Comparison of Epsilometer test and agar dilution method in detecting the sensitivity of Helicobacter pylori to metronidazole / 北京大学学报(医学版)

OBJECTIVE@#Agar dilution method (ADM) was used as the golden standard to evaluate the consistency of Epsilometer test (E-test) in detecting the sensitivity of Helicobacter pylori (H. pylori) to metronidazole.@*METHODS@#From August 2018 to July 2020, patients with H. pylori infection treated for the first time in Peking University Third Hospital for gastroscopy due to dyspepsia were included in this study. Gastric mucosas were taken from the patients with H. pylori infection. H. pylori culture was performed. Both the ADM and E-test were applied to the antibiotic susceptibility of H. pylori to metro-nidazole, and the consistency and correlation between the two methods were validated.@*RESULTS@#In the study, 105 clinical isolates of H. pylori were successfully cultured, and the minimum inhibitory concentration ≥ 8 mg/L was defined as drug resistance. Both ADM and the E-test showed high resistance rates to metronidazole, 64.8% and 62.9%, respectively. Among them, 66 drug-resistant strains were detected by ADM and E-test, and 37 were sensitive strains, so the consistency rate was 98.1%. Two strains were evaluated as drug resistance by ADM, but sensitive by the E-test, with a very major error rate of 1.9%. There was zero strain sensitive according to ADM but assessed as resistant by the E-test, so the major error rate was 0%. Taking ADM as the gold standard, the sensitivity of E-test in the detection of metronidazole susceptibility was 97.1% (95%CI: 0.888-0.995), and the specificity was 100% (95%CI: 0.883-1.000). Cohen's kappa analysis showed substantial agreement, and kappa coefficient was 0.959 (95%CI: 0.902-1.016, P < 0.001). Spearmans correlation analysis confirmed this correlation was significant (r=0.807, P < 0.001). The consistency evaluation of Bland-Altman method indicated that it was good, and there was no measured value outside the consistency interval. In this study, cost analysis, including materials and labor, showed a 32.2% higher cost per analyte for ADM as compared with the E-test (356.6 yuan vs. 269.8 yuan).@*CONCLUSION@#The susceptibility test of H. pylori to metronidazole by E-test presents better agreement with ADM. Because it is less expensive, less labor intensive, and more rapid, it is an easy and reliable method for H. pylori susceptibility testing.

Evaluation of the in vitrofungicidal activity of the dynamized essential oil of Aloysia polystachyabefore and after freezing

The method of preserving substances of natural origin should not only maintain the microbiological safety of the product but also the integrity of its therapeutic potential. Essential oils obtained from plants are complex mixtures of substances and it issuggested to keep them under refrigeration for better conservation. On the other hand, homeopathic mother tincture prepared from plant is kept at room temperature. Aim: This work aimed to evaluate if the freezing process changes the in vitro antifungal activity potential of the homeopathic preparation Aloysia polystachya1CH against Candida albicans. Methodology:The inoculum of C. albicansATCC 10231 was cultivated in culture médium Sabouroud (Himedia®), standardized on a spectrometer and distributed in a 96-well plate. Then, A. polystachya1CH was added to the wells, prepared accordingtothe Brazilian Homeopathic Pharmacopoeia (FHB, 3rd edition) from A. polystachya essencial oil. An aliquot of this homeopathic preparation was frozen and after 40 days it was submitted to the same methodology for evaluation of the antifungal activity. After incubation, the plates were read with triphenyltetrazolic (TTC) (Vetec®). Results and discussion: The results of the in vitroevaluation showed that the freezing process retained the antifungal activity of the dynamized essential oil of A. polystachya1CH against C. albicans. Conclusion: Under the conditions evaluated in this study, the freezing method presented as a viable method of conservation of dynamized plant material.

Veterinary Biotypology: a review and new possibilities

The truths surrounding medical practices are seasonally challenged by innovative concepts that can aggregate changing procedures in many degrees. The Galtonian eugenics issues supported the pure-breed idea in dictatorial governments, and introduced mesological studies, turning possible to join genetic concepts to the physiology and psychology of the human organism. Following human medicine, more therapeutic models need to forthcoming in domestic animals. The companionship necessity and the highly responsive behavior have addressed the domestication of dogs and their relationship to owners, to an endpoint that both share the same pathologies. Thus, traditional human concepts of biotypology could be extended to companion animals. Grauvogl (1811-1877) proposeda simple biochemical correlation between physiological states and the miasmas of sick individuals (oxygenoid -syphilis, hydrogenoid -sycosis, carbo-nitrogenoid -psora). Antoine Nebel (1870-1954) correlated biochemical status with the musculoskeletal system and behavior as well. Leon Vannier (1880-1963) model, whose morphophysiological distortions and behavioral inconsistencies were explained by the carbon element and variations in its bonds with phosphorus or fluor radicals was another attempt to categorize and predefine physiology states. Following the advent of structural and functional identification of thyroid hormone in the 1940s, Henri Bernard described the neuro-morphofunctional plasticity of individuals guided by their predominant embryonic leaflet and consequent hormonal diseases. Methods:This work is a narrative review with the purpose of describing and discussingthe legacy ofbiotypology studies and their applicability in dog therapy, and proposinga new homeopathic approach in veterinary medicine based on the miasmas, also contributing to the scarcelyavailable literature. Results:Based on cellular exchanges and consequent metabolic rate, animals can be classified into psoric (no evidence of clinical signs, stable behavior, and adequate exonerative cellular processes); sycotic (cellular dysfunction with alterations in oxidative phosphorylation processes allowing accumulation of cellular toxins such as reactive oxygen and nitrogen species; clinically culminating in chronic inflammations in noble organs, and purulent discharges; unstable and polarized behavior) and syphilitic (whose cellular alterations have reached the molecular level, reducing protein expression and determining cellular toxicity and loss of function; indifferentbehavior). Generalities such as temperature influence, weight, thirst and feedingshall also be considered. Discussion and Conclusion: Thismodel could benefit stray animals, newly adopted or even from shelters, whose actual behavior is unknown, and the search for the Simillimum may be impaired.

