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ABSTRACT: The tetra-primer amplification refractory mutation system PCR (T-ARMS-PCR) is a fast and economical means of assaying SNP's, requiring only PCR amplification and subsequent electrophoresis for the determination of genotypes. To improve the throughput and efficiency of T-ARMS-PCR, we combined T-ARMS-PCR with a chimeric primer-based temperature switch PCR (TSP) strategy, and used capillary electrophoresis (CE) for amplicon separation and identification. We assessed this process in the simultaneous genotyping of four breast cancer-and two cervical cancer risk-related SNPs.
A total of 24 T-ARMS-PCR primers, each 5'-tagged with a universal sequence and a pair of universal primers, were pooled together to amplify the 12 target alleles of 6 SNPs in 186 control female blood samples. Direct sequencing of all samples was also performed to assess the accuracy of this method.
Of the 186 samples, as many as 11 amplicons can be produced in one single PCR and separated by CE. Genotyping results of the multiplex T-ARMS-PCR were in complete agreement with direct sequencing of all samples.
This novel multiplex T-ARMS-PCR method is the first reported method allowing one to genotype six SNPs in a single reaction with no post-PCR treatment other than electrophoresis. This method is reliable, fast, and easy to perform.
PLoS ONE 01/2013; 8(4):e62126. · 4.09 Impact Factor
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ABSTRACT: A new, rapid, and high-throughput method was developed for simultaneous detection of 11 human papillomavirus (HPV) genotypes including nine high-risk types (HPV16, 18, 31, 33, 35, 39, 52, 58, and 66) and two low-risk types (HPV6 and 11) in a single tube by multiplex PCR based on a GenomeLab Gene Expression Profiler (GeXP) analyzer (GeXP-PCR). Eleven sets of chimeric primers were used to initiate the PCR, and one pair of universal primers was used for the subsequent cycles of the PCR. The specificity of GeXP-PCR for each HPV type was examined with clinical samples of single type HPV infection tested previously. The sensitivity of GeXP-PCR was evaluated by performing the assay on serial 10-fold dilutions of cloned PCR products. The GeXP-PCR achieved a sensitivity of 100 copies when all of the 11 pre-mixed plasmids containing HPV targets were present. Analyses of 124 clinical specimens using the GeXP-PCR demonstrated that the GeXP-PCR assay had comparable sensitivity and specificity to those of reported multiple PCR assay and an increased detection of HPV 11 in samples with mixed infections. In conclusion, the GeXP-PCR is a fast, sensitive, and high throughput method for the detection of multiple HPV infections.
Journal of Medical Virology 06/2012; 84(6):957-63. · 2.82 Impact Factor
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Hao Wan,
Yan Li,
Jing Liu,
Xue-qin Xie,
Zai-hua Wei,
Wei Wang, Miao Wang,
Jia-yi Sun,
Lan-ping Qin,
Jun Liu,
Yue Qi,
Dong Zhao
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ABSTRACT: To explore the characteristics of status and different populations of prehospital death associated with acute coronary events among young adults in Beijing.
Data of acute coronary events of hospitalization or death were obtained from the Hospital Discharge Information System from Beijing Public Health Information Center and Death Register System from Beijing Center for Disease Control in Beijing. The total case fatality rate of acute coronary events and proportion of prehospital coronary heart disease (CHD) death were compared upon gender, area, occupation and marital status among people aged between 25 - 45 years old.
A total of 3489 cases were identified during 2007 to 2009 with acute coronary events (male: 3183, female: 306), with a mean age of (40.5 ± 4.3) years old. The 3-years' overall mortality was 26.0%, with female's higher than male's (51.0% vs 23.6%, P < 0.05); and it was higher in rural area than in urban areas (28.9% vs 22.9%, P < 0.05). Ninety-five percent of death due to acute coronary events occurred prehospital, with the proportion of 95.2% in male and 94.2% in female. Among the people with different occupations, self-employed people had the highest rate of prehospital death. Majority of prehospital deaths (64.8%) occurred at home.
More than 90% of deaths caused by acute coronary events among young adults aged between 25-45 years old occurred before been admitted into hospital, and the site of prehospital deaths was mainly at home.
Zhonghua nei ke za zhi [Chinese journal of internal medicine] 04/2012; 51(4):274-8.
