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Congenital brucellosis in infants is well described but occurs rarely and very few cases have been reported till now. Acquired brucellosis in young infants has rarely been reported. A 5-week-old infant weighing 3340 gm, presented to the emergency with complaints of fever, abdominal distension, lethargy, and decreased feeding for five days. A clinical diagnosis of septicemia was made. Laboratory parameters were suggestive of septicemia. There was a history of ingestion of unpasteurized buffalo抯 milk. Empirical antibiotics started after withdrawing the blood culture. Blood culture came positive for Brucella anthropi. Ultrasonography of the abdomen showed a small abscess in the left iliac fossa measuring 44� mm. The antibiotics were changed to piperacillin-tazobactam and amikacin as per the blood culture and sensitivity report. Supportive care was given, and the baby gradually improved. The child was discharged on oral rifampicin and trimethoprim-sulfamethoxazole for 4 weeks and is being followed up and is doing well. Young infants may acquire Brucella infection from breastmilk or ingesting unpasteurized animal milk. Prompt diagnosis and treatment is the key to management. Exclusive breastfeeding is the best way to prevent not only animal milk-acquired Brucella but most other types of infection.
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Brucellosis in humans is one of the most common zoonoses. The infection is caused by various species of Brucella bacteria. Brucella infections frequently affect several organ systems with vague clinical symptoms. Humans typically acquire brucellosis through direct contact with infected animals or consumption of contaminated animal products like unpasteurized dairy. It poses significant clinical challenges due to its varied presentation. The disease manifests with a range of nonspecific symptoms including fever, sweating, fatigue, joint and muscle pain, and loss of appetite. Brucellosis can also affect various organs such as the liver, spleen, heart, and nervous system. Diagnosis often involves blood cultures, serological tests, and polymerase chain reaction (PCR) assays to detect the bacteria or antibodies produced by the immune system. This case report presents the clinical course of patient X, a 58-year-old female who succumbed to death following an unusual presentation of brucellosis. Despite initial diagnostic challenges, the identification of Brucella melitensis in her blood marked a critical turning point. The case highlights the importance of considering Brucellosis in patients with persistent fever and multi-organ involvement, emphasizing the need for early recognition and appropriate management.
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Objective To compare the safety and clinical efficacy of lesion removal combined with percutaneous pedicle screw fixation with classical posterior lesion removal in the treatment of lumbar brucelli spondylitis(LBS)by unilateral biportal endoscopic technique with transforaminal lumbar interbody fusion(UBE-LIF)technique.Methods The clinical data of 32 patients with LBS admitted by the Department of Spine and Orthopedics of Gansu Provincial Hospital of Traditional Chinese Medicine from January 2020 to January 2022 were retrospectively analyzed,and the clinical data of the 32 LBS patients were divided into 15 cases in the UBE-LIF group and 17 cases in the posterior group.The general data,surgery-related indexes,and postoperative pathological HE staining of the two groups were recorded and analyzed.The patients'clinical recovery was assessed according to their erythrocyte sedimentation rate(ESR)and C-reactive protein(CRP),low back pain visual analogue score(VAS),Japanese Orthopaedic Association(JOA)score,and Oswestry Dysfunction Index(ODI)preoperative,1 week after surgery,1,3,6 months and 1 year after surgery.Lumbar lordosis angle(LL)and intervertebral space height(DH)were measured by imaging before surgery and at the last follow-up,and intervertebral bone graft fusion was assessed using Suk grading criteria.Results Both groups successfully completed the operation and no serious postoperative complications occurred.There were no significant differences in gender,age,surgical segment,operation time,preoperative ESR and CRP,preoperative VAS,JOA score and ODI index,preoperative LL and DH(P＞0.05).The intraoperative blood loss,postoperative drainage,postoperative getting out of bed,and postoperative hospital stay in UBE-LIF group were significantly lower than those in the posterior group(P＜0.001).Pathological examination of diseased tissues was performed during surgery,all of which was consistent with brucellosis changes.Patients in both groups were followed up for 12-18 months,with an average of 14.8 months.The VAS,JOA score,and ODI index at all postoperative time points in the two groups were significantly improved compared with the preoperative period(P＜0.05).The difference between the two groups was significantly greater than that in the postoperative group:VAS score was lower in UBE-LIF group than in the posterior group(P＜0.01),CRP in both groups was higher than that in the preoperative group,and the elevation level was significantly lower in UBE-LIF group than in the posterior group(P＜0.001).There was no significant difference in ESR between the two groups compared with that before surgery(P＞0.05).There were no significant differences in VAS,JOA score,ODI index,CRP or ESR between the remaining time points after surgery(P＞0.05).At the last follow-up,imaging examination showed that the overall fusion rate of intervertebral bone graft in UBE-LIF group was 93.3％and 94.1％in the posterior group,without significant difference(x2=0.246,P=0.884).LL and DH were significantly improved in both groups compared with preoperative ones(P＜0.01),and the two groups did not significantly differ before and after surgery(P＞0.05).Conclusion Both surgical treatments for LBS are safe effect.Compared with posterior lesion removal bone graft fusion internal fixation,UBE-LIF technology combined with percutaneous pedicle screw internal fixation has the advantages of clear intraoperative vision,less blood loss,faster early postoperative recovery,and shorter postoperative hospital stay,and thus is a feasible surgical method for the minimally invasive treatment of LBS.
