The application and epidemiological research of xTAG-GPP multiplex PCR in the Diagnosis of children persistent and chronic diarrhea

Background Persistent and chronic diarrhea is difficult to treat, and infection is still the main causes. Clearly infected pathogens are essential for treatment. In this study, we investigate the application value of xTAG gastrointestinal pathogen panel (xTAG GPP) multiplex PCR in the early diagnosis of children with persistent and chronic diarrhea and understand the epidemiology of intestinal diarrhea pathogens. Methods One hundred and ninety-nine specimens were collected from Nanjing Children's Hospital Affiliated to Nanjing Medical University (Nanjing, China). Comparing the xTAG GPP multiplex PCR Assay with the traditional methods (culture, rapid enzyme immunoassay chromatography, microscopic examination) and madding the statistical analysis. Results The positive rate of 199 patients with diarrhea specimens was 72.86% (145/199). The virus detection rate was 48.7%, Rotavirus A was the most common organism detected (34.6%), concentrated in winter, popular in children. Secondly, Norovirus GI/GII (20.6%). The positive rate of bacteria was 40.2%, Campylobacter (22.11%, 44/199) was most frequently detected. With C. difficile toxins A/B and Salmonella detected 44 and 17 samples, respectively. Infections with Shigella occurred 4 times, E. coli O157 was only detected once. There were three samples with parasitic (1.51%), two samples were positive for Entamoeba histolytica, one for Cryptosporidium. Adenovirus40/41, STEC, ETEC, Giardia, Yersinia enterocolitica and Vibriocholera were not detected. Totally 86 (43.2%) infected specimens with single pathogen were detected. There were 57 co-infections (28.64% of samples) of viruses and/or bacteria and/or parasites. Co-infections involved 29 double infections (23.62%) samples, 9 triple infections (4.52%) and 2 quadruple infections (0.5%). Norovirus GI/GII was found to have the highest involvement in co-infections 30(15.08%).


Background
Diarrhea continues to be a health burden world-wide, especially in children living in developing countries. It is estimated that in these regions it is responsible for 2.5 million infant deaths annually, with a mortality rate of 4.9 per 1,000 children and anannual incidence of 3 episodes per child among children under 5 years of age [1,2]. Most of the diarrheal illnesses are acute, lasting no more than 7 days; however, about 3%-19% of the acute episodes lasting more than two weeks, which called persistent and chronic diarrhea [3]. Persistent and chronic diarrhea cases are difficult to treat, their treatment cost is higher, and a case fatality rate as high as 60% has been reported [4]. It is more important to determine the cause of chronic diarrhea by a systematic approach, because it can provide the most suitable therapy and give a good prognosis. The causes of chronic diarrhea are divided into infectious and non-infectious causes. In the developed countries, the incidence of noninfectious based diseases (food allergy, enteropathy or inflammatory) is increasing. However, in developing and industrialized countries, the most common and most important cause of persistent and chronic diarrhoea is still enteric infection [5,6]. It is crucial for timely and effective treatment of infectious diarrhea in the rapid identification of pathogens, because appropriate antimicrobial therapy and/or isolation measures to prevent the spread of infectious patients from healthy people can shorten the disease and reduce some bacteria and parasites infection incidence, and can help the invasive infection [7,8]. The gold standard for the diagnosis of infectious chronic diarrhea pathogens is the culture of pathogens, but this method takes a long time (72 hours) and requires higher fecal Thus, the purpose of our study was to explore the distribution of enteropathogens in persistent and chronic diarrheal patients in Nanjing, China, and to further evaluate the performance and applicability of xTAG GPP in identifying pathogens in these children.

Sample collection
A total of 199 stool samples prospectively collected from 199 diarrhoeic children mainly under 5 years of age (85.93%, Table 1) which including 103 simple of diarrhea and 88 secondary diarrhea (colitis, pneumonia and tumor associated) that attending the Nanjing Children's Hospital Affiliated to Nanjing Medical University (Nanjing, China). The study protocol was approved by the ethics committee of the Children's Hospital of Nanjing Medical University (Nanjing, China). Written informed consent was obtained from the proband and their parents. One sample was received from each patient. Inclusion criteria patients with diarrhoeic present watery and/or loose and/or mucous and/or blood stools with ≥3 instances within a 24-hour period. Patients with inflammatory bowel diseases were excluded from the study. Stool samples were sent to the Department of Microbiology for investigation. 5g of fresh stool samples were collected into empty tube were placed in Carye Blair transport Medium for bacterial culture. Stool specimens were then stored at -80°C until processing with the multiplex PCR tests. Unqualified samples (samples volume < 5g, swabs not preserved in Carye Blair Medium) were rejected and resubmission requested.

Routine diagnostic methods
Stool culture for Salmonella and Shigella was performed using Salmonella-Shigella agar plates and Hektoen enteric agar plates. To detect toxigenic Clostridium difficile A and B toxins, norovirus GI/GII, realtime reverse transcription-polymerase chain reaction (RT-PCR) assays were performed on the 7500 real-time PCR platform (Applied Biosystems, Foster City, CA). Rotavirus were detected directly in stool samples with rapid enzyme linked immunosorbent assay (ELISA) tests: Diagnostic Kit for Rotavirus. All assays were carried out in accordance with the instructions. We looked for Entamoeba histolytica and Giardialiamblia by microscopic examination of fresh stools.

