Epidemiological and etiological characteristics of Vibrio parahaemolyticus strains causing foodborne disease outbreaks in Guangdong Province from 2017 to 2020 / 中华预防医学杂志

Objective: To study the epidemiological and pathogenic characteristics of Vibrio parahaemolyticus isolated from outbreaks cases in Guangdong Province, 2017-2020. Methods: Epidemiological characteristics of 87 outbreak events caused by Vibrio parahaemolyticus were analyzed. Strains were serotyped, and then analyzed by pulsed-field gel electrophoresis (PFGE). Results: The food-borne disease outbreak caused by Vibrio parahaemolyticus was found in 16 cities. 44.8% (39/87) and 37.9% (33/87) of the outbreaks occurred in hotels, restaurants and school canteens, respectively. Improper food processing and storage (40.2%, 35/87) and cross contamination caused by indiscriminate raw and cooked food (25.3%, 22/87) were the main causes of food-borne disease outbreaks of Vibrio parahaemolyticus. The main serotypes of patient derived strains were O3:K6 (87.5%) and O4:KUT (22.5%). The similarity value between O3:K6 type isolates was 65.5%-100.0%, and the PFGE pattern similarity value of O4:KUT type isolates was 66.5%-100.0%. Conclusion: Outbreaks caused by Vibrio parahaemolyticus are widely distributed in Guangdong province. It is necessary to strengthen the publicity and education on the correct handling of food in hotels, restaurants, schools, and unit canteens. O3:K6 and O4:KUT serotypes are the main serotypes of the outbreak. There is genetic diversity among the epidemic strains.

Kinetic Characteristics of Neutralizing Antibody Responses Vary among Patients with COVID-19 / 生物医学与环境科学（英文）

Objective@#The coronavirus disease 2019 (COVID-19) pandemic continues to present a major challenge to public health. Vaccine development requires an understanding of the kinetics of neutralizing antibody (NAb) responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).@*Methods@#In total, 605 serum samples from 125 COVID-19 patients (from January 1 to March 14, 2020) varying in age, sex, severity of symptoms, and presence of underlying diseases were collected, and antibody titers were measured using a micro-neutralization assay with wild-type SARS-CoV-2.@*Results@#NAbs were detectable approximately 10 days post-onset (dpo) of symptoms and peaked at approximately 20 dpo. The NAb levels were slightly higher in young males and severe cases, while no significant difference was observed for the other classifications. In follow-up cases, the NAb titer had increased or stabilized in 18 cases, whereas it had decreased in 26 cases, and in one case NAbs were undetectable at the end of our observation. Although a decreasing trend in NAb titer was observed in many cases, the NAb level was generally still protective.@*Conclusion@#We demonstrated that NAb levels vary among all categories of COVID-19 patients. Long-term studies are needed to determine the longevity and protective efficiency of NAbs induced by SARS-CoV-2.

Analysis of the brucella pathogen and its molecular genotype in Guangdong province / 中国地方病学杂志

Objective To analysis the etiology and molecular classification of brucella strains isolated in Guangdong province in 2010.Methods The strains of 19 brucella were verified and identified by some methods including traditional biology phenotype confirmation,PCR amplification and pulsed field gel electrophoresis (PFGE).Results On phenotype level,4 strains were brucella melitensis biovar 1,2 strains were brucella suis biovar 3,and the rest were brucella melitensis biovar 3,which were specific B genes positive strains,and the PFGE typing similar values ranging from 67.9％ to 100％.In addition to the four strains from Zhuhai for the outbreak,the homology was 100％,and the rest were sporadic cases.Conclusions Brucella cases,in Guangdong province,are highly sporadic and dispersed outbreaks.Compared with a few years ago,it shows species diversification,and brucella melitensis biovar 3 is still the dominant serotype.PFGE can be used to distinguish the three species of brucella,but it can't effectively distinguish the allotypes.