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Objective:To analyze the mutation characteristics of rpoB gene in rifampicin-resistant Brucella strains. Methods:DNA of 4 rifampicin-resistant Brucella strains (JSY-26, G-9, WSY-13 and AW-3) isolated from Xinjiang Uygur Autonomous Region was selected, rifampicin rpoB gene was amplified by PCR and its nucleotide sequence was sequenced. The rpoB gene sequences of rifampicin-resistant Brucella standard strain (RB51) and sensitive strain (ALT-8) were used as reference, the mutation sites and types of the rpoB gene inside and outside the rifampicin resistance determination region (RRDR) of the 4 rifampicin-resistant Brucella strains were analyzed by Mega 7.0 software. Results:Through sequence alignment, both JSY-26 and WSY-13 strains underwent a single base point mutation at the RRDR 1 576 bp of the rpoB gene, with the base changing from guanine (G) to adenine (A). The G-9 strain underwent a single base point mutation at the RRDR 1 606 bp of the rpoB gene, with the base changing from cytosine (C) to A. The AW-3 strain showed 5 mutations of 3 types outside rpoB gene RRDR at 2 536, 2 537, 2 626, 2 636 and 2 654 bp, namely 3 insertion mutations [thymine (T) insertion once and C insertion twice], 1 deletion mutation (C deletion), and 1 single base point mutation (from G to C mutation).Conclusion:The RRDR mutations in the rpoB gene of the rifampicin-resistant Brucella strains are mainly characterized by single base point mutations, while multiple insertion and deletion mutations occur outside the RRDR.
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La endocarditis bacteriana secundaria a la infección por Brucella spp., en este caso B. melitensis, como complicación de la brucelosis humana tiene una incidencia baja y, aunque es la presentación clínica con la que se asocia más frecuentemente la mortalidad, no todos los casos son letales, si son tratados oportunamente. Se describe el caso clínico de una endocarditis bacteriana por B. melitensis, diagnosticada en un adulto por el aislamiento del microorganismo en el hemocultivo. Paciente del sexo masculino, de 40 años, con antecedentes de realizar partos en el ganado bovino y consumir leche no pasteurizada. Acudió al médico por presentar durante siete días de evolución de las siguientes manifestaciones clínicas: fiebre, mialgias, artralgias, tos seca y pérdida de peso (15 kg). El hemograma informa: leucopenia, trombocitopenia y anemia; mientras que en un ecocardiograma transesofágico se observó vegetación en la válvula aórtica con una disminución de la función sistólica y en el hemocultivo se aisló B. melitensis. Debido a estos antecedentes, se inició el tratamiento antibacteriano con rifampicina, doxiciclina y gentamicina. El paciente se recuperó y tuvo una evolución clínica satisfactoria. La brucelosis es una enfermedad infrecuente. Debe considerarse en toda persona con fiebre de foco desconocido que resida en zonas endémicas o esté expuesto al cuidado de animales de granja. En esta enfermedad se impone un diagnóstico y tratamiento preciso, por ser una complicación con alta letalidad.
Bacterial endocarditis, secondary to Brucella spp. infection, in this case by B. melitensis, as a complication of human brucellosis has a low incidence. Although it is the clinical presentation most frequently associated with mortality, not all cases are lethal if timely treatment is provided. We describe a clinical case of bacterial endocarditis due to B. melitensis in a 40-year-old male patient with a history of conducting cattle deliveries and consuming unpasteurized milk, diagnosed after isolating the microorganism in blood culture. He presented with the following clinical manifestations after seven days of evolution: fever, myalgias, arthralgias, dry cough and weight loss (15 kg). The hemogram revealed leukopenia, thrombocytopenia, and anemia; while a transesophageal echocardiogram showed vegetation on the aortic valve with decreased systolic function, and B. melitensis was isolated in a blood culture. Considering this medical history, antibacterial treatment was initiated with rifampicin, doxycycline and gentamicin. The patient recovered and had satisfactory clinical evolution. Brucellosis is a rare disease. It should be considered in any person with a fever of unknown origin who lives in endemic areas or is exposed to the care of farm animals. Endocarditis is a highly lethal complication of human brucellosis; therefore, it requires a precise diagnosis and treatment.