High Dilutions of Drugs show Distinct Variation from each other in their Electronic Spectra

Drugs at high dilution (HD) produce therapeutic effect on man, animals and plants. Experimental evidence shows that free water molecules and hydrogen bond strength of OH groups constitute the physical basis of HDs which are otherwise devoid of original drug molecules. HDs are produced in aqueous EtOH by serial dilution of a substance with mechanical agitation or succussion in each step, and are called potencies. Three potencies 6 cH, 12 cH and 30 cH of two drugs Anacardium orientale and Natrum muriaticum(NaCl) and their mother tincture (MT) are used in this study. Electronic spectra of these MTs and potencies, all in 90% EtOH, were taken in the wavelength region of 190 nm ­350 nm. The objective is to find out any additional physical-chemical entities in potencies besides the aforesaid two factors. It was reported earlier that charge transfer (CT) interaction accompanies potentization of drugs. This study focused on the CT interaction. The results indicate that spectral pattern and absorbance intensities of the test samples vary from each other. Natm 6cH (absorbance 0.30 at 196.53nm), 12cH (abs. 0.06 at 196.53nm) and 30cH (abs. 1.32 at 196.5nm). Anac 6cH (abs. 0.33 at 203nm), 12cH (abs. 0.61 at 208nm) and 30cH (abs. 0.09 at 200.67nm). The spectrum of each potency shows two peaks. The 2nd peak at higher wave length belongs to CT interaction. Anac 6cH suc, 7cH unsuc. Insersections at 197.14nm with abs. 0.05, and 290nm with abs. 0.01. Anac 12cH suc, 13cH unsuc. Intersections at 196.93nm with abs. 0.06, and 273nm with abs. 0.00. Anac 30cH suc, 31cH unsuc. Intersections at 194.42nm with abs. -0.05, 238.03nm with abs. -0.01, 252.15nm with abs. -0.002, and 261nm with abs. 0.004. Natm 6cH suc, 7cH unsuc. Intersection at 199.44nm with Abs -0.11. Natm 12cH suc, 13cH unsuc. Instersection at 200.48nm with abs. -0.11. Natm 30cH suc, 31cH unsuc. Intersection at 204.24nm with abs. -0.08. Potentization involves CT interaction in consecutive potencies. Water and EtOH do not form a homogeneous mixture and have aggregates of EtOHand water molecules. CT interactions occur in these individual aggregates and are mostly inter molecular within EtOH or water. These aggregates vary from each other in the test samples. The spectra of test samples were analysed for margin of error (MOE). The MOE is very small (0.001-0.002%), and for this reason the difference between the spectra is significant. Besides that the intersection between consecutive spectra vary in number and position. It is concluded that water and EtOH aggregates and their relative distribution constitute additional physical-chemical basis of potencies.

Assessment of colistin resistance in Gram negative bacteria from clinical samples in resource-limited settings

Objective: To find a proper method to assess colistin resistance in multidrug resistant Gram negative bacteria (MDR-GNB) on a routine basis in resource limited settings. Methods: Clinical samples were processed. MDR-GNB were identified and were examined for colistin resistance by colistin broth elution method, colistin agar method, and colistin disk elution screening method. Broth microdilution method was used the gold standard. Results: A total of 10 235 clinical samples were processed, in which 857 (8.4%) MDR-GNB were identified. The very significant errors, categorical agreement, major errors, positive predictive values, negative predictive values, specificity and sensitivity of all the phenotypic methods were 5.5%, 0%, 94.4%, 100%, 99.6%, 100% and 94.4%, respectively for the detection of colistin resistance. The colistin elution screening method was cheap and easy to perform with similar results to broth microdilution method. Conclusions: All the evaluation methods for colistin resistance showed similar results. So the laboratories can choose any method for detection of colistin resistance. However, we recommend colistin disk elution screening method because, it is easy and cheap and can be performed in limited resources.