Surveillance and analysis on the pathogenic features of Salmonella in Guangdong province in 2010 / 中华预防医学杂志

<p><b>OBJECTIVE</b>In order to better understand the nature of Salmonella infection in diarrheal patients in Guangdong province, the study analyzed the serum types, antibiotic resistance and molecular determinants of the isolated Salmonella strains.</p><p><b>METHODS</b>In year 2010, 8405 diarrhea patients from 16 surveillant hospital in Guangzhou, Zhongshan, Dongguan, Zhuhai, Maoming, Yangjiang and Jiangmen cities in Guangdong province, were recruited in the study. A total of 8405 fecal specimen were collected and subjected to Salmonella isolation and culture. The isolated Salmonella strains were further analyzed via serotyping, antimicrobial susceptibility testing, and PFGE. The χ(2) test was applied to compare the differences between the isolated Salmonella strains in different seasons and districts. BioNumerics software was used to analyze the PFGE results in order to determine the correlation between different Salmonella strains.</p><p><b>RESULTS</b>The positive rate of the surveillant Salmonella in Guangdong province was 3.58% (301/8405) in 2010; with the gender ratio at 1.34:1 (166/124). Salmonella infection was found in all age groups, and most in infants, accounting for 57.48% (173/301). The isolated rates of Salmonella were separately 3.48% (61/1751), 4.97% (134/2695), 3.08% (73/2370) and 2.08% (33/1589) in the four seasons; and the difference was statistically significant (χ(2) = 27.29, P < 0.01). The isolated rates of Salmonella in different regions were as follows: Zhuhai 15.43% (25/162), Maoming 7.53% (18/239), Dongguan 6.51% (39/599), Yangjiang 3.64% (14/385), Zhongshan 3.03% (70/2309), Guangzhou 2.90% (126/4349) and Jiangmen 2.49% (9/362). The difference between regions was statistically significant (χ(2) = 100.75, P < 0.01). Except one strain of the isolated Salmonella cannot be serotyped, the other 300 strains were divided into 42 serotypes, of which Salmonella typhimurium and Salmonella enteritidis were dominant, account for 45.18% (136/301) and 10.96% (33/301) respectively. Although over 85% of Salmonella were sensitive to cephalosporin, ACSSuT resistance patterns (defined as resistance to at least ampicillin, chloramphenicol, streptomycin, sulfamethoxazole and tetracycline) reached 34.88% (105/301), the highest resistant rate was found in serotype Salmonella typhimurium, as high as 65.44% (89/136). 136 strains of Salmonella typhimurium were divided into 51 PFGE types, showed great genetic diversity. 33 strains of Salmonella enteritidis were divided into 18 PFGE types. The strains with same PFGE pattern may have different drug-resistant patterns, and vice versa.</p><p><b>CONCLUSION</b>Salmonella typhimurium and Salmonella enteritidis were the dominant serotypes causing infectious diarrhea in Guangdong province. Cephalosporin was the primary choice in clinical medicine. However, Salmonella typhimurium was resistant to drug most seriously in Guangdong province. There was no significant correlation between Salmonella resistance patterns and PFGE type.</p>

Automated ribotyping of Salmonella and Staphylococcus aureus in food poisoning of Guangdong province / 中华流行病学杂志

Objective To understand the genetic polymorphism of Salmonella and Staphylococcus aureus in Guangdong province, as well as to explore methods for identifying and tracing the source of these two foodbome pathogens. Methods Using the automated ribotyping system, two foodbome pathogens were tested with either EcoR Ⅰ or Pvu Ⅱ restriction enzymes. BioNumerics software was then applied for image analysis, database establishment and other corresponding analysis. Results Digestion of 32 Salmonella isolates with Pvu Ⅱ yielded 19 different ribotypes,and digestion of 14 Salmonella isolates with EcoR Ⅰ yielded 2 different ribotypes. Staphyloccus aureus isolates showed greater genetic diversity, whereas EcoR Ⅰ digestion of 49 different isolates yielded 31 different ribotypes. Conclusion Unique Salmonella and Staphylococcus aureus isolates could be identified through ribotyping. Although Salmonella serotyping and ribotyping were not strongly correlated, the combination of both restriction enzymes could be used to more effectively identify the genetic relationship among different strains as well as the source of food poisoning. Thus, not only could the genetic relationships amongst the different strains be inferred through ribotyping skills, the source of food poisoning and mode of transmission could also be determined under the use of this method.

