Clinical Significance of Real Time PCR Panel in Children With Acute Gastrointestinal Symptoms
Phase: NA
Conditions: Gastroenteritis Acute, Gastrointestinal Symptoms, Abdominal Pain/ Discomfort, Vomiting in Infants and/or Children
Sponsor: University of Oulu

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Cover Page for ClinicalTrials.gov 
 
Document 
Study Protocol 
 
Official study title 
Clinical significance of real time PCR panel in children with acute gastrointestinal symptoms 
 
Document Date 
October 16, 2025 
 

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Clinical significance of real time PCR panel in children with acute gastroin-
testinal symptoms – RealCAGI 
Joni Kalermo, Suvi Mattila, Kimmo Halt, Hilla Pöyry, Minna Honkila, Niko Paalanne, 
Vesa Mäki-Koivisto, Laura Savolainen, Terhi Ruuska-Loewald 
 
Abstract 
Acute gastroenteritis is one of the most common reasons for pediatric emergency visits in both 
general and pediatric emergency departments. For most of the children rehydration is the only 
therapy needed. However, a range of bacterial pathogens may need accurate diagnosis and tar-
geted antimicrobial therapy. Use of molecular multiplex testing has increased detection of patho-
gens in children with acute gastrointestinal symptoms. The PCR tests currently available enable 
rapid identification of gastrointestinal pathogens, with the test results often being available during 
the day sample was taken. However, it is unclear which of the patients are most likely to benefit 
from testing. Also, there is considerable uncertainty about the cost-effectiveness of the multiplex 
panels used to test for suspected infectious gastroenteritis in hospital and community settings. We 
have demonstrated that acute gastrointestinal symptoms are one of the most common diagnoses 
and a major cost in high-income population. 
 
We hypothesize that real time multiplex PCR testing for gastrointestinal pathogens at pedi-
atric emergency department setting could allow earlier initiation of appropriate antibiotics, 
reduce use of unnecessary antibiotics and improve identification of conditions in need for 
follow-up. 
 
To assess the diagnostic performance of alternative sample types, we investigate the use of 
rectal swab and vomit in detecting gastrointestinal pathogens. 
 
 
To estimate the usefulness of real time multiplex PCR testing, we will conduct investigator-driven 
academic clinical trial with randomized controlled study setting conducted at the Pediatric Emer-
gency Department of Oulu University Hospital. For eligibility, children aged under 16 years arriving 
to pediatric emergency due to acute gastrointestinal symptoms will be assessed. After obtaining 
the written consent, fecal specimens will be collected by the nurses from the first stool after arriving 
to hospital. The pediatric nurses also collect rectal swab samples from each child. Vomit is also 
collected, if vomiting occurs. 
 
The composite primary outcome consists of three outcomes which are evaluated using medical 
records: 1) correctly targeted antimicrobial treatment, 2) untargeted antimicrobial treatment and 3) 

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identification of conditions that require specific follow-up. Secondary outcomes, evaluated by medi-
cal records and electronic survey sent to families two weeks after the study visit, include: propor-
tion of correctly targeted antimicrobial treatment, proportion of untargeted antimicrobial treatment, 
proportion of conditions in need for hospitalization or specific follow-up, time needed for clinician to 
receive the results of the samples, length of hospital stay, time to correct diagnosis, resolution of 
symptoms, laboratory and radiology costs, total costs, need for surgical consultation and proportion 
of patients needing surgical procedure, proportion of unscheduled revisits and proportion of cor-
rectly used hospital infection control measures. 
 
In addition to the clinical trial conducted with fecal samples, we aim to assess the usability of rectal 
swab samples and vomit samples in identification of gastrointestinal pathogens. 
 
1. BACKGROUND 
 
Diarrheal diseases are the second leading infectious cause of mortality globally, after lower respir-
atory infections, among children younger than 5 years. Although childhood mortality due to diar-
rhea has declined substantially during previous decades, diarrhea is responsible for 500 000 
deaths annually (GBD 2017). Acute gastroenteritis is one of the most common reasons for pediat-
ric emergency visits in both general and pediatric emergency departments (Pöyry et al. 2021, 
Yoong et al. 2021). For most of the children rehydration is the only therapy needed. However, a 
range of bacterial pathogens may need accurate diagnosis and targeted antimicrobial therapy 
(Guarino et al. 2014). 
 
