[Preliminary evaluation of immunogenicity and protective effect of multicomponent recombinant protein vaccine EPRHP014 against tuberculosis].

Objective: To evaluate the immunogenicity and protective effect of a multicomponent recombinant protein vaccine EPRHP014 constructed independently and provide a scientific basis for developing new tuberculosis (TB) vaccine and effective prevention and control of TB. Methods: Three full-length Mycobacterium (M.) tuberculosis protein antigens (EsxH, Rv2628, and HspX) and two epitope-predicted and optimized epitope-dominant protein antigens (nPPE18 and nPstS1) were selected, from which five protein antigens were used to construct a protein antigen composition EPRHP014, including a fusion expression multi-component protein antigen (EPRHP014f) and a multi-component mixed protein antigen (EPRHP014m) formed with the five single protein using clone, purification, and purification respectively. Multicomponent protein vaccines EPRHP014f and EPRHP014m were prepared with aluminum adjuvant, and the BCG vaccine was used as a control. ELISA detected the titer of serum-specific antibodies, the secretion of various cytokines was detected by ELISpot and Luminex, and immune protection was observed by the M. tuberculosis growth inhibition test in vitro. The results were statistically analyzed by t-test or rank sum test, and P<0.05 was considered a statistically significant difference. Results: Mice Immunized with EPRHP014m and EPRHP014f could produce highly effective IgG antibodies and their subtypes IgG1 and IgG2a, and the antibody titers were similar to those of mice immunized with BCG, with no statistical significance (P>0.05). The number of spot-forming cells (SFC) secreting IFN-γ and IL-4 induced by EPRHP014f group was significantly higher than those by EPRHP014m group and BCG group (P<0.05), but there was no significant difference in the number of SFC for IFN-γ and IL-4 induced between EPRHP014m group and BCG group (P>0.05). The secretion levels of GM-CSF and IL-12p70 induced by the EPRHP014m group were higher than those of the BCG group (P<0.05), but there was no significant difference in the levels of IL-6 and IL-10 induced between EPRHP014m group and BCG group (P>0.05). There was no significant difference in the secretions of IL-6, IL-10, IL-12, and GM-CSF between the EPRHP014f and BCG groups (P>0.05). EPRHP014m group, EPRHP014f group, and BCG group had obvious antibacterial effects in vitro, and the difference was insignificant (P>0.05). Conclusion: Both EPRHP014f and EPRHP014m can induce strong humoral and cellular immune responses in mice after immunization, and have a strong ability to inhibit the growth of M. tuberculosis in vitro, indicating that the antigen composition EPRHP014 has good potential in the development and application of TB vaccine.

Characterization of internal transcribed spacer-1 and apical membrane antigen-1 sequences provides insights into the genetic diversity of Eimeria tenella strains.

Coccidiosis is a major recurring problem in the poultry industry and is caused by infection of one or more of the seven Eimeria species known to infect chickens, with Eimeria tenella among the best studied and economically important. Studies on the genetic diversity of E. tenella strains is essential for the development of universally acceptable diagnostic markers and vaccines against the disease. Eimeria tenella internal transcribed spacer-1 (ITS-1) and apical membrane antigen-1 (AMA-1) sequences from different parts of the world are available in the public domain and therefore provide suitable comparative markers for genetic diversity study. In this study, the ITS-1 and AMA-1 sequence diversity of two local E. tenella strains, namely EtNSN6 and EtSGR6 were characterized. Both ITS-1 and AMA-1 sequences for EtNSN6 and EtSGR6 were retrieved by mapping to their respective genome sequences generated using next generation sequencing. Multiple sequence alignment of the ITS-1 and AMA-1 sequences with selected homologous sequences revealed the presence of a total of five and 13 single nucleotide polymorphisms (SNPs) respectively. All SNPs appeared to occur at random and did not show any unique pattern based on geographical regions while no insertions and deletions (indels) was found to occur in the aligned sequences. However, unique bases that defined certain strains were detected. Phylogenetics analyses performed with Maximum Parsimony (MP) and Maximum Likelihood (ML) methods revealed similar topology for the internal groups with all the E. tenella ITS-1 and AMA-1 sequences grouped in the same clade supported by high bootstrap confidence. This confirmed that both EtNSN6 and EtSGR6 samples are E. tenella strains. Sequence comparison and phylogenetics analyses further suggest the possibility of low genetic diversity among E. tenella strains.