Multiplex PCR assays for 15 pathogens detection/Molecular diagnostic assay
Total nucleic acids were extracted from the stool samples using the NucleoSpin® Virus Kit (MACHEREY-NAGEL, Germany) according to the manufacturer's instructions. An internal control (bacteriophage MS2) was included in each specimen to control the quality of the detection process.
The RT-PCR reactions and subsequent hybridization step were performed according to the instructions in the GPP manual. Negative and positive controls were included in all runs of the GPP assay. The data were acquired on the Luminex 200 analyzer and data analysis was carried out using TDAS GPP version 1.11 (xTAG Data Analysis Software).

Demographic and clinical parameters of patients with persistent and chronic diarrhea
The demographic and clinical characteristics of the 199 patients are summarized in Table 1. One hundred and ninety-nine stool samples were prospectively collected from 199 diarrhoeic children mainly under 5 years (85.93%, 171/199), mean age was 12.93±15.86 months. The percent of boys (58.29%, 116/199) was slightly higher in comparison to girls (41.71%, 83/199). There were 163 persistent diarrhea cases and 28 chronic case, the majority were the inpatients (88.44%, 176/199) during the study period, no deaths were reported. Of the 199 stool specimens submitted to laboratories, watery/loose stool (n=139, 72.78%) were the most common type, mucus/bloody stool were less about 27.22% (52/199).

Comparison of the xTAG GPP and conventional detection
Among the enteropathogens that can be detected by xTAG GPP, 5 enteropathogens cannot be detected by routine detection(STEC, ETEC, Adenovirus 40/41, Yersinia enterocolitica and Campylobacter), so in this study, the specificity and sensitivity of this method for the diagnosis of these five enteropathogens are not compared. As shown in Table 4 enteropathogens Giardia and Vibriocholera have not been detected positive samples in our samples by both xTAG GPP and the routine assays, so it is impossible to evaluate the sensitivity of these enteropathogens. The overall sensitivity and specificity of xTAG GPP for the diagnosis of intestinal pathogens were 96.3% and 98.2% respectively, which were significantly higher than those of conventional detection. The sensitivity and specificity of this method to individual pathogens are shown in Table 4.

Age and sex distribution of children with enteropathogens
The prevalence of enteropathogens among sex groups was compared, 75.86% male patients and 57 (68.67%) female patients were positive for enteropathogens. The distribution of enteropathogens was similar in both boys and girls (Table 5), with Rotavirusas A the most common pathogen detected at 39.66% and 27.71%, respectively, followed by Campylobacter and Norovirus GI/GII, there was also no significance in co-infection (p>0.05). The distribution of virus, parasite and co-infection were similar in the three age group (0-12months, 12-60 months and ≥60 months ), the P value were 0.73, 0.724 and 0.76 respectively (Table 5). Rotavirus A was the most common enteropathogen in patients 0-12 months (37.9%, ) and 12-60 months (33.33%) in age, while Campylobacter was the most frequent enteropthogen in patients ≥60 months ( 28.6%, 8/18 ). In this study, Bacteria infections were the most common in the 12-60 months age group (57.1%) compared with the other age groups (33.3%-46.4%).

Seasonal distribution of children with enteropathogens
In this study, the seasonal curve of virus had a peak in winter and trough in summer, Rotavirus A was

Discussion
There are few data that simultaneously describe the prevalence of bacterial and viral pathogens in persistent and chronic diarrhea children in China. In our research, the persistent and chronic diarrhea have related to the sex and age, common exist in the boy especially under 2 years old children, in our study, the boy was 116 (58.29%), 0-1 years patient was 129 (64.82%), this was similar with the research that the morbidity age was 4 months to 1 year [3]. Patient with watery and/or loose were predominate more than the mucoid/bloody group consider that the most sample were collected in winter (Table 4).
In this study, the overall sensitivity and specificity of, xTAG GPP were 96.3% and 98.2% respectively, which were significantly different from those of routine detection. At the same time there were significant differences in single or mixed intestinal enteropathogen infection (P < 0.001) ( Table 4). In the present study, the xTAG GPP method efficiently detected about 57(28.64%) out of 199 children showed multiple positive results (co-infection) ( Table 2), this figure is higher than that was relatively high compared with the positive result also detected by xTAG GPP in Deng J et al previous study [13].
Norovirus GI/GII was found to have the highest involvement in co-infections in our study.
In our study, Rotavirus A was the most common pathogen in children with chronic diarrhea in spring and autumn, followed by Norovirus. Previous reports have shown that Rotavirus A is the most common virus that causes diarrhea in children [15]. Moreover, Norovirus is an important cause of diarrhea in adults and children [16].
This result was similar to other studies conducted previously in China [17][18][19] as well as other countries prior to the introduction of rotavirus vaccination [20,21].
In our study, 4 samples with positive Salmonella culture had no positive results in xTAG GPP, existing false negative results. This phenomen is consistent with some previously reported results [13,22].
The cause of the failure for Salmonella pathogen needs further sequence analysis or qPCR assay investigation. Special attention should be paid to the occurrence of Campylobacter, in our study we detected a high frequency of 25% (43/199) by the xTAG GPP assay, because in China the detection of Campylobacter is rarely requested in patients with diarrhea.

Conclusions
In conclusion, our research shows that xTAG GPP has very good sensitivity and specificity in detecting

Availability of data and materials
All data generated or analysed during this study are included in this published article.

Competing interests
The authors declare that they have no competing interests.

Funding
This work was supported by the National Natural Science Foundation of China (grant number: 81570470). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Authors' contributions
CLW and XYZ researched the topic, analyzed the data, and was a major contributor in writing the    Figure 1 Seasonal distribution of children with enteropathogens detected by xTAG GPP assay.