Performance evaluation of serum progesterone measurement by ID-LC/MS/MS candidate reference methods and their clinical application value / 中华检验医学杂志

Objective:To establish a candidate reference method for serum progesterone using isotope dilution liquid chromatography tandem mass spectrometry (ID-LC/MS/MS) in our laboratory, validate the analytic performance of five clinical routine detection systems to explore the comparability of serum progesterone detection by different detection systems.Methods:A candidate reference method for serum progesterone using ID-LC/MS/MS method was established. The sample was pretreated by liquid-liquid extraction method, and the reversed phase liquid phase separation in positive ion mass spectrometry mode was used to detect progesterone in human serum, and the detection time of a single sample was controlled within 5 minutes by gradient elution. In order to improve the accuracy of the method, the bracketing calibration method (BCM) was used to establish the standard curve. The sensitivity, accuracy, precision and specificity of BCM and classical calibration curve method were evaluated according to CLSI C62-A, EP15-A2, EP6-A2 and EP9-A3, and the analytical performance and comparability of five clinical routine progesterone detection systems were evaluated,compared with ID-LC/MS/MS method, the bias at medical decision level 2 and 25 ng/ml was evaluated to see if they were <1/2TEa (12.5%).Results:The limit of detection (LOD) of ID-LC/MS/MS was 0.005 ng/ml. The recoveries of BCM method and classical calibration curve method are 97.95%-101.58% and 96.88%-110.70%, respectively. The measurement results of BCM method for certified reference materials are within its declared uncertainty range. The intra-and inter-assay coefficient of variation ( CV) of BCM method was less than 3.0%, which was better than that of classical calibration curve method ( CV: 2.48%-9.33%). The precision and linear range of the five clinical routine detection systems can meet the detection requirements. The measurement bias of detection system 1, 3 and 5 at 25 ng/ml of medical decision level was less than 1/2TEa, and the measurement bias at 2 ng/ml of medical decision level was more than 1/2TEa. The measurement bias of detection system 2 and 4 at two medical decision levels was less than 1/2TEa. Conclusion:The candidate reference method for serum progesterone ID-LC/MS/MS established in our laboratory meets the requirements of the reference method. BCM has better detection performance than classical calibration curve method. The precision and linearity of the five progesterone clinical detection systems are satisfactory. The five clinical detection systems could meet the clinical requirements at the medical determination level of 25 ng/ml, however, only two of the five clinical detection systems meet the clinical requirements at the medical determination level of 2 ng/ml.

Analytical performance of a candidate reference measurement procedure for serum 17α-hydroxyprogesterone based on liquid chromatography tandem mass spectrometry / 中华检验医学杂志

Objective:To determine the analytical performance of a candidate reference measurement procedure for 17α-hydroxyprogesterone in human serum by liquid chromatography-tandem mass spectrometry (LC-MS/MS).Methods:The serum spiked with a deuterium-labeled internal standard was extracted from serum from individual undergoing physical examination by liquid-liquid extraction with n-hexane/ethyl acetate (3∶2, v/v), separated by C18 reversed-phase chromatography and detected by positive electrospray ionization mass spectrometry. According to the Clinical and Laboratory Standards Institute (CLSI) C62-A documents, the analytical performance including linearity, limit of detection,limit of quantitation,relative matrix effect,precision and trueness,carry-over and specificity was evaluated.Results:The linear range of 17α-hydroxyprogesterone by LC-MS/MS was 0.21-119.67 μg/L. The limit of detection and limit of quantitation were 5.218 ng/L and 0.116 μg/L. The relative matrix effects were -0.02%, -0.40% and -0.90% for sera and solution mixtures in 3 different ratios (50∶50, 80:20 and 20∶80). The coefficients of variation ( CVs) of intra-assay were 1.73%-2.11%, 0.98%-1.71%, 0.47%-0.87% at 0.164 μg/L, 14.81 μg/L, 81.63 μg/L and the CVs of inter-assay were 1.82%, 1.03%, 0.80% at above three concentrations. The average recovery rates of 3 levels (0.5, 20 and 100 μg/L) were 100.4%, 101.7%, 102.8%, respectively. The measured values of GBW09829 of National Institute of Metrology were within the specified uncertainty range. Conclusion:The candidate reference measurement procedure for 17α-hydroxyprogesterone in human by LC-MS/MS is established with good accuracy and precision, which can be clinically used for measurement traceability.

High dilutions of a drug of COVID-19 origin show difference in ranks

High dilutions (HDs) of drugs, used in Homeopathy, are prepared in aqueous EtOH (ethanol) through serial dilution accompanying mechanical agitation or succussion, and are called potencies. The potencies from the rank 12 onwards are too dilute to contain any original drug molecules. Do the potency ranks show any difference from each other? Do serial dilution and succussion contribute to the difference in potency ranks? This study aims to address these two questions. The throat swab of a Covid-19 patient was preserved and diluted with aqueous EtOH 90% to prepare the mother tincture (MT) and five different potencies of Covid named Covidinum. These potencies and their solvent media were analysed by electronic and vibrational spectroscopy. Charge transfer (CT) and proton transfer interactions occur during preparation of the potencies. The FT-IR spectra of all the test samples after normalization show difference from each other with respect to O-H stretching and bending (v2) bands. Serial dilution and succussion contribute to the observed difference in ranks and CT interactions.

