The present review describes the current status of multiplex quantitative real time polymerase chain reaction(q PCR) assays developed and used globally for detection and subtyping of hepatitis viruses in body fluids. ...The present review describes the current status of multiplex quantitative real time polymerase chain reaction(q PCR) assays developed and used globally for detection and subtyping of hepatitis viruses in body fluids. Several studies have reported the use of multiplex q PCR for the detection of hepatitis viruses, including hepatitis A virus(HAV), hepatitis B virus(HBV), hepatitis C virus(HCV), hepatitis D virus(HDV), and hepatitis E virus(HEV). In addition, multiplex q PCR has also been developed for genotyping HBV, HCV, and HEV subtypes. Although a single step multiplex q PCR assay for all six hepatitis viruses, i.e., A to G viruses, is not yet reported, it may be available in the near future as the technologies continue to advance. All studies use a conserved region of the viral genome as the basis of amplification and hydrolysis probes as the preferred chemistries for improved detection. Based on a standard plot prepared using varying concentrations of template and the observed threshold cycle value, it is possible to determine the linear dynamic range and to calculate an exact copy number of virus in the specimen. Advantages of multiplex q PCR assay over singleplex or other molecular techniques in samples from patients with co-infection include fast results, low cost, and a single step investigation process.展开更多
Objective To establish and modify quantitative real-time polymerase chain reaction(qPCR)-based serotyping assays to distinguish 97 pneumococcal serotypes.Methods A database of capsular polysaccharide(cps)loci sequence...Objective To establish and modify quantitative real-time polymerase chain reaction(qPCR)-based serotyping assays to distinguish 97 pneumococcal serotypes.Methods A database of capsular polysaccharide(cps)loci sequences was generated,covering 97 pneumococcal serotypes.Bioinformatics analyses were performed to identify the cps loci structure and target genes related to different pneumococcal serotypes with specific SNPs.A total of 27 novel qPCR serotyping assay primers and probes were established based on qPCR,while 27 recombinant plasmids containing serotype-specific DNA sequence fragments were constructed as reference target sequences to examine the specificity and sensitivity of the qPCR assay.A panel of pneumococcal reference strains was employed to evaluate the capability of pneumococcal serotyping.Results A total of 97 pneumococcal serotyping assays based on qPCR were established and modified,which included 64 serotypes previously reported as well as an additional 33 serotypes.Twenty-seven novel qPCR serotyping target sequences were implemented in the pneumococcal qPCR serotyping system.A total of 97 pneumococcal serotypes,which included 52 individual serotypes and 45 serotypes belonging to 20 serogroups,could not be identified as individual serotypes.The sensitivity of qPCR assays based on 27 target sequences was 1–100 copies/μL.The specificity of the qPCR assays was 100%,which were tested by a panel of 90 serotypes of the pneumococcal reference strains.Conclusion A total of 27 novel qPCR assays were established and modified to analyze 97pneumococcal serotypes.展开更多
BACKGROUND Extended-spectrumβ-lactamase(ESBL)-producing Escherichia coli(E.coli)are among the main pathogens in urinary tract infections(UTIs)among kidney transplant patients(KTPs).AIM To estimate the prevalence of E...BACKGROUND Extended-spectrumβ-lactamase(ESBL)-producing Escherichia coli(E.coli)are among the main pathogens in urinary tract infections(UTIs)among kidney transplant patients(KTPs).AIM To estimate the prevalence of ESBL-producing E.coli in KTPs and to evaluate the most prevalent serotypes and antibacterial susceptibility patterns of isolated bacteria in Tehran,Iran.METHODS A total of 60 clinical isolates of uropathogenic E.coli were collected from 3 kidney transplant centers from April to May 2019.Antimicrobial susceptibility testing was performed by the disk diffusion method as recommended by the Clinical Laboratory and Standards Institute.The serotyping of E.coli isolates was performed by the slide agglutination method.The presence of blaTEM,blaSHV,and bla CTX-M genes was evaluated by polymerase chain reaction.RESULTS The frequency of ESBL-producing E.coli in KTPs was found to be 33.4%.All of the 60 E.coli isolates were found to be susceptible to doripenem(100%)and ertapenem(100%).High resistance rates to ampicillin(86%),cefotaxime(80%),and cefazolin(77%)were also documented.The most frequent serotypes were serotype I(50%),serotype II(15%),serotype III(25%),and serotype VI(10%).The gene most frequently found was blaTEM(55%),followed by blaCTX-M(51%)and blaSHV(41%).CONCLUSION Molecular analysis showed that blaTEM was the most common ESBL-encoding gene.The high resistance toβ-lactams antibiotics(i.e.,ampicillin,cefotaxime,and cefazolin)found in E.coli from KTPs with UTIs remains a serious clinical challenge.Further efforts to control ESBL-producing E.coli should include the careful use of all antibiotics as well as barrier precautions to reduce spread.展开更多
Bovine Respiratory Disease (BRD) causes a severe form of pneumonia in all age of cattle. This study was designed to investigate the distribution of capsular types, serotypes, and virulence-associated genes of the majo...Bovine Respiratory Disease (BRD) causes a severe form of pneumonia in all age of cattle. This study was designed to investigate the distribution of capsular types, serotypes, and virulence-associated genes of the major bacterial pathogens from BRD outbreak samples in Ethiopia. In this study 166 samples were collected from clinically sick (<i>n</i> = 107) and pneumonic lung tissue (<i>n</i> = 59). Laboratory assay confirmed isolation of <i>M. haemolytica</i> 37 (22.29%), <i>P. multocida</i> 25 (15.06%), <i>B. trehalosi</i> 12 (7.23%), and <i>H. somni</i> 15 (9.04%). PCR assay of <i>P. multocida</i> capsular typing revealed 21 (84.0%) cap A (<i>hyaD-hyaC</i>) and 4 (16.0%) cap D (<i>dcbF</i>) strains. <i>M. haemolytica</i> serotypes belonged to A: 1, A: 2, and A: 6 from 26 (70.27%), 4 (10.81%), and 7 (18.92%) isolates, respectively. <i>P. multocida</i> biotyping showed isolation of A: 1, A: 2, and A: 3 from 3 (14.29%), 2 (9.52%), and 16 (76.19%) isolates, respectively. <i>M. haemolytica</i> harbored more than 60% <i>ssa</i> gene, and 90.91% <i>sodA</i> while <i>FbpA</i>, <i>TbpA</i>, and <i>lktC</i> genes were found in all isolates. Likewise, all <i>P. multocida</i> exhibited <i>toxA</i>, <i>FbpA</i>, <i>TbpA</i>, and <i>pmSLP</i> genes. The current finding showed that <i>M. haemolytica</i> serotype A: 1 is frequently associated with BRD followed by <i>P. multocida</i> biotype A: 3. These two isolates harbored diverse virulence-associated genes and presented the pathogenic potential of the current isolates. Thus, investigation of pathogenic strains of BRD, virulence genes distribution, and molecular epidemiology of the disease from wider areas of the country are essential. Hence, continuous outbreak surveillance and molecular approaches are indispensable in designing efficient prevention strategies.展开更多
Streptococcus suis serotype 2(S.suis 2)is a zoonotic pathogen that clinically causes severe swine and human infections(such as meningitis,endocarditis,and septicemia).In order to cause widespread diseases in different...Streptococcus suis serotype 2(S.suis 2)is a zoonotic pathogen that clinically causes severe swine and human infections(such as meningitis,endocarditis,and septicemia).In order to cause widespread diseases in different organs,S.suis 2 must colonize the host,break the blood barrier,and cause exaggerated inflammation.In the last few years,most studies have focused on a single virulence factor and its influences on the host.Membrane vesicles(MVs)can be actively secreted into the extracellular environment contributing to bacteria-host interactions.Gram-negative bacteria-derived outer membrane vesicles(OMVs)were recently shown to activate host Caspase-11-mediated non-canonical inflammasome pathway via deliverance of OMV-bound lipopolysaccharide(LPS),causing host cell pyroptosis.However,little is known about the effect of the MVs from S.suis 2(Gram-positive bacteria without LPS)on cell pyroptosis.Thus,we investigated the molecular mechanism by which S.suis 2 MVs participate in endothelial cell pyroptosis.In this study,we used proteomics,electron scanning microscopy,fluorescence microscope,Western blotting,and bioassays,to investigate the MVs secreted by S.suis 2.First,we demonstrated that S.suis 2 secreted MVs with an average diameter of 72.04 nm,and 200 proteins in MVs were identified.Then,we showed that MVs were transported to cells via mainly dynamin-dependent endocytosis.The S.suis 2 MVs activated NLRP3/Caspase-1/GSDMD canonical inflammasome signaling pathway,resulting in cell pyroptosis,but it did not activate the Caspase-4/-5 pathway.More importantly,endothelial cells produce large amounts of reactive oxygen species(ROS)and lost their mitochondrial membrane potential under induction by S.suis 2 MVs.The results in this study suggest for the first time that MVs from S.suis 2 were internalized by endothelial cells via mainly dynamin-dependent endocytosis and might promote NLRP3/Caspase-1/GSDMD pathway by mitochondrial damage,which produced mtDNA and ROS under induction,leading to the pyroptosis of endothelial cells.展开更多