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Humans , Male , Adult , Gentamicins/therapeutic use , Brucella melitensis/pathogenicity , Endocarditis, Bacterial/complicationsABSTRACT
Objective:To elucidate the diagnostic utility of clinical features and radiomics characteristics derived from magnetic resonance imaging T2-weighted fat-suppressed images (T2WI-FS) in differentiating brucellosis spondylitis from pyogenic spondylitis.Methods:Clinical records of 26 patients diagnosed with Brucellosis Spondylitis and 23 with Pyogenic Spondylitis were retrospectively reviewed from Xinjiang Medical University First Affiliated Hospital between January 2019 and December 2021. Confirmatory diagnosis was ascertained through histopathological examination and/or microbial culture. Demographic characteristics, symptoms, clinical manifestations, and hematological tests were collected, followed by a univariate analysis to discern clinically significant risk factors. For the radiomics evaluation, preoperative sagittal T2WI-FS images were utilized. Regions of interest (ROIs) were manually outlined by two adept radiologists. Employing the PyRadiomics toolkit, an extensive array of radiomics features encompassing shape, texture, and gray-level attributes were extracted, yielding a total of 1,500 radiomics parameters. Feature normalization and redundancy elimination were implemented to optimize the predictive efficacy of the model. Discriminatory radiomics features were identified through statistical methods like t-tests or rank-sum tests, followed by refinement via least absolute shrinkage and selection operator (LASSO) regression. An integrative logistic regression model incorporated selected clinical risk factors, radiomics attributes, and a composite radiomics score (Rad-Score). The diagnostic performance of three models clinical risk factors alone, Rad-Score alone, and a synergistic combination were appraised using a confusion matrix and receiver operating characteristic (ROC) analysis.Results:The cohort comprised 49 patients, including 36 males and 13 females, with a mean age of 53.79±13.79 years. C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) emerged as significant clinical risk factors ( P<0.005). A total of seven discriminative radiomics features (logarithm glrlm SRLGLE, exponential glcm Imc1, exponential glcm MCC, exponential gldm SDLGLE, square glcm ClusterShade, squareroot glszm SALGLE and wavelet.HHH glrlm Run Variance) were isolated through LASSO regression. Among these selected features, the square glcmClusterShade feature exhibited the best performance, with an area under the curve (AUC) value of 0.780. It demonstrated a sensitivity of 68.8%, specificity of 94.4%, accuracy of 82.4%, precision of 91.7%, and negative predictive value of 0.773. Furthermore, the logarithm glrlm SRLGLE feature had an AUC of 0.736, sensitivity of 68.8%, specificity of 72.2%, accuracy of 76.5%, precision of 72.2%, and negative predictive value of 0.812. The exponential glcm Imc1 feature had an AUC of 0.736, sensitivity of 50.0%, specificity of 94.4%, accuracy of 73.5%, precision of 88.9%, and negative predictive value of 0.680. Three diagnostic models were constructed: the clinical risk factors model, the radiomics score model, and the integrated model (clinical risk factors+radiomics score), which showed AUC values of 0.801, 0.818, and 0.875, respectively. Notably, the integrated model exhibited superior diagnostic efficacy. Conclusion:The amalgamation of clinical and radiomics variables within a sophisticated, integrated model demonstrates promising efficacy in accurately discriminating between Brucellosis Spondylitis and Pyogenic Spondylitis. This cutting-edge methodology underscores its potential in facilitating nuanced clinical decision-making, precise diagnostic differentiation, and the tailoring of therapeutic regimens.
