| Background:Acute infectious diarrhea was a common disease with a high incidence rate in our country.83.6 million people sufferd it ever year. Athough its incidence rate decreased recently, with the reforming and opening, urbanizing the countryside, especially the development of industry food processing and entertainment, in rural area, the connecting area between city and village, where the people were from different areas and with different backgrounds, the incidence rate wasn't low. Diarrhea breaked out sometimes. So making surveillance of diarrhea in these areas secularly, systemly and in a planned way was significant and important to controlling and preventing.Virus was the major pathogen of diarrhea, and Rotavirus (RV), Norovirus(NV), Astrovirus, Sapovirus were the main pathogens. RV infected children who were under 5 years old mainly. In China over half of diarrhea in children were caused by RV. In autumn and winter it was popular usually. At the same time, many researches in and out of our country pointed out that NV was an important pathogen of acute gastroenteritis. NV caused preveillings and outbreakings in every age group, especially in travelers.Enterovirus type 71 and CoxsachievirusA 16 were the main pathogens caused Hand-foot-and-mouth disease (HFMD). Their transmissions were through the fecal-oral cycle, respiratory tract and close contacting mainly. Although EV71 and CoxA16 didn't cause diarrhea mainly, they hermited in intestine, it may point a new way of researching their characterization and pathogenesis if we can detect them from dirrahea patient's stool.Objectives:1. Analyzing the infection of rotavirus (RV), norovirus (NV) in the monitoring community, including the season,age,sex distributions, and the gene type of the prevailing trend.2. Probing the clinic characters and main risks of diarrhea caused by RV, NV in the community.3. Comprehending the infection of enterovirus type 71 (EV71) and coxsachievirusA 16 (CoxA16) in diarrhea patients, and provide a new idea to research their pathogenesis.Methods:1. Monitoring samples:We collected stool samples in a community health service station in Baiyun district, Guangzhou, and made questionnaires with the patients (or their parents) from May,2010 to April,2011.2. Evaluating TD-PCR:We estabished and refined the procedure of TD-PCR, which can detect NV,EV71 and CoxA16 at the same time. 3. Sensitivity:We searched the sensitivity of the TD-PCR, by means of ten folds serial dilution and ultraviolet spectrophotometry.4. Sepicificity:We made gene sequence analyzing to the amplifying products of TD-PCR, and inspecting its specificity by amplifying RV, Sapovirus SV, Astrovirus AstV, Salmonella typhi, Yersinia enterocolitica Ye, Laribacter hongkongensis LH.5. Data were put in by Epidata 2.1, and statistic analyses were performed using SPSS software version 13.0(SPSS Chicago,IL). We used chisquare test for categorical data. A P value less than 0.05 was considered statistically signigicant.Results:1.We collected 244 of which 241 were available in the sentinel hospital from May,2010 to Apirl,2011. Diarrhea was sporadic there and the summit of incidence rate appeared in Jun, Oct and Nov. There were 161 men and 83 women. The oldest was 59, the youngest was just 7 days after the birth, and most of them were under 3 years old (89.75%).2. The monitoring results:In the 241 samples,120 were all of the 4 viruses detected negatively(49.8%), only one virus detected positive were 107(44.4%), inter infection with two types of virus were 13 samples(5.4%), and only one child infected with NV, EV71 and CoxA16, no one infected with the 4 types of virus. RV (+) were 104 samples, NV(+) 28, EV71(+) 3, CoxA16(+) 1.11 patients suffered with RV and NV, and 2 children suffered with RV and EV71. Most of the positive samples come from children who were under 3 years old, and there were no significant differences among different age groups..3. Results of RV:104 were positive,of which 71 were male,and 33 were female,the rate of sex was 2.15:1. There were no significant differences between male and female(χ2=0.429,P=0.513). The oldest patient was 59 years old, and the youngest was just 41 days, most of the patients were under 3 years old. There were no significant differences among different age groups (χ2=9.940,P=0.077). Most of the patients (RV+) were detected in August or Novenber, the detecting rate of RV were 20.19%(21/104) and 23.08%(24/104) respectively.4. Results of NV:28 were positive, half of which were male. The oldest patient was 22 years old, and the youngest was just 48 days, most of the patients were under 3 years old. There were no significant differences among different age groups (χ2=7.843,P=0.165). Most of the patients (RV+) were detected in August, the rate of RV was 64.29%(18/28).5. Risks analyzing:There no significant differences between Group A (no virus was detected) and Group B (at lest one virus between RV and NV were deteced) in basic human information, characters of stool, clinc symptoms and epidemiological attaching history. But in diagnosing time, patients'occupation and whether vomiting significant differences were found.6. The reaction systems of TD-PCR which can detect NV,EV71 and CoxA16 at the same time were GoTaq Green Master Mix (2×) 12.5μl (including remixed GoTaq DNA polymase,dNTPs,MgCl2 and buffer), template (cDNA) 2μl, upstream primer (10mM) 0.5μl, downstream primer(lOmM) 0.5μl, and added ddH2O to 25μl. The procedures of reaction were as fallows:94℃for 2 min; 20 cycles of 94℃45s, various annealing temperatures (55～45℃) for 45s and 72℃for 30s; and 75℃for 10min.7. TD-PCR was designed and the data suggested that TD-PCR was more sensitive than regular PCR. The minimum level of testing the virus of NV, EV71 and CoxA16 were 4.775μg/ml,2.360μg/ml,43.273μg/ml respectively. Other pathogens didn't show positive results by parallel tests. In clinic samples detecting, the incidence rate of NV by TD-PCR was 29.55%(65/220), while the regular PCR was 25%(55/220),χ2=8.1,P=0.002; the rate of EV71 by TD-PCR was 34.2% (25/73), while the regular PCR was 24.66%(18/73),χ2=5.14,P=0.016; the rate of CoxA16 by TD-PCR was 46.58%(34/73), while the regular was 27.40% (20/73),χ2=10.87,P=0.001.8.3 EV71 and 1 CoxA16 positive samples were detected by TD-PCR from diarrhea patients' stools.Conclusions:1. Results of community monitoring:RV (+) 104 samples, NV (+) 28, EV71 (+) 3, CoxA 16(+) 1, and there were inter infections.Most of the positive samples come from children who were under 3. But the incidence rates among different age and sex groups did not differ significantly.2. There no significant differences between Group A (no virus was detected) and Group B (at lest one virus between RV and NV were deteced) in basic human information, characters of stool, clinc symptoms and epidemiological attaching history. But in diagnosing time, patients' occupation and whether vomiting significant differences were found.3. Successfully establishing TD-PCR technique for detecting NV, EV71 and CoxA 16, and providing a reliable way to quickly and accurately detecting related virus from diarrhea and HFMD patients' stool.4. EV71 and CoxA 16 were detected from diarrhea patients' stools who has diahhea symptoms only. Maybe low level of virus of EV71 and CoxA16 in patients caused diarrhea. |
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