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Xue-qin Xie,
Xiu-ying Zhang,
Dong Zhao,
Wei Wang, Miao Wang,
Mo-ning Guo,
Jia-yi Sun,
Jian-peng Zheng,
Yue Qi,
Jun Liu,
Hao Wan,
Jing Liu
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ABSTRACT: To examine the distribution and trends of hospitalization rates for coronary heart disease (CHD) from 2007 to 2009 in Beijing.
We calculated hospitalization rates for CHD using data from Beijing Hospital Discharge Information System. Information of census registered population in Beijing was obtained from Beijing Municipal Bureau of Statistics. CHD includes acute myocardial infarction, unstable angina and other forms of CHD. Age-standardized hospitalization rates for CHD per 100 000 population aged 25 years or more were calculated.
During 2007 - 2009, a total of 248 049 patients aged 25 years or more hospitalized in Beijing with the primary discharge diagnosis of CHD were enrolled, of whom 73.7% were permanent registered Beijing citizens. The average hospitalization rate for CHD in 2007 - 2009 was 651.2/100 000 for the permanent residences in Beijing (741.2/100 000 in men, 560.9/100 000 in women). The highest average hospitalization rate (671.9/100 000) was seen in exurban area compared to other areas in Beijing. The average hospitalization rate for acute myocardial infarction, unstable angina, and other CHD was 126.4/100 000, 226.4/100 000 and 298.4/100 000, respectively. The hospitalization rate for CHD increased 18.1% from 2007 to 2009 (from 598.1/100 000 to 706.5/100 000). The same trend was seen in women (20.2%) and men (16.6%). The hospitalization rates of CHD in the urban, suburban, and exurban areas of Beijing all increased in the three years, and the greatest increase (36.6%) was found in exurban area. Hospitalization rates of acute myocardial infarction and unstable angina increased 24.5% and 55.3%, respectively, in the three years, while hospitalization rates of other CHD decreased 5.7%.
The hospitalization rate of CHD is higher in men than in women in Beijing. The hospitalization rates for CHD increased from the observation period, especially in those living in exurban area. Awareness of the magnitudes and trends of CHD hospitalization rates is of great importance in evaluating the burden of cardiovascular disease, allocating and utilizing health care resources, and estimating the health insurance for Beijing.
Zhonghua xin xue guan bing za zhi [Chinese journal of cardiovascular diseases] 03/2012; 40(3):188-93.
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Jia-yi Sun,
Jing Liu,
Xue-qin Xie,
Zai-hua Wei,
Wei Wang, Miao Wang,
Yue Qi,
Jun Liu,
Mo-ning Guo,
Xiu-ying Zhang,
Hao Wan,
Dong Zhao
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ABSTRACT: To survey the incidence of acute coronary events and its trend in three years, and explore the distribution of the incidence across Beijing residents aged 25 years and more from 2007 to 2009.
The present study incorporated and linked the routinely collected data from the Hospital Discharge Information System and Cause of Death Register System in Beijing, estimated the incidence of acute coronary events, and analyzed the distribution of the incidence across gender, age groups and regions. Acute coronary event was defined as non-fatal myocardial infarction and death from coronary heart disease. Numbers of residents by age, gender and area were obtained from the Beijing Statistics Bureau.
A total of 68 390 acute coronary events were identified among permanent residents of Beijing aged 25 years and more from 2007 to 2009. The age-standardized incidence was 166.4 per 100 000 people in overall population, with 218.5 in males and 115.2 in females. The age-standardized incidence was 144.3, 154.7, and 195.8 per 100 000 people in urban, suburban, and exurban area, respectively. The incidence was the highest in Huairou district (263.8 per 100 000), while was the lowest in Haidian district (121.5 per 100 000). The age-standardized incidence was 158.4, 169.4, and 171.2 per 100 000 in 2007, 2008, and 2009, respectively. The age-standardized incidence increased by 8.1% in 2009 compared to 2007, increase in men (11.1%) was greater than in women (2.5%). The incidence increased significantly with age in each year. The incidence raised by 30.3% in 2009 compared to 2007 for men aged 35 - 44 years. In 2009, the incidence was 146.7, 155.9, and 207.4 per 100 000 people in urban, suburban, and exurban area, respectively. The rates increased by 3.2% in both urban and suburban areas, and 16.4% in exurban areas in 2009 compared to 2007.