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Objective To investigate the clinical features of Brucella meningitis combined with anti-N-methyl-D-aspartate receptor(NMDAR)encephalitis.Methods The clinical data of one patient diagnosed by the Department of Neurology of Nanjing Second Hospital in May 2022 with brucella meningitis combined with anti-NMDAR encephalitis were reported,and its clinical characteristics were summarized and analyzed through literature search.Results The patient was a 33-year-old man with headache,vomiting,weakness in both lower extremities,and fever 1 week later.Brain MRI showed abnormal signals in the deep white matter of the bilateral frontal lobes,and mild enhancement of the basal ganglia and pia mater.CSF suggested an increase in nucleated cells,mainly monocytes,increased protein,and decreased glucose.CSF culture showed Brucella,Brucella IgG antibody(+),Brucellosis tiger red plate agglutination test(+),test tube agglutination test was 1∶25,and temperature decreased after anti-brucella treatment.Patients presented with unresponsiveness,cognitive decline,and speech impairment in the 22nd day of the course of the disease,CSF pathogenic microorganism metagenomic testing suggested Brucella malta,and CSF autoimmune antibodies suggested moderate positivity for antiglutamate receptor(NMDAR type).Symptoms improved with high-dose shock hormone therapy.Conclusions CSF etiology metagenomic testing can improve the diagnosis rate of Brucella meningitis.Brucella may be used as a predisposing factor for anti-NMDAR encephalitis,when the patient's condition is recurrent,and cognitive decline,speech disorders and other symptoms,it is necessary to be alert to the possibility of promoting anti-NMDAR encephalitis,and relevant antibody detection should be improved in time to start immunotherapy as soon as possible.
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Brucellosis is a zoonotic disease caused by Brucella. Its typical clinical manifestations include fever, chills, fatigue, bone and muscle pain, etc. Brucellosis can affect multiple organs and tissues, of which spine is the most common affected part, forming Brucella spondylitis. Due to the clinical manifestations and imaging characteristics of infected individuals being similar to other spinal diseases, it is easy to cause misdiagnosis, missed diagnosis, and mistreatment. This article reviews the latest research progress in clinical manifestations, imaging examination, diagnosis, differential diagnosis, and treatment of Brucella spondylitis both domestically and internationally, in order to provide reference for the diagnosis and treatment of Brucella spondylitis.
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Objective:To study the clinical characteristics of patients with brucella bloodstream infection in different age groups, and provide a basis for clinicians to take targeted diagnosis and treatment measures. Methods:Demographic data and general condition (age, sex, occupation, location, onset season, source of infection, clinical stage), clinical characteristics (main clinical symptoms and complications), and laboratory test results (routine and pathogenic tests) of adult patients with brucella bloodstream infection admitted to the Affiliated Hospital of Chengde Medical College from January 2015 to January 2020 were collected. According to the age stratification standards recommended by the World Health Organization, the patients were divided into a young group (18 - 44 years old), a middle-aged group (45 - 59 years old), and an elderly group (≥60 years old), and various indicators among different age groups were compared and analyzed. Results:A total of 75 patients were included, including 15 cases (20.00%) in the young group, 37 cases (49.33%) in the middle-aged group, and 23 cases (30.67%) in the elderly group. Among them, 61 cases (81.33%) were males and 14 cases (18.67%) were females, with statistically significant differences in gender ratios among different age groups (χ 2 = 7.28, P = 0.021). The majority of patients were farmers (64 cases, 85.33%), and 92.00% (69/75) of the patients came from rural areas. The main sources of infection were infected cattle and sheep, and contaminated food (39 cases, 52.00%). The main season of onset was spring and summer (45 cases, 60.00%). The clinical staging was mainly in the acute phase (66 cases, 88.00%). In terms of clinical symptoms, the young group of patients had no symptoms of low back pain, while the incidence rates of low back pain in the middle-aged and elderly groups were 35.14% (13/37) and 30.43% (7/23), respectively. There was a statistically significant difference between the three groups (χ 2 = 6.98, P = 0.031). In terms of complications, there were no cases of concurrent spondylitis in the young group of patients. The incidence rates of spondylitis in the middle-aged and elderly groups were 32.43% (12/37) and 34.78% (8/23), respectively. There was a statistically significant difference among the three groups (χ 2 = 6.86, P = 0.032). In terms of routine laboratory examinations, there were statistically significant differences in the proportion of blood lymphocytes and albumin levels among patients of different age groups ( F = 3.41, 3.27, P = 0.038, 0.044). In terms of pathogenic examination, there was a statistically significant difference in the median alarm time for positive blood culture among patients of different age groups ( H = 9.54, P = 0.008), with the middle-aged group having the longest (66.24 h) and the elderly group having the shortest (58.80 h). Conclusions:The clinical characteristics of patients with brucella bloodstream infection vary among different age groups, middle-aged and elderly patients are prone to low back pain symptoms, accompanied by spondylitis. Clinicians should pay attention to the patient's own characteristics and provide targeted diagnosis and treatment.
