| According to WHO report, about 2 billion cases of diarrhea occured every year, with more common in developing countries due to the poor sanitation. The global analysis of children during 2000 to 2010 manifests that diarrhea ranked No.3 of the causes of death in children under five years old, only after pneumonia and perinatal diseases. A substantial reduction in deaths caused by diarrhea has been observed over the last decade. However, diarrhea was still one of the leading causes of death and disability in the developing world. In China, the incidence of infectious diarrhea ranked the top three of infectious diseases. Documenting the etiology of diarrhea and developing fast and accute methods for causative agents screening are essential for development of prevention and treatment strategies and vaccine design.Diarrhea was defined as sudden onset of watery diarrhea over 3 times within 24 hours, companied by vomiting, fever and respiratory symptoms or not. There are two types of diarrhea, infectious and not infectious. Virus, bacteria and parasite are all related to infectious diarrhea. Pathogens are including Adenovirus, Norovirus, Rotavirus, Campylobacter, Escherichia coli 0157, Salmonella, Shigella, Vibrio cholerae, Yersinia enterocolitica, Cryptosporidium, Entamoeba histolytica and Giardia. Rapid, specific and high-throughput laboratory technology for diarrhea-associated pathogens detection is very helpful to assist the clinical medication and control diarrhea outbreak.Traditional diagnostic method for diarrheal pathogens is bacterial culture, which is time consuming, labor intensive and relatively insensitive. The immunological methods, including latex agglutination test, enzyme-linked immunoassay, radiation immunoassay, immune colloidal gold method, immune chromatography, etc., have been widely used in clinical laboratory. However, the strict requirements for monoclonal antibody and lack of integration in most system limit the development of fast and throughput immunological methods for diagnosis. Molecular biotechnology, such as reverse transcription-polymerase chain reaction (RT-PCR) and realtime RT-PCR (rRT-PCR), has provided information on detection and evaluation of bacterial or viral regarding to infectious diarrhea prevalence. Furthermore, molecular biotechnology can be used for further genotyping of viral epidemiological studies. In a word, molecular biotechnology method has obvious advantages in terms of sensitivity nd specificity. Most conventional (RT-) PCR assays are monoplex, for which use single set of primer and can detect only one target virus. These methods are potentially expensive and resource intensive, since diarrheal is caused by several viruses with similar clinical manifestions and the inability to culture some of these viruses, such as Norovirus and Sapovirus, in vitro. In addition with the gradually mature of gene chip technology, (RT-) PCR assays are changing from monoplex to multiplex meaning that incorporating different sets of specific primers for two or more targets in one reaction tube, simultaneous amplification of different target nucleic acids in a single test become true. Due to the reduction in labor and reagent costs and running time, the multiplex molecular methods are powerful tools in clinical and epidemiological studies.Part Ⅰ. Distrisbution of Enteropathogens in Guangzhou from September 2013 to October 2014Infectious diarrhea caused by a wide range of pathogens including bacteria, viruses and parasites. Etiology diagnosis depends on clinical laboratory detection of diarrhea stool specimens, which routinely been culture of bacteria, immune colloidal gold method of virus and microscopic examination of parasite. However, these methods are labor intensive and relatively insensitive. And for diarrhea etiology investigation at home and abroad, due to the limitations of detection methods, epidemiological reports are concentrated in one kind of viral or bacterial pathogens. According to the white paper on the Guangzhou economic society, Guangzhou has become the fifth city of tens of millions of population size after Chongqing, Shanghai, Beijing and Tianjin since the end of 2013, which means that once diarrhea outbreak will be a heavy burden to the development of society and economy. At the same time, the research into the causes for diarrhea in Guangzhou is still inadequate. Therefore, it is necessary to monitor diarrhea etiology in Guangzhou using advanced detection methods.Completing in approximately 5h, the xTAG Gastrointestinal Pathogen Panel (xTAG GPP) assay based on the technology of liquid suspension chip detects in one assay the most common diarrhea-causing pathogens and toxins, namely Adenovirus 40/41, Norovirus genogroup Ⅰ/Ⅱ, Rotavirus A, Clostridium difficile toxin A/B, Campylobacter sp., Escherichia coli 0157, Enterotoxigenic E. coli heat-labile enterotoxin/heat-stable enterotoxin, Salmonella sp., Shiga-toxin producing E. coli, Shiga-like toxin (Stx)1/2, Shigella sp., Vibrio cholerae, Yersinia enterocolitica, Cryptosporidium sp., Entamoeba histolytica and Giardia sp.. The performance and serviceability of xTAG GPP for detection of enteropathogens in diarrheal patients had been evaluated in our lab previously. In the study, a total of 289 stool specimens collected from diarrhea patients during September 2013 to October 2014 in Zhujiang hospital Guangzhou were detected by xTAG GPP.The results showed that the detection rate of positive samples was 74.4% (215/289), in which bacteria and virus accounted for 47.4% and 35.0% respectively. Bacterial diarrhea occured throughout the year and all age groups, while viral diarrhea has the obvious trend of high incidence in cold season and 1 to 3 years-old age group. The difference between bacterial and viral diarrhea in clinical symptoms is that the former is more likely to have positive results of fecal white blood cell (WBC) and/or red blood cell (RBC) and/or occult blood test (OB). Nevertheless, the latter was more likely causing vomiting. In addition, the