Etiologic detection and epidemiological analysis of one suspected case of diphtheria in Guangdong province / 中华预防医学杂志

<p><b>OBJECTIVE</b>To clarify the diagnosis of one suspected case of diphtheria in Guangdong province by epidemiological analysis and etiologic detection.</p><p><b>METHODS</b>On July 6th 2010, the corynebacterium diphtheria was detected from the nasal secretions of one nasopharyngeal carcinoma patient in a college-town hospital in Guangzhou City, Guangdong Province. The patient and the close contacts were asked to participate in the epidemiological survey; and their nasopharyngeal swabs (3 samples) and the nasal secretions of the patient (1 sample) were collected. The bacteria of the samples were isolated and cultured by blood plate and agar loefflera. The smears of positive strains were dyed and identified by BioMerieux API Coryne biochemical card. Gene tox of β-Corynebacteriophage, Corynebacterium diphtheriae was tested by PCR method, the aliphatic acid was analyzed by gas chromatography method and the Corynebacterium diphtheriae (CMCC 38009) was selected as positive control.</p><p><b>RESULTS</b>The patient had not gone out, neither had been visited. The patient denied history of vaccines or the immunizations. From the survey on patient's family members and close contacts, no similar symptoms had been found. One strain of Corynebacterium diphtheriae was isolated from the patient's nasal secretions, Gram positive and shape diversified. After cultured by agar loefflera and Gram-dyed and Neisser-dyed, one end or both two ends of the strain showed typical metachromatic granule. API Coryne was identified to Corynebacterium diphtheriae mitis/belfanti (99.4%). The result of gas chromatography method also indicated Corynebacterium diphtheriae. No Corynebacterium diphtheriae was isolated from the nasopharyngeal swabs, neither of the patient nor of the close contacts. The gene tox of β-Corynebacteriophage, Corynebacterium diphtheriae was negative according to the PCR test.</p><p><b>CONCLUSION</b>The isolated Corynebacterium diphtheriae did not produce toxin as there was no biological structure gene of toxin. The patient was a health carrier of nontoxic Corynebacterium diphtheriae.</p>

Surveillance on Salmonella infection in Guangdong province, 2008-2009 / 中华流行病学杂志

Objective To understand the infection of Salmonella (S.) in patients with diarrhea and outbreaks caused by Salmonella to identify the serotypes, resistance to antibiotics and PFGE types of the strains from the surveillance program in Guangdong province. Methods S. strains from patients with diarrhea were detected, and all the positive strains collected in routine and outbreak surveillance programs, were tested by serum agglutination, antibiotic susceptibility and PFGE.Results 71 S. strains were isolated from 1922 stool samples in 2008, with positive rate as 3.7%.85 S. strains were isolated from 2110 stool samples in 2009, with positive rate as 4.0%. All the 156 strains were divided into 37 serotypes, with S. serotype typhimurium and enteritidis as the most common serotypes. 10 incidents of food poisoning were detected, of which 4 were caused by enteritidis and 3 by typhimurium. A suspected outbreak by enteritidis was discovered and under epidemiological investigation. The findings indicated that 2 of the 4 patients from this outbreak were infected with identical enteritidis isolates. 80% of the 229 isolates were found susceptible to cephalosporins and quinoione and 59.3% of them were muitiresistant to the antibiotics. Conclusion S. enteritidis and S. typhimurium were the most common serotypes that caused infectious diarrhoea and food poisoning in Guangdong province.

Etiologic characteristics of Vibrio parahaemolyticus strains causing outbreaks and sporadic cases in Guangdong,2009 / 中华流行病学杂志

Objective To study the serotypes,virulence features and molecular characteristics of Vibrio parahaemaolyticus isolated in food poisoning cases and surveillance program on diarrhea patients in Guangdong,2009.Methods 95 Vibrio parahaemolyticus strains from food poisoning cases and 15 strains from surveillance program on diarrhea patients were serotyped and detected for tdh(thermostable direct hemolysin,tdh)and trh(tdh-related hemolysin gene,trh)by PCR.81 sero-variant Vibrio parahaemolyticus strains were selected through PFGE subtyping.Results There were 15 Vibrio parahaemolyticus strains isolated from surveillance program on diarrhea patients and 95 strains were isolated from 11 Vibrio parahaemolyticus-caused food poisoning cases in 2009.Among these strains,O3:K6(46.67％ and 44.21％)and O4:K8(33.33％ and 28.42％)were the dominant serotypes,but not the 7 food-borne strains.There were 93(84.54％)tdh +trh-,13(11.81％)tdh-trh-,and 3(3.65％)tdh+ trh + strains.The similarity value was between 57.7％ to 100.0％ of the 81 strains after PFGE sub-typing method and 36 PFGE subtypes were identified.PFGE001 and PFGE029 appeared to be the dominant subtypes.Conclusion O3:K6 and O4:K8 were the most dominant serotypes in Vibrio parahaemolyticus-caused diarrhea and food poisoning cases in Guangdong and tdh were detected in most of the strains.Dominant PFGE subtypes were causing both sporadic and outbreak cases in different areas in Guangdong province.

