ingredients absorbed into blood and their metabolites of Xihuang pill (XHP) were conducted using high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF-MS/MS) technology. Network pharmacology was used to explore the potential anticancer mechanisms of the ingredients against glioma, and their specific mechanisms were validated through molecular docking and experimental verification. SD rats were intragastrically administered with XHP, and rat serum samples were collected. Ingredients absorbed into blood and their metabolites were identified based on the retention time of chromatographic peaks, accurate molecular mass, characteristic fragment ions, and comparisons with reference substances and literature data. PharmMapper and SwissTarget Prediction databases were used to obtain the targets of the XHP-medicated serum, while GeneCards, OMIM, PharmGKB, TTD, and DrugBank databases were used to obtain glioma disease targets. The "component-target" network relationship diagram was constructed using Cytoscape 3.9.1 software. The protein-protein interaction (PPI) network diagram was constructed using the STRING database, and the targets were analyzed using GO and KEGG analyses. Molecular docking was used to verify the binding ability of core targets with their corresponding compounds in XHP-medicated serum. The potential mechanism of the anti-glioma effect of 11-keto-β-boswellic acid (KBA), a representative component of XHP-medicated serum, was verified using CCK-8 and Western blot assays. A total of 40 compounds were identified in the XHP-medicated serum, including 28 prototype components and 12 metabolites. The network pharmacology results showed that elemonic acid, 3-acetyl-β-boswellic acid, KBA, α-boswellic acid, and other 5 compounds might be the active ingredients of XHP-medicated serum in the treatment of glioma. Glutathione reductase (GSR), glucose-6-phosphate dehydrogenase (G6PD), ATP-citrate lyase (ACLY), aldo-keto reductase family 1 member B1 (AKR1B1) and glutaredoxin (GLRX) were identified as key targets, involving pathways such as glutathione metabolism and the pentose phosphate pathway. Further cell experiments showed that KBA significantly inhibited the proliferation of T98G cells with an IC50 of 30.96 μmol·L-1, and KBA (30 μmol·L-1) significantly downregulated the protein expression levels of GSR in T98G cells. In summary, XHP-medicated serum may exert its anti-glioma effect by regulating GSR and G6PD-targeted pathways involved in glutathione metabolism. These results provide valuable evidence for further investigating the mechanism of XHP in treating glioma. The animal welfare and experimental procedures were approved by the Ethical Committee of Laboratory Animals at Nanjing University of Chinese Medicine (approval No. ACU221001).

Mechanism of Yi-xin-yin oral liquid according to homotherapy for heteropathy theory based on UHPLC-Q-TOF/MS combined with network pharmacology and molecular docking techniques / 中国临床药理学与治疗学

AIM: To predict the core targets and related signaling pathways of Yi-xin-yin oral liquid for the treatment of arrhythmia, heart failure and myocarditis based on UHPLC-Q-TOF/MS, network pharmacology, molecular docking methods, cell experiments, according to the“homotherapy for heteropathy”theory in traditional Chinese medicine. METHODS: UHPLC-Q-TOF / MS was used to analyze and identify the chemical composition of Yi-xin-yin oral liquid Extract and the blood-absorbing components of rats oral administrated with Yi-xin-yin oral liquid extract, which compounds were applied in the databases searching for the potential targets (TCMSP, SwissTargetPrediction) and disease targets (OMIM, Genecard). Venn diagram was used for target intersection, and the subsequent protein-protein interaction network obtained core targets by STRING11.5 database, and then construct a "disease-component-target" network by cytoscape3.9.0. Finally, DAVID database was used to analysis GO function and KEGG enrichment analysis of core targets, and molecular docking validation was performed using Autodock vina software. And, validated with H9c2 cells for potential active ingredients and targets. RESULTS: A total of 156 compounds were identified from Yi - xin-yin Oral Liquid extract; 34 compounds were identified from rat serum, including 6-gin-gerol, isoliquiritigenin, glycyrrhizic acid and other compounds, and 139 intersecting targets were obtained. The KEGG pathway enrichment analysis mainly involved the TNF signaling pathway, IL-17 signaling pathway, MAPK signaling pathway, PI3K-Akt signaling pathway and so on. The TNF and IL-6 targets were selected for molecular docking with the main compounds, and the docking results were good (less than -5 kcal/mol). In vitro cellular experiments have shown that Yi-xin-yin oral liquid can exert therapeutic effects by regulating TNF and IL-6. CONCLUSION: The main potential active ingredients of Yi-xin-yin oral liquid may be isoliquiritigenin, glycyrrhetinic acid, calycosin-7-glucoside, salvianolic acid B, and 6-gingerol, which mainly act on TNF, IL-6 and other targets to regulate specific signaling pathways and exert therapeutic effects.