In previous observational studies, use of molecular multiplex testing for all children who presented 
to the ED with acute gastroenteritis has increased detection of pathogens and decreased return 
visits (Pavia et al. 2023). However, it is unclear which of the patients most likely to benefit from 
testing. Also, there is considerable uncertainty about the cost-effectiveness of the multiplex panels 
used to test for suspected infectious gastroenteritis in hospital and community settings (Freeman et 
al. 2017). 
 
Collection of fecal samples can be challenging and time consuming as children do not necessarily 
defecate during emergency visit. Finding the pathogens with recto-anal swab, that could be ana-
lyzed with real time multiplex PCR could significantly decrease the time to correct diagnosis. Rectal 
swabs have been reported to be reasonable option as sample collection method for real time PCR 
in children with or without diarrhea (Kabayiza et al. 2013) and in adults with norovirus infection (Si-
dler et al. 2014). Similarly, vomit samples have been reported eligible in identification of noroviral 
infection. (McHugh et al. 2018). The literature concerning usability of rectal swabs or vomit sam-
ples with multiplex PCR is however lacking. 
 
We have demonstrated that acute gastrointestinal symptoms are one of the most common diagno-
ses and a major cost in high-income population (Pöyry et al. 2021). 
 
 
 
2. RESEARCH HYPOTHESIS AND AIMS OF THE STUDY 

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Clinical significance of real time PCR panel for gastrointestinal pathogens 
 
 
We hypothesize that real time multiplex PCR testing for gastrointestinal pathogens at pe-
diatric emergency department setting could enhance initiation of appropriate antibiotics, 
decrease use of inappropriate antibiotics and increase identification of conditions in 
need for a particular follow-up or hospitalization. 
 
 
 
The aim of the study is to evaluate the clinical significance of real time multiplex gastrointestinal 
PCR panel at pediatric emergency department. 
 
Diagnostic study about alternative sample types for detecting gastrointestinal patho-
gens 
 
We also hypothesize that rectal swab samples are a rapid and competent sample 
collection technique in identification of gastrointestinal pathogens with multiplex PCR in 
children with acute gastrointestinal symptoms. 
 
 
 
Based on earlier literature, we hypothesize that rectal swab samples are rapid and competent 
sample collection technique in identification of gastrointestinal pathogens with multiplex PCR in 
children with acute gastrointestinal symptoms (Kabayiza et al. 2013). We also hypothesize that as 
in identification of norovirus, vomit samples could be utilized with multiplex PCR testing as well 
(McHugh et al. 2018). We aim to evaluate the sensitivity and specificity of rectal swab and vomit 
samples in relation to gold standard fecal samples. 
 
 
3. METHODS 
 
3.1 Clinical significance of real time PCR panel for gastrointestinal pathogens (RCT) 
 
Study design and oversight 
 
This is an investigator-driven academic clinical trial with randomized controlled study setting con-
ducted at the Pediatric Emergency Department of Oulu University Hospital. The samples are pro-
cessed in the NordLab clinical microbiology laboratory at Oulu University Hospital, where the diag-
nostic device is already in clinical use. The device has been as acquired by NordLab under na-
tional procurement legislation. 
 
Study population and inclusion criteria 

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For eligibility, children aged under 16 years arriving in pediatric emergency due to acute gastroin-
testinal symptoms (vomiting and/ or diarrhea and/or abdominal pain and/or watery stool) will be as-
sessed. Pediatric nurses in the ED screen children for eligibility upon arrival. Exclusion criteria are 
the need for cardiopulmonary resuscitation, immediate transfer to the intensive care unit, haemato-
oncological disease or other severe immunosuppression, bloody diarrhea or a clinical suspicion of 
typhoid/paratyphoid fever. Otherwise, patients with comorbidities or prior antibiotic treatment are 
included. The multiplex PCR test is used only in children admitted to the study or those that meet 
the exclusion criteria. 
 
Randomization 
 
Before initiation of the study, a biostatistician who is not involved in the data collection creates a 
randomization list using a computer-generated random sequence of numbers, randomization being 
performed in permuted blocks of sizes varying randomly between 4 and 6. After obtaining written 
informed consent from each child’s legal guardian and from children older than 6 years, the ED 
nurse opens the next opaque randomization envelope to assign the participant to either the inter-
vention or control group (Figure 1). 
 
 
 
Figure 1. Randomized, controlled trial study flow chart regarding the clinical benefit of active test-
ing. 
 