Surveillance program on and the distribution related to the virulence-associated genes of Vibrio cholerae in estuary of Pearl River / 中华流行病学杂志

Objective To understand the distribution,molecular characteristics and virulence genes of the O1 and O139 Vibrio cholerae isolates from the Pearl River Estuary water.Methods Vibrio cholerae isolates collected from the Pearl River estuary waters from January 2009 to December 2010,were tested by PCR for eight virulence-related genes,including cholera toxin(ctxA),zonula occludens toxin(zot),accessory cholera enterotoxin(ace),hemolysin(hlyA),toxin-coregulated pilus (tcpA),outer membrane protein(ompU),and the regulatory protein genes(tcpⅠ,toxR).Genetic relation was assessed by pulsed-field gel electrophoresis(PFGE)and the patterns were clustered by BioNumerics.Results From 1152 aquatic samples,69 isolates were identified,including 41 Inaba,18 Ogawa and 10 O139.All the isolates showed ctxA negative,while the hlyA and toxR genes were positive in all the isolates.34.15％(14/41)of the Inaba strains were hlyA + toxR + ompU + ace + zot + tcpI+,while 66.67％(12/18)belonged to Ogawa strains and 70％(7/10)of the O139 strains were hlyA + toxR+.Through PFGE analysis,the O1 isolates formed three clusters in this study.The patterns of O1 isolates differed widely,with the similarity as 72.8％-100.0％,while the patterns of O139 isolates having the similarity of 69.9％-95.5％.Conclusion The non-toxigenic O1 and O139 V.cholerae had a wide distribution in the environment of Pearl River estuary water during the nonepidemic period of cholera.All the aquatic isolates presented diversities on the related virulent genes.

Comparative study on the phenotypic characteristics and molecular typing of foodborne Vibrio parahaemolyticus in Guangdong province / 中华流行病学杂志

Objective To understand the phenotypic characteristics of foodbome Vibrio parahaemolyticus in Guangdong province through carrying out a comprehensive comparison including pulse field gel electrophoresis,ribotyping and serotyping.Methods 74 different Vibrio parahaemolyticus strains isolated from seafood and cases due to food poisoning in Guangdong province were under serotyping and susceptibility testing,in addition to the testing of direct heat hemolysin(tdh)and the heat hemolysin-related hemolysin hormone(trh)via PCR.Ribosomal genotyping(ribotyping)with EcoR Ⅰ restriction enzyme was utilized on 74 different Vibrio parahaemolyticus isolates,whereas pulsed-field gel electrophoresis(PFGE)with the Not Ⅰ restriction enzyme was used on 74 different Vibrio parahaemolyticus isolates.BioNumerics software was used to compare the isolates from different sources,times and places in order to elicit the correlation between different strains.Results Although Vibrio parahaemolyticus was 100.00％ sensitive to chloramphenicol,it still presented different levels of resistance against 13 other antibiotics.Among the 74 different strains of Vibrio parahaemolyticus under testing,24.32％ showed positive for the tdh virulence gene,whereas 4.05％ positive for trh.74 different Vibrio parahaemolyticus strains were found to belong to 26 serotypes,where the O5:K17 and O2:K28 serotypes were dominant in those isolates that causing seafood-poisoning.The O3:K6 serotype was found to be the dominant of those isolates that causing food-poisoning.Based on ribosomal genotyping,the 74 Vibrio parahaemolyticus isolates were divided into 62 different ribotypes,whereas the 74 strains of Vibrio parahaemolyticus were divided into 67 different PFGE types,thus exhibiting considerable genetic diversities of the strains.Conclusion Majority of the isolates causing food-poisoning carried tdh virulence gene.PFGE was shown to have the highest resolution,followed by ribotyping with serotyping being the lowest,where the combination of the three could improve the resolution.