The incidence of acute coronary events increased from 2007 to 2009 among the permanent residents of Beijing aged 25 years and over, especially in young men, and people living in the exurban areas.
Zhonghua xin xue guan bing za zhi [Chinese journal of cardiovascular diseases] 03/2012; 40(3):194-8.
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Xiumei Hu,
Yong Zhang,
Xiaomian Zhou,
Banglao Xu,
Mengjie Yang, Miao Wang,
Chen Zhang,
Jin Li,
Ruyin Bai,
Wenbo Xu,
Xuejun Ma
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ABSTRACT: Hand, foot, and mouth disease (HFMD) is a contagious enteroviral disease occurring primarily in young children and caused by enterovirus 71 (EV71), coxsackievirus A16 (CVA16), and other serotypes of coxsackievirus and echovirus. In this study, a GeXP analyzer-based multiplex reverse transcription (RT)-PCR assay (GeXP assay) consisting of chimeric primer-based PCR amplification with fluorescent labeling and capillary electrophoresis separation was developed to simultaneously identify nine serotypes of enteroviruses associated with HFMD in China, including EV71, CVA16, CVA4, -5, -9, and -10, and CVB1, -3, and -5. The RNAs extracted from cell cultures of viral isolates and synthetic RNAs via in vitro transcription were used to analyze the specificity and sensitivity of the assay. The GeXP assay detected as little as 0.03 tissue culture infective dose (TCID(50)) of EV71 and CVA16, 10 copies of panenterovirus, EV71, CVA16, CVB1, and CVB5, and 100 copies of 10 (including panenterovirus) premixed RNA templates. A total of 180 stool specimens collected from HFMD patients and persons suspected of having HFMD were used to evaluate the clinical performance of this assay. In comparison with the results of conventional methods, the sensitivities of the GeXP assay for detection of panenterovirus, EV71, and CVA16 were 98.79% (163/165), 91.67% (44/48), and 91.67% (33/36), respectively, and the specificities were 80.00% (12/15), 98.48% (130/132), and 100% (144/144), respectively. The concordance of typing seven other serotypes of enteroviruses with the results of conventional methods was 92.59% (25/27). In conclusion, the GeXP assay is a rapid, cost-effective, and high-throughput method for typing nine serotypes of HFMD-associated enteroviruses.
Journal of clinical microbiology 11/2011; 50(2):288-93. · 4.16 Impact Factor
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ABSTRACT: A simple, rapid, sensitive, qualitative, colorimetric loop-mediated isothermal amplification (LAMP) with hydroxynaphthol blue dye (HNB) was established to detect high-risk human papillomavirus (HPV) genotypes 16, 18, 45, 52, and 58. All initial validation studies with the control DNA proved to be type specific. The colorimetric type-specific LAMP assay could achieve a sensitivity of 10 to 100 copies at 63°C for 65 min, comparable to that of real-time PCR. In order to evaluate the reliability of HPV type-specific LAMP, the assay was further evaluated with HPV DNAs from a panel of 294 clinical specimens whose HPV status was previously determined with a novel one-step typing method with multiplex PCR. The tested panel comprised 108 HPV DNA-negative samples and 186 HPV-DNA-positive samples of 14 genotypes. The results showed that the sensitivity of HPV type-specific LAMP for HPV types 16, 18, 45, 52, and 58 was 100%, 100%, 100%, 100%, and 100%, respectively, and the specificity was 100%, 98.5%, 100%, 98.8%, and 99.2%, respectively, compared with a novel one-step typing method with multiplex PCR. No cross-reactivity with other HPV genotypes was observed. In conclusion, this qualitative and colorimetric LAMP assay has potential usefulness for the rapid screening of HPV genotype 16, 18, 45, 52, and 58 infections, especially in resource-limited hospitals or rural clinics of provincial and municipal regions in China.