Sample Collection and Pathogen Detection 
After obtaining the written consent, fecal specimens will be collected by the nurses from the first 
stool after arriving in hospital. We collect fecal samples from all the children in both groups (n = 

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526) following the IVD (in vitro diagnostic) protocols. Also, all fecal samples will be processed ac-
cording to IVD protocols to ensure reliability of the results. The pediatric nurses also collect rectal 
swab samples from all children. If vomiting occurs, vomit samples are also collected. 
 
Fecal specimens will be collected by the pediatric nurses from the first stool after arriving in the 
hospital. Sterile swab such as Copan Regular Flocked Swab 502CS01 will be used to insert the 
specimen into Cary-Blair medium (Copan FecalSwab) according to manufacturer’s instructions. 
Rectal swab samples and vomit samples are collected using the same swab-kits. Swab is inserted 
approximately 2 cm into the rectum and rotated 3-4 times before removal. In case of vomiting, the 
swab is rotated in the gastric secretion. The specimens are then inserted into Cary-Blair medium 
solution, similarly to fecal samples. 
 
Fecal samples will be tested with QIAstat-Dx gastrointestinal panel 2, which can detect 13 gastro-
intestinal bacterial species, 5 viral pathogens and 4 different parasitic species with results ready 
within 70 minutes (Table 1). Positive test result for Clostridium Difficile is complemented with an 
addendum “Clostridium Difficile is part of normal gut microbiota in children under 2 years of age”. 
Following the IVD protocols, only fecal sample results are available to care taking physicians. Rec-
tal swab and vomit results are only used for the methodological analysis (Figure 2). Threshold cy-
cle (Ct) values for all samples will be recorded to estimate pathogen loads in different samples. 
 
Study results are available to the clinicians mostly within a few hours from receiving the fecal sam-
ple in the intervention group. In the control group the results are delayed until 72 hours. By analyz-
ing fecal samples in both groups, we ensure safety, transparency and enable a pathogen-specific 
impact analysis. 
 
If the child is admitted to hospital and the test is considered necessary the following day, a new 
sample may be taken in addition to study samples. 

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Table 1. Pathogens detected by the QIAstat-Dx Gastrointestinal Panel 2 
 
 
 
 
 
 
 
Follow-up 
Primary outcome and most secondary outcomes will be assessed using medical record review. For 
selected secondary outcomes, we will follow the patients with electronic survey sent via e-mail two 
weeks after the study visit. The survey is established, and the answers are retained on the Red-
CAP -platform. With the survey we estimate recovery of the child (duration of symptoms, return to 
day care or school). If the symptoms have not resolved by two weeks, an automized follow-up 
questionnaire will follow four weeks after the study visit. 
 
Sample size calculation 
With a composite primary outcome consisting of 1) use of correctly targeted antibiotic treatment, 2) 
avoiding incorrect antibiotic treatment and 3) identification of conditions in need for a precise fol-
low-up, we regard a clinically significant difference if the proportion of children with a positive com-
posite outcome is 20 % in the intervention group and 10 % in the control group. With an α error of 
5% and a statistical power of 80 % and using 1:1 randomization, with an assumption of 20 % loss 
in retaining requested fecal samples, we need 263 children in intervention group and 263 controls, 
for a total of 526 children. We use all children with alternative sample types (rectal swab or vomit) 
for the diagnostic analysis. 

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Primary outcome 
To assess the test’s impact on clinical decision making, primary outcome composite includes three 
questions: 
 
1) Was correctly targeted antimicrobial treatment started 
2) Was untargeted antimicrobial treatment avoided 
3) Was specific follow-up planned due to risk of complications (e.g. EHEC and hemolytic uremic 
syndrome) 
 
within 72 hours from initial contact in Pediatric Emergency Room? 
 
When any of these questions is answered YES, the composite variable is considered positive. Anti-
microbial treatment is considered appropriate if a bacterial pathogen has been found and the pa-
tient meets criteria for severe gastroenteritis (aged under 1 year or immunocompromised or hospi-
talized due to gastroenteritis). 
 
Secondary outcomes are proportion of correctly targeted antimicrobial treatment, proportion of con-
ditions in need for hospitalization or specific follow-up, time needed for clinician to receive the re-
sults of the samples, length of hospital stay, time to correct diagnosis, resolution of symptoms, la-
boratory and radiology costs, total costs, need for surgical consultation and proportion of patients 
needing surgical procedure, proportion of unscheduled revisits, proportion of correctly isolated pa-
tients or correct use of personal protective equipment and the number of consultations for pediatric 
infectious diseases specialist. 
 