Characterization of internal transcribed spacer-1 and apical membrane antigen-1 sequences provides insights into the genetic diversity of Eimeria tenella strains

@#Coccidiosis is a major recurring problem in the poultry industry and is caused by infection of one or more of the seven Eimeria species known to infect chickens, with Eimeria tenella among the best studied and economically important. Studies on the genetic diversity of E. tenella strains is essential for the development of universally acceptable diagnostic markers and vaccines against the disease. Eimeria tenella internal transcribed spacer-1 (ITS-1) and apical membrane antigen-1 (AMA-1) sequences from different parts of the world are available in the public domain and therefore provide suitable comparative markers for genetic diversity study. In this study, the ITS-1 and AMA-1 sequence diversity of two local E. tenella strains, namely EtNSN6 and EtSGR6 were characterized. Both ITS-1 and AMA-1 sequences for EtNSN6 and EtSGR6 were retrieved by mapping to their respective genome sequences generated using next generation sequencing. Multiple sequence alignment of the ITS-1 and AMA-1 sequences with selected homologous sequences revealed the presence of a total of five and 13 single nucleotide polymorphisms (SNPs) respectively. All SNPs appeared to occur at random and did not show any unique pattern based on geographical regions while no insertions and deletions (indels) was found to occur in the aligned sequences. However, unique bases that defined certain strains were detected. Phylogenetics analyses performed with Maximum Parsimony (MP) and Maximum Likelihood (ML) methods revealed similar topology for the internal groups with all the E. tenella ITS-1 and AMA-1 sequences grouped in the same clade supported by high bootstrap confidence. This confirmed that both EtNSN6 and EtSGR6 samples are E. tenella strains. Sequence comparison and phylogenetics analyses further suggest the possibility of low genetic diversity among E. tenella strains.

Three-year follow-up of a Traumatic Hemipelvectomy Survivor: A Case Report.

The management of a patient with traumatic hemipelvectomy is complex. We report the acute management and rehabilitation of a 21-year-old patient as well as her prosthesis modification. She was able to return to society as a K3 level ambulator.

A novel approach to a rare case of non-islet cell hypoglycaemia.

SUMMARY: Insulin autoimmune syndrome (IAS) is a rare cause of non-islet cell hypoglycaemia. Treatment of this condition is complex and typically involves long-term use of glucocorticoids. Immunotherapy may provide an alternative in the management of this autoimmune condition through the suppression of antibodies production by B-lymphocyte depletion. We present a case of a 62-year-old male, with refractory hypoglycaemia initially presenting with hypoglycaemic seizure during an admission for acute psychosis. Biochemical testing revealed hypoglycaemia with an inappropriately elevated insulin and C-peptide level and no evidence of exogenous use of insulin or sulphonylurea. Polyethylene glycol precipitation demonstrated persistently elevated free insulin levels. This was accompanied by markedly elevated anti-insulin antibody (IA) titres. Imaging included CT with contrast, MRI, pancreatic endoscopic ultrasound and Ga 68-DOTATATE position emission tomography (DOTATATE PET) scan did not reveal islet cell aetiology for hyperinsulinaemia. Maintenance of euglycaemia was dependent on oral steroids and dextrose infusion. Complete resolution of hypoglycaemia and dependence on glucose and steroids was only achieved following treatment with plasma exchange and rituximab. LEARNING POINTS: Insulin autoimmune syndrome (IAS) should be considered in patients with recurrent hyperinsulinaemic hypoglycaemia in whom exogenous insulin administration and islet cell pathologies have been excluded. Biochemical techniques play an essential role in establishing high insulin concentration, insulin antibody titres, and eliminating biochemical interference. High insulin antibody concentration can lead to inappropriately elevated serum insulin levels leading to hypoglycaemia. Plasma exchange and B-lymphocyte depletion with rituximab and immunosuppression with high dose glucocorticoids are effective in reducing serum insulin levels and hypoglycaemia in insulin autoimmune syndrome (IAS). Based on our observation, the reduction in serum insulin level may be a better indicator of treatment efficacy compared to anti-insulin antibody (IA) titre as it demonstrated greater correlation to the frequency of hypoglycaemia and to hypoglycaemia resolution.