Effect of Gegen Qinliantang on Fecal Short-chain Fatty Acids in Rats with Antibiotic-associated Diarrhea Based on Targeted Metabonomics / 中国实验方剂学杂志

ObjectiveTo explore the impact of Gegen Qinliantang（GQT） on the fecal short-chain fatty acids（SCFAs） metabolism in antibiotic-associated diarrhea（AAD） through targeted metabolomics. MethodA total of 240 SD rats were randomly divided into six groups（n=40， half male and half female）， including blank group， model group， bifidobiogen group（0.15 g·kg-1）， and GQT high-， medium-， and low-dose groups（10.08， 5.04， 2.52 g·kg-1）， except for the blank group， clindamycin（250 mg·kg-1） was given to all groups by gavage for modeling every day for 7 d. After successful modeling， each administered group was gavaged with the corresponding dose of the drug， and the blank and model groups were gavaged with an equal volume of normal saline solution， 1 time/d， for 14 d. At 0， 3， 7， 14 d after the drug intervention， eight rats were randomly selected from each group， respectively. Gas chromatography-time-of-flight mass spectrometry（GC-TOF-MS） was used to perform targeted metabolomic analysis of SCFAs in the feces of rats， and partial least squares-discriminant analysis（PLS-DA） was applied to compare the differences in metabolic profiles between groups at different treatment times， and to compare the changes in the contents of SCFAs in rat feces between groups. ResultPLS-DA results showed that the blank group could be clearly distinguishable from the model group， with GQT exhibiting a closer proximity to the blank group after 7 d of treatment. After further analyzing the composition of SCFAs， it was found that the proportion of acetic acid increased and the proportions of butyric acid， valeric acid， hexanoic acid and isovaleric acid decreased in the model group compared with the blank group. After the treatment with GQT， the proportions of butyric acid， isobutyric acid， valeric acid， and isovaleric acid increased， and the proportions of acetic acid， propionic acid and caproic acid decreased. Subsequent differential analysis revealed that GQT could significantly improve the content of butyric acid， and had a certain retrogressive effect on the contents of valeric acid and hexanoic acid. ConclusionThe medium dose group of GQT can improve the contents of SCFAs in AAD feces after 7 days of treatment， which may be related to the improvement of the composition ratio of SCFAs and the contents of butyric acid， valeric acid and caproic acid.

Investigation of Effect of Different Processed Products of Atractylodes chinensis Rhizoma on Rats with Spleen Deficiency Based on Pharmacodynamics and Metabolomics / 中国实验方剂学杂志