Journal of clinical microbiology 08/2011; 49(10):3545-50. · 4.16 Impact Factor
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ABSTRACT: A sensitive reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for rapid visual detection of human enterovirus 71 subgenotype C4 (EV71-C4) and Coxsackievirus A16 (CVA16) infection, respectively. The reaction was performed in one step in a single tube at 65°C for 60 min with the addition of the hydroxynaphthol blue (HNB) dye prior to amplification. The detection limits of the RT-LAMP assay were 0.33 and 1.58 of a 50% tissue culture infective dose (TCID(50)) per reaction based on 10-fold dilutions of a titrated EV71 or CVA16 strain, respectively. No cross-reaction was observed with Coxsackievirus A (CVA) viruses (CVA2, 4, 5, 7, 9, 10, 14, and 24), Coxsackievirus B (CVB) viruses (CVB1,2,3,4, and 5) or ECHO viruses (ECHO3, 6, 11, and 19). The assay was further evaluated with 47 clinical stool specimens diagnosed previously with EV71, CVA16 or other human enterovirus infections. Virus isolates from stool samples were confirmed by virus neutralization testing and sequencing. RT-LAMP with HNB dye was demonstrated to be a sensitive and cost-effective assay for rapid visual detection of human EV71-C4 and CVA16.
Journal of virological methods 08/2011; 175(2):283-6. · 2.13 Impact Factor
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Xiu-Mei Hu,
Yong Zhang,
Bang-Lao Xu,
Meng-Jie Yang, Miao Wang,
Chen Zhang,
Jin Li,
Ru-Yin Bai,
Xiao-Mian Zhou,
Wen-Bo Xu,
Xue-Jun Ma
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ABSTRACT: A multiplex RT-PCR assay based on GeXP system was developed in order to detect simultaneously human enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) and other coxsackieviruses (CVA4, 5, 9 and 10, CVB1, 3 and 5). Enterovirus detection was performed with a mixture of 12 pairs of oligonucleotide primers including one pair of published primers for amplifying all known pan-enterovirus genomes and eleven primer pairs specific for detection of the VP1 genes of EV71, C A16, CVA4, CVA5, CVA9, CVA10, CVB1, CVB3 and CVB5, respectively. The specificity of multiplex RT-PCR system was examined using enterovirus cell cultures and positive strains identified previously from hand-foot-and-mouth disease (HFMD) patients. Serial dilution of titrated EV71 and C A16 cell cultures and in vitro transcripted RNA of enterovirus VP1 regions were used to detect the sensitivity of the multiplex RT-PCR system. The limit of detection for this multiplex RT-PCR system was 10(0.5) TCID50/microL for EV71 and C A16 cell cultures and 1000 copies for in vitro transcripted RNA of nine viruses per assay. This multiplex RT-PCR assay is a rapid, sensitive and specific assay for the diagnosis of common enterovirus infection in cases of HFMD outbreak and is also potentially useful for molecular epidemiological investigation.
Bing du xue bao = Chinese journal of virology / [bian ji, Bing du xue bao bian ji wei yuan hui] 07/2011; 27(4):331-6.
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ABSTRACT: To establish a new and rapid GeXP based multiplex PCR assay for the detection and typing of human papillomavirus 6, 11, 31, 33 and 52.
Nucleotide sequences of HPV6, HPV11, HPV31, HPV33 and HPV52 from NCBI were obtained and compared. Genotype-specific primers were then designed and the sensitivity and specificity of multiple PCR assay was evaluated. Optimized assay was further validated with 30 clinical specimens collected from the cervical secretions of patients.
A GeXP based multiplex PCR was developed for sensitive detection and reliable differentiation of five HPV genotypes (HPV6, 11, 31, 33 and 52),
A GeXP based multiplex PCR assay is demonstrated to be a new and rapid technique for simultaneous detection and typing of 5 different human papillomaviruses.
Zhonghua shi yan he lin chuang bing du xue za zhi = Zhonghua shiyan he linchuang bingduxue zazhi = Chinese journal of experimental and clinical virology 02/2011; 25(1):69-72.