Antimicrobianos en neonatología. Parte II: Recomendaciones para la dilución y administración de antimicrobianos utilizados en el recién nacido / Antimicrobials in neonatology. Part II: Recommendations for the dilution and administration of antimicrobials in the newborn

Resumen Los antimicrobianos corresponden al grupo de medicamentos más utilizados en Unidades de Cuidados Intensivos Neonatales; no obstante, su uso ha sido asociado a constantes errores de medicación en la práctica clínica. Paradojalmente, aún no existe consenso en torno a la administración adecuada de estos medicamentos y existen importantes brechas de conocimiento en torno a los procesos de dosificación, administración y manipulación de antimicrobianos en esta población. Con el fin de mejorar el uso de antimicrobianos, disminuir errores y optimizar los resultados clínicos en el recién nacido, la presente revisión tiene por objetivo entregar recomendaciones y servir de guía para la correcta preparación de aquellos antimicrobianos de mayor relevancia en neonatología.

Abstract Antimicrobials are among the most commonly prescribed classes of medications in Neonatal Intensive Care Units; however, its use has been constantly associated with a number of medication errors in clinical practice. In contrast to this situation, there is no common agreement when it comes to determining the right dosing, administration, or handling of antibiotics in this population. In order to help improve the use of antibiotics, decrease the rate of medication errors and optimize clinical results in the newborn, this review aims to provide recommendations to support and guide the correct preparation of some of the most relevant antibiotics used in neonatal wards.

Evaluation of rapid identification methods for foodborne diarrhea Campylobacter and detection of drug resistance / 中华检验医学杂志

Objective:To explore and evaluate a appropriate suitable method for detection of Campylobacter and antibiotic sensitivity test for foodborne diarrhea in clinical laboratories. Methods:Pre-experiment:a total number of 400 fecal samples of patients with foodborne diarrhea were prospectively collected from the intestinal disease clinic of Beijing Tongren Hospital from September 2017 to January 2018. Double-hole filtration culture method and modified cefoperazone charcoal deoxycholate (CCD) agar culture method were used for fecal culture in micro-aerobic environment for 48 hours, and then suspicious colonies were identified by matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Meanwhile, C. jejuni and C. coli were detected by real-time quantitative polymerase chain reaction(qPCR). Large sample verification: 2 062 fecal samples of patients with foodborne diarrhea in three hospitals of different levels in different areas of Beijing were collected for qPCR detection and culture from April 2018 to March 2019. The antimicrobial sensitivity test (AST) of C. jejuni and C. coli was performed according to the disk diffusion method and agar dilution method recommended by Clinical and Laboratory Standards Institute and National Antimicrobial Resistance Monitoring System for Enteric Bacteria. The results of the three detection methods and the consistency of the two antibiotic sensitivity tests were compared. Results:In the pre-experiment, the positive rates of Campylobacter ( jejuni/coli) detected of qPCR, double-hole filtration culture and modified CCD agar culture were 9.0% (36/400), 5.0% (20/400)and 3.5% (14/400), and the difference was statistically significant ( P<0.01). The samples with negative result of qPCR were negative by both culture methods. The total positive rates of Campylobacter detected by qPCR was 8.1% (168/ 2 062)including 7.0% (144/2 062) for C. jejuni and 1.2% (24/2 062) for C. coli. The samples with positive qPCR results were cultured by double-hole filtration culture method and the positive rate was 61.9%(104/168), among which, the positive rate of C. jejuni and C. coli were 58.3%(84/144) and 83.3%(20/24) respectively, which was not significantly different from the detection rate and culture positive rate in the pre-test ( P>0.1). The resistance rates of C. jejuni and C. coli to ciprofloxacin were 94.0%(94/100) and 100.0%(24/24) and to erythromycin were 6.0%(6/100) and 33.3%(8/24). The results from two antibiotic sensitivity test methods were consistent (Kappa>0.75). Conclusions:qPCR is rapid, sensitive and easy to operate, so it is suitable for routine development in clinical laboratories. The double-hole filtration culture method is beneficial to the acquisition of strains and is essential for the further study of Campylobacter. There was no significant difference between agar dilution method and disk diffusion method in antibiotic sensitivity test. Campylobacter showed a very high resistance rate to quinolones, which was no longer suitable for the treatment of Campylobacter foodborne diarrhea in Beijing area. Macrocyclic lipid antibiotics should be preferred.