3.2 Diagnostic analysis 
 
For the diagnostic analysis, we will assess the usability of rectal swab and vomit samples for de-
tecting gastrointestinal pathogens within both arms of the RCT. (Figure 2) We will conduct a diag-
nostic study, in which the PCR testing of the fecal samples is considered the gold standard. We will 
calculate the sensitivity and specificity of rectal swab and vomit samples analyzed with the PCR 
device in relation to the fecal sample results. Also, we will collect a VAS score on how unpleasant 
the rectal swab sample collection was in relation to the collection of fecal samples. The VAS 
scores are questioned from children 7-15 years of age, and from parents of children younger than 
7 years of age. We will also calculate the time to the result as compared to the use of fecal sam-
ples in same patients. 

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Figure 2. Diagnostic analysis regarding the detection of pathogens in alternative sample types. 
 
 
 
4. IMPLEMENTATION 
Schedule 
The study will be conducted at the Pediatric Emergency Room at the Oulu University Hospital. Re-
cruiting of patients will start in 2025. We estimate that we can recruit 5 patients weekly. Therefore, 
the estimated duration of the recruiting process will be approximately 2 years until 2027. However, 
recruiting is continued at least for one epidemiological year to maximize coverage of different epi-
demics and causative gastrointestinal pathogens. Results of the study will be analyzed and pub-
lished during 2028. 
 
5. ETHICAL CONSIDERATIONS 
The study will not include any additional blood samples. Apart from the possible transportation of 
fecal samples received at home, there will be no additional visits to hospital due to the study. The 
equipment used in the study is already in clinical use. We collect written informed consent from all 
participants and participating is voluntary. Before initiation of the study, we will request approval 
from Ethical Committee of Oulu University Hospital, Finland. 

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6. RISK ASSESMENT AND POTENTIAL IMPACT 
This is a low-risk project. Most challenging part of this study is recruiting the children. However, 
gastrointestinal symptoms are one of the most common reasons for pediatric emergency visits. 
The study does not contain any painful procedures. We have an experienced study nurse with 
whom we have successfully recruited thousands of study patients in numerous projects in case the 
pediatric nurses need any support in the recruiting process. We also have a highly experienced 
and well-funded study group with background of several successfully conducted clinical studies in 
emergency department settings. 
 
This study is based on a relevant hypothesis and focuses on one of the major patient groups in pe-
diatric emergencies. The randomized controlled setting is scientifically relevant. If successful, it 
could improve identification of conditions in need for antibiotic treatment or follow-up and on the 
other hand, decrease testing and revisits in conditions they aren’t necessary in. Also, it could re-
duce unnecessary use of antibiotics in children and therefore reduce long term adverse effects on 
gut microbiome and children’s health. 
 
7. FINANCIAL CONSIDERATIONS 
 
This is an independent academic investigator driven project. Financially, the study contains only 
low risk. As the QIAstat-Dx Gastrointestinal Panel 2 is already actively in clinical use at Oulu Uni-
versity Hospital, the fecal sample testing in the active group of the RCT (N = 263) is carried out as 
a part of accustomed hospital patient care. To purchase the rest of the cartridges needed for the 
study, procurement contract is made with Qiagen Finland. These analysis costs will be covered 
partly with grants received by the pediatric infectious diseases study group and partly with support 
of The Association of Friends at the University Children's Hospitals (Lastenklinikoiden kummit). 
Doctoral researcher’s work is funded by personal grants. The study nurse is already hired by the 
study group partly with state research funding (VTR) received by the study group, so there will be 
no additional personnel related costs. 
 
8. STUDY GROUP 
 
• 
Joni Kalermo, MD, PhD student 
• 
Suvi Mattila, MD, PhD, Co-supervisor of PhD thesis 
• 
Kimmo Halt MD, PhD 
• 
Hilla Pöyry MD, PhD 
• 
Minna Honkila MD, PhD 
• 
Niko Paalanne MD, PhD 
• 
Terhi Ruuska-Loewald, MD, PhD, Professor, Principal supervisor of PhD thesis 
 
All members of the study group are affiliated to Research Unit of Clinical Medicine, University of 
Oulu and Department of Pediatrics and Adolescent Medicine, Oulu University Hospital. 
 
9. AMENDMENTS 
 
Added “pediatric infectious diseases specialist consultations” to secondary outcomes (16.10.2025). 
 

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