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Objectives:To evaluate the values of sagittal CT image histogram features in the differential diagnosis of brucella spondylitis(BS)and pyogenic spondylitis(PS).Methods:The data of 40 BS patients[25 males,15 females;age:51.6±13.0 years old;body mass index(BMI):23(20,28)kg/m2,the BS group]and 33 PS patients[13 males,20 females;age:50.8±16.7 years old;BMI:23(20,26)kg/m2,the PS group]who underwent CT examination of the spine in our hospital and were confirmed through pathology and/or etiology were collected.The region of interest(ROI)was delineated on each level of the sagittal CT images of the two groups of patients by using the 3D Slicer platform and grayscale global histogram analysis was performed.The clinical data were compared using chi square test,independent sample t-test,and Mann Whitney U test between the two groups of patients;Univariate analysis,correlation analysis,and multivariate analysis were used in sequence to identify the histogram features with significant differences between the two groups(including 10％percentile,1％percentile,25％percentile,5％percentile,median,minimum,skewness,and variance);Logistic regression and the screened features were combined for modeling,and receiver operating characteristic(ROC)curves were drawn and areas under the curve(AUC)were calculated to compare the discriminative ability of each histogram feature.Results:There was no statistically significant difference in age,gender,and BMI between the two groups of patients(P＞0.05).Among the histogram parameters,10％percentile value,1％percentile value,25％percentile value,5％percentile value,median,minimum value,skewness,and variance were statistically different between the two groups(P＜0.05).The 10％percentile value displayed the best diagnostic performance,with an AUC value of 0.824 and a specificity of 0.893.The combined model had an AUC value of 0.860 and a specificity of 0.946.Conclusions:Based on 10％percentile value of CT grayscale histogram and joint model,PS and BS can be distinguished effectively,providing a basis for accurately distinguishing the two diseases in clinical practice.
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Using bioinformatics methods to predict the physical and chemical properties,spatial structure,and T and B cell epitopes of Brucella Periplasmic Protein(EipB),and to explore the immune effects of T and B dominant epitope peptides,for Brucella Periplasmic Protein(EipB)Lay the foundation for the screening of molecular peptide vaccine against bacillosis.Online websites and bioinformatics-related databases were used to analyse the physicochemical properties,transmembrane structural domains,signal peptide sequences,secondary and tertiary structures,hydrophilicity/hydrophobicity and T and B antigenic epitopes of the EipB protein.And select the B and T epitopes with the highest antigenicity(located at 121-137 and 95-110 respectively)to connect with hemocyanin,and after immunizing the mice,detect the production of IgG,IgM,IgA and IgE an-tibodies by ELISA;Flow cytometry was used to detect the secretion of cytokines in T cells;The brucellosis EipB protein is composed of 279 amino acids,is weakly basic,has a molecular mass of 31 kDa and is relatively stable;contains a signal peptide sequence and a transmembrane structural domain;has a secondary structure with a-helix,β-turn,extended linkage and irregular coi-ling;has a distinct hydrophilic region,and the protein contains 21 dominant B-cell epitope peptides in amino acid regions,the amino acid regions in which these dominant B-cell epitopes are located correspond to regions of higher hydrophilicity.Contains 56 CD4+T cell epitopes and 39 CD8+T cell epitopes.After immunizing mice with T-B dominant cell epitope combination,antibodies such as IgG,IgM,IgA and IgE in serum increased significantly;T cells produced IFN-γ and other cytokines significantly increased.1)Bioinformatics method was used to predict that EipB pro-tein contains dominant B and T cell epitopes.2)Combined immunization of mice with dominant B-T dominant epitope peptides can induce a higher level of immune response in mice,providing a reference for the screening of brucellosis molecular peptide vaccines.
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Brucellosis is a zoonosis with worldwide distribution,which is caused by bacteria of the genus Brucella.The in-cidence of brucellosis has recently increased each year,and caused great losses in terms of public health,animal husbandry and the social economy.Doxycycline plus rifampicin or streptomycin,the preferred antibiotic regimen,plays a key role in brucello-sis treatment.Because of the use of antibacterial drugs,Brucella has developed antimicrobial resistance,thus challenging clini-cal treatment.This article reviews research progress in drug resistance of Brucella from cattle and sheep(mainly Brucella abortus and Brucella melitensis)and Brucella from humans(mainly Brucella melitensis)from 2013 to 2023,including the types of antibiotic resistance,the rates of antibiotic resistance and the possible mechanism of antibiotic resistance,to provide a comprehensive reference for clinical the treatment of brucellosis and study of the drug resistance mechanisms of Brucella.
