Acute diarrhea is common, and in the general population its incidence ranges from 0.6 to 0.8 episodes of illness per person, annually [1,2]. Acute diarrhea is usually self-limited (lasting 1 to 3 days) [3], and thus, adequate fluid and electrolyte replacement are often sufficient for its management. Nevertheless, acute diarrhea remains an important cause of morbidity and mortality, particularly in developing countries, and a significant cause of economic loss in industrialized countries [4].

Infection is the most common cause of acute diarrhea. In the past, microbiological examinations were performed, mainly by stool culture or enzyme immunoassay, to identify the causative bacterial agent. Unfortunately, the diagnostic yield of stool culture is very low (ranging from 1.5% to 5.6%) and its minimum turnaround time ranges from 48 to 76 hours [5]. Moreover, enzyme immunoassay is labor intensive and has low sensitivity [6].

In contrast, stool polymerase chain reaction (PCR) testing is more sensitive than standard stool culture [7-9], has a short (approximately 1 day) turnaround time, and can simultaneously detect multiple organisms.

For these reasons, conventional methods are likely to be replaced by stool multiplex PCR. The recent adoption of the test supports this belief. However, there are still concerns about the usefulness of stool multiplex PCR in a clinical setting. Accordingly, we undertook this study to verify the outcomes and clinical relevance of stool multiplex bacterial PCR testing, by analyzing its results and clinical findings.

This retrospective study was conducted at Dongguk University Ilsan Hospital between August 1, 2014 and November 31, 2016. We initially searched for patients of all ages hospitalized for acute diarrhea and diarrhea-associated symptoms (fever, dehydration, or restlessness) who underwent stool multiplex bacterial PCR testing. We defined acute diarrhea as the passage of ≥ 3 loose or watery stool within 24 hours, or of ≥ 1 bloody or mucoid stools [10]. Exclusion criteria were as follows: hospital visits were more than 2 weeks after diarrhea onset, underlying gastrointestinal diseases including inflammatory bowel disease and gastrointestinal cancer, or a lack of clinical information (Fig. 1).

Detailed clinical findings and laboratory parameters were obtained by reviewing electronic medical records. Collected variables were as follows: age, sex, concomitant disease, fever, maximum diarrhea frequency per day, bloody diarrhea and vomiting, blood test (serum leukocyte, C-reactive protein [CRP], blood urea nitrogen, and creatinine), the use of empirical antibiotics, stool test (microscopic examination of white blood cells [WBCs], occult blood, and culture), stool multiplex bacterial PCR, and duration of hospital stay. Fever was defined as a tympanic temperature of ≥ 38.3°C [11,12]. Severe acute diarrhea was defined as diarrhea with fever, acute kidney injury (prerenal azotemia), bloody diarrhea (grossly or a positive stool occult blood test), or frequent diarrhea (passage of ≥ 6 unformed stools per 24 hours) [13]. A Charlson comorbidity index score of ≥ 2 was defined as a “severe underlying disease,” including cerebrovascular disease, cardiovascular disease, pulmonary disease, liver cirrhosis, chronic kidney disease, and malignancy [14]. Doctors prescribed empirical antibiotics when they did not expect the diarrhea to be self-limited before stool multiplex PCR results became available.

Stool multiplex PCR has attracted considerable interest as a diagnostic tool for the early detection of causative pathogens in patients with diarrhea. This diagnostic tool has several advantages over conventional methods, which include increased sensitivity; decreased turnaround time; broad coverage without the need to select specific tests; and reduced sample volume requirements. Several multiplex PCR test kits are now commercially available. The Seeplex Diarrhea-ACE (Seegene) kit, which was used for this study, has received conformite europeenne marking for use in Europe [17]. Previous studies have compared these multiplex PCR tests against conventional detection methods. However, few studies have addressed their clinical relevance.

In this study, the overall prevalence of patients with detected pathogens by multiplex PCR test was 31.2% (226/725). A search of the literature revealed a wide range of values have been previously reported (30% to 70%) [18-20], perhaps because of inter-study differences between numbers, types (bacteria, virus, or protozoa) of targeted pathogens and between the characteristics of enrolled patients. This study showed that the positive rate of multiplex PCR testing was higher than that of stool culture (32.7% vs. 3.3%, p < 0.01). Evidently, smaller numbers of targeted pathogens (three species) result in lower diagnostic yields for stool culture, although this result reflects clinical practice, where miscellaneous pathogens cannot be cultured because of cost or workload.