Establishment of a candidate reference method for plasma normetanephrine and its application in the external quality assessment program / 中华检验医学杂志

Objectives:To establish a candidate reference method of isotope dilution liquid chromatography tandem mass spectrometry (ID-LC/MS/MS) for the determination of human plasma normetanephrine, and to evaluate the performance of the method. The method was used to quantify the samples of the external quality assessment program, and to initially evaluate the detection status of plasma normetanephrine.Methods:The isotope standard solution of normetanephrine was selected as the internal standard, the gravimetric method was used for sampling, and the standard curve method was used for quantification. Protein precipitation combined with weak cation solid phase extraction was used for pretreatment, and ultra-high liquid chromatography-coupled triple quadrupole mass spectrometry was used for LC/MS analysis. According to the relevant EP documents, the specificity, matrix effect, detection limit, quantification limit, precision, accuracy, and uncertainty of the method were estimated. This method is used to quantify the samples of the 2020 National Center for Clinical Laboratories external quality assessment program of normetanephrine. Taking the average value of this method as the target value, the optimal allowable total error standard of biological variation as the evaluation limit, the quality of the laboratory testing was evaluated.Results:The method had good specificity, and the interferences and matrix effects did not affect the detection results. The detection limit and quantification limit of plasma normetanephrine were 1.08 pg/g and 3.54 pg/g, respectively. The intra-batch coefficient of variation ( CV) and total CV were 0.43%-1.10% and 0.61%-1.42%, respectively. The relative recovery rates were 98.5%～101.9%. The relative expansion uncertainty of the four plasma samples were 3.10%, 2.34%, 2.16%, and 1.73%, respectively. The results of the external quality assessment program showed that the pass rates of the 202013 and 202014 samples were 80% and 85%, respectively. Conclusions:The study established a candidate reference method of ID-LC/MS/MS for the measurement of plasma normetanephrine. The method is accurate, precise and simple, and is expected to be used as a reference method for the determination of plasma normetanephrine, and can be applied to quantify the samples of the external quality assessment program.

Comparison of well diffusion, disc diffusion and broth dilution methods for antimicrobial activity of Andrographis paniculata herbal gel against acne-associated pathogen

Aims@#Acne is a common skin disease among teenagers and also affects other ages. It occurs when the oil and dead skin cells plug into the hair follicles and causing pimples or whitehead. Although antibiotics have been used for many years in treating acne, the widespread use of it has led to the development of bacterial resistant, which resulted in unsuccessful treatment. Thus, in this study, Andrographis paniculata (AP) herbal formulation gel is proposed in order to determine its effectiveness in treating acne. Three different methodologies were used to compare the antimicrobial effect of A. paniculata herbal gel against acne-associated pathogens. @*Methodology and results@#Well diffusion, disc diffusion and broth dilution methods were applied to evaluate the antimicrobial effect of AP herbal gel at concentrations of 1.5% (w/w), 2.5% (w/w) and 5.0% (w/w) onto selected pathogens associated with acne which consisted of Staphylococcus epidermidis, Staphylococcus aureus, Propionibacterium acnes and Candida albicans. Among the three methods, broth dilution showed the best antimicrobial effect towards all microorganisms used. AP herbal gel at concentration 2.5% (w/w) showed the optimum antimicrobial effect of S. aureus and C. albicans, while 5.0% (w/w) exhibited the best antimicrobial activities for P. acnes and S. epidermidis. @*Conclusion, significance and impact of study@#Broth dilution method appears to be a reliable method for the determination of antimicrobial effects for the pathogens tested. In addition, AP herbal formulation gel has great potential to treat acne effectively.

New Derivatives of (E)-3-(5-((substitutedphenylamino)methyl)-1,3,4-thiadiazol-2-yl)-2-styryl quinazolin-4(3H)-one: Searching for New Antifungal and Antibacterial Agents

Objective: The objective of the paper was to evaluate the antifungal and antibacterial potential of new derivatives of ((E)-3-(5-((substitutedphenylamino)methyl)-1,3,4-thiadiazol-2-yl)-2-styryl quinazolin-4(3H)-one. Materials and Methods: Various syntheses of (E)-3-(5-(substitutedaminomethyl)-1,3,4-thiadiazol-2-yl)-2-styrylquinazolin-4(3H)-one derivatives have been synthesized by reacting 2-substituted benzoxazin-4-one with (E)-2-(4-Substituedstyryl)-4H-benzo[d] [1,3]oxazin-4-one. All synthesized compounds have been characterized by the infrared, 1HNMR, and mass spectral analysis. Proposed compounds have been evaluated for antifungal and antibacterial activity. The antimicrobial activity of synthesized compounds (QNM-1 to QNM-15) has been carried through the serial dilution method. For bacterial screening, bacterial species were taken includes Staphylococcus aureus (MTCC-96), Bacillus subtilis (MTCC-441), Pseudomonas aeruginosa (MTCC-424), and Escherichia coli (MTCC-40). Norfloxacin (1-Ethyl-6-fluoro-1,4,dihydro-4-oxo-7-(1-piperazinyl)-3-quinoline carboxylic acid) was used as the standard drug for antibacterial study. For antifungal screening, the following fungal species were used includes Aspergillus niger (MTCC-281), Candida albicans (MTCC-227), and Fusarium oxysporum (MTCC-284). Clotrimazole was selected as a standard drug for antifungal study. Results and Discussion: QNM-1, QNM-2, QNM-3, QNM-5, QNM-7, QNM-9, QNM-12, QNM-14, and QNM-15 were the most active compounds [Table 1] in the synthesized series which were active against both Gram-positive and Gram-negative organisms by the antibacterial screening. In the case of antibacterial activity, the presence of electronegative group (Cl, Br, F, and NO2) at both R may enhance the activity when they are p-substituted, but the compounds QNM-6 (R1=-C6H5Br (o); Ar=-C6H5), QNM-10 (R1 = -C6H5F (o); Ar= -C6H5F), QNM-11 (R1 =-C6H5NO2 (p); Ar=-C6H5F), and QNM-4 (R1 =-C6H5F (m); Ar=-C6H5) with given substitution may result in diminishing the activity. In case of antifungal activity, compounds QNM-1, QNM-5, QNM-7, QNM-9, QNM-11, QNM-12, QNM-14, and QNM-15 were the most active compounds in the synthesized series which were active against both Gram-positive and Gram-negative organisms. In that series, compounds QNM-14, QNM-11, QNM-5, and QNM-7 have shown the highest activity. Compounds QNM-3, QNM-6, QNM-10, and QNM-13 have the least active. This result has also concluded that o-substituted compounds, i.e., -C6H5Cl(o), -C6H5Cl (m), -C6H5Br(o), -C6H5F (o), -C6H5F (p) at R1 position my resulted in diminishing or lower the activity