Objective:To determine the distribution,phenotypic and genetic background of extended spectrumβ-lactamases(ESBL)-producing Klebsiella(K.)pneumoniae clinical isolates associated with K1 and K2 serotypes in two selecte...Objective:To determine the distribution,phenotypic and genetic background of extended spectrumβ-lactamases(ESBL)-producing Klebsiella(K.)pneumoniae clinical isolates associated with K1 and K2 serotypes in two selected hospitals in Malaysia.Methods:A total of 192 K.pneumoniae isolates were collected and subjected to antibiotic susceptibility,hypermucoviscosity test and multiplex PCR to detect the presence of K1-and K2-serotype associated genes.Multilocus sequence typing(MLST)was performed on ESBL-producing K.pneumoniae isolates presented with K1 and K2 serotypes,followed by phylogenetic analysis.Results:A total of 87 out of 192(45.3%)of the K.pneumoniae isolates collected were ESBL producers.However,only 8.3%(16/192)and 10.9%(21/192)of the total isolates were detected to carry K1-and K2-serotype associated genes,respectively.Statistical analysis showed that K1 and K2 capsular serotypes were not significantly associated with ESBL phenotype(P=0.196).However,they were significantly associated with hypervirulent,as demonstrated by the positive string test(P<0.001).MLST analysis revealed that ST23 as the predominant sequence type(ST)in the K1 serotype,while the ST in the K2 serotype is more diverse.Conclusions:Although the occurrence of ESBL-producing isolates among the hypervirulent strains was low,their coexistence warrants the need for continuous surveillance.MLST showed that these isolates were genetically heterogeneous.展开更多
[Objective]The aim was to provide a theoretical basis for preparation of polyclonal antibodies and monoclonal antibody that of type specificity, as well as Foot-and-mouth Disease Virus （FMDV） typing.[Method] The rec...[Objective]The aim was to provide a theoretical basis for preparation of polyclonal antibodies and monoclonal antibody that of type specificity, as well as Foot-and-mouth Disease Virus （FMDV） typing.[Method] The recombinant plasmid pGEM-CP1 that contained VPI gene of FMDV of serotype C was used as template for the VP1 and its C terminus coding fragments of FMDV of serotypes C amplification. The coding fragments of VP1 and its C terminus were respectively cloned into prokaryotic expression vector for prokaryotic expression and the reactionogenicity was detected. The purified fusion protein of FMDV VP1 and its C terminus of serotype C were used to construct the indirect ELISA method to detect positive sera of four serotypes A, O, C and Asia 1 of guinea pig and determine the cross reactivity of FMDV antibody of VP1 and its C terminus of serotype C with other three serotypes. [ Result] The recombinant prokaryotic expression plasmids of PPRO-CVP1 and pPRO-CVPlc were constructed, FMDV VP1 and its C terminus of serotype C were expressed in high level, and the molecular weight of target proteins was 33 kD and 20 kD respectively. Western blot result showed that the fusion protein of VP1 and its C terminus could react with the positive sera of guinea pig of the same serotype. ELISA results revealed that VP1 and its C terminus of serotype C are type-specific and no cross-reactivities were shown between guinea pig positive sera of FMDV of serotype C with the other three serotypes, and the C terminus showed better type-specificity. [ Conclusion] FMDV specific antigen of serotype C was obtained.展开更多
Background: Since 2021, high-risk Human Papilloma Virus (HR-HPV) testing has been the recommended screening test for cervical cancer for all settings;either used alone in a “test and treat” strategy, or with a triag...Background: Since 2021, high-risk Human Papilloma Virus (HR-HPV) testing has been the recommended screening test for cervical cancer for all settings;either used alone in a “test and treat” strategy, or with a triage test, with or without biopsy, before treatment. Cameroon has rolled out immunization against HPV 16 and 18, but studies show a higher prevalence of non-16/18 HR-HPV types. Objectives: Determine the prevalence of precancerous lesions, in women with HR-HPV infection and evaluate association of digital cervicography (DC) VIA/VILI positivity with HPV serotype, as a measure of their contribution to precancer and cancer incidence. Methodology: The study was cross-sectional, descriptive, and analytic. It took place at the Etoug-Ebe and Ekoudoum Baptist Hospitals in Yaoundé, during the period April-September 2022. We reviewed the records of women screened for cervical cancer between February 2020 and December 2021 and evaluated the prevalence of lesions on digital cervicography (DC) with VIA/VILI for women positive for HR-HPV serotypes. The data were analyzed using SPSS version 20.0 for Windows. P values Results: We identified 315 cases with a positive HR-HPV deoxyribonucleic acid (DNA) test, 224 (71.1%) had a DC VIA/VILI triage test done. Of these, 30 (13.4%) women had a positive DC VIA/VILI, with five women (2.2%) having lesions suggestive of cancer. Out of 11 cases positive for HPV 16 alone, 05 (45.5%) had a positive DC VIA/VILI test. Of the 14 cases positive for HPV 18 alone, 03 (21.4%) had a positive VIA/VILI, meanwhile only 19 (10.7%) of the 177 cases positive for non-16/18 HPV had a positive VIA/VILI test. Conclusion: A high proportion of women (13.4%) with HR HPV had a positive DC VIA/VILI, with a significant proportion (2.2%) having lesions suggestive of invasive cervical cancer HR-HPV serotype was associated with DC VIA/VILI positivity;HPV 16 had the strongest association (45.5%), followed by HPV 18 (21.4%), and non-16/18 HR-HPV (10.7%), suggesting a decreasing order of oncogenicity.展开更多
Background and Objectives: Dengue is an arbovirosis caused by the dengue virus with 04 serotypes. The aim of the study was to characterise the four serotypes (DENV-1, DENV-2, DENV-3 and DENV-4) of the dengue virus cir...Background and Objectives: Dengue is an arbovirosis caused by the dengue virus with 04 serotypes. The aim of the study was to characterise the four serotypes (DENV-1, DENV-2, DENV-3 and DENV-4) of the dengue virus circulating in Ouagadougou, Burkina Faso. Methods: This was a descriptive analytical study that included 2833 patients and was carried out from January 2021 to December 2022. Rapid diagnosis of dengue was performed using the “Dengue Duo (AgNS1/IgM/IgG)” kit (SD Bioline, Korea). Viral RNA was extracted using the QIAGEN RNA RNeasy Plus Mini Kit (Quiagen, Germany) and virus serotypes were identified using the DENGUE Real-TM Genotype PCR kit (Sacace biotechnologies, Italy). Platelet counts were also performed using the XN-1500 Sysmex. Results: The prevalence of acute infections (NS1Ag positive) by TDR was 5.7% (162/2833), with the peak of dengue virus infection occurring between October and November. On the other hand, the AgNS1+ samples tested by RT-PCR were 53.7% positive for dengue virus;this shows the extent of probable cross-reactions with rapid diagnostic tests and false positives. Serotype 1 accounted for 52.6%, 28.4% had serotype 3, 16.8% had serotype 2 and 2.1% had serotype 4. We found cases of co-infection with DENV-1 and DENV-2 in two patients, co-infection with DENV-1 and DENV-3 in three patients, co-infection with DENV-1 and DENV-4 in one patient, co-infection with DENV-3 and DENV-4 in one patient and co-infection with three serotypes, DENV-1, DENV-2 and DENV-3 in one patient. Conclusion: The study showed that all four serotypes of the dengue virus were circulating in Ouagadougou. Serotype 1 was predominant.展开更多
Goals: The aim of this study was to determine the antibiotic resistance profile of serotypes of Streptococcus pneumoniae strains circulating in Bangui. Methodology: A prospective and analytical analysis was carried ou...Goals: The aim of this study was to determine the antibiotic resistance profile of serotypes of Streptococcus pneumoniae strains circulating in Bangui. Methodology: A prospective and analytical analysis was carried out at the National Laboratory of Clinical Biology and Public Health from 2017 to 2022. The strains came from our study on the contribution to the study of antibiotic sensitivity of Streptococcus pneumoniae strains. The multiplex PCR test was used for its cost-effectiveness in terms of amplifiers which can be purified in order to be sequenced. It also makes it possible to detect several germs as well as their serotypes. For a PCR reaction, several elements are involved in the reaction medium or Master Mix. These are the desoxyribonucleotides (dNTPs), the magnesium ions (MgCl2) and the primers. A set of 14 primers divided into 3 classes were used. Class 1 primers served as an internal control by targeting the cpsA gene. It is a highly conserved gene found in capsular loci characterized to date. The primers of the second class were used to target specific serotypes by specific reactions (out of six possibilities). The group reaction was carried out using the primers of the third class in order to carry out an initial screening of the samples and to classify the pneumococcal isolates. Related serotypes were grouped based on the amplification of common genes. Using the technique of electrophoresis on agarose gel and an ultraviolet radiation device, the migration bands are then visualized and analyzed. The data collected had been entered into Excel 2010 and analyzed with Epi info 7. The exact Fischer chi2 test at the 5% threshold, the relative risk and its 95% confidence interval were used to compare the proportions and determine the associations. Results: 187 antibiotic-resistant strains of Streptococcus pneumoniae were collected. The average frequency of serotypes 1, 9A, 4 and untypeable identified were 43.59%, 18.18%, 18.27% and 39.57% respectively. The frequency of serotype 1 was predominant for the age group over five years old with 56.88%. The male sex was predominant with 55.08% for serotype 1. Resistance to penicillin and gentamicin for serotype 1 during this study, for the age group under 5 years old, was 77%. For serotypes 19A and 4, tetracycline resistance was predominant with 20% for the age group under 5 years. The resistance to penicillin and gentamicin of non-typeable serotypes was 33% for the age group under 5 years old. For the age group over 5 years old, resistance to erythromycin predominated at 37%. The distribution of serotypes by sex depending on antibiotic resistance was variable. There was a statistically significant association between identified serotypes and antibiotic resistance (p Conclusion: The study determined serotypes 1, serotypes 19A, serotypes 4 and non-typeable serotypes. These results would be due to the quality of vaccination or poor protection of vaccines.展开更多