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Objective:To learn about the genotyping of human Brucella isolated from Sichuan Province. Methods:BCSP31-PCR and AMOS-PCR were used to identify the genus and biotype of the 66 strains isolated from confirmed human brucellosis cases in Sichuan Province from 2014 to 2020, respectively. The isolated strains were genotyped by multi-locus sequence typing (MLST)-9. The sequence type (ST) was compared trough the online MLST database. A minimum spanning tree (MST) was constructed to cluster the newly discovered and known ST using the BioNumerics software version 7.6.Results:The 66 strains isolated from human cases of brucellosis in Sichuan Province from 2014 to 2020 were Brucella, and 65 of them were Brucella melitensis while one strain was Brucella abortus. The MLST method identified three known STs (ST-8, ST-39 and ST-2) and one newly type (ST-101). Among them, ST-8 was the main ST in Sichuan Province (90.91%, 60/66), another 4 strains of Brucella melitensis were ST-39, and 1 strain of Brucella abortus was ST-2. The newly type ST-101 was isolated from Leshan City in 2019, belonging to the Brucella melitensis and closely related to the evolution of ST-8. Conclusion:Brucella melitensis is the main epidemic Brucella strain in Sichuan Province, ST-8 is predominant genotype, with a small amount of ST-39, ST-101 and ST-2.
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Objective:A nested-PCR assay is developed to detect and identify the genomic DNA of Brucella vaccine A19 strain. Methods:The whole genomic sequences of Brucella vaccine A19 strain and other Brucella spp. strains were compared and analyzed. The primers were designed by nucleotide difference sites. The nested-PCR assay was established to detect and identify Brucella vaccine A19 strain. The genomic DNA of Brucella vaccine A19 strain was extracted and diluted. The diluted template DNA was tested for sensitivity of using nested-PCR assay. And the specificity of nested-PCR assay was tested for the genomic DNA of other Brucella spp. strains and non- Brucella spp. strains. Results:The minimum detection limit of the nested-PCR assay was 3.43 fg. The nested-PCR assay established for amplification of Brucella vaccine A19 strain showed 246 bp electrophoresis bands, while other Brucella spp. strains showed 314 bp electrophoresis bands, and non- Brucella spp. strains did not produce electrophoresis bands. Conclusions:The nested-PCR assay established has the characteristics of high sensitivity and specificity. It can be detected when there is one copy of Brucella vaccine A19 strain genomic DNA in the reaction system. This method is particularly suitable for the detection and identification of trace genomic DNA of Brucella vaccine A19 strain in sample.
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Objective:To observe multiple locus variable-number tandem repeat analysis (MLVA) typing of Brucella isolated from Himalayan marmot in Qinghai-Tibet Plateau of Qinghai Province, and to explore the relationship between the strains and strains previous isolated from Qinghai Province. Methods:Blood samples of Himalayan marmot were collected in Qinghai-Tibet Plateau of Qinghai Province from March 2019 to October 2020. Pathogens were isolated and cultured from Brucella antibody positive samples identified by using the rose bengal test (RBT). Conventional biological methods and molecular biological methods (BCSP31-PCR and AMOS-PCR) were used for strain identification. At the same time, MLVA method was used to genotype the isolated strains, and cluster analysis was used to analyze the genetic relationships between the strains based on the genotype of 70 Brucella isolated from different hosts in Qinghai Province. Results:A total of 1 466 blood samples of Himalayan marmot were collected from Qinghai-Tibet Plateau. Two strains of Brucella were isolated and cultured from 64 RBT-positive samples, named QH2013054 and QH2013062, respectively. They were identified as Brucella ovis biotype Ⅲ by conventional and molecular biological methods. The MLVA genotyping results showed that QH2013054 and QH2013062 were different at the Bru16 locus, indicating different MLVA genotypes. Cluster analysis showed that strain QH2013054 had the same MLVA genotype as 7 strains, among which 6 strains were from 3 farmers and 3 sheep from the same family in Gonghe County, and 1 strain was from a farmer in Menyuan Hui Autonomous County. The strain QH2013062 had the same MLVA genotype as 4 strains, including 3 strains from 3 farmers in Menyuan Hui Autonomous County and 1 strain from a farmer in Tu Autonomous County of Huzhu. Conclusions:The strains of Brucella isolated from Himalayan marmot in Qinghai-Tibet Plateau of Qinghai Province have the same MLVA genotype as some strains of Brucella isolated from humans and sheep in Qinghai Province. It is speculated that the host humans, sheep and Himalayan marmot in Qinghai-Tibet Plateau may have a common source of infection.