Vertebrae Destruction with Cauda Equina Syndrome Secondary to Spinal Gouty Arthritis: A Case Report.

Gouty arthritis commonly affects peripheral joints and is associated with hyperuricaemia. Spinal manifestations of gouty arthritis are not common, and majority of published articles worldwide were case reports. This is a case report of spinal gouty arthritis that presented with spinal vertebrae destruction and cauda equina syndrome. The magnetic resonance imaging (MRI) showed destruction of L5/S1end plates with cystic collection mimicking infective changes. The tissue histological examination confirmed presence of urate crystal needles that displayed negative double refraction on light microscopy. Spinal gouty arthritis is part of the differential diagnoses in gouty arthritis patients.

[Cellular immunity evaluation of five mycobacterium tuberculosis recombinant proteins and their compositions].

Objective: The cellular immunity of 5 Mycobacterium tuberculosis recombinant proteins and their compositions was evaluated. Method: A total of 88 fresh venous blood from peripheral heparin anticoagulant population, 42 of which were from tuberculosis patients treated by The Tuberculosis Prevention and Treatment Center of Changping District, Beijing, and 46 of healthy volunteers were provided by the Infection Diseases of Chinese Center for Disease Control and Prevention. Healthy volunteers without a history of tuberculosis exposure and any clinical signs and symptoms. Using the Mycobacterium tuberculosis standard strain H37Rv DNA as a template, complete genes of the selected 5 recombinant proteins Rv3874, Rv3875, Rv2031c, Rv1411c and Rv3418c by PCR amplified; 5 recombinant proteins were cloned, expressed and purified as stimulants by genetic recombination and protein purification techniques, and the effector T cell enzyme-linked immunospot assay (ELISPOT) was used to detect cellular immunity in the population. Results: The recombinant proteins Rv3874, Rv3875, Rv2031c, Rv1411c and Rv3418c were successfully cloned, expressed and purified; And the sensitivities were 50.00%, 71.43%, 69.04%, 73.81% and 76.19%, and the specificities were 86.96%, 76.09%, 71.74%, 39.13% and 36.96%. In addition, the positive predictive value, negative predictive value, area under the curve and Youden index were 52.46% to 77.78%, 62.96% to 74.47%, 0.511 to 0.754 and 0.129 to 0.475, respectively. Except for Rv1411c and Rv3418c, the number of spot-forming cell (SFC) detected by Rv3874, Rv3875 and Rv2031c in tuberculosis patients was higher than healthy volunteers, and the differences were statistically significant (P<0.001). Among the 26 compositions composed of 5 recombinant proteins, the sensitivity was 80.95% to 95.24%, and the specificity was 68.89% to 24.44%. As the number of recombinant proteins in the composition increases, the sensitivity gradually increased, but the specificity decreased. Conclusion: The recombinant proteins of Mycobacterium tuberculosis Rv3874, Rv3875 and Rv2031c have strong ability to stimulate T cells to produce immune response, and have certain antigenicity. The efficacy of Rv1411c and Rv3418c alone as diagnostic antigens is not ideal, and the composition composed of multi-component antigens has certain application value. This article provides experimental evidence for the immune diagnosis of tuberculosis and the preparation of new anti-tuberculosis vaccines.

[Analysis on drug resistance-associated mutations of multi-drug resistant Mycobacterium tuberculosis based on whole-genome sequencing in China].