Simultaneous determination of 15 mycotoxins in peanuts by ultra high performance liquid chromatography-tandem mass spectrometry with QuEChERS EMR-Lipid approach and stable isotope dilution / 上海预防医学

Objective To develop a method for the simultaneous determination of 15mycotoxins in peanuts by ultra high performance liquid chromatography-tandem mass spectrometry with QuEChERS EMR-Lipid approach and stable isotope dilution. Methods The samples were extracted by 2% formic acid acetonitrile-water (50 : 50, V/V) and then purified with QuEChERS EMR-Lipid approach.The mycotoxins were fully separated on a pentafluorophenyl column under a gradient elution with methonal-0.01%formic acid aqueous solution.The mycotoxins were analyzed by UPLC-MS/MS with multiple reaction monitoring (MRM) mode and quantified by isotope internal standard method. Results Fifteen mycotoxins had good linear relationship in the certain correlation ranges with the correlation coefficients all above 0.995 and the detection limits were 0.1－10 μg/kg.The mean recoveries ranged from 81.2% to 115.3% with RSD (n=6) varying from 2.1% to 10.7%. Conclusion The method is simple, highly sensitive, practical, and proves to be suitable for quantitative analysis of 15 mycotoxins in peanuts.

Leishmania tropica: The comparison of two frequently-used methods of parasite load assay in vaccinated mice

Objective: To compare limiting dilution assay and real-time PCR methods in Leishmania tropica parasite load measurement in vaccinated mice. Methods: BALB/c mice were vaccinated by Leishmania tropica soluble Leishmania antigen or recombinant Leishmania tropica stress-inducibleprotein-1 with/without adjuvant. After three vaccinations, mice were challenged by Leishmania tropica promastigotes. Two months after challenge, the draining lymph nodes of mice footpad were removed and parasite load was assayed by limiting dilution assay and real-time PCR methods. Limiting dilution assay was done by diluting tissue samples to extinction in a biphasic medium. For real-time PCR, DNA of the lymph nodes was extracted, equal dilutions of each sample were prepared and hot-start real-time PCR was done using appropriate primers. The data of the two methods were compared by appropriate statistical methods. Results: Both methods were able to measure different levels of parasite load in vaccinated/unvaccinated mice. In addition, wherever parasite load of a group was estimated high (or low) by one method, the estimated parasite load by another method was the same, although statistically significant differences were found between some groups. Conclusions: Both methods lead to approximately similar results in terms of differentiating parasite load of the experimental groups. However, due to the lower errors and faster process, the real-time PCR method is preferred.

Japanese encephalitis virus: Biological clones from a clinical isolate quasispecies show differing neurovirulence in vitro and in a mouse model

@#The Japanese encephalitis virus (JEV), a leading cause of encephalitis, exists as quasispecies in clinical isolates. Using a limiting dilution method combined with immunohistochemistry to detect viral antigens, 10 biological clones were isolated and purified from a clinical JEV isolate (CNS138/9) derived from an autopsy brain. These biological clones were tested for neurovirulence in SK-N-MC and NIE-115 neuronal cells, and a 2-week-old, footpad-infected, JE mouse model. Nine clones were found to be neurovirulent; one clone neuroattenuated. Although further studies are needed to determine genotypic differences, if any, in these clones, the limiting dilution purification and neurovirulence testing methods described herein should be useful for phenotypic studies of quasispecies of neurotropic viruses in general, and JEV and other flaviviruses in particular.

Leishmania tropica: The comparison of two frequently-used methods of parasite load assay in vaccinated mice

Objective: To compare limiting dilution assay and real-time PCR methods in Leishmania tropica parasite load measurement in vaccinated mice.Methods: BALB/c mice were vaccinated by Leishmania tropica soluble Leishmania antigen or recombinant Leishmania tropica stress-inducible protein-1 with/without adjuvant. After three vaccinations, mice were challenged by Leishmania tropica promastigotes. Two months after challenge, the draining lymph nodes of mice footpad were removed and parasite load was assayed by limiting dilution assay and real-time PCR methods. Limiting dilution assay was done by diluting tissue samples to extinction in a biphasic medium. For real-time PCR, DNA of the lymph nodes was extracted, equal dilutions of each sample were prepared and hot-start real-time PCR was done using appropriate primers. The data of the two methods were compared by appropriate statistical methods. Results: Both methods were able to measure different levels of parasite load in vaccinated/unvaccinated mice. In addition, wherever parasite load of a group was estimated high (or low) by one method, the estimated parasite load by another method was the same, although statistically significant differences were found between some groups. Conclusions: Both methods lead to approximately similar results in terms of differentiating parasite load of the experimental groups. However, due to the lower errors and faster process, the real-time PCR method is preferred.