Development of a DNA microarray for detecting 8 common species of food-borne bacterial pathogens in south China / 南方医科大学学报

<p><b>OBJECTIVE</b>To prepare a DNA Microarray that can detect 8 common species of food borne bacterial pathogens in south China.</p><p><b>METHODS</b>All the 70mer oligo probes were designed on the characteristic genome loci of the 8 species of food borne bacterial pathogens. Eight subarrays corresponding to the 8 food borne bacterial pathogens were spotted onto the slide and integrated into a pan-array on the chip. A number of identified and known bacterial samples from the storage bank were selected for the validation test.</p><p><b>RESULTS</b>Based on the PPR ranking, for LM sub-array, the PPR of the 3 Listeria bacteria LM, Lin and Liv was 68.8%, 51.8% and 59.6%, respectively, while that of the non-Listeria bacterial samples was all below 43%. For VC sub-array, the PPR of VC sample was 54.1% and that of the non-VC bacterial samples was lower than 17.2%. For VP sub-array, the PPR was 66.7% for VP sample and below 24.2% for non-VP bacterial samples. For Sal sub-array, the PPR was 55.9% for Sal sample and below 50.5% for non-Sal bacterial samples. For Shi sub-array, the PPR of Shi sample and the non-Shi bacterial samples was 53.8% and below 36.6%, respectively. For SA sub-array, the PPR of SA sample and non-SA bacterial samples was 65.2% and below 22.7%, respectively. For CJ sub-array, the PPR of the 2 Campylobacter bacteria CJ and CC were 88.2% and 58.8%, respectively, and that of the non-Campylobacter bacterial samples was lower than 35.3%. For EC sub-array, the PPR of EC sample was 47.9%, and that of the non-EC bacterial samples was lower than 41.6%. Evaluation of the Biosafood-8 chip developed in this study by 18 biological samples from different origins demonstrated its good specificity and accuracy in the identification of the pathogens.</p><p><b>CONCLUSION</b>The chip we developed can clearly differentiate the target food borne pathogenic bacteria and non-target bacteria and allows specific and accurate identification of the species of the tested bacteria isolates.</p>

Molecular characters of epidemic cerebrospinal W135 Neisseria meningitidis strain firstly isolated in Guangdong province / 中华预防医学杂志

<p><b>OBJECTIVE</b>To analyze the molecular characters of the W135 Neisseria meningitidis strain firstly isolated from patients in Guangdong province.</p><p><b>METHODS</b>Biochemical profile by using the API NH system (bio-Merieux, France) was used for confirmation,and sero-grouping of the meningococcal isolates including one serogroup W135, one serogroup C and three serogroups of a Neisseria meningitidis isolated from patients in Guangdong province in recent two years were performed. The subtype was determined after amplifying porA and porB respectively from the genome DNA of Neisseria meningitidis. Multilocus sequence typing (MLST) was performed for determining the allele profiles and the sequence types (STs). The polygenetic tree was obtained by analyzing the allele profiles with program Splits tree online. The molecular characters of the serogroup W135 Neisseria meningitidis was analyzed by its evolution relationship and the variable regions in porA and porB which encoding the outer membranes proteins (OMPs).</p><p><b>RESULTS</b>The subtype determined by porA variable regions of the serogroup W135 Neisseria meningitidis was P1.5,2, which was one of the most invasive types. The types of variable regions (VRs) I, IV, V, VII with porB were 1, 1, 1, 17, and there was no VI and VIII in porB. The allele profile of the W135 strain in this study was 2, 123, 4, 3, 8, 4, 6, and its sequence type was ST-2960, which belonged to ST-11/ET-37 clone complex. The subtypes of the serogroup C and serogroup A strains were P1.20, while their types of VR IV were all 7, and they all hadn't VR VII in porB. The strain serogroup C belonged to ST-4821 clone complex, and the 3 serogroup A strains belonged to ST-5 clone complex.</p><p><b>CONCLUSION</b>The molecular character of the serogroup W135 Neisseria meningitidis should be the same with the strains isolated in foreign country, and be different from the epidemic types in the area. This serogroup W135 Neisseria meningitis isolated from patients in Guangdong for the first time was thought to be a new type appearing in the local area.</p>