Objective: To analyze the resistance mutational profiles of multi-drug resistant Mycobacterium tuberculosis in China and the correlation between major mutation types and genotypes based on the whole-genome sequencing data. Methods: Search and download of the genome-wide sequencing data of M. tuberculosis published in China by August 2019 on NCBI database were conducted. Mutation frequency of drug resistance-related gene loci based on whole-genome sequencing was used to predict the molecular susceptibility of strains, and the correlation between mutation types and genotypes was analyzed. Results: According to the results of molecular resistance and susceptibility profiles, 1 024 MDR strains were identified from 2 019 M. tuberculosis strains. The major mutation types of resistance-related genes to common drugs were katG S315T (73.2%, isoniazid), rpoB S450L (63.1%, rifampicin), rpsL K43R (70.0%, streptomycin), embB M306V (37.4%, ethambutol), pncA_promoter T (-11)C (7.9%, pyrazinamide), gyrA A90V (32.3%, fluoroquinolones), rrs A1401G (67.7%, second-line injection drugs), fabG1_promoter C (-15) T (87.0%, Ethionamide), folC I43T (30.4%, P-aminosalicylic acid). Among them, the frequencies of katG S315T, embB M306V, rpsL K43R, gyrA A90V in lineage 2 were significantly higher than those in lineage 4, and folC I43T was only found in lineage 2. The proportion of katG S315T was significantly higher in the ancient Beijing genotype compared to the modern genotype, in contrast, the proportion of rpsL K43R was significantly higher in modern Beijing genotype, the differences were significant (all P<0.05). Conclusions: The results showed the main mutation types of resistance-related genes of MDR strains to many commonly used anti-tuberculosis drugs in China based on whole-genome sequencing, providing a basis for the development of sensitive and specific rapid molecular detection methods. At the same time, it was also found that the major mutation types of MDR-related genes were related to the genotype of the strains.

[Analysis on drug sensitivity spectrum of 167 multidrug-resistant Mycobacterium tuberculosis in China].

Objective: To investigate the drugs-sensitivity spectrum of multidrug-resistant tuberculosis (MDR-TB) in China and provide a scientific evidence for the drug selection in clinical therapy and the control of MDR-TB. Methods: A total of 167 strains of MDR-TB were included in this study. Every strain was genotyped by lysX gene sequencing and their sensitivity to 13 different anti-TB drugs was tested by using MicroDST(TM) and BACTEC(TM) MGIT 960(TM) liquid-culturing method. The association between drug resistance and genotypes as well as cross drug resistance was also analyzed. The results were analyzed by means of the comparison of enumeration data between two groups with χ(2) test. Results: The overall resistance rate of 167 MDR-TB strains to 11 anti-TB drugs, except isoniazide and rifampicin, was 95.81%, the rates of pre-extensive drug-resistance (pre-XDR) and extensive drug-resistance were 31.14%(52/167) and 6.59% (11/167), respectively. The streptomycin resistance rate of Beijing genotypes was significantly higher than that of the non-Beijing genotypes ( χ(2)=30.682, P<0.05), while the pre-XDR proportion in Beijing genotypes was lower than that in non-Beijing genotypes (χ(2)=5.332, P<0.05). The resistance rates of Ofloxacin and Pyrazinamide in the modern Beijing genotype were significantly higher than those in classical ones (χ(2)=4.105 and χ(2)=3.912, P<0.05). In addition, the cross-resistance rate to rifampicin and rifabutin was 86.23%. A significant difference in drug-resistance rate to rifabutin was seen among groups with different levels of rifampicin resistance (χ(2)=45.912, P<0.05). There was positive correlation not only between ofloxac resistance and moxifloxac resistance, but also between amikacin resistance and kanamycin resistance, with the coefficient of 0.87 and 0.91, respectively. Conclusions: In this study, we observed that there were high incidences of the resistance to 11 anti-TB drugs in 167 clinical MDR-TB strains and the cross resistance phenomena between drugs of the same type were quite serious. The majority of MDR-TB strains belonged to Beijing genotype, which was highly associated with streptomycin resistance.