Optimized Tube Dilution Technique and Sole Carbon Utilization Assay for Anti-leptospiral In Vitro Screening of Plant Extracts

Introduction@#Leptospirosis is one of the neglected reemerging zoonoses that is of public health concern globally. The need to discover novel therapeutic alternatives for leptospirosis through screening for and elucidating the mechanism/s of the anti-leptospiral activity of plant extracts is therefore necessary. This study analyzes the optimized tube dilution technique and the BiologTM sole carbon utilization phenotype microarray as screening tool for anti-leptospiral activity of plant extracts. @*Methods@#The suitability of the optimized tube dilution technique was evaluated by determining the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and motility inhibition property of a plant extract and an antimicrobial control (pen G) against 4 dominantly circulating Leptospira serovars/serogroup in the Philippines. Likewise, the suitability of the BiologTM sole carbon utilization assay was evaluated using a plant extract and selected antimicrobials against L. interrogans serovar Manilae strain K64 and L. interrogans serovar Losbanos strain K37. @*Results@#The MIC, MBC, and motility inhibition property of a plant extract and the antibiotic controls as well as its effect on the carbon utilization phenome of the Leptospira serovars gave consistent results, within and between several runs. With standard deviation = 0 for all serovars. The MIC and MBC of the antimicrobial control (pen G), the positive control, was 10 ug/ml. The growth control (leptospires without treatment), the negative control, showed presence of motile leptospires. The MIC and the MBC of the test plant extract was 250 ug/ml – 500 ug/ml. Results of the carbon utilization phenome or pattern of carbon utilization were consistent within the 3 replicates and between two runs. @*Conclusion@#The optimized tube dilution technique and the BiologTM sole carbon utilization assay is a potential in vitro screening tool for determining anti-leptospiral activity of plant extracts.

False-negative result in molecular diagnosis of SARS-CoV-2 in samples with amplification inhibitors / Resultado falso negativo no diagnóstico molecular de SARS-CoV-2 em amostras com inibidores de amplificação

ABSTRACT Introduction: Although reverse transcription-polymerase chain reaction (rRT-PCR) is the gold standard method for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), some factors, such as the presence of amplification inhibitors, lead to false-negative results. Objective: Here we describe the differences between rRT-PCR results for SARS-CoV-2 infection in normal and diluted samples, simulating the need for dilution due to the presence of amplification inhibitors. Material and method: Viral ribonucleic acid (RNA) from samples of nasopharyngeal swabs from 20 patients previously detected as "Negative" and 21 patients detected as "Positive" for SARS-CoV-2 was performed with the EasyExtract DNA-RNA kit (Interprise®). The rRT-PCR was performed with the OneStep/COVID-19 kit (IBMP), with normal and diluted (80 µl of H2O RNAse free) samples, totaling 82 tests. Results: The results indicate that there is an average variation (a < 0.05) delaying the Cq between the results of amplification of the internal control (IC), N gene (NG), and ORF1ab (OF), 1.811 Cq, 3.840 Cq, and 3.842 Cq, respectively. Discussion: The extraction kit does not completely purify the inhibitor compounds; therefore, no amplified product result may occur. In this study, we obtained a 19.04% false-negative diagnosis after sample dilution; this process reduces the efficiency of rRT-PCR to 29.8% in detecting SARS-CoV-2. Conclusion: Knowing the rRT-PCR standards of diluted samples can assist in the identification of false-negative cases and, consequently, avoid incorrect diagnosis.

RESUMEN Introducción: Aunque la reacción en cadena de la polimerasa con transcriptasa reversa en tiempo real (rRT-PCR) sea el método de referencia para detección del coronavirus tipo 2 del síndrome respiratorio agudo grave (Sars-CoV-2), algunos factores como la presencia de inhibidores de amplificación conducen a resultados falsos negativos. Objetivo: Describimos las diferencias entre los resultados de rRT-PCR para infección por Sars-CoV-2 en muestras normales y diluidas, simulando la necesidad de dilución debido a la presencia de inhibidores de amplificación. Material y método: La extracción de ácido ribonucleico (ARN) viral de muestras de hisopos nasofaríngeos de 20 pacientes previamente detectados como "negativos" y 21 pacientes detectados como "positivos" para Sars-CoV-2 se realizó con el kit Easy Extract DNA-RNA (Interprise®). La rRT-PCR se realizó con el kit OneStep/Covid-19 (IBMP), con muestras normales y diluidas (80 µl de H2O libre de ARNasa), totalizando 82 pruebas. Resultados: Los resultados indican que hay una variación media (a < 0,05) retrasando el ciclo de cuantificación (Cq) entre los resultados de amplificación del control interno (CI), gen N (GN) y ORF1ab (OF) de 1,811 Cq, 3,840 Cq y 3,842 Cq. Discusión: El kit de extracción no purifica completamente los compuestos inhibidores; por lo tanto, puede ocurrir no amplificación. Obtuvimos un diagnóstico falso negativo de 19,04% después de la dilución de la muestra; ese proceso reduce la eficiencia de la rRT-PCR hacia 29,8% en la detección de Sars-CoV-2. Conclusión: Conocer los patrones de la rRT-PCR de muestras diluidas puede ayudar en la identificación de casos falsos negativos y, por consiguiente, evitar un diagnóstico equivocado.