[Objective] This study aimed to establish a method for quantitative detection of mRNA transcriptional level of SS2 adhesive related-factors of Streptococcus suis serotype 2 （SS2） by fluorescent quantitative PCR. []V...[Objective] This study aimed to establish a method for quantitative detection of mRNA transcriptional level of SS2 adhesive related-factors of Streptococcus suis serotype 2 （SS2） by fluorescent quantitative PCR. []Vlethod] The gene fragments en- coding SS2 adhesive related-factors MRP, FBPS and CPS2J and a housekeeping gene aroA were amplified by reverse transcription PCR from the total RNA of SS2, cloned, and sequenced. The recombinant plasmids containing the target genes were constructed, and used as templates in Real-time PCR. [Result] Dynamic curves, stan- dard curves and melting curves of the adhesive related-factors and aroA were ob- tained by the optimized Real-time PCR system. The standard curves showed a good linear relationship between template copy number and circulation number, and the correlation coefficients （FF） of the standard curves were over 0.995. Also, these as- says were highly specific a^d there was single specific melting peak for every gene. Moreover, the assays were highly sensitive and had a detection limit of 1.0×102 copies in 1 μl of initial templates. Finally, it was highly repeatable and had a coeffi- cient of variation less than 2% for intra-assay. [Conclusion] This study will provide a way to reveal the adhesion mechanism of SS2 to different host cells at molecular level.展开更多
Nutritional value of vegetables and high prices of meat and meat originated food compel common people to consume plant originated food particularly salad vegetables. Microbial population of vegetable surfaces contains...Nutritional value of vegetables and high prices of meat and meat originated food compel common people to consume plant originated food particularly salad vegetables. Microbial population of vegetable surfaces contains a large number of pathogenic bacteria including members of Enterobactereace like Escherichia coli (E. coli). A survey was conducted in three major markets of Rawalpindi, Pakistan. Tomato, lettuce, cabbage and cucumber samples were collected from three shops of each market. Each vegetable was analysed as unwashed and washed for total coliforms, faecal coliforms and E. coli by FAO (Food Quality Manual). About two hundred and fifty E. coli isolates were preserved, serotyped for presence of O157 serotype. Total coliforms, faecal coliforms and E. coli count exceeded the permissible limits in most samples. The highest Total coliforms were associated with cabbage (3.78 log10 cfu/g). Cucumber was the least contaminated by Total coliforms (2.15 log10 cfu/g). E. coli was detected in tomato, lettuce, cucumber and cabbage. Washed samples showed reduced bacterial population. Seventy six isolates of E. coli were biochemically characterized and serotyped for O157 antigen. A majority of strains could not be identified by serotyping. These findings conclude with high potentially pathogenic microbial load on salad vegetables and urge for preventive action on priority basis.展开更多
Campylobacter is one of the most common bacterial enteropathogens of food borne origin in industrialized countries with C. jejuni being the most common species followed by C. coli. The prevalence of Campylobacters in ...Campylobacter is one of the most common bacterial enteropathogens of food borne origin in industrialized countries with C. jejuni being the most common species followed by C. coli. The prevalence of Campylobacters in and around Chandigarh, India was studied by phenotypic and genotypic methods. Fecal samples from 1145 diarrheal patients and 102 healthy subjects from hospital and community were cultured on Campylobacter media and identified by Gram stain, biochemical investigations and serotyping. Molecular identification of Campylobacter isolates was done using specific primers to unique regions of 16S rRNA, Campylobacter jejuni (hipO), Campylobacter coli (aspK), Campylobacter lari (glyA) and Campylobacter upsaliensis (lpxA) genes. Identification of specific genes to look for resistance to nalidixic acid, ciprofloxacin, tetracyclin and streptomycin was also done. Campylobacters were isolated from 2.6% of patients with diarrhea. Campylobacteriosis was more prevalent in children ≤5 years old and during summer season. The most frequent serotypes were S:27, B:2, Z5:52 and V:32. All the Campylobacters isolated by culture were confirmed genotypically by identification of 16S rRNA, hipO and aspK genes. Of the 30 isolates, 27 were C. jejuni and 3 were C. coli. No C. lari or C. upsaliensis were detected. Antibiotic resistance was 40% for nalidixic acid, 23.3% for ciprofloxacin, 50% for tetracyclin and 20% for streptomycin. Campylobacter prevalence is low in the region with C. jejuni being the most common species. A high degree of resistance was found for nalidixic acid and tetracyclin but moderate for ciprofloxacin and streptomycin.展开更多
Campylobacter is one of the most common food-borne bacterial enteropathogens. We planned to investigate the prevalence and antibiotic resistogram of Campylobacter in poultry in and around Chandigarh. Poultry samples (...Campylobacter is one of the most common food-borne bacterial enteropathogens. We planned to investigate the prevalence and antibiotic resistogram of Campylobacter in poultry in and around Chandigarh. Poultry samples (n = 127) were obtained from slaughter houses/retail outlets and cultured microaerophilically on Campylobacter media. The isolates were identified phenotypically and by molecular investigation. Identification of specific genes to look for resistance to nalidixic acid, ciprofloxacin, tetracyclin and streptomycin was also done. Campylobacter was isolated from 57/127 (44.9%) of the samples. The most frequent serotypes identified were B: 2, S: 27, Z5: 52 and Z7: 57. All culture isolates (100%) were reconfirmed as Campylobacter by 16S rRNA polymerase chain reaction. Molecular identification of isolates revealed the presence of C. jejuni in 45 (79.0%), C. coli in 1 (1.8%) and co-infection of C. coli and C. jejuni in 11 (19.3%). No C. lari and C. upsaliensis were detected. Antibiogram typing showed nalidixic acid resistance in 36.8%, ciprofloxacin resistance in 35.0% and 31.5% resistance for both streptomycin and tetracyclin. A high level of Campylobacter prevalence was found among the poultry with C. jejuni being the most commonly isolated species. Resistance to major antibiotics among Campylobacter isolates from poultry was also very high. The study of prevalence of Campylobacter in poultry and its resistance to major antibiotics will help to plan risk burden strategies throughout the food chain.展开更多
A sandwich format immunochromatographic assay for detecting foot-and-mouth disease virus (FMDV) serotypes was developed. In this rapid test,affinity purified polyclonal antibodies from Guinea pigs which were immunized...A sandwich format immunochromatographic assay for detecting foot-and-mouth disease virus (FMDV) serotypes was developed. In this rapid test,affinity purified polyclonal antibodies from Guinea pigs which were immunized with sucking-mouse adapted FMD virus (A/AV88(L) strain) were conjugated to colloidal gold beads and used as the capture antibody,and affinity purified polyclonal antibodies from rabbits which were immunized with cell-culture adapted FMD virus (A/CHA/09 strain) were used as detector antibody. On the nitrocellulose membrane of the immunochromatographic strip,the capture antibody was laid on a sample pad,the detector antibody was printed at the test line(T) and goat anti-guinea pigs IgG antibodies were immobilized to the control line(C). The lower detection limit of the test for a FMDV 146S antigen is 11.7ng/ml as determined in serial tests after the strip device was assembled and the assay condition optimization. No cross reactions were found with FMDV serotype C,Swine vesicular disease (SVD),Vesicular stomatiti svirus (VSV) and vesicular exanthema of swine virus (VES) viral antigens with this rapid test. Clinically,the diagnostic sensitivity of this test for FMDV serotypes A was 88.7% which is as same as an indirect-sandwich ELISA. The specificity of this strip test was 98.2% and is comparable to the 98.7% obtained with indirect-sandwich ELISA. This rapid strip test is simple,easy and fast for clinical testing on field sites; no special instruments and skills are required,and the result can be obtained within 15 min. To our knowledge,this is the first rapid immunochromatogarpic assay for serotype A of FMDV.展开更多