Status of pyrethroid resistance in Aedes (Stegomyia) aegypti (Linneaus) from dengue hotspots in Klang Valley, Malaysia.

The continued absence of an effective and safe tetravalent dengue vaccine and the lack of specific anti-viral treatment have made mosquito vector control using chemical insecticides as the mainstream for dengue prevention and control. However, the long-term use of chemical insecticides may induce resistance. Hence detection of insecticide resistance in dengue vectors is crucially important in ensuring the insecticide-based intervention in dengue control program is still effective and reliable. In this study, the susceptibility status of Aedes aegypti from five selected dengue hotspots in Klang Valley, Malaysia against pyrethroids was determined by employing the World Health Organization (WHO) protocol of adult bioassay. Four types of pyrethroids were tested against adult female Aedes aegypti to determine the knockdown rate, post 24-h adult mortality and resistance ratio. All field-collected Aedes aegypti strains were resistant to the four pyrethroids tested, except for the Taman Sungai Jelok (TSJ) strain. Permethrin exhibited the lowest knockdown rate against Aedes aegypti, followed by deltamethrin, cyfluthrin and lambda-cyhalothrin. This present study indicated the widespread of pyrethroid resistance in Aedes aegypti in Klang Valley, indicating the needs of implementing alternative measures in vector control program. The data in this study can be utilised as an input for insecticide resistance management of Aedes aegypti in Malaysia.

Status of pyrethroid resistance in Aedes (Stegomyia) aegypti (Linneaus) from dengue hotspots in Klang Valley, Malaysia

@#The continued absence of an effective and safe tetravalent dengue vaccine and the lack of specific anti-viral treatment have made mosquito vector control using chemical insecticides as the mainstream for dengue prevention and control. However, the long-term use of chemical insecticides may induce resistance. Hence detection of insecticide resistance in dengue vectors is crucially important in ensuring the insecticide-based intervention in dengue control program is still effective and reliable. In this study, the susceptibility status of Aedes aegypti from five selected dengue hotspots in Klang Valley, Malaysia against pyrethroids was determined by employing the World Health Organization (WHO) protocol of adult bioassay. Four types of pyrethroids were tested against adult female Aedes aegypti to determine the knockdown rate, post 24-h adult mortality and resistance ratio. All field-collected Aedes aegypti strains were resistant to the four pyrethroids tested, except for the Taman Sungai Jelok (TSJ) strain. Permethrin exhibited the lowest knockdown rate against Aedes aegypti, followed by deltamethrin, cyfluthrin and lambda-cyhalothrin. This present study indicated the widespread of pyrethroid resistance in Aedes aegypti in Klang Valley, indicating the needs of implementing alternative measures in vector control program. The data in this study can be utilised as an input for insecticide resistance management of Aedes aegypti in Malaysia.

[Application of ARIMA model in predicting the incidence of tuberculosis in China from 2018 to 2019].

Objective: Autoregressive integrated moving average (ARIMA) model was used to predict the incidence of tuberculosis in China from 2018 to 2019, providing references for the prevention and control of pulmonary tuberculosis. Methods: The monthly incidence data of tuberculosis in China were collected from January 2005 to December 2017. R 3.4.4 software was used to establish the ARIMA model, based on the monthly incidence data of tuberculosis from January 2005 to June 2017. Both predicted and actual data from July to December 2017 were compared to verify the effectiveness of this model, and the number of tuberculosis cases in 2018-2019 also predicted. Results: From 2005 to 2017, a total of 13 022 675 cases of tuberculosis were reported, the number of pulmonary tuberculosis patients in 2017 was 33.68% lower than that in 2005, and the seasonal character was obvious, with the incidence in winter and spring was higher than that in other seasons. According to the incidence data from 2005 to 2017, we established the model of ARIMA (0,1,2)(0,1,0)(12). The relative error between the predicted and actual values of July to December 2017 fitted by the model ranged from 1.67% to 6.80%, and the predicted number of patients in 2018 and 2019 were 789 509 and 760 165 respectively. Conclusion: The ARIMA (0, 1, 2)(0, 1, 0)(12) model well predicted the incidence of tuberculosis, thus can be used for short-term prediction and dynamic analysis of tuberculosis in China, with good application value.