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Objective:To establish a quantitative real-time PCR detection system for Brucella S2 vaccine strain. Methods:Based on the differences in the entire genome sequence between Brucella S2 vaccine strain and other reference strains of Brucella, primers and probes were designed to establish a quantitative real-time PCR detection system for Brucella S2 vaccine strain. The DNA of 22 reference strains of Brucella and 8 non- Brucella control strains were obtained from the National Institute for Infectious Disease Control and Prevention of the Chinese Center for Disease Control and Prevention. At the same time, environmental samples were obtained from the brucellosis vaccine manufacturers, and bacterial DNA from environmental samples was extracted using a blood/tissue genomic DNA extraction kit. The obtained DNA was pre-amplified by conventional PCR, and then subjected to quantitative real-time PCR secondary amplification (nested fluorescence quantitative PCR) using the amplified PCR product as a template. The specific fluorescence curve and corresponding number of cycles (Ct value) were observed, and the sensitivity was tested. Results:The quantitative real-time PCR detection system established did not detect specific fluorescence curves (without Ct values) for 21 reference strains of Brucella and 8 non- Brucella control strains, except for S2 vaccine strains. The established detection system had a minimum detection limit of 4.34 fg (genomic DNA) for detecting the DNA of Brucella S2 vaccine strain; DNA of Brucella S2 vaccine strain was detected in 3 of the 14 environmental samples collected. Conclusion:The quantitative real-time PCR detection system established can detect Brucella S2 vaccine strain in samples, with good sensitivity and specificity.
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Objective:To investigate the effect of recombinant lipoproteins of Brucella outer membrane protein 16, 19 (L16 and L19) on the expression of immune regulatory factors in human monocytic leukemia cell line (THP-1 cells). Methods:THP-1 cells activated with phorbol ester (PMA) were used as an in vitro experimental cell model, and a group design was used to co-culture L16, L19 and THP-1 cells (L16 stimulated group, L19 stimulated group), respectively. THP-1 cells activated with PMA were used as the control group. When co-cultured for 4 hours, immunofluorescence staining (IFS) and Western blotting were used to detect whether L16 and L19 entered the cells, respectively; when co-cultured for 12, 24 hours, real-time fluorescent quantitative PCR was used to measure the mRNA expression levels of interferon regulatory factor 1 (IRF-1) and trans activator protein of major histocompatibility complex class Ⅱ (CⅡTA); Western blotting was used to detect the protein expression levels of T cell immunoglobulin mucin-3 (Tim-3) and γ interferon receptor 1 (IFNGR1). Results:When co-cultured for 4 hours, L16 and L19 were observed entering THP-1 cells in the L16 stimulated group and L19 stimulated group, respectively. When co-cultured for 12 hours, the expression level of IRF-1 mRNA in the L16 stimulated group (0.16 ± 0.15) was significantly lower than that in the control group (1.00 ± 0.00, P < 0.05). When co-cultured for 24 hours, the expression level of CⅡTA mRNA in the L16 stimulated group (0.17 ± 0.10) was significantly lower than that in the control group (1.00 ± 0.00, P < 0.05). When co-cultured for 12 and 24 hours, there were no statistically significant differences in the expression levels of IRF-1 and CⅡTA mRNA between the L19 stimulated group and the control group ( P > 0.05). Western blotting results showed that there were statistically significant differences in the expression levels of INFGR1 and Tim-3 protein among the control group, L16 stimulated group, and L19 stimulated group after co-cultured for 12 and 24 hours ( F = 50.92, 6.80, 148.73, 156.57, P < 0.05). Among them, when co-cultured for 12 hours, the expression level of INFGR1 protein in the L16 and L19 stimulated groups were significantly lower than that in the control group, and the L19 stimulated group was higher than the L16 stimulated group ( P < 0.05), and the expression level of Tim-3 protein in the L19 stimulation group was higher than that in the control group ( P < 0.05). When co-cultured for 24 hours, the expression level of INFGR1 protein in the L16 and L19 stimulated groups were lower than that in the control group, and the L19 stimulated group was higher than that in the L16 stimulated group ( P < 0.05); and the expression level of Tim-3 protein in the L16 stimulated group was higher than that in the control group and L19 stimulated group ( P < 0.05). Conclusions:Brucella L16 can downregulate the expression levels of IRF-1 and CⅡTA mRNA in THP-1 cells. Both L16 and L19 can downregulate IFNGR1 and upregulate Tim-3 protein expression levels.