The pathogenesis of HBsAg （＋）/HBsAb （＋） double positive hepatitis B virus infection was investigated by simulating HBsAg/HBsAb coexistence in vitro and establishing HBsAg/HBsAb double positive model in vivo. Euk...The pathogenesis of HBsAg （＋）/HBsAb （＋） double positive hepatitis B virus infection was investigated by simulating HBsAg/HBsAb coexistence in vitro and establishing HBsAg/HBsAb double positive model in vivo. Eukaryotic expression plasmids PCI-SY, PCI-adw, PCI-adr, PCI-ayw, which expressed S gene product of different serotypes, were constructed and transfected into HepG2 cells. Recombinant proteins were purified from the transfected cells. At the same time, HBsAg mouse antiserum was obtained by immunizing mice with PCI-SY plasmid. HBsAg/HBsAb coexistence was simulated using these antigens and antiserum. Furthermore, the expression plasmids expressing different serotypes of S gene product including PCI-adw, PCI-adr, and PCI-ayw were injected into mice via tail vein. HBsAg and HBsAb in mice sera were tested at the first and 7th day respectively after antigen plasmids injection. Both in vitro simulation and in vivo animal models demonstrated that HBsAg antigen and HBsAb of the same serotypes Could not coexist, but HBsAg antigen and HBsAb of different serotype could coexist. HBsAg/HBsAb double positive hepatitis B virus infection could be due to infection of viruses of different serotypes.展开更多
BACKGROUND Hand,foot,and mouth disease(HFMD)has become one of the most common infectious diseases in China.Before 2016,the primary causal serotypes were enterovirus A71(EV-A71)and coxsackievirus A16(CV-A16).Following ...BACKGROUND Hand,foot,and mouth disease(HFMD)has become one of the most common infectious diseases in China.Before 2016,the primary causal serotypes were enterovirus A71(EV-A71)and coxsackievirus A16(CV-A16).Following the introduction of EV-A71 vaccines in China since 2016,the situation could change.CV-A6 has recently replaced EV-A71 and CV-A16 in some areas of China.However,the epidemiological characteristics of central China remain unknown.AIM To investigate the clinical symptoms and pathogen spectrum of HFMD in Shiyan City,central China,in recent years.METHODS The epidemiological,clinical,and laboratory data from HFMD cases reported to the Shiyan Center for Disease Control and Prevention between January 2016 and December 2020 were analyzed.196 throat swab specimens were collected from hospitalized HFMD patients between January 2018 and December 2020.To detect and genotype enteroviruses,real-time reverse transcription-polymerase chain reaction and sequencing of the 5'-untranslated region were used.In Shiyan,168 laboratory-confirmed HFMD cases were studied using a logistic regression model to determine the effect of predominant enterovirus serotypes.Based on the logistic regression model,the least absolute shrinkage and selection operator model was used to analyze the correlation between CV-A6 infection and various clinical characteristics in HFMD patients in Shiyan.RESULTS From 2016 to 2020,35840 HFMD cases were reported in Shiyan.The number of cases decreased by 48.4%from 2016 to 2017.Approximately 1.58-fold increases were found in 2018 and 2019 when compared to the previous year,respectively.In 2020,a decrease of about 85.5%was reported when compared to 2019.The most common serotypes shifted from EV-A71 and CV-A16(about 60%-80%in 2016 and 2018)to others(more than 80.0%in 2017,2019,and 2020).EV-A71 lost its dominance in 2017 in Shiyan.Among 196 confirmed HFMD cases,85.7%tested positive for enterovirus,with CV-A6 being the most common serotype(121/168,72.0%).The positive rates for CV-A16 and CVA10 were 4.8%and 3.0%,respectively.There was no EV-A71 discovered.Infection with CV-A6 was linked to fever,myocardial damage,increased creatine kinase MB isoenzyme,and lactate dehydrogenase levels.CONCLUSION CV-A6 was the most common enterovirus serotype in Shiyan City,replacing EV-A71 and CV-A16 as the HFMD pathogen.Developing vaccines against CV-A6 or multiple pathogens,as well as rising CV-A6 surveillance,will help prevent HFMD in central China.展开更多
216 avian pathogenic Escherichia coli （APEC） isolates were obtained from poultry with colibacillosis in different areas of China. Among them, 195 were serotyped as 078, 088, and 093. Thirteen virulence-associated ge...216 avian pathogenic Escherichia coli （APEC） isolates were obtained from poultry with colibacillosis in different areas of China. Among them, 195 were serotyped as 078, 088, and 093. Thirteen virulence-associated genes, including fimC, iucD, iss, tsh, fyuA, irp2, eaeA, hlyE, colV, papC, stx2f, vat, and astA, were submitted to PCR amplification. The fimC gene was the most prevalent with a detection rate of 93.6%, followed by iucD （70.8%）, iss （58.8%）, and tsh （51.4%） in APEC isolates. The detection rate of high pathogenicity islands （HPI）-associatedfyuA and irp2 genes were both 44.9%, with no LEE （the locus of enterocyte effacement） island-associated gene eaeA detected. In terms of distribution patterns of the 13 virulence-associated genes, 5 isolates harborbed 10 genes, 19 isolates contained onlyfimC gene, and only 4 isolates had no virulence-associated gene detected. Different correlations of the virulence-associated genes with O serotypes were also investigated and 50% 078 isolates had a gene distribution patterns of fimC^＋iucD^＋irp2^＋fyuA^＋iss^＋colV^＋tsh^＋.展开更多
Objective:To identify the circulating serotypes of human echovinis in Malaysia from 2002 in 2013.Methods:A toial of 31 retrospective samples from non-polio acute flacid paralysis,hand-food-and-mouth disease,viral meni...Objective:To identify the circulating serotypes of human echovinis in Malaysia from 2002 in 2013.Methods:A toial of 31 retrospective samples from non-polio acute flacid paralysis,hand-food-and-mouth disease,viral meningitis and enterovirus cases were subjected to amplification of partial VPI gene by RT-PCR.Results:Sequencing and phylogeneiic analysis of the partial sequences identified presence of human echovinis and human coxsackie viruses.It was found that echovinis 11 was the commonly circulating serotype followed by echovinis6.echovinis 7.echovinis 3.echovinis 9.echovinis 30 and echovinis I in decreasing order.Additionally two types of human coxsackie virus isolates were detected which were coxsackie A24 and B3.Condusions:From the findings,there is a possibility that echovinis 11 is the predominant serotype among Malaysian patients with echovinis infection.However,a larger sample size will yield a more confident result to support this evidence.____________________展开更多
Hepatitis-hydropericardium syndrome(HHS)is an infectious disease caused by fowl adenovirus serotype 4(FAdV-4).Several structural and non-structural proteins of FAdV-4 have been expressed in Escherichia coli and baculo...Hepatitis-hydropericardium syndrome(HHS)is an infectious disease caused by fowl adenovirus serotype 4(FAdV-4).Several structural and non-structural proteins of FAdV-4 have been expressed in Escherichia coli and baculovirus expression system to develop candidate subunit vaccines.However,the protective efficiency of baculovirus-expressed penton base protein has not been assessed.In this study,two recombinant capsid proteins,penton base and fiber-2,were constructed.And then,penton base and fiber-2 were administrated alone or together to specific pathogen-free(SPF)chickens at 14 days of life and boosted at 28 days of life.At 42 days of life,the immunized groups and the control group were challenged with FAdV-4 virulent strain.Results show that inoculating penton base or penton base+fiber-2 provided 100%protection to the chickens.All groups vaccinated with the recombinant protein produced detectable antibodies and showed no apparent lesions.Thus,baculovirus-expressed penton base protein is a promising candidate subunit vaccine.展开更多
文摘The present review describes the current status of multiplex quantitative real time polymerase chain reaction(q PCR) assays developed and used globally for detection and subtyping of hepatitis viruses in body fluids. Several studies have reported the use of multiplex q PCR for the detection of hepatitis viruses, including hepatitis A virus(HAV), hepatitis B virus(HBV), hepatitis C virus(HCV), hepatitis D virus(HDV), and hepatitis E virus(HEV). In addition, multiplex q PCR has also been developed for genotyping HBV, HCV, and HEV subtypes. Although a single step multiplex q PCR assay for all six hepatitis viruses, i.e., A to G viruses, is not yet reported, it may be available in the near future as the technologies continue to advance. All studies use a conserved region of the viral genome as the basis of amplification and hydrolysis probes as the preferred chemistries for improved detection. Based on a standard plot prepared using varying concentrations of template and the observed threshold cycle value, it is possible to determine the linear dynamic range and to calculate an exact copy number of virus in the specimen. Advantages of multiplex q PCR assay over singleplex or other molecular techniques in samples from patients with co-infection include fast results, low cost, and a single step investigation process.