[Identification and drug susceptibility testing of Mycobacterium thermoresistibile and Mycobacterium elephantis isolated from a cow with mastitis].

Objective: To understand the etiological characteristics and drug susceptibility of Mycobacterium thermoresistibile and Mycobacterium elephantis isolated from a cow with mastitis and provide evidence for the prevention and control of infectious mastitis in cows. Methods: The milk sample was collected from a cow with mastitis, which was pretreated with 4% NaOH and inoculated with L-J medium for Mycobacterium isolation. The positive cultures were initially identified by acid-fast staining and multi-loci PCR, then Mycobacterium species was identified by the multiple loci sequence analysis (MLSA) with 16S rRNA, hsp65, ITS and SodA genes. The drug sensitivity of the isolates to 27 antibiotics was tested by alamar blue assay. Results: Two anti-acid stain positive strains were isolated from the milk of a cow with mastitis, which were identified as non-tuberculosis mycobacterium by multi-loci PCR, and multi-loci nucleic acid sequence analysis indicated that one strain was Mycobacterium thermoresistibile and another one was Mycobacterium elephantis. The results of the drug susceptibility test showed that the two strains were resistant to most antibiotics, including rifampicin and isoniazid, but they were sensitive to amikacin, moxifloxacin, levofloxacin, ethambutol, streptomycin, tobramycin, ciprofloxacin and linezolid. Conclusions:Mycobacterium thermoresistibile and Mycobacterium elephantis were isolated in a cow with mastitis and the drug susceptibility spectrum of the pathogens were unique. The results of the study can be used as reference for the prevention and control the infection in cows.

[Cloning expression and serological evaluation on Mycobacterium tuberculosis four new antigens].

Objective: To evaluate the serological diagnostic value of Mycobacterium (M.) tuberculosis four new antigens Rv0432, Rv0674, Rv1566c and Rv1547. Methods:Rv0432, Rv0674, Rv1566c and Rv1547 were amplified from M. tuberculosis strain H37Rv genomic DNA by using PCR, among which Rv1547 was divided into two segments for amplification (Rv1547-1 and Rv1547-2). The segments were cloned into expression vector PET-32a while the recombinant proteins were purified by affinity chromatography. Serums were incubated with BL21 (DE3) proteins. Antibodies IgG against M. tuberculosis were tested with 151 serum samples (41 healthy people and 110 TB patients) by using ELISA. The diagnostic efficiency of antigens was analyzed by means of receiver operating characteristic curve. Difference of the objective proteins in TB patients and healthy controls was compared by t-test. Results: Recombinant antigens Rv0432, Rv0674, Rv1566c, Rv1547-1 and Rv1547-2 were successfully expressed and purified. Results from ELISA showed that the sensitivity, specificity, positive predictive value, negative predictive value, Youden index and area under the curve of Rv0432, Rv0674, Rv1566c, Rv1547-1 and Rv1547-2, as 43.64%-92.73%, 80.49%-92.68%, 0.92-0.94, 0.38-0.80, 0.363-0.732 and 0.649-0.915. All the objective proteins showed significantly higher antibody levels in TB patients, when compared to the healthy controls (P<0.000 1). Conclusion: The newly identified antigens Rv0432, Rv0674, Rv1566c, Rv1547-1 and Rv1547-2 all performed well when being used for TB serological diagnosis, thus were expected to be new candidate antigens used for TB diagnosis.

[Identification and evaluation of T cell epitopes of Rv0585c from Mycobacterium tuberculosis].