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Objective:To study the multi-locus sequence typing (MLST) gene characteristics of Brucella isolates in Guizhou Province. Methods:Brucella strains, which were isolated from 2017 to 2021 in Guizhou Province (preserved in the Bacterial and Viral Seed Bank of Guizhou Center for Disease Control and Prevention) were identified Brucella and species/types by BCSP31-PCR and AMOS-PCR methods, respectively. MLST method was used for genotyping, and Biometrics 8.0 software was used for cluster analysis of the typing results. Results:A total of 32 strains of Brucella were isolated in Guizhou Province and identified as Brucella melitensis ( B.melitensis) by BCSP31-PCR and AMOS-PCR methods. These strains were classified into 2 ST types (ST8 and ST39) by MLST method, with 28 strains of ST8 type(87.5%) and 4 strains of ST39 type (12.5%). The 28 strains of ST8 type were distributed in 7 cities (prefectures) of Guizhou Province, while the 4 strains of ST39 type were only found in Qianxinan Buyi and Miao Autonomous Prefecture. The cluster analysis results showed that ST8 and ST39 types strains were clustered in a group with the reference strain of B.melitensis, and there was only one nucleotide site difference between ST39 and ST8 types in the glk gene, indicating a close genetic relationship. Conclusions:B.melitensis is the main pathogen of the brucellosis epidemic in Guizhou Province in recent years. ST8 is the dominant MLST genotype in Guizhou Province.
ABSTRACT
Objective:To establish a real-time quantitative PCR assay to detect the copy number of IS711 transposase gene (orfA) in Brucella genome, and the assay is applied to identify the species and biovars of Brucella. Methods:To establish an orfA gene copy number detection system based on Taqman real-time quantitative PCR technique. Primers and probes of bcsp31 and orfA genes were designed, the contents of bcsp31 gene and orfA gene in the same strain with the same DNA concentration were simultaneously detected by real-time quantitative PCR assay, and cycle number (CT value) of the two genes were obtained. According to the differences of CT values of bcsp31 gene and orfA gene, the copy number of orfA gene in Brucella genome was calculated. At the same time, the DNA of Brucella 16M strain was double decreasing dilution to verify the stability of the detection system. Results:A real-time quantitative PCR assay was used to detect bcsp31 gene and orfA gene simultaneously, when the DNA concentration difference of 16M strain was 2 times, the mean difference of CT values measured was 1.00, 95% confidence interval was 0.95-1.05, standard deviation was 0.17, and coefficient of variation was 0.17. The orfA gene copy number of 30 Brucella strains was detected by this detection system. It was found that there were 6, 9, and 7 copy numbers in the biovars 1-3 of Brucella melitensis, respectively. The strain of Brucella suis biovar 2 had 10 copy numbers, which were different from those of the other 4 strains of biovars 1, 3-5. There were 37 copy numbers in Brucella ovis strain. The copy numbers were stable at 5-6 copies in 8 biovars (1-7, 9) of Brucella abortus strains. Conclusions:A real-time quantitative PCR assay for detection of orfA gene copy number in Brucella DNA has been established. This method could identify some Brucella species and biovars strains.
ABSTRACT
@#Abstract: Objective To investigate the clinical characteristics and incidence of Brucella encephalitis and meningitis in children. Methods We report the clinical data of a child with Brucella melitensis meningitis in children, and summarize the incidence, diagnosis methods and treatment of Brucella encephalitis or meningitis in children, taking into account the relevant domestic and foreign literature from January 2014 to December 2020. Results A 4-year-old girl was admitted to the hospital with status epilepticus on March 15, 2021 because of interrupted right limb numbness for 16 hours and convulsions for 2 hours. She had 2 non-febrile convulsions three months before admission and was diagnosed with epilepsy. This incident was acute, accompanied by low fever, with epilepsy as the main manifestation. Cerebrospinal fluid test suggested central nervous system infection, but the nature of infection could not be determined by routine and biochemistry of cerebrospinal fluid.The cerebrospinal fluid next generation sequencing confirmed that the pathogen of the infection was B. melitensis, which was further verified by the peripheral blood antibody test. After effective antibiotics combined with a full course of treatment, the patient recovered after six months of treatment. A total of 60 articles were retrieved in the database, including 29 in Chinese. During this period, a total of 7 cases of brucellosis in children with nervous system involvement were reported, one of which was a case report, and the other 6 cases were mentioned in the comprehensive analysis of children with brucellosis. Conclusions Brucella encephalitis or meningitis in children has a low incidence and various clinical features, which are easy to be misdiagnosed or missed.