ObjectiveTo clarify the differences in the efficacy and mechanism of different processed products of Atractylodes chinensis rhizoma by the pharmacodynamics and metabolomics studies of raw， bran-fried and rice water-processed products on rats with spleen deficiency. MethodSixty male SD rats were randomly divided into blank group， model group， raw product group（3.75 g·kg-1）， bran-fried product group（3.75 g·kg-1）， rice water-processed product group（3.75 g·kg-1） and Shenling Baizhusan group（6.7 g·kg-1）， with 10 rats in each group. The method of excessive fatigue+improper diet was used to establish a spleen deficiency model in rats. After the end of modeling， except for the blank and model groups， each dosing group was given the corresponding drug suspension， the immune organ coefficients of each group of rats were examined， the levels of interleukin-6（IL-6）， tumor necrosis factor-α（TNF-α）， immunoglobulin G（IgG）， amylase（AMS）， motilin（MTL）， gastrin（GAS）， Na+-K+-adenosine triphosphatase（ATPase）， aquaporin 2（AQP2）， AQP3 and AQP8 in rats were measured by enzyme-linked immunosorbent assay（ELISA）. Ultra high performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry（UPLC-Q-TOF-MS） combined with orthogonal partial least squares-discriminant analysis（OPLS-DA） were used to search for biomarkers in the plasma samples of spleen-deficient rats by using two criteria［P<0.05 and variable importance in the projection（VIP） value>1］， and to compare the different modulatory effects of the three decoction pieces on the splenic-deficient biomarkers， and metabolic pathway analysis was conducted through the Kyoto Encyclopedia of Genes and Genomes（KEGG） database. ResultCompared with the blank group， the thymus index and spleen index of rats in the model group were significantly decreased（P<0.05）， the levels of IL-6， TNF-α， IgG and AQP2 were significantly increased（P<0.05）， the levels of AMS， GAS， MTL， AQP3， AQP8 and Na+-K+-ATPase were significantly decreased（P<0.05）. Compared with the model group， raw products， bran-fried products and rice water-processed products all increased thymus index and spleen index（P<0.05）， decreased IL-6， TNF-α， IgG and AQP2 levels（P<0.05）， and increased AMS， GAS， MTL， AQP3， AQP8 and Na+-K+-ATPase levels to different degrees. A total of 176 differential metabolites were screened in the model group compared with the blank group， of which 75， 72 and 84 biomarkers were called back by the raw products， bran-fried products and rice water-processed products， respectively（P<0.05， P<0.01）. Raw products of A. chinensis rhizoma mainly affected glycine， serine and threonine metabolism. Bran-fried products mainly affected alanine， aspartate and glutamate metabolism， D-arginine and D-ornithine metabolism. Rice water-processed products mainly affected glycine， serine and threonine metabolism， alanine， aspartate and glutamate metabolism， citrate cycle， thiamine metabolism， D-arginine and D-ornithine metabolism. ConclusionRaw products， bran-fried products and rice water-processed products of A. chinensis rhizoma all have good spleen strengthening effects， among which the effects of bran-fried products and rice water-processed products were stronger. Meanwhile， raw products has the strongest dryness， followed by bran-fried products， and the weakest dryness of rice water-processed products. The three decoction pieces are able to significantly modulate metabolic abnormalities in spleen-deficient rats， and the mechanism may be related to amino acid metabolism such as glycine， serine and threonine metabolism as well as alanine， aspartate and glutamate metabolism.

Coinfección por Aggregatibacter aphrophilus y SARS-CoV-2 con desenlace fatal / Fatal outcome in coinfection by Aggregatibacter aphrophilus and SARS-CoV-2 / Coinfecção por Aggregatibacter aphrophilus e SARSCoV- 2 com desfecho fatal

Resumen Aggregatibacter aphrophilus es un cocobacilo gram negativo de requerimientos nutricionales exigentes, que obliga al microbiólogo a utilizar métodos de identificación no convencionales. Se comunica el caso de una paciente que fue internada con un cuadro clínico caracterizado por fiebre, cefalea y dificultad respiratoria por COVID-19. De las muestras de hemocultivos se rescataron colonias cocobacilares gram negativas de lento crecimiento, las cuales fueron finalmente identificadas como A. aphrophilus mediante MALDI-TOF MS (Matrix-assisted laser desorption ionization time-of-flight mass spectrometry). Dado que en nuestro medio la identificación de este tipo de gérmenes es dificultosa, es fundamental y se recomienda formar una red de laboratorios que den respuesta a las necesidades diagnósticas y tecnológicas.

