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BY-NC-ND 3.0 license Open Access Published by De Gruyter August 1, 2019

Detection of Salmonella, Campylobacter, Shiga toxin-producing E. coli and Shigella/EIEC by culture and a multiplex PCR panel in pediatric patients with acute diarrheal illness

Ozlem Koyuncu Ozyurt, Ana Laura Ferrandi Vilas Boas Bertocco, Larissa Alessandra Bourdeth Pereira, Luisa Paganelli Jimenes, Hatice Yazisiz, Betil Ozhak, Dilara Ogunc, Levent Donmez, Filiz Gunseren, Aygen Yilmaz and Gozde Ongut

Abstract

Background

Acute diarrhea is the second most common cause of child deaths worldwide. We investigated Salmonella species (spp.), Campylobacter spp., Shiga toxin-producing Escherichia coli (STEC) and Shigella spp./enteroinvasive E. coli (EIEC) in stool samples of 741 pediatric patients with acute diarrheal illness.

Methods

Between January 2017 and October 2017, 741 stool specimens were tested by the BD Max Enteric Bacterial Panel (EBP) assay and conventional culture.

Results

Campylobacter spp., Salmonella spp. and E. coli O157:H7 were detected in 10 (1.3%), 20 (2.7%) and two (0.3%) stool specimens by culture, respectively. Campylobacter spp., Salmonella spp., Shiga toxin and Shigella/EIEC were detected in 46 (6.2%), 37 (5.0%), 20 (2.7%) and three (0.4%) stool specimens by the BD Max EBP assay, respectively. The percentage of detected pathogens was 4.3% (32/741) by culture. Use of the BD Max EBP assay identified an additional 112 pathogens, thereby increasing the frequency to 15.1% (112/741). Empirically, 58 patients received antibiotics and 43 patients underwent probiotic and/or symptomatic therapy.

Conclusions

We concluded that nucleic acid amplification testing markedly improves the detection rates of bacterial stool pathogens and offers rapid results.

Reviewed Publications:

Ahmad-NejadP.Edited by:GhebremedhinB.


Introduction

Diarrheal diseases are the fourth largest disease burden worldwide, accounting for 5% of the total disease burden in children globally [1]. It is the second most common cause of childhood deaths worldwide and kills around 500,000 children under 5 years every year [2]. According to the Turkish Statistical Institute’s Turkey Health Survey report in 2012, diarrhea with a rate of 27.9% was the most common disease in children between 0 and 6 years [3].

Diarrhea is a common symptom of gastrointestinal infections due to a wide variety of pathogens, including bacteria, viruses and protozoa. Although viruses are the main cause of diarrhea, the most common bacterial causes are Salmonella, Shigella, Campylobacter and Shiga toxin-producing Escherichia coli (STEC). It is not always possible to distinguish viral, parasitic or bacterial diarrhea on the basis of clinical grounds alone. Detection of the etiological agents of acute bacterial diarrhea is important for the management of the patient and for public health interventions [4].

The aim of this study was to determine the frequency of Salmonella spp., Campylobacter spp., STEC and Shigella spp./enteroinvasive E. coli (EIEC) in pediatric patients with acute diarrheal illness using both conventional culture methods and BD Max Enteric Bacterial Panel (EBP) assay (Becton Dickinson Diagnostics, Baltimore, MD, USA).

Materials and methods

All pediatric patients who were admitted to the Department of Pediatrics at Akdeniz University Hospital between 1 January 2017 and 31 October 2017 were included in the study according to the following criteria: (i) children between the ages 0 and 6 years and (ii) who had an acute diarrheal illness. Acute diarrhea is defined as the passage of three or more loose or liquid stools per day, or more frequently than normal for the individual. Duplicate specimens from the same patient were not enrolled.

Culture

Fresh stool specimens were inoculated onto MacConkey agar, Xylose-Lysine-Deoxycholate agar for Salmonella and Shigella, and incubated at 35±2 °C for 24 h in an aerobic incubator. Lactose and xylose nonfermenting colonies with or without black centers on these media were screened phenotypically on triple sugar iron agar, motility medium, urea agar, Simmons’ citrate agar and lysine iron agar. Suspected colonies were tested using the Wellcolex™ Color Salmonella Rapid Latex Agglutination Test Kit and Wellcolex™ Color Shigella (ThermoFisher, Dartford, UK).