基金supported by a grant from Beijing Municipal Natural Science Foundation [L212011]National Institute for Communicable Disease Control and Prevention,Chinese Center for Disease Control and Prevention [131031102000210003&102393230020020000002]。
文摘Objective To establish and modify quantitative real-time polymerase chain reaction(qPCR)-based serotyping assays to distinguish 97 pneumococcal serotypes.Methods A database of capsular polysaccharide(cps)loci sequences was generated,covering 97 pneumococcal serotypes.Bioinformatics analyses were performed to identify the cps loci structure and target genes related to different pneumococcal serotypes with specific SNPs.A total of 27 novel qPCR serotyping assay primers and probes were established based on qPCR,while 27 recombinant plasmids containing serotype-specific DNA sequence fragments were constructed as reference target sequences to examine the specificity and sensitivity of the qPCR assay.A panel of pneumococcal reference strains was employed to evaluate the capability of pneumococcal serotyping.Results A total of 97 pneumococcal serotyping assays based on qPCR were established and modified,which included 64 serotypes previously reported as well as an additional 33 serotypes.Twenty-seven novel qPCR serotyping target sequences were implemented in the pneumococcal qPCR serotyping system.A total of 97 pneumococcal serotypes,which included 52 individual serotypes and 45 serotypes belonging to 20 serogroups,could not be identified as individual serotypes.The sensitivity of qPCR assays based on 27 target sequences was 1–100 copies/μL.The specificity of the qPCR assays was 100%,which were tested by a panel of 90 serotypes of the pneumococcal reference strains.Conclusion A total of 27 novel qPCR assays were established and modified to analyze 97pneumococcal serotypes.
基金Supported by Research Department of School of Medicine Shahid Beheshti University of Medical Sciences,No.17920,and accepted by the ethic committee,Code.IR.SBMU.MSP.REC.1398.349.
文摘BACKGROUND Extended-spectrumβ-lactamase(ESBL)-producing Escherichia coli(E.coli)are among the main pathogens in urinary tract infections(UTIs)among kidney transplant patients(KTPs).AIM To estimate the prevalence of ESBL-producing E.coli in KTPs and to evaluate the most prevalent serotypes and antibacterial susceptibility patterns of isolated bacteria in Tehran,Iran.METHODS A total of 60 clinical isolates of uropathogenic E.coli were collected from 3 kidney transplant centers from April to May 2019.Antimicrobial susceptibility testing was performed by the disk diffusion method as recommended by the Clinical Laboratory and Standards Institute.The serotyping of E.coli isolates was performed by the slide agglutination method.The presence of blaTEM,blaSHV,and bla CTX-M genes was evaluated by polymerase chain reaction.RESULTS The frequency of ESBL-producing E.coli in KTPs was found to be 33.4%.All of the 60 E.coli isolates were found to be susceptible to doripenem(100%)and ertapenem(100%).High resistance rates to ampicillin(86%),cefotaxime(80%),and cefazolin(77%)were also documented.The most frequent serotypes were serotype I(50%),serotype II(15%),serotype III(25%),and serotype VI(10%).The gene most frequently found was blaTEM(55%),followed by blaCTX-M(51%)and blaSHV(41%).CONCLUSION Molecular analysis showed that blaTEM was the most common ESBL-encoding gene.The high resistance toβ-lactams antibiotics(i.e.,ampicillin,cefotaxime,and cefazolin)found in E.coli from KTPs with UTIs remains a serious clinical challenge.Further efforts to control ESBL-producing E.coli should include the careful use of all antibiotics as well as barrier precautions to reduce spread.
文摘Bovine Respiratory Disease (BRD) causes a severe form of pneumonia in all age of cattle. This study was designed to investigate the distribution of capsular types, serotypes, and virulence-associated genes of the major bacterial pathogens from BRD outbreak samples in Ethiopia. In this study 166 samples were collected from clinically sick (<i>n</i> = 107) and pneumonic lung tissue (<i>n</i> = 59). Laboratory assay confirmed isolation of <i>M. haemolytica</i> 37 (22.29%), <i>P. multocida</i> 25 (15.06%), <i>B. trehalosi</i> 12 (7.23%), and <i>H. somni</i> 15 (9.04%). PCR assay of <i>P. multocida</i> capsular typing revealed 21 (84.0%) cap A (<i>hyaD-hyaC</i>) and 4 (16.0%) cap D (<i>dcbF</i>) strains. <i>M. haemolytica</i> serotypes belonged to A: 1, A: 2, and A: 6 from 26 (70.27%), 4 (10.81%), and 7 (18.92%) isolates, respectively. <i>P. multocida</i> biotyping showed isolation of A: 1, A: 2, and A: 3 from 3 (14.29%), 2 (9.52%), and 16 (76.19%) isolates, respectively. <i>M. haemolytica</i> harbored more than 60% <i>ssa</i> gene, and 90.91% <i>sodA</i> while <i>FbpA</i>, <i>TbpA</i>, and <i>lktC</i> genes were found in all isolates. Likewise, all <i>P. multocida</i> exhibited <i>toxA</i>, <i>FbpA</i>, <i>TbpA</i>, and <i>pmSLP</i> genes. The current finding showed that <i>M. haemolytica</i> serotype A: 1 is frequently associated with BRD followed by <i>P. multocida</i> biotype A: 3. These two isolates harbored diverse virulence-associated genes and presented the pathogenic potential of the current isolates. Thus, investigation of pathogenic strains of BRD, virulence genes distribution, and molecular epidemiology of the disease from wider areas of the country are essential. Hence, continuous outbreak surveillance and molecular approaches are indispensable in designing efficient prevention strategies.