Objective: To investigate the human T cell epitopes of Mycobacterium (M.) tuberculosis Rv0585c protein antigen and their immunogenicity and provide evidence for the development of specific tuberculosis immune diagnostic techniques and tuberculosis vaccine. Methods: We synthesized peptides from M. tuberculosis Rv0585c protein antigen predicted by TE-predict and IEDB human T cell epitope prediction tool. The cellular immunoreactivity of the predicted peptides was evaluated through ELISpot assay with the peripheral blood monouclear cells (PBMC) of clinical tuberculosis patients. In animal experiments, BALB/c mice were respectively immunized with high dose (100 µg/mice) and low dose (50 µg/mice) of the peptides of Rv0585c, at the same time, high dose (50 µg/mice) and low dose (20 µg/mice) of Ag85B protein were used in positive control group. The levels of IFN-γ, IL-2, IL-4 and IL-10 were tested with ELISA kit respectively. Results: By means of bioinformatics technique, 66 human T cell epitopes of Rv0585c were predicted, from which9 peptides concentrated epitopes were synthesized for the animal immune experiments. Peptides P10110, P10112 and P10117 were confirmed to be antigenic. The sensitivity and specificity of P10110, P10112 and P10117 were 14.00%, 12.00%, 6.00% and 100.00%, 100.00%, 97.96% respectively when they were used as diagnostic reagents of tuberculosis. The sensitivity and specificity were 22.00% and 97.96% when the epitopes were combined together. The results of animal immunity test showed that high levels of cytokines IFN-γ, IL-2, IL-4 and IL-10 were induced by high and low dose of P10110, and high levels of IFN-γ、IL-2 and IL-10 were induced by high and low dose of P10112, which were much higher than that in negative controls, respectively (P<0.001). Conclusion: Rv0585c, including its human T cell epitopes, has good immunogenicity and immunoreactivity, stimulating the body to produce a stronger cellular immune response and has better potential application value in cellular diagnosis of tuberculosis and the development of new type of tuberculosis vaccine.

[A preliminary study on the molecular characteristics of D-cycloserine resistance of Mycobacterium tuberculosis].

Objective: To investigate the relationship between D-cycloserine resistance and the gene mutations of alrA, ddlA and cycA of Mycobacterium (M.) tuberculosis, as well as the association between D-cycloserine resistance and spoligotyping genotyping. Methods: A total of 145 M. tuberculosis strains were selected from the strain bank. D-cycloserine resistant phenotypes of the strains were determined by the proportion method and the minimal inhibitory concentration was determined by resazurin microtiter assay. PCR amplification and DNA direct sequencing methods were used for the analysis of gene mutations. Relationship between the resistance phenotype and genotype was analyzed by chi-square test. Results: Of the 145 clinically collected strains, 24 (16.6%) of them were D-cycloserine resistant and 121 (83.4%) were sensitive. There were only synonymous mutations noticed on alrA, ddlA and cycA in sensitive strains. Of the 24 D-cycloserine resistant strains, 3 (12.5%) isolates' cycA and 1 (4.2%) isolates' alrA happened to be non-synonymous mutations, in which the codes were 188, 318 and 508 of cycA, and 261 of alrA, respectively. Results on drug sensitivity tests confirmed the minimal inhibitory concentration of the mutant strains were all increased to some degrees. The D-cycloserine resistant rates of 88 Beijing genotype and 57 non-Beijing genotype strains were 20.5% and 10.5% , respectively, but with no statistically significant difference (χ(2) =2.47, P>0.05). Conclusions: The non-synonymous mutations of alrA and cycA might contribute to one of the mechanisms of M. tuberculosis D-cycloserine resistance. M. tuberculosis Beijing genotype or non-Beijing genotype was not considered to be associated with the D-cycloserine resistance.

[Analysis on drug resistance of Mycobacterium tuberculosis and influencing factors in six provinces of China].