Screening for E. coli O157:H7 in stool was performed using MacConkey agar with sorbitol. Sorbitol-negative colonies were identified as E. coli by matrix-assisted laser desorption/ionization (MALDI)-time of flight mass spectrometry (TOF MS) (Bruker Daltonik GmbH, Bremen, Germany) and then tested using Wellcolex E. coli O157:H7 (ThermoFisher, Dartford, UK). Screening for Campylobacter spp. in stool was performed using Campylobacter selective agar (Becton Dickinson GmbH, Heidelberg, Germany) and incubated under microaerobic condition at 42 °C for 7 days. Suspected colonies were identified by Gram stain examination of the colony along with the oxidase test and MALDI-TOF MS.

BD Max EBP automated PCR

The BD Max EBP is a commercial multiplex nucleic acid amplification assay designed for the detection of Salmonella spp., Shigella spp., Campylobacter spp. (Campylobacter jejuni and Campylobacter coli) and Shiga-like toxin genes (stx1 and/or stx2) in stool specimens with the BD Max system. The primer sets used by the BD Max EBP multiplex real-time polymerase chain reaction (PCR) assay are: spaO (gene that encodes the surface presentation of antigens) for Salmonella spp., ipaH (gene that encodes the invasion plasmid antigen) for Shigella spp. and EIEC, stx1 and stx2 (genes that encode the Shiga toxins) for Shiga toxin-producing organisms and tuf (gene that encodes the elongation factor for protein synthesis) for C. coli and C. jejuni. These primer sets cannot differentiate the two Campylobacter species and stx1 and stx2 from each other. Fresh stool samples were tested daily using the BD Max EBP assay, according to the manufacturer’s instructions.

Ethical considerations

This study was approved by the Akdeniz University School of Medicine Ethical Committee of Clinical Research (Decision number: 70904504/291/434).

Statistical analysis

A chi-squared (χ2) test was used. A value of p<0.05 was considered to be significant.

Results

Between January and October 2017, 741 stool specimens were tested by the BD Max EBP assay and conventional culture.

Culture results: Ten (1.3%) specimens were positive for Campylobacter spp., 20 (2.7%) were positive for Salmonella spp. and two (0.3%) were positive for E. coli O157:H7. These were also positive with the BD Max EBP assay. Coinfection was not detected by culture.

BD Max EBP assay results: Forty-six (6.2%) specimens were positive for Campylobacter spp., 37 (5.0%) were positive for Salmonella spp., 20 (2.7%) were positive for Shiga toxin and three (0.4%) were positive for Shigella/EIEC. In addition, Campylobacter and stx1, stx2 were detected simultaneously in three specimens (0.4%).

The percentage of investigated pathogens was 4.3% (32/741) by culture. Use of the BD Max EBP assay identified an additional 112 pathogens, thereby increasing the frequency to 15.1% (112/741). Difference between the two percentages was statistically significant (p<0.05). The number of investigated pathogens according to the age groups is shown in Table 1.

Table 1:

The number of investigated pathogens according to the age groups.

Age groups0123456Total
nn=246n=151n=91n=77n=67n=64n=45n=741
n%n%n%n%n%n%n%n%
Culture
 EHEC10.410.700.000.000.000.000.020.3
Campylobacter10.432.066.600.000.000.000.0101.3
Salmonella20.853.322.256.557.500.012.2202.7
Shigella/EIEC00.000.000.000.000.000.000.000.0
PCR
stx1/stx252.096.022.222.623.000.000.0202.7
Campylobacter41.6127.91617.611.357.534.7511.1466.2
Salmonella20.8106.688.8810.469.023.112.2375.0
Shigella/EIEC00.021.300.011.300.000.000.030.4
Stx1/Stx2+Campylobacter00.021.311.100.000.000.000.030.4

  1. n, number; EHEC, enterohemorrhagic E. coli; EIEC, enteroinvasive E. coli; Stx, Shiga toxin.