Abstract Aggregatibacter aphrophilus is a gram-negative coccobacillus with demanding nutritional requirements, which forces the microbiologist to use unconventional typification methods. The case of a patient who was admitted with a clinical history characterised by fever, headache and respiratory distress due to COVID-19 was reported. Slow-growing gram-negative coccobacillary colonies were recovered from the blood culture samples, which were finally typed as A. aphrophilus using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Identifying this organism is difficult in our setting. As it is considered essential, building a network of laboratories that give response to diagnostic and technological needs is highly recommended.

Resumo Aggregatibacter aphrophilus é um cocobacilo gram-negativo com pedidos nutricionais exigentes, o que obriga o microbiologista a utilizar métodos de tipagem não convencionais. É relatado o caso de um paciente que deu entrada com quadro clínico caracterizado por febre, cefaleia e desconforto respiratório devido à COVID-19. Colônias de cocobacilos gram-negativas de crescimento lento foram recuperadas das amostras de hemoculturas, que foram finalmente identificadas como A. aphrophilus usando MALDI-TOF MS (Matrix-assisted laser desorption ionization time-of-flight mass spectrometry). Devido a que a identificação deste tipo de germes é difícil no nosso meio, considera-se essencial e se recomenda construir uma rede de laboratórios que respondam às necessidades diagnósticas e tecnológicas.

Identificación directa con MALDI-TOF desde el frasco de hemocultivo a partir de subcultivos con incubación corta. Estudio multicéntrico / Direct identification with MALDI-TOF from the blood culture bottle from subcultures with short incubation. Multicenter study / Identificação direta com MALDI-TOF do frasco de hemocultura de subculturas com incubação curta. Estudo multicêntrico

Resumen El objetivo de este trabajo fue evaluar el rendimiento de la identificación realizada con MALDI-TOF a partir de la incubación de 3-5 h de subcultivos de hemocultivos positivos monomicrobianos que se comparó con la obtenida con la incubación de 24 h de los mismos. En dos hospitales se utilizó el sistema Vitek-MS (bioMérieux, Francia) y en uno el sistema Micro- Flex LT (Bruker, Daltonics). A partir de la incubación corta, MALDI-TOF identificó correctamente a 5/5 de las levaduras, a 91,1% (153/168) de las bacterias gram positivas, a 96,7% (119/123) de los bacilos gram negativos y a 93,6% (277/296) del total de cepas. La identificación por medio de MALDI-TOF a partir de una corta incubación de los subcultivos de los hemocultivos en medio sólidos es un método práctico, sencillo y confiable.

Abstract The objective of this work was to evaluate the performance of the identification carried out with MALDI-TOF from the 3-5 h incubation of subcultures of monomicrobial positive blood cultures that was compared with that obtained with the 24 h incubation of the same subcultures. The Vitek-MS system (bioMérieux, France) was used in two hospitals and the Micro-Flex LT system (Bruker, Daltonics) in one. With a short incubation, MALDI-TOF correctly identified 5/5 of the yeasts, 91.1% (153/168) of the gram-positive bacteria, 96.7% (119/123) of the gram-negative bacilli and 93.6% (277/296) of the total strains. Identification by means of MALDI-TOF with a short incubation of subcultures of blood cultures in solid media is a practical, simple and reliable method.

Resumo O objetivo deste trabalho foi avaliar o desempenho da identificação realizada com MALDI-TOF a partir de 3 a 5 h de incubação de subculturas de hemoculturas positivas monomicrobianas que foi comparada com a obtida com a incubação de 24 h das mesmas. O sistema Vitek-MS (bioMérieux, França) foi utilizado em dois hospitais e o sistema Micro-Flex LT (Bruker, Daltonics) em um. A partir da incubação curta, o MALDI-TOF identificou corretamente 5/5 das leveduras, 91,1% (153/168) das bactérias gram positivas, 96,7% (119/123) dos bacilos gram-negativos e 93,6% (277/296) das cepas totais. A identificação por meio de MALDI-TOF a partir de uma incubação curta das subculturas das hemoculturas em meio sólido é um método prático, simples e confiável.