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ABSTRACT: A simple, rapid and sensitive colorimetric loop mediated isothermal amplification (LAMP) method was established to detect HPV6 and HPV 16 respectively. The method employed a set of four specially designed primers that recognized six distinct sequences of HPV6-E6 or HPV16-E7 for amplification of nucleic acid under isothermal conditions at 63 degrees C for one hour. The amplification process of LAMP was monitored by the addition of HNB (hydroxy naphthol blue) dye prior to amplification. A positive reaction was indicated by a color change from violet to sky blue and confirmed by real-time turbidimeter and agarose electrophoresis. Thirteen cervical swab samples having single infection with 13 different HPV genotypes were examined to evaluate the specificity. A serial dilution of a cloned plasmid containing HPV-E6 or HPV-E7 gene was examined to evaluate the sensitivity. The results showed that no cross-reaction with other HPV genotypes was observed. The colorimetric LAMP assay could achieve a sensitivity of 1000 copies, 10-20 times lower than that of real-time PCR. The assay was further evaluated with 62 clinical specimens and consistent results were obtained compared with the detection using Kai Pu HPV Genotyping Kit. We concluded that this colorimetric LAMP assay had potential usefulness for the rapid screening of the HPV6 or HPV16 infection in the laboratories and hospitals of provincial and municipal region in China.
Bing du xue bao = Chinese journal of virology / [bian ji, Bing du xue bao bian ji wei yuan hui] 01/2011; 27(1):64-70.
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Jun Han,
Xue-Jun Ma,
Wen-Bo Xu,
Jun-Feng Wan,
Qun Li,
Chan Tian,
Chen Gao, Miao Wang,
Liu-Ying Tang,
Yong Zhang,
Cao Chen,
Yan-Hai Wang,
Feng Wang,
Yong-Jun Gao,
De-Xin Li,
Xiao-Ping Dong
The Journal of infection 11/2010; 62(1):107-8. · 4.13 Impact Factor
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ABSTRACT: A novel application of the GeXP genetic analysis system for the differential detection of pandemic influenza A H1N1 from seasonal influenza A H1N1 and H3N2 is described. The assay was evaluated using identified influenza viruses and clinical samples. The results indicate that the assay is both highly sensitive and specific for the detection of the pandemic influenza A H1N1 virus with a detection limit of 10 copies per reaction superior to that of assays in use currently. The assay is able to detect potential mixed infections. This technique has the potential to provide both a powerful method to enhance surveillance of influenza and a platform for investigating the differentiation of other similar pathogens.
Journal of virological methods 05/2010; 168(1-2):255-8. · 2.13 Impact Factor
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Xue-Jun Ma,
Yue-Long Shu,
Kai Nie,
Meng Qin,
Da-Yan Wang,
Rong-Bao Gao, Miao Wang,
Le-Ying Wen,
Feng Han,
Shu-Mei Zhou,
Xiang Zhao,
Yan-Hui Cheng,
De-Xin Li,
Xiao-Ping Dong
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ABSTRACT: A sensitive reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for rapid visual detection of pandemic influenza A H1N1 virus infection. The reaction was performed in one step in a single tube at 65 degrees C for 60 min with the addition of hydroxynaphthol blue (HNB) dye prior to amplification. The detection limit of the RT-LAMP assay was approximately 60 copies, and no cross-detection was observed. The assay was evaluated further with 50 clinical specimens diagnosed clinically with seasonal influenza or pandemic influenza A H1N1 virus infection. RT-LAMP with HNB dye was demonstrated to be a sensitive and easy assay for rapid detection of pandemic influenza A H1N1 virus.
Journal of virological methods 04/2010; 167(2):214-7. · 2.13 Impact Factor
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Meng Qin,
Da-Yan Wang,
Fang Huang,
Kai Nie,
Mei Qu, Miao Wang,
Feng Han,
Xiang Zhao,
Yan-Hui Cheng,
Yue-Long Shu,
Xue-Jun Ma
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ABSTRACT: In this study, we established a rapid and sensitive multiplex Real-time reverse transcription polymerase chain reaction (mrtRT-PCR) to simultaneously detect the novel human influenza A H1N1 virus, human seasonal influenza A H1N1 and H3N2. This assay had three pairs of primer to target the conserved regions of the HA gene for each of the HA types including novel H1N1, seasonal H1N1 and seasonal H3N2, and one pair of primer designed to detect the internal control-RnaseP gene. This assay was performed in one-step in one tube. To validate the specificity of the multiplex Real-time RT-PCR assay, different human influenza virus including human seasonal influenza A H1N1 and H3N2, human influenza B and reference A/California/07/2009 (H1N1) sw1 was tested. To evaluate the sensitivity of the assay, serial dilutions of RNA from in vitro transcription of the novel human influenza A H1N1 HA gene was tested. Finally this assay was evaluated with clinical samples from 54 fever patients diagnosed with novel influenza A H1N1 or seasonal H1/H3 or HB infection either by real-time PCR recommended by the WHO or HI assay by the National Influenza Center. Our results showed that the assay could achieve a sensitivity of 20 RNA copies of novel influenza A H1N1 with high specificity and could detect potential mixed co-infection. In conclusion, this multiplex real-time RT-PCR assay combines both rapidity and sensitivity for not only detecting the novel human influenza A H1N1 virus, but also monitoring the human seasonal influenza A H1N1 and H3N2 simultaneously.