Out of 107 patients with PCR positivity, clinical records of 100 patients were available. Empirically, 32 patients received antibiotics other than metronidazole, 26 patients received metronidazole and 43 patients underwent probiotic and/or symptomatic therapy. There was no statistically significant difference between culture results and metronidazole usage, antibiotic usage, probiotic and/or symptomatic therapy (p>0.05). Symptoms and management of patients are given in Table 2.

Table 2:

Symptoms and management of patients.

Total patient numberan (%)
Culture result (n=100)
 Positive27(27.0)
 Negative73(73.0)
Empirical therapy
 Not determined13(13.0)
 Yes87(87.0)
Metronidazole26(26.0)
Antibacterial agent32(32.0)
Probiotics/symptomatic43(43.0)
Symptoms
 Fever (n=92)51(55.4)
 Nausea (n=91)14(15.4)
 Vomiting (n=91)29(31.9)

  1. aPatients with medical records.

Although none of the patients had protozoa or helminths detected in feces, 26 patients were treated with metronidazole. Management of patients according to the culture results is given in Table 3.

Table 3:

Management of patients according to the culture results.

ManagementCulture result
Positive, n (%)Negative, n (%)Total, n (%)
Metronidazole usage
 Used6 (22.2)20 (27.4)26 (26.0)
 Not used21 (77.8)53 (72.6)74 (74.0)
Antibiotic usage
 Used10 (37.0)22 (30.1)32 (32.0)
 Not used17 (63.0)51 (69.9)68 (68.0)
Probiotic and/or symptomatic therapy
 Used8 (29.6)35 (47.9)43 (43.0)
 Not used19 (70.4)38 (52.1)57 (57.0)
Total27 (100.0)73 (100.0)100 (100.0)

While PCR results are obtained in 3 h, this time for culture may extend up to several days depending on the pathogen. We could not give the growth time for each pathogen in the culture, but the time for results for any given pathogen was not as short as 3 h.

Discussion

We found at least one positive laboratory result for enteric bacteria in 109 (14.7%) patients. PCR was positive in all 109 (100%) patients, but only 32 (29.4%) of them were culture positive. Salmonella spp. was the most common bacteria detected by culture while Campylobacter spp. was mostly detected by PCR.

In a study, Hascelik et al. [5] investigated stool specimens of 677 children less than 15 years of age. Campylobacter was found in 72 (10.6%) of the samples. Shigella spp. and Salmonella spp. were isolated in 26 (3.8%) and 11 (1.6%) children, respectively.

In another study of 82 children with suspected acute bacterial gastroenteritis, the detection rates of Salmonella spp. and C. jejuni under 5 years of age were 19.5% and 15.9%, respectively. Shigella spp. was not detected in any of the stool samples [6].

In a study of 339 stool samples taken from children aged 0–5 years, Salmonella spp. was isolated in 14 (4.1%) and Shigella spp. in 11 (3.2%), while E. coli O157 was detected in only one (0.3%) sample [7].

In our study, Salmonella spp. was isolated in 20 (2.7%), C. jejuni in 10 (1.3%) and E. coli O157:H7 in two (0.3%) samples. Shigella spp. was not detected.

There are a number of reasons for the difference in the frequency of pathogens detected in culture including, but not limited to, different culture methods, ordering practices and geographical differences.

Campylobacter spp., which are fastidious organisms, are probably the most difficult for clinical laboratories to detect because of specimen transport and specific culture requirements [8]. Jansen et al. [1] demonstrated that culture-based methods miss a substantial proportion of Campylobacter infections. One of the benefits of nucleic acid amplification tests (NAATs) is the ability to detect low levels of fastidious organisms despite poor growth. In a study, the BD MAX EBP assay detected 100% of Campylobacter spp. at 1×103 colony forming unit (CFU) despite a culture detection rate of 43.8% [9]. Many studies have also shown the value of molecular tests for the detection of Campylobacter species [10].