genotyping experiment in positive viral infectious specimens indicates that Rotavirus G2P[8] and Norovirus GII.4 are the prevalent strains.Part Ⅱ. Development a Multiplex RT-PCR Method for Detecting 5 Viruses Related to DiarrheaDiarrhea etiology diagnosis is vital for the clinical treatment and correct medication. Differentiation between bacterial and viral diarrhea by a specific, rapid and sensitive diagnostic tool may help to effectively improve the clinical effect, to avoid the abuse of antibiotics and to limit economic losses and unnecessary pain to the patient. Conventional method in Clinical laboratory for the detection of bacterial diarrhea is fecal culture, which takes long time about 3-5 days and requires higher experimental technology to operators. On the other hand, clinical laboratory test for viral diarrhea usually use the immune colloidal gold method. Although immunological detective method is quick and easy to operate, unsatisfactory specificity and sensitivity and the limitation of covering viral pathogens spectrum can easily cause misdiagnosis. The xTAG Gastrointestinal Pathogen Panel (xTAG GPP) assay based on the technology of liquid suspension chip has a sensitive, specific and high-throughput multiplex detection capability. However, this method has not yet been routinely used in clinical laboratory due to its high-cost. Therefore, we aim to develope a multiplex RT-PCR for viral pathogens detecting simultaneously for the rapid and accurate diagnosis of viral diarrhea.The multiplex RT-PCR was developed for texting Norovirus genogroup I and II, Adenovirus, Sapovirus and Astrovirus simultaneously. Primers for five targets were from previously published. The optimizing of the multiplex RT-PCR system included the concentration of primers and proper annealing temperature. The nucleic acid of non-target virus, including Rotavirus, Enterovirus71, Coxackie A16, Salmonella, Shigella, Clostridium Difficile Bacteria, Escherichia Coli 0157, were used to verify the specificity of the multiplex RT-PCR. A total of 107 feces samples from patients with diarrhea were collected and tested in parallel by both multiplex RT-PCR and xTAG GPP for Adenovirus, Norovirus genogroup I and II as well as by both multiplex RT-PCR and monoplex RT-PCR for Astrovirus and Sapovirus.The multiplex RT-PCR showed high consistency with xTAG GPP even for co-infections samples, with the Kappa value of 0.885(P=0.000). Compared to xTAG GPP, the sensitivity and specificity of the multiplex RT-PCR were 80.8% and 100% respectively for Norovirus genogroup I and Ⅱ, Adenovirus. As compared to monoplex RT-PCR, the multiplex RT-PCR showed highest consistency with a Kappa value of 1.0(P=0.000) for Sapovirus and Astrovirus. The detection limit and accuracy of multiplex RT-PCR were 104opies/μL～106 copies/μL and the sequences of virus positive samples shared high identity (97%-100%) with the targets.Part III. The genotype analysis of astrovirus and sapovirus infection among people with acute diarrhea in Guangzhou, ChinaHuman viral diarrhea is caused by a number of viruses, including noroviruse, rotaviruse, adenoviruse, astroviruse and sapoviruse. Currently, the rotavirus, norovirus and adenovirus infectious diarrhea in the domestic and overseas has detailed epidemiological data. Although astrovirus and sapovirus are two of the major causative agents for diarrhea, there is relatively little information on the ecology and epidemiology of these viruses on account of low detection rates. Human astrovirus (HAstV), in the genus Mamastrovirus of the family Astroviridae, have a single-stranded positive-sense RNA genome of approximately 6.8 to 7.2 kb in length comprised of three open reading frames (ORFs):ORF1a, ORF1b, and ORF2. Based on nucleotide sequence analysis of a partial region of ORF2, HAstV are divided into eight genotypes (HAstV 1-8) that have been demonstrated for their good correlation with eight serotypes. The sapovirus (SaV) as well as norovirus is a distinct genus within the family Caliciviruses. SaV possess a positive-sense ssRNA genome of approximately 7.5 kb in length, and have two ORFs. At present, SaV are classified formally into 5 genogroups (G I-GV) based on phylogenetic analysis of the full-length VP1 sequence, among which only genogroups I, Ⅱ, IV and V are known to infect human. The objective of the study was to investigate the incidence of virus infection in patients with diarrhea in Zhujiang Hospital of Guangzhou during October 2013 to September 2014, and further identify the genotypes of HAstV and SaV.A total of 273 stool samples were collected from 273 people who suffered from diarrhea and attended to Zhujiang Hospital of Southern University from October 2013 to September 2011 in this study. Astrovirus, sapovirus, norovirus (G I and GⅡ) and adenovirus were tested using the multiplex reverse transcriptase polymerase chain reaction (multiplex RT-PCR). Positive samples for astrovirus and sapovirus were sent for gene sequencing. Of the 273 samples,45 were positive, yielding all the enteropathogens detected, including 9 (3.3%,9/273) Astrovirus,4 (1.5%,4/273) sapovirus, one (0.4%,1/273) norovirus G Ⅰ,29 (10.6%,29/273) norovirus GⅡ, and 2 (0.7%,2/273) adenovirus. Sequencing result for astrovirus and sapovirus showed identification of astrovirus-1, astrovirus-2, astrovirus-4, astrovirus-8 and sapovirus GI genotypes.In summary, we first presented an etiology investigation of diarrhea patients in Guangzhou from September 2013 to October 2014 using xTAG GPP based on the suspension arrays for detecting 15 enteropathogens. Secondly, a multiplex RT-PCR was developed and evaluated by comparing with xTAG GPP. It was indicated a rapid diagnostic tool for the detection of the major viral pathogens related to diarrhea in clinical laboratory with high specialty, sensitivety and high throughput. Finally, using the multiplex RT-PCR, we further investigated the genetype distribution of astrovirus and sapovirus in diarrhea patients of Guangzhou. |
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