Bing du xue bao = Chinese journal of virology / [bian ji, Bing du xue bao bian ji wei yuan hui] 03/2010; 26(2):97-102.
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Kai Nie,
Da-Yan Wang,
Meng Qin,
Rong-Bao Gao, Miao Wang,
Shu-Mei Zou,
Feng Han,
Xiang Zhao,
Xi-Yan Li,
Yue-Long Shu,
Xue-Jun Ma
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ABSTRACT: A simple, rapid and sensitive colorimetric Reverse Transcription Loop Mediated Isothermal Amplification (RT-LAMP) method was established to detect human influenza A H1N1 virus. The method employed a set of six specially designed primers that recognized eight distinct sequences of the HA gene for amplification of nucleic acid under isothermal conditions at 65 degrees C for one and half hour. The amplification process of RT-LAMP was monitored by the addition of HNB (Hydroxy naphthol blue) dye prior to amplification. A positive reaction was indicated by a color change from violet to sky blue and confirmed by agarose electrophoresis. The specificity of the RT-LAMP assay was validated by cross-reaction with different swine and human influenza virus including human seasonal influenza A /H1N1 A /H3N2, influenza B and swine A /H1N1. The sensitivity of this assay was evaluated by serial dilutions of RNA molecules from in vitro transcription of human influenza A H1N1 HA gene. The assay was further evaluated with 30 clinical specimens with suspected pandemic influenza A H1N1 virus infection in parallel with RT-PCR detection and 26 clinical specimens with seasonal influenza virus infection. Our results showed that the RT-LAMP was able to achieve a sensitivity of 60 RNA copies with high specificity, and detection rate was comparable to that of the RT-PCR with the clinical samples of pandemic influenza A H1N1 infection. The RT-LAMP reaction with HNB could also be measured at 650nm in a microplate reader for quantitative analysis. Thus, we concluded that this colorimetric RT-LAMP assay had potential for the rapid screening of the human influenza A H1N1 virus infection in National influenza monitoring network laboratories and sentinel hospitals of provincial and municipal region in China.
Bing du xue bao = Chinese journal of virology / [bian ji, Bing du xue bao bian ji wei yuan hui] 03/2010; 26(2):81-7.
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ABSTRACT: EV71 is associated with the fatal cases of brain stem encephalitis during large HFMD outbreaks from 1998 to 2008. EV71 may continuously shed from upper respiratory tracts and feces of HFMD patients for relatively long time after recovery. However, the persistence of viruses in the patients' secretions and excretions is not clear.
Serial throat swabs and feces of 34 definitely diagnosed patients, including 30 mild cases and 4 severe cases, were traced and collected with the interval of 2 to 4 days for up to 32 and 48 days, respectively, and tested by a nested RT-PCR.
The EV-71 specific sequences were identified by a Nested RT-PCR in all specimens of 0-4 days, and 5-8 days. The positive rates of EV71 in throat swabs dropped markedly to 42.86% during 9-12 days, and maintained at 20-30% during 13-24 days, while that in feces reduced to 71.43% during 9-12 days, and maintained roughly 20% till 37-40 days. EV71 nucleotide of 36.36% cases disappeared simultaneously both in throats and feces, 39.39% cases showed longer persistence of EV71 nucleotides in feces, and 21.21% were longer in throats. The longest duration of shedding observed was 24 days for throat swabs and 42 days for fecal specimens.
EV71 shedding from respiratory tract may continue for nearly four weeks after onset, but its excretion through feces can persist more than five weeks.
BMC Infectious Diseases 01/2010; 10:178. · 3.12 Impact Factor