基金supported by the National Natural Science Foundation of China(U22A20520)the Innovation Team Project of Modern Agricultural Industrial Technology System of Guangdong Province,China(2023KJ119)the Natural Science Foundation Program of Guangdong Province,China(2023A1515012206)。
文摘Streptococcus suis serotype 2(S.suis 2)is a zoonotic pathogen that clinically causes severe swine and human infections(such as meningitis,endocarditis,and septicemia).In order to cause widespread diseases in different organs,S.suis 2 must colonize the host,break the blood barrier,and cause exaggerated inflammation.In the last few years,most studies have focused on a single virulence factor and its influences on the host.Membrane vesicles(MVs)can be actively secreted into the extracellular environment contributing to bacteria-host interactions.Gram-negative bacteria-derived outer membrane vesicles(OMVs)were recently shown to activate host Caspase-11-mediated non-canonical inflammasome pathway via deliverance of OMV-bound lipopolysaccharide(LPS),causing host cell pyroptosis.However,little is known about the effect of the MVs from S.suis 2(Gram-positive bacteria without LPS)on cell pyroptosis.Thus,we investigated the molecular mechanism by which S.suis 2 MVs participate in endothelial cell pyroptosis.In this study,we used proteomics,electron scanning microscopy,fluorescence microscope,Western blotting,and bioassays,to investigate the MVs secreted by S.suis 2.First,we demonstrated that S.suis 2 secreted MVs with an average diameter of 72.04 nm,and 200 proteins in MVs were identified.Then,we showed that MVs were transported to cells via mainly dynamin-dependent endocytosis.The S.suis 2 MVs activated NLRP3/Caspase-1/GSDMD canonical inflammasome signaling pathway,resulting in cell pyroptosis,but it did not activate the Caspase-4/-5 pathway.More importantly,endothelial cells produce large amounts of reactive oxygen species(ROS)and lost their mitochondrial membrane potential under induction by S.suis 2 MVs.The results in this study suggest for the first time that MVs from S.suis 2 were internalized by endothelial cells via mainly dynamin-dependent endocytosis and might promote NLRP3/Caspase-1/GSDMD pathway by mitochondrial damage,which produced mtDNA and ROS under induction,leading to the pyroptosis of endothelial cells.
基金supported by the Ministry of Higher Education under the Fundamental Research Grant Scheme(FRGS/1/2021/SKK0/UPM/02/8)the Universiti Putra Malaysia Research University Grant Scheme(GP/IPS/2021/9702000).
文摘Objective:To determine the distribution,phenotypic and genetic background of extended spectrumβ-lactamases(ESBL)-producing Klebsiella(K.)pneumoniae clinical isolates associated with K1 and K2 serotypes in two selected hospitals in Malaysia.Methods:A total of 192 K.pneumoniae isolates were collected and subjected to antibiotic susceptibility,hypermucoviscosity test and multiplex PCR to detect the presence of K1-and K2-serotype associated genes.Multilocus sequence typing(MLST)was performed on ESBL-producing K.pneumoniae isolates presented with K1 and K2 serotypes,followed by phylogenetic analysis.Results:A total of 87 out of 192(45.3%)of the K.pneumoniae isolates collected were ESBL producers.However,only 8.3%(16/192)and 10.9%(21/192)of the total isolates were detected to carry K1-and K2-serotype associated genes,respectively.Statistical analysis showed that K1 and K2 capsular serotypes were not significantly associated with ESBL phenotype(P=0.196).However,they were significantly associated with hypervirulent,as demonstrated by the positive string test(P<0.001).MLST analysis revealed that ST23 as the predominant sequence type(ST)in the K1 serotype,while the ST in the K2 serotype is more diverse.Conclusions:Although the occurrence of ESBL-producing isolates among the hypervirulent strains was low,their coexistence warrants the need for continuous surveillance.MLST showed that these isolates were genetically heterogeneous.
基金Supported by Key Projects in the National Science &Technology Pillar Program during the Eleventh Five-year Plan Period (2006BAD06A14)~~
文摘[Objective]The aim was to provide a theoretical basis for preparation of polyclonal antibodies and monoclonal antibody that of type specificity, as well as Foot-and-mouth Disease Virus （FMDV） typing.[Method] The recombinant plasmid pGEM-CP1 that contained VPI gene of FMDV of serotype C was used as template for the VP1 and its C terminus coding fragments of FMDV of serotypes C amplification. The coding fragments of VP1 and its C terminus were respectively cloned into prokaryotic expression vector for prokaryotic expression and the reactionogenicity was detected. The purified fusion protein of FMDV VP1 and its C terminus of serotype C were used to construct the indirect ELISA method to detect positive sera of four serotypes A, O, C and Asia 1 of guinea pig and determine the cross reactivity of FMDV antibody of VP1 and its C terminus of serotype C with other three serotypes. [ Result] The recombinant prokaryotic expression plasmids of PPRO-CVP1 and pPRO-CVPlc were constructed, FMDV VP1 and its C terminus of serotype C were expressed in high level, and the molecular weight of target proteins was 33 kD and 20 kD respectively. Western blot result showed that the fusion protein of VP1 and its C terminus could react with the positive sera of guinea pig of the same serotype. ELISA results revealed that VP1 and its C terminus of serotype C are type-specific and no cross-reactivities were shown between guinea pig positive sera of FMDV of serotype C with the other three serotypes, and the C terminus showed better type-specificity. [ Conclusion] FMDV specific antigen of serotype C was obtained.
文摘Background: Since 2021, high-risk Human Papilloma Virus (HR-HPV) testing has been the recommended screening test for cervical cancer for all settings;either used alone in a “test and treat” strategy, or with a triage test, with or without biopsy, before treatment. Cameroon has rolled out immunization against HPV 16 and 18, but studies show a higher prevalence of non-16/18 HR-HPV types. Objectives: Determine the prevalence of precancerous lesions, in women with HR-HPV infection and evaluate association of digital cervicography (DC) VIA/VILI positivity with HPV serotype, as a measure of their contribution to precancer and cancer incidence. Methodology: The study was cross-sectional, descriptive, and analytic. It took place at the Etoug-Ebe and Ekoudoum Baptist Hospitals in Yaoundé, during the period April-September 2022. We reviewed the records of women screened for cervical cancer between February 2020 and December 2021 and evaluated the prevalence of lesions on digital cervicography (DC) with VIA/VILI for women positive for HR-HPV serotypes. The data were analyzed using SPSS version 20.0 for Windows. P values Results: We identified 315 cases with a positive HR-HPV deoxyribonucleic acid (DNA) test, 224 (71.1%) had a DC VIA/VILI triage test done. Of these, 30 (13.4%) women had a positive DC VIA/VILI, with five women (2.2%) having lesions suggestive of cancer. Out of 11 cases positive for HPV 16 alone, 05 (45.5%) had a positive DC VIA/VILI test. Of the 14 cases positive for HPV 18 alone, 03 (21.4%) had a positive VIA/VILI, meanwhile only 19 (10.7%) of the 177 cases positive for non-16/18 HPV had a positive VIA/VILI test. Conclusion: A high proportion of women (13.4%) with HR HPV had a positive DC VIA/VILI, with a significant proportion (2.2%) having lesions suggestive of invasive cervical cancer HR-HPV serotype was associated with DC VIA/VILI positivity;HPV 16 had the strongest association (45.5%), followed by HPV 18 (21.4%), and non-16/18 HR-HPV (10.7%), suggesting a decreasing order of oncogenicity.
文摘Background and Objectives: Dengue is an arbovirosis caused by the dengue virus with 04 serotypes. The aim of the study was to characterise the four serotypes (DENV-1, DENV-2, DENV-3 and DENV-4) of the dengue virus circulating in Ouagadougou, Burkina Faso. Methods: This was a descriptive analytical study that included 2833 patients and was carried out from January 2021 to December 2022. Rapid diagnosis of dengue was performed using the “Dengue Duo (AgNS1/IgM/IgG)” kit (SD Bioline, Korea). Viral RNA was extracted using the QIAGEN RNA RNeasy Plus Mini Kit (Quiagen, Germany) and virus serotypes were identified using the DENGUE Real-TM Genotype PCR kit (Sacace biotechnologies, Italy). Platelet counts were also performed using the XN-1500 Sysmex. Results: The prevalence of acute infections (NS1Ag positive) by TDR was 5.7% (162/2833), with the peak of dengue virus infection occurring between October and November. On the other hand, the AgNS1+ samples tested by RT-PCR were 53.7% positive for dengue virus;this shows the extent of probable cross-reactions with rapid diagnostic tests and false positives. Serotype 1 accounted for 52.6%, 28.4% had serotype 3, 16.8% had serotype 2 and 2.1% had serotype 4. We found cases of co-infection with DENV-1 and DENV-2 in two patients, co-infection with DENV-1 and DENV-3 in three patients, co-infection with DENV-1 and DENV-4 in one patient, co-infection with DENV-3 and DENV-4 in one patient and co-infection with three serotypes, DENV-1, DENV-2 and DENV-3 in one patient. Conclusion: The study showed that all four serotypes of the dengue virus were circulating in Ouagadougou. Serotype 1 was predominant.