Actinomyces europaeus (Gleimia europaea) asociado con absceso cerebral: comunicación de tres casos / Actinomyces europaeus (Gleimia europaea) associated with brain abscess: A reportof three cases

Resumen El absceso cerebral es una infección focal caracterizada por acumulación de pus enel parénquima cerebral; su diagnóstico es de urgencia debido a la alta mortalidad que acarrea.Presentamos tres casos de pacientes con abscesos cerebrales con foco otogénico de origen poli-microbiano, que presentaron en común el aislamiento de Actinomyces europaeus, agente nodescrito hasta el momento en esta localización. A. europaeus fue identificado por la metodo-logía convencional, por espectrometría de masas por desorción/ionización asistida por matriz(MALDI-TOF MS) y por secuenciación del gen ARNr 16S. La sensibilidad antibiótica se evaluó porel método epsilométrico. Todos los aislados presentaron sensibilidad a penicilina, vancomicinay linezolid, mientras que la sensibilidad a clindamicina y eritromicina fue variable. La iden-tificación por MALDI-TOF MS permitió arribar a nivel de especie de forma rápida y confiabley dar una respuesta oportuna y efectiva, evitando el retraso en el tratamiento, lo que sueleincrementar la morbimortalidad del cuadro clínico.

Orquiepididimitis por Haemophilus parainfluenzae: caso clínico y revisión de la literatura / Orchiepididymitis due to Haemophilus parainfluenzae: case report and review of the literature / Orquiepididimite por Haemophilus parainfluenzae: caso clínico e revisão da literatura

Resumen Haemophilus parainfluenzae forma parte de la microbiota normal de la cavidad oral y del tracto respiratorio superior. Es un reconocido agente causal de endocarditis y, con menor frecuencia, de enfermedades como neumonía, sepsis, osteomielitis, celulitis, meningitis y gastroenteritis aguda. Aquí se presenta un caso de orquiepididimitis en un joven adulto donde H. parainfluenzae, confirmado por espectrometría de masas (MALDI-TOF MS), fue el único patógeno detectado. Este caso contribuye a valorar el rol de H. parainfluenzae como patógeno humano, aislado a partir de sitios diferentes del torrente sanguíneo y las vías respiratorias.

Abstract Haemophilus parainfluenzae is part of the normal microbiota of the oral cavity and the upper respiratory tract. It is a recognised causal agent of endocarditis and, less frequently, of diseases such as pneumonia, sepsis, osteomyelitis, cellulitis, meningitis, and acute gastroenteritis. A case of orchiepididymitis in a young adult is reported, where H. parainfluenzae, confirmed by mass spectrometry (MALDI-TOF MS), was the only pathogen detected. This case contributes to assess the role of H. parainfluenzae as a human pathogen, isolated from sites other than the bloodstream and the respiratory tract.

Resumo Haemophilus parainfluenzae faz parte da microbiota normal da cavidade oral e do trato respiratório superior. É um reconhecido agente causal de endocardite e, menos frequentemente, de doenças como pneumonia, sepse, osteomielite, celulite, meningite e gastroenterite aguda. Aqui é relatado um caso de orquiepididimite em um adulto jovem onde H. parainfluenzae, confirmado por espectrometria de massa (MALDI-TOF MS), foi o único patógeno detectado. Este caso contribui para avaliar o papel do H. parainfluenzae como patógeno humano, isolado de outros locais que não sejam a corrente sanguínea e o trato respiratório.

Microascus gracilis y otomicosis / Microascus gracilis and otomycosis

Microascus spp, teleomorfo de Scopulariopsis, es un hongo saprofito encontrado normalmente en suelo, alimentos, vegetales e incluso en ambientes interiores. Considerado un contaminante ambiental, se caracteriza por la resistencia intrínseca a los antifúngicos disponibles. Existen escasas referencias de infecciones por Microascus gracilis, asociándose como causa de eumicetoma o enfermedad diseminada en pacientes sometidos a trasplante pulmonar. Presentamos un caso de otomicosis por M. gracilis con el fin de considerar la búsqueda de hongos en los cultivos óticos y poner de relevancia el poder patógeno y colonizador de este agente.