In previous studies, bloody diarrhea and frequent diarrhea were more commonly presented by patients with acute bacterial diarrhea [21,22]. In this study, although severe acute diarrhea, defined as diarrhea with one of severe symptoms, showed no significance in the multivariate analysis, bloody diarrhea and frequent diarrhea were more common among patients with a positive multiplex PCR test (p = 0.02, p < 0.01, respectively). These findings suggest that a significant correlation exists between stool bacterial multiplex PCR test results and the severe symptoms of acute diarrhea. Vomiting tended to be more frequent in the negative group (p = 0.08), perhaps because vomiting is common in diarrheal diseases of viral origin [23,24] and the multiplex PCR test used in this study did not include viral genera. The presence of WBCs in stool suggests an inflammatory process caused by bacterial spp., such as Salmonella spp., Shigella spp., C. jejuni, or C. difficile. In this study, the WBCs detection rate was higher in the positive multiplex PCR group (p = 0.02).

The empirical antibiotics were prescribed by attending doctors according to patients’ overall condition such as the frequency of diarrhea, bloody diarrhea, and fever and, as above mentioned, these severe symptoms were relatively more common in acute bacterial diarrhea, which would explain why that the taking of antibiotics showed positive correlation with the positive result of stool multiplex bacterial PCR. The administration of empirical antibiotics was decided on without the confirmation of multiplex PCR results, and we were not able to discover whether a negative multiplex PCR result might lead to taking patients off antibiotics. Furthermore, the duration of hospital stays as a prognostic factor showed no difference between the two groups according to the multiplex PCR results. Therefore, so far, it is hard to say whether multiplex PCR has certain usefulness in clinical practice for acute diarrhea. Nevertheless, the multiplex PCR showed a high detection rate and had a positive correlation with symptoms associated with bacterial etiology and with the use of empirical antibiotics. These results indicate that multiplex PCR has advantages for identifying the culprit bacterial pathogen and for providing additional information on the appropriateness of antibiotics. These advantages could allow bacterial multiplex PCR to take over much of the role of stool WBC and culture testing for acute diarrhea. Consequently, increasing the use of bacterial multiplex PCR could reduce the use of stool WBC and culture testing. However, concerns about the significance of certain pathogens targeted by multiplex PCR and about the cost-effectiveness of the technique still remain, because stool multiplex PCR is much more expensive than stool WBC testing and stool culture [25].

Campylobacter infection is recognized as a major cause of gastroenteritis [26-28]. Previous epidemiologic studies using stool cultures have reported that the most common pathogen for acute diarrhea was Salmonella, followed by Campylobacter. However, in this study, Campylobacter spp. was the most common bacterial pathogen detected by stool multiplex PCR in acute diarrhea patients (39.9%), perhaps because stool PCR testing increased the Campylobacter spp. detection rate. Buchan et al. [29] reported that PCR testing had a higher sensitivity (100%) than culture (76.9%) for Campylobacter spp. (C. jejuni and C. coli). Although the possibility of a false-positive should also be considered, the multiplex PCR test could show the real etiological importance of Campylobacter spp. in acute diarrhea, as validated by previous studies. In addition, these previous studies reported that the true positive rate of a PCR test for Campylobacter spp. was high (83.3%) by repeating different PCR tests; they found a similar proportion of Campylobacter spp. on detected pathogens by stool multiplex PCR (39.5%) [30,31].

In this study, we found Campylobacter spp. to be significantly associated with frequent diarrhea and high CRP (p < 0.01). Furthermore, the use of empirical antibiotics was more common in Campylobacter-positive patients (p < 0.01). On the other hand, hospital stays were no different in the multiplex PCR positive and negative groups (p = 0.09). As mentioned above, in discussing the utility of stool multiplex PCR testing, these results suggest that the multiplex PCR test can increase the detection rate of Campylobacter spp. as a pathogen for acute diarrhea and provide additional information on the appropriate antibiotics, despite the observation that Campylobacter positivity was not associated with prognosis.