文摘Goals: The aim of this study was to determine the antibiotic resistance profile of serotypes of Streptococcus pneumoniae strains circulating in Bangui. Methodology: A prospective and analytical analysis was carried out at the National Laboratory of Clinical Biology and Public Health from 2017 to 2022. The strains came from our study on the contribution to the study of antibiotic sensitivity of Streptococcus pneumoniae strains. The multiplex PCR test was used for its cost-effectiveness in terms of amplifiers which can be purified in order to be sequenced. It also makes it possible to detect several germs as well as their serotypes. For a PCR reaction, several elements are involved in the reaction medium or Master Mix. These are the desoxyribonucleotides (dNTPs), the magnesium ions (MgCl2) and the primers. A set of 14 primers divided into 3 classes were used. Class 1 primers served as an internal control by targeting the cpsA gene. It is a highly conserved gene found in capsular loci characterized to date. The primers of the second class were used to target specific serotypes by specific reactions (out of six possibilities). The group reaction was carried out using the primers of the third class in order to carry out an initial screening of the samples and to classify the pneumococcal isolates. Related serotypes were grouped based on the amplification of common genes. Using the technique of electrophoresis on agarose gel and an ultraviolet radiation device, the migration bands are then visualized and analyzed. The data collected had been entered into Excel 2010 and analyzed with Epi info 7. The exact Fischer chi2 test at the 5% threshold, the relative risk and its 95% confidence interval were used to compare the proportions and determine the associations. Results: 187 antibiotic-resistant strains of Streptococcus pneumoniae were collected. The average frequency of serotypes 1, 9A, 4 and untypeable identified were 43.59%, 18.18%, 18.27% and 39.57% respectively. The frequency of serotype 1 was predominant for the age group over five years old with 56.88%. The male sex was predominant with 55.08% for serotype 1. Resistance to penicillin and gentamicin for serotype 1 during this study, for the age group under 5 years old, was 77%. For serotypes 19A and 4, tetracycline resistance was predominant with 20% for the age group under 5 years. The resistance to penicillin and gentamicin of non-typeable serotypes was 33% for the age group under 5 years old. For the age group over 5 years old, resistance to erythromycin predominated at 37%. The distribution of serotypes by sex depending on antibiotic resistance was variable. There was a statistically significant association between identified serotypes and antibiotic resistance (p Conclusion: The study determined serotypes 1, serotypes 19A, serotypes 4 and non-typeable serotypes. These results would be due to the quality of vaccination or poor protection of vaccines.
基金Supported by National Natural Science Foundation of China(31072155)Natural Science Foundation of Jiangsu Province(BK2010068)+1 种基金Fund for Independent Innovation of Agricultural Science in Jiangsu Province[CX(11)2060]Special Fund for Agroscientific Research in the Public Interest(201303041)~~
文摘[Objective] This study aimed to establish a method for quantitative detection of mRNA transcriptional level of SS2 adhesive related-factors of Streptococcus suis serotype 2 （SS2） by fluorescent quantitative PCR. []Vlethod] The gene fragments en- coding SS2 adhesive related-factors MRP, FBPS and CPS2J and a housekeeping gene aroA were amplified by reverse transcription PCR from the total RNA of SS2, cloned, and sequenced. The recombinant plasmids containing the target genes were constructed, and used as templates in Real-time PCR. [Result] Dynamic curves, stan- dard curves and melting curves of the adhesive related-factors and aroA were ob- tained by the optimized Real-time PCR system. The standard curves showed a good linear relationship between template copy number and circulation number, and the correlation coefficients （FF） of the standard curves were over 0.995. Also, these as- says were highly specific a^d there was single specific melting peak for every gene. Moreover, the assays were highly sensitive and had a detection limit of 1.0×102 copies in 1 μl of initial templates. Finally, it was highly repeatable and had a coeffi- cient of variation less than 2% for intra-assay. [Conclusion] This study will provide a way to reveal the adhesion mechanism of SS2 to different host cells at molecular level.
文摘Nutritional value of vegetables and high prices of meat and meat originated food compel common people to consume plant originated food particularly salad vegetables. Microbial population of vegetable surfaces contains a large number of pathogenic bacteria including members of Enterobactereace like Escherichia coli (E. coli). A survey was conducted in three major markets of Rawalpindi, Pakistan. Tomato, lettuce, cabbage and cucumber samples were collected from three shops of each market. Each vegetable was analysed as unwashed and washed for total coliforms, faecal coliforms and E. coli by FAO (Food Quality Manual). About two hundred and fifty E. coli isolates were preserved, serotyped for presence of O157 serotype. Total coliforms, faecal coliforms and E. coli count exceeded the permissible limits in most samples. The highest Total coliforms were associated with cabbage (3.78 log10 cfu/g). Cucumber was the least contaminated by Total coliforms (2.15 log10 cfu/g). E. coli was detected in tomato, lettuce, cucumber and cabbage. Washed samples showed reduced bacterial population. Seventy six isolates of E. coli were biochemically characterized and serotyped for O157 antigen. A majority of strains could not be identified by serotyping. These findings conclude with high potentially pathogenic microbial load on salad vegetables and urge for preventive action on priority basis.
文摘Campylobacter is one of the most common bacterial enteropathogens of food borne origin in industrialized countries with C. jejuni being the most common species followed by C. coli. The prevalence of Campylobacters in and around Chandigarh, India was studied by phenotypic and genotypic methods. Fecal samples from 1145 diarrheal patients and 102 healthy subjects from hospital and community were cultured on Campylobacter media and identified by Gram stain, biochemical investigations and serotyping. Molecular identification of Campylobacter isolates was done using specific primers to unique regions of 16S rRNA, Campylobacter jejuni (hipO), Campylobacter coli (aspK), Campylobacter lari (glyA) and Campylobacter upsaliensis (lpxA) genes. Identification of specific genes to look for resistance to nalidixic acid, ciprofloxacin, tetracyclin and streptomycin was also done. Campylobacters were isolated from 2.6% of patients with diarrhea. Campylobacteriosis was more prevalent in children ≤5 years old and during summer season. The most frequent serotypes were S:27, B:2, Z5:52 and V:32. All the Campylobacters isolated by culture were confirmed genotypically by identification of 16S rRNA, hipO and aspK genes. Of the 30 isolates, 27 were C. jejuni and 3 were C. coli. No C. lari or C. upsaliensis were detected. Antibiotic resistance was 40% for nalidixic acid, 23.3% for ciprofloxacin, 50% for tetracyclin and 20% for streptomycin. Campylobacter prevalence is low in the region with C. jejuni being the most common species. A high degree of resistance was found for nalidixic acid and tetracyclin but moderate for ciprofloxacin and streptomycin.
文摘Campylobacter is one of the most common food-borne bacterial enteropathogens. We planned to investigate the prevalence and antibiotic resistogram of Campylobacter in poultry in and around Chandigarh. Poultry samples (n = 127) were obtained from slaughter houses/retail outlets and cultured microaerophilically on Campylobacter media. The isolates were identified phenotypically and by molecular investigation. Identification of specific genes to look for resistance to nalidixic acid, ciprofloxacin, tetracyclin and streptomycin was also done. Campylobacter was isolated from 57/127 (44.9%) of the samples. The most frequent serotypes identified were B: 2, S: 27, Z5: 52 and Z7: 57. All culture isolates (100%) were reconfirmed as Campylobacter by 16S rRNA polymerase chain reaction. Molecular identification of isolates revealed the presence of C. jejuni in 45 (79.0%), C. coli in 1 (1.8%) and co-infection of C. coli and C. jejuni in 11 (19.3%). No C. lari and C. upsaliensis were detected. Antibiogram typing showed nalidixic acid resistance in 36.8%, ciprofloxacin resistance in 35.0% and 31.5% resistance for both streptomycin and tetracyclin. A high level of Campylobacter prevalence was found among the poultry with C. jejuni being the most commonly isolated species. Resistance to major antibiotics among Campylobacter isolates from poultry was also very high. The study of prevalence of Campylobacter in poultry and its resistance to major antibiotics will help to plan risk burden strategies throughout the food chain.