Several limitations of molecular assays such as the BD MAX EBP should be considered. These tests detect specific genetic targets and they are unable to detect pathogens for which a specific target is lacking. Likewise, the laboratories cannot do susceptibility and epidemiologic testing without an isolate. Despite these limitations, the BD MAX EBP is a useful diagnostic tool, which has a higher sensitivity at low levels of concentration for enteric pathogens compared to culture.

In our study, the percentage of investigated pathogens was 4.3% (32/741) by culture. Use of the BD Max EBP assay identified an additional 80 pathogens, thereby increasing the frequency to 15.1% (112/741).

Liu and colleagues [2] using quantitative molecular diagnostic methods in the Global Enteric Multicenter Study found that the burdens for Shigella spp. and Campylobacter spp. were estimated to be 2 times greater than previously reported.

Harrington et al. [11] reported that the combined prevalence of all four targets in prospective specimens increased from 3.9% based on culture for Salmonella spp., Campylobacter spp., Shigella spp. and the Shiga toxin enzyme immunoassay (EIA) to 6.2% with the additional positive results detected by the BD Max EBP assay.

In our study, it was found that 26 of the 100 patients with PCR positivity received metronidazole for possible protozoal infection. This shows that physicians cannot distinguish bacterial and parasitic infections only by clinical symptoms. In children, antibiotics are often used empirically for acute gastroenteritis; however, the indications for antibiotic treatment in this setting have not been standardized so far. The application of molecular diagnostic methods may ensure specific antibiotic treatment which may prevent overuse of antibiotics, associated drug resistance and adverse events.

Diagnostic testing for enteric pathogens has rapidly evolved in the past decade. Although culture independent diagnostic tests are still most commonly being used for Campylobacter and STEC, the Foodborne Diseases Active Surveillance Network found the highest percentage increase in use for Shigella and Salmonella compared with the previous 3-year average. They concluded that the most likely reason for this change was the implementation of newly available DNA-based syndrome panels in participating laboratories [12].

The Infectious Diseases Society of America (IDSA) recommends routine stool enteric pathogen culture or NAAT for the laboratory diagnosis of infectious diarrhea caused by Salmonella enterica, Shigella spp. and Campylobacter spp. When the clinical or epidemiologic history suggests a possible Shiga toxin-producing organism, diagnostic approaches should be applied that detect Shiga toxin (or the genes that encode them) and distinguish E. coli O157:H7 from another STEC in stool [4].

Although the same target is used for the detection of Shigella and EIEC in PCR assay, we did not specifically identify EIEC which may be considered as a limitation of our study. Unfortunately, we could not confirm the discordant results between culture and PCR with an additional test. Even though nucleic acid amplification testing markedly improved the detection rates of bacterial pathogens, we could not determine the clinical relevance of these results as we could not follow up the patients.

Compared with culture, NAAT offers rapid results and enhanced sensitivity. It can also reduce unnecessary treatment and enable contact investigations.

Previous studies have shown that the BD Max EBP is a sensitive and specific assay for the diagnosis of enteric pathogens. PCR testing detects bacterial DNA, not viable organisms, and positive NAAT results must therefore be interpreted in conjunction with clinical presentation. When PCR is used for diagnosis, culture is required for antimicrobial susceptibility testing for PCR-positive samples.

In conclusion, NAAT markedly improves the detection rates of bacterial stool pathogens and offers rapid identification. Therefore, we recommend using NAAT with culture to achieve optimal results.

Conclusions

In our study, Salmonella was most commonly detected by culture while Campylobacter was most commonly identified by BD Max. The percentage of investigated pathogens was 4.3% (32/741) by culture. Use of the BD Max EBP assay identified an additional 112 pathogens, thereby increasing the frequency to 15.1% (112/741).


*Correspondence: Prof. Betil Ozhak, MD, Faculty of Medicine, Department of Medical Microbiology, Akdeniz University, Antalya, Turkey, Phone: (+90) 2422496914, Fax: (+90) 2422496903

  1. Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

  2. Research funding: None declared.

  3. Employment or leadership: None declared.

  4. Honorarium: None declared.

  5. Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.

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Received: 2019-03-11
Accepted: 2019-07-08
Published Online: 2019-08-01
Published in Print: 2019-08-27

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