RESUMO Introdução: Embora a reação em cadeia da polimerase de transcrição reversa (rRT-PCR) seja o método padrão-ouro para detecção de coronavírus da síndrome respiratória aguda grave 2 (SARS-CoV-2), alguns fatores como a presença de inibidores de amplificação levam a resultados falso negativos. Objetivo: Descrevemos as diferenças entre os resultados de rRT-PCR para infecção por SARS-CoV-2 em amostras normais e diluídas, simulando a necessidade de diluição devido à presença de inibidores de amplificação. Material e método: A extração de ácido ribonucleico (RNA) viral de amostras de suabes nasofaríngeos de 20 pacientes previamente detectados como "negativos" e 21 pacientes detectados como "positivos" para SARS-CoV-2 foi realizada com kit o EasyExtract DNA-RNA (Interprise®). A rRT-PCR foi realizada com o kit OneStep/COVID-19 (IBMP), com amostras normais e diluídas (80 µl de H2O RNAse-free), totalizando 82 testes. Resultados: Os resultados indicam que existe uma variação média (a < 0,05) atrasando o Cq entre os resultados de amplificação do controle interno (CI), gene N (GN) e ORF1ab (OF) de 1,811 Cq, 3,840 Cq e 3,842 Cq, respectivamente. Discussão: O kit de extração não purifica completamente os compostos inibidores, portanto, pode ocorrer não amplificação. Obtivemos um diagnóstico falso negativo de 19,04% após a diluição da amostra; esse processo reduz a eficiência da rRT-PCR para 29,8% na detecção de SARS-CoV-2. Conclusão: Conhecer os padrões da rRT-PCR de amostras diluídas pode auxiliar na identificação de casos falso negativos e, consequentemente, evitar um diagnóstico incorreto.

Corynebacterium pseudotuberculosis biovar ovis: evaluación de la sensibilidad antibiótica in vitro / Corynebacterium pseudotuberculosis biovar ovis: Evaluation of antibiotics susceptibility in vitro

Resumen Los objetivos de este trabajo fueron estudiar la sensibilidad antibiótica de aislamientos de Corynebacterium pseudotuberculosis procedentes de pequeños rumiantes e investigar la presencia de integrones que contienen genes de resistencia. Se estudiaron 15 aislamientos de diferentes fuentes por los métodos de difusión y dilución. Por el método de difusión, amoxicilina-clavulánico, ampicilina, cefotaxima, cefoxitina, ciprofloxacina, cloranfenicol, eritromicina, estreptomicina, gentamicina, imipenem, kanamicina, norfloxacina, penicilina, rifampicina, tetraciclina, trimetroprima-sulfametoxazol y vancomicina fueron activos frente al 100% de los aislamientos, mientras que amicacina presentó resultados variables. En los aislamientos que desarrollaron frente a amicacina se investigó la presencia de integrones de clase 1. El resultado fue negativo, sugiriendo la ausencia del integrón. Utilizando el método de dilución, los antibióticos más activos correspondieron a los grupos de cefalosporinas, gluco-péptidos, macrólidos, quinolonas y tetraciclinas. Se demostró menor actividad de p-lactámicos y aminoglucósidos. No se registró variabilidad en los perfiles antibióticos en los aislamientos procedentes de diferentes fuentes.

Abstract The aims of this work were to study the antibiotic susceptibility in Corynebacterium pseudotuberculosis isolated from small ruminants and to determine the presence of integrons that contain resistance genes. Fifteen isolates of different sources were analysed using the diffusion and the dilution methods. When the diffusion method was performed, amoxicillin-clavulanic, ampicillin, cefotaxime, cefoxitin, ciprofloxacin, chloramphenicol, erythromycin, streptomycin, gentamicin, imipenem, kanamycin, norfloxacin, penicillin, rifampicin, tetracycline, trimethoprim-sulfamethoxazole and vancomycin were effective against the 100% of isolates, while amikacin showed variable results. The isolates that were able to grow with amikacin, were studied in relation to the presence of integron class 1. The result was negative, suggesting the absence of integron. Using dilution method, the antibiotics belonging to the cephalosporin, glycopeptide, macrolide, quinolone, and tetracycline groups were the most active ones for the C. pseudotuberculosis biovar ovis isolates. Less activity of p-lactam and aminoglycosides were observed. There was no observation of variability in the antibiotic patterns in the strains coming from different sources.