OBJECTIVE: To analyze the drug-resistance of clinical Mycobacterium tuberculosis strains isolated from the tuberculosis(TB)patients in six provinces in China and related risk factors, and provide evidences for the effective prevention and treatment of drug resistant TB. METHODS: Six provinces were selected from China. The background information of the TB patients was investigated with questionnaire survey, and the drug susceptibilities of the clinical M. tuberculosis strains to isoniazid, rifampin, ethambutol and streptomycin were tested by means of the proportional drug susceptibility test. Then the results and related risk factors were analyzed with software SPSS 20.0. RESULTS: The overall drug resistant rate and multi drug-resistant(MDR)rate were 23.42% and 13.51% respectively. The overall drug resistant rate and MDR rate in Beijing, Jilin, Hunan, Henan, Shaanxi, Xinjiang were 21.50%, 12.24%, 36.27%, 42.86%, 27.78%, 24.39% and 4.67%, 8.16%, 24.51%, 26.53%, 15.28%, 14.15%, respectively. The χ(2) analysis results showed that the differences in single drug-resistant rate, overall drug resistant rate and MDR rate in these provinces had significant differences(P=0.000). The univariate statistical analysis results showed that the retreatment for TB and TB treatment history were the risk factors associated with drug resistance(P<0.05). CONCLUSION: The drug resistance of TB was very serious in China, but the TB drug resistance varied with province. The preventive intervention should be strengthened against all the major risk factors associated with the drug resistance for the better prevention and control of TB.

[Analysis on the polymorphisms of human B cell epitopes of 5 specificity antigens in Mycobacterium tuberculosis in China].

OBJECTIVE: To study the diversities of human B cell epitopes of five proteins (Mpt83, Mpt70, LpqH, PstS3, GroES) in Mycobacterium tuberculosis isolates from China. METHODS: We selected 179 strains isolated from patients with tuberculosis in different regions of China, and cultured these strains by L-J medium. The gene sequences of these 5 proteins were amplified and sequenced by PCR. The variations (single nucleotide polymorphisms, SNP) of the DNA sequences were compared and analyzed according to the immune epitope database (IEDB). The synonymous mutation rate (dS), non-synonymous mutation rate (dN) and dN/dS values were calculated with MEGA6 software. RESULTS: Among the 179 clinical MTBC isolates, 7 SNP mutations were found in the mpt83gene. The dN/dS value of the whole mpt83 gene was 0.88. One SNP mutation was found in the mpt70 gene. The dN/dS values of the whole pstS3 gene and non-B epitopes areas were 1.74 and 1.40 respectively. But no mutation was found in the groES gene. CONCLUSION: The mpt83, lpqH and pstS3 genes of 179 MTBC strains contain gene SNPs and human B cell epitopes diversities, while the coding gene and human B cell epitopes of protein mpt70 and groES were conservative.

Sequence analysis of the PIP5K locus in Eimeria maxima provides further evidence for eimerian genome plasticity and segmental organization.

Commercial flocks infected by Eimeria species parasites, including Eimeria maxima, have an increased risk of developing clinical or subclinical coccidiosis; an intestinal enteritis associated with increased mortality rates in poultry. Currently, infection control is largely based on chemotherapy or live vaccines; however, drug resistance is common and vaccines are relatively expensive. The development of new cost-effective intervention measures will benefit from unraveling the complex genetic mechanisms that underlie host-parasite interactions, including the identification and characterization of genes encoding proteins such as phosphatidylinositol 4-phosphate 5-kinase (PIP5K). We previously identified a PIP5K coding sequence within the E. maxima genome. In this study, we analyzed two bacterial artificial chromosome clones presenting a ~145-kb E. maxima (Weybridge strain) genomic region spanning the PIP5K gene locus. Sequence analysis revealed that ~95% of the simple sequence repeats detected were located within regions comparable to the previously described feature-rich segments of the Eimeria tenella genome. Comparative sequence analysis with the orthologous E. maxima (Houghton strain) region revealed a moderate level of conserved synteny. Unique segmental organizations and telomere-like repeats were also observed in both genomes. A number of incomplete transposable elements were detected and further scrutiny of these elements in both orthologous segments revealed interesting nesting events, which may play a role in facilitating genome plasticity in E. maxima. The current analysis provides more detailed information about the genome organization of E. maxima and may help to reveal genotypic differences that are important for expression of traits related to pathogenicity and virulence.