基金Financial supported by the Gansu ProvincialSci. & Tech. Department (1002NKDA037)
文摘A sandwich format immunochromatographic assay for detecting foot-and-mouth disease virus (FMDV) serotypes was developed. In this rapid test,affinity purified polyclonal antibodies from Guinea pigs which were immunized with sucking-mouse adapted FMD virus (A/AV88(L) strain) were conjugated to colloidal gold beads and used as the capture antibody,and affinity purified polyclonal antibodies from rabbits which were immunized with cell-culture adapted FMD virus (A/CHA/09 strain) were used as detector antibody. On the nitrocellulose membrane of the immunochromatographic strip,the capture antibody was laid on a sample pad,the detector antibody was printed at the test line(T) and goat anti-guinea pigs IgG antibodies were immobilized to the control line(C). The lower detection limit of the test for a FMDV 146S antigen is 11.7ng/ml as determined in serial tests after the strip device was assembled and the assay condition optimization. No cross reactions were found with FMDV serotype C,Swine vesicular disease (SVD),Vesicular stomatiti svirus (VSV) and vesicular exanthema of swine virus (VES) viral antigens with this rapid test. Clinically,the diagnostic sensitivity of this test for FMDV serotypes A was 88.7% which is as same as an indirect-sandwich ELISA. The specificity of this strip test was 98.2% and is comparable to the 98.7% obtained with indirect-sandwich ELISA. This rapid strip test is simple,easy and fast for clinical testing on field sites; no special instruments and skills are required,and the result can be obtained within 15 min. To our knowledge,this is the first rapid immunochromatogarpic assay for serotype A of FMDV.
基金supported by grants from National Key Basic Research Program of China (No.2005CB522900)College Science and Technology Research Program of Anhui Province (No.KJ2008B300)
文摘The pathogenesis of HBsAg （＋）/HBsAb （＋） double positive hepatitis B virus infection was investigated by simulating HBsAg/HBsAb coexistence in vitro and establishing HBsAg/HBsAb double positive model in vivo. Eukaryotic expression plasmids PCI-SY, PCI-adw, PCI-adr, PCI-ayw, which expressed S gene product of different serotypes, were constructed and transfected into HepG2 cells. Recombinant proteins were purified from the transfected cells. At the same time, HBsAg mouse antiserum was obtained by immunizing mice with PCI-SY plasmid. HBsAg/HBsAb coexistence was simulated using these antigens and antiserum. Furthermore, the expression plasmids expressing different serotypes of S gene product including PCI-adw, PCI-adr, and PCI-ayw were injected into mice via tail vein. HBsAg and HBsAb in mice sera were tested at the first and 7th day respectively after antigen plasmids injection. Both in vitro simulation and in vivo animal models demonstrated that HBsAg antigen and HBsAb of the same serotypes Could not coexist, but HBsAg antigen and HBsAb of different serotype could coexist. HBsAg/HBsAb double positive hepatitis B virus infection could be due to infection of viruses of different serotypes.
基金Supported by the Hubei Province Health and Family Planning A Scientific Research Project,No.WJ2017M220the Wuhan Health Bureau Scientific Research Fund,No.WX19C11+2 种基金the Joint Precision Medical Research Fund From Taihe Hospital,No.2016JZ10the Shiyan COVID-19 Pilot Emergency Scientific Research Project,No.20Y19the Wuhan Children's Hospital Research Project,No.2017FE007.
文摘BACKGROUND Hand,foot,and mouth disease(HFMD)has become one of the most common infectious diseases in China.Before 2016,the primary causal serotypes were enterovirus A71(EV-A71)and coxsackievirus A16(CV-A16).Following the introduction of EV-A71 vaccines in China since 2016,the situation could change.CV-A6 has recently replaced EV-A71 and CV-A16 in some areas of China.However,the epidemiological characteristics of central China remain unknown.AIM To investigate the clinical symptoms and pathogen spectrum of HFMD in Shiyan City,central China,in recent years.METHODS The epidemiological,clinical,and laboratory data from HFMD cases reported to the Shiyan Center for Disease Control and Prevention between January 2016 and December 2020 were analyzed.196 throat swab specimens were collected from hospitalized HFMD patients between January 2018 and December 2020.To detect and genotype enteroviruses,real-time reverse transcription-polymerase chain reaction and sequencing of the 5'-untranslated region were used.In Shiyan,168 laboratory-confirmed HFMD cases were studied using a logistic regression model to determine the effect of predominant enterovirus serotypes.Based on the logistic regression model,the least absolute shrinkage and selection operator model was used to analyze the correlation between CV-A6 infection and various clinical characteristics in HFMD patients in Shiyan.RESULTS From 2016 to 2020,35840 HFMD cases were reported in Shiyan.The number of cases decreased by 48.4%from 2016 to 2017.Approximately 1.58-fold increases were found in 2018 and 2019 when compared to the previous year,respectively.In 2020,a decrease of about 85.5%was reported when compared to 2019.The most common serotypes shifted from EV-A71 and CV-A16(about 60%-80%in 2016 and 2018)to others(more than 80.0%in 2017,2019,and 2020).EV-A71 lost its dominance in 2017 in Shiyan.Among 196 confirmed HFMD cases,85.7%tested positive for enterovirus,with CV-A6 being the most common serotype(121/168,72.0%).The positive rates for CV-A16 and CVA10 were 4.8%and 3.0%,respectively.There was no EV-A71 discovered.Infection with CV-A6 was linked to fever,myocardial damage,increased creatine kinase MB isoenzyme,and lactate dehydrogenase levels.CONCLUSION CV-A6 was the most common enterovirus serotype in Shiyan City,replacing EV-A71 and CV-A16 as the HFMD pathogen.Developing vaccines against CV-A6 or multiple pathogens,as well as rising CV-A6 surveillance,will help prevent HFMD in central China.
基金the National Natural Science Foundation of China (30800822)Jiangsu Prov-ince Natural Science Foundation (BK2006070)Jiangsu Education Department (VK0410190)
文摘216 avian pathogenic Escherichia coli （APEC） isolates were obtained from poultry with colibacillosis in different areas of China. Among them, 195 were serotyped as 078, 088, and 093. Thirteen virulence-associated genes, including fimC, iucD, iss, tsh, fyuA, irp2, eaeA, hlyE, colV, papC, stx2f, vat, and astA, were submitted to PCR amplification. The fimC gene was the most prevalent with a detection rate of 93.6%, followed by iucD （70.8%）, iss （58.8%）, and tsh （51.4%） in APEC isolates. The detection rate of high pathogenicity islands （HPI）-associatedfyuA and irp2 genes were both 44.9%, with no LEE （the locus of enterocyte effacement） island-associated gene eaeA detected. In terms of distribution patterns of the 13 virulence-associated genes, 5 isolates harborbed 10 genes, 19 isolates contained onlyfimC gene, and only 4 isolates had no virulence-associated gene detected. Different correlations of the virulence-associated genes with O serotypes were also investigated and 50% 078 isolates had a gene distribution patterns of fimC^＋iucD^＋irp2^＋fyuA^＋iss^＋colV^＋tsh^＋.
基金supported by Institute for Medical Research Operational Grant(Vol:040300/OS27403)
文摘Objective:To identify the circulating serotypes of human echovinis in Malaysia from 2002 in 2013.Methods:A toial of 31 retrospective samples from non-polio acute flacid paralysis,hand-food-and-mouth disease,viral meningitis and enterovirus cases were subjected to amplification of partial VPI gene by RT-PCR.Results:Sequencing and phylogeneiic analysis of the partial sequences identified presence of human echovinis and human coxsackie viruses.It was found that echovinis 11 was the commonly circulating serotype followed by echovinis6.echovinis 7.echovinis 3.echovinis 9.echovinis 30 and echovinis I in decreasing order.Additionally two types of human coxsackie virus isolates were detected which were coxsackie A24 and B3.Condusions:From the findings,there is a possibility that echovinis 11 is the predominant serotype among Malaysian patients with echovinis infection.However,a larger sample size will yield a more confident result to support this evidence.____________________
基金supported by the National Key Research and Development Program of China (2016YFD0500801)
文摘Hepatitis-hydropericardium syndrome(HHS)is an infectious disease caused by fowl adenovirus serotype 4(FAdV-4).Several structural and non-structural proteins of FAdV-4 have been expressed in Escherichia coli and baculovirus expression system to develop candidate subunit vaccines.However,the protective efficiency of baculovirus-expressed penton base protein has not been assessed.In this study,two recombinant capsid proteins,penton base and fiber-2,were constructed.And then,penton base and fiber-2 were administrated alone or together to specific pathogen-free(SPF)chickens at 14 days of life and boosted at 28 days of life.At 42 days of life,the immunized groups and the control group were challenged with FAdV-4 virulent strain.Results show that inoculating penton base or penton base+fiber-2 provided 100%protection to the chickens.All groups vaccinated with the recombinant protein produced detectable antibodies and showed no apparent lesions.Thus,baculovirus-expressed penton base protein is a promising candidate subunit vaccine.