MLST typing of Streptococcus suis isolated from clinical patients in Guangdong Province in 2005 / 南方医科大学学报

Intensive surveillance of human S.suis infection was carried out in July and August of 2005 in Guangdong Province, which coincided with the Sichuan outbreak. Five isolated cases of human infections were identified during this period, from which 5 S. suis serotype 2 isolates were recovered. MLST analysis showed that these 5 isolates shared identical sequences of 6 MLST housekeeping genes except for one point mutation found within the thrA gene fragment, a neutral mutation (TTA to TTG) in the third nucleotide (360 nt) of the codon for leucine. MLST analysis identified 2 sequence types in the Guangdong sporadic infection. Three Guangdong isolates L-SS002, L-SS003 and L-SS005 belonged to ST7, while the other two isolates L-SS004 and L-SS006 belonged to ST1, but they all belonged to ST1 clonal complex. This finding represents a striking feature that differs from the Sichuan outbreak caused by a single ST7 SS2 clone. The 3 isolates of ST7 were probably imported from Sichuan Province, while the origin of the other 2 isolates of ST1 still remain to be clarified.

The etiologic characteristics of Vibrio cholerae in Guangdong province in 2007 / 中华流行病学杂志

Objective To analyze the etiologic characteristics of Vibrio cholerae in Guangdong province in 2007.Genetic relationship was observed including among predominated biotype isolates from different areas within the province and among same biotypes isolates from cholera cases and regular surveillance.Methods Isolates from cholera cases and through environmental surveillance were typed by sero-and phage-typings.Similarity of molecular fingerprinting was analyzed through comparing the pulsed field gel electrophoresis(PFGE)pattern of predominated biotype isolates,and those of the same biotype isolates from cholera cases and environment surveillance,respectively.In addition,genetic relationship was determined by clustering analysis,using bionumerics software.Results In total,31 isolates from cholera cases were collected and subtyped for 3 serogroups.V.cholerae O1 El Tor Inaba phage 1d was the predominant biotype which causing most of the cases in Guangdong province in 2007.Data from cluster analysis showed that the similarity among Inaba phage 1d strains from different areas were from 94.5% to 100%.However.16 isolates were collected from environment surveillance programs and the predominated biotype could not be found.Additionally,the biotype distribution of cases isolates was not consistent with those isolates through surveillance.High phylogenetic diversity was observed for the same biotypes isolates from cases and surveillance samples.Conclusion Our data showed that V.cholerae O1 El Tor Inaba phage 1d was the predominated biotype with multi-clone coexisting and circulating in Guangdong province in 2007.It also appeared to be the characteristics of cholera in the non-epidemic period,suggesting that it was necessary to enhance the alert surveillance programs for cholera epidemic based on the molecular typing techniques.

Molecular typing of Listeria monocytogenes isolated from foodstuff in Guangdong province by pulsed-field gel electrophoresis / 中华流行病学杂志

<p><b>OBJECTIVE</b>To establish molecular typing of Listeria monocytogenes isolates by pulsed-field gel electrophoresis (PFGE) for studying the epidemiologic characteristics of Listeria monocytogenes isolated from foodstuff in Guangdong province and to build up PFGE typing database of Listeria monocytogenes isolates for identifying the infectious resource of the outbreaks and other epidemiologic investigation.</p><p><b>METHODS</b>"Standardized Protocol for Molecular Subtyping of Listeria monocytogenes by PFGE" was followed. BioNumerics software was applied on image analysis, database establishment, comparative and corresponding analysis.</p><p><b>RESULTS</b>107 Listeria monocytogenes isolates were typed by PFGE, 41 PFGE types were observed among the isolates. The PFGE types were dispersive among these isolates. Listeria monocytogenes isolates were most frequently isolated in raw chicken while the most PFGE types were found in this type of food. The positive rate was relatively high in cold and iced foods. Only 1-2 DNA fragment difference occurred in 26 Listeria monocytogenes isolates by PFGE, so high degree of relatedness remained among these isolates. There were unique PFGE patterns in the regions of Shaoguan and Huizhou. From time to time, a number of isolates remained close relationship.</p><p><b>CONCLUSION</b>PFGE typing of the 107 Guangdong Listeria monocytogenes isolates demonstrated relative genetic diversity but a number of the isolates showed close relatedness.</p>