Campylobacter selective agar preparation and the time needed for microaerobic incubation (2 days at 37°C) produce a considerable workload. For these reasons, routine stool cultures in our institution have not included Campylobacter spp. The stool multiplex PCR test for Campylobacter spp. could reduce the labor required to culture. Furthermore, early identification of Campylobacter as a causative pathogen offers advantages in terms of antibiotic selection. In particular, delayed stool culture turnaround times often result in the indiscriminate use of broad-spectrum antibiotics, such as fluoroquinolones. Its indiscriminate use has resulted in a high resistance rate of Campylobacter spp. against fluoroquinolones. The short turnaround time of stool multiplex PCR testing would provide more opportunity to use macrolide in cases of Campylobacter-associated diarrhea [32] and decrease the risk of hemolytic uremic syndrome associated with broad-spectrum antibiotics in cases of Shiga toxin-producing, E. coli-associated diarrhea [33]. Obviously, investigation on the prevalence of macrolide-resistant Campylobacter spp. should also be carried out.

Campylobacter infection can cause severe, prolonged or relapsing illness and bacteremia in immune-compromised patients. In a study in the United States, the incidence of Campylobacter infection was found to be 40 times higher in people with acquired immunodeficiency syndrome than in the general population [34]. However, in this study, somewhat unexpectedly, patients positive for Campylobacter spp. had a lower prevalence of severe underlying disease. This may have been due to lower consumptions of raw poultry and milk, the main sources of Campylobacter in the food chain [35], and less frequent travel by patients with severe underlying disease [36].

In this study, the detection rate of multiplex PCR test for C. perfringens, a common cause of food poisoning, was relatively higher than that by stool culture in former studies. In a study about the limit of detection according to a bacterial load of stool, the stool PCR test had a higher or equal sensitivity for C. perfringens and Campylobacter spp. than did stool culture [37,38]. Thus, a stool multiplex PCR test would be helpful in detecting C. perfringens as a pathogen in acute diarrhea. Although not all of them can be defined as a community-acquired C. difficile infection, this study suggests an increased prevalence of community-acquired C. difficlie infection and virulence because of an increased use of antibiotics. Further study using the PCR test is therefore warranted for community-acquired C. difficlie infection, especially in pediatric patients, because 44.4% (20/45) of positive C. difficile toxin findings were younger than 15 years old.

This study has several limitations. First, other than Campylobacter spp., few pathogens were detected by multiplex PCR. A larger sample is needed to establish the clinical relevance of testing for other pathogens. Second, unfortunately, the exact collection time of stool multiplex bacterial PCR tests could not be identified because of the limitations of retrospective study. Stool specimens might be collected after use of empirical antibiotics in real clinical situation. In addition, the quality of stool samples could not be controlled either. These factors might explain the relatively low pathogen detection rate of multiplex PCR. Third, multiplex PCR using ipaH and vif as target genes cannot differentiate Shigella spp. from enteroinvasive E. coli. Duplex, real-time PCR using the lacY gene, a gene encoding lactose permease, and the β-glucuronidase gene could be used for the discrimination in further study [39]. Fourth, it remains unclear whether the pathogens detected by stool multiplex PCR were actually the cause of acute diarrhea. Despite this limitation, we focused on evaluating the clinical usefulness of stool multiplex PCR in a real clinical setting, and we believe our findings shed light on the relevance and clinical utility of broadly based multiplexed PCR testing for the detection of potentially colonizing pathogens/pseudo pathogens. To establish more accurately the meaning of a positive PCR test result, we suggest that a study be undertaken to compare the clinical characteristics of PCR-positive/culture-positive and PCR-positive/culture-negative acute diarrhea patients.

In conclusion, although it is uncertain whether a stool bacterial multiplex PCR test influences disease prognosis, the test results correlated with severe symptoms and the use of empirical antibiotics. Thus, the stool bacterial multiplex PCR test might be a useful tool for identifying pathogens and providing additional information on appropriate antibiotics in acute diarrhea cases, especially in those with a Campylobacter infection.