Microascus spp, a teleomorph of Scopulariopsis, is a saprophytic fungus normally found in soil, food, vegetables, and even indoors. Considered an environmental pollutant, it is characterized by its intrinsic resistance to available antifungals. There are few references to infections by Microascus gracilis, associating it as a cause of eumycetoma or disseminated disease in lung transplant recipients. We present a case of otomycosis caused by M. gracilis, to consider the search for fungi in ear cultures and highlight the pathogenic and colonizing power of this agent.

An overview of mycetoma and its diagnostic dilemma: Time to move on to advanced techniques

The neglected tropical disease mycetoma can become extremely devastating, and can be caused both by fungi and bacteria; these are popularly known as eumycetoma and actinomycetoma respectively. The classical triad of the disease is subcutaneous swelling, multiple discharging sinuses and the presence of macroscopic granules. The present study aims to highlight the existing diagnostic modalities and the need to incorporate newer and more advanced laboratory techniques like pan fungal/pan bacterial 16S rRNA gene polymerase chain reaction (PCR) and sequencing, Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA). It is important for the medical team to be aware of the various diagnostic options (both existing and future), so that diagnosis of such a debilitating disease is never missed, both by clinicians and microbiologists/pathologists. The newer diagnostic methods discussed in this article will help in rapid, accurate diagnosis thus facilitating early treatment initiation, and decreasing the overall morbidity of the disease. In the Indian context, newer technologies need to be made available more widely. Making clinicians aware and promoting research and development in mycetoma diagnostics is the need of the hour.

A STUDY ON PATTERN AND CLINICAL PROFILES OF CONGENITAL HEART DISEASES IN TERTIARY CARE CENTRE, KAKINADA.

BACKGROUND: Congenital heart disease is an abnormality of anatomical structure of the heart that is of paramount importance. The incidence is approximately 8 per 1000 live births. CHD not only contributes to signi?cant mortality and morbidity but also causes tremendous psychological stress and economical burden to whole family. OBJECTIVE: To study the pattern and clinical pro?le of congenital heart diseases in a tertiary care hospital and to aid for early stdetection and quick referral of children with congenital heart disease In this study, done between January 1 2020 to RESULTS: june 31st 2021, 54 children with con?rmed congenital heart disease were observed. ACHD was the most common type of CHD and of them, VSD was the most common type, followed by ASD. Based on sex distribution, 31 were male (55.5%) and 23 were female (44.5%), with a male: female ratio of 1.25:1. Most children belonged in the age group 6m – 1year (46.2%) followed by (42.5%) in 30days – 6months age. The common clinical presentation observed in this study was breathlessness. The common complication observed in the current study was heart failure followed by malnourishment and recurrent LRTI.

Acid-fast bacteria as causative agents of skin and soft tissue infections: case presentations and literature review

ABSTRACT Acid-fast bacteria can be implicated in skin and soft tissue infections. Diagnostic identification can be challenging or not feasible by routine laboratory techniques, especially if there is no access to the Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) technology. Here, we present two cases of skin and soft tissue infections caused by two different acid-fast bacteria, Nocardia brasiliensis and Mycobacterium marinum. They both grew on Löwenstein-Jensen medium, Sabouraud agar medium and blood agar medium. Both bacteria appeared acid-fast by Ziehl-Neelsen stain and Gram-positive by Gram stain. The identification was performed by MALDI-TOF MS and gene analysis. N. brasiliensis and nontuberculous mycobacterium M. marinum represent rare pathogens that cause severe skin and soft tissue infections. Failure to identify the causative agent and subsequent inappropriate or inadequate treatment may lead to severe complications or even disseminated disease, especially in immunocompromised individuals.