Multiplex PCR test for detection of enteropathogens in an infant

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Fig. 1. Schematic of target-enriched multiplex PCR (A) and workflow of sample processing on multiplex gastrointestinal panel (B).

Although the conventional stool culture is the well-established diagnostic tool for enteric pathogens, its effectiveness is very low, with reporting times ranging from two to four days, and it may be less sensitive than molecular methods.^4,5 Furthermore, widespread use of antibiotics can adversely affect the growth of potential pathogens in bacterial culture, rendering false-positive or false-negative results. PCR amplification has emerged as a useful tool to detect pathogen DNA and RNA rapidly (four to six hours) with higher sensitivity and specificity. Multiplex PCR-based tests for the detection of enteric pathogens in a single stool specimen are well suited to clinical purposes.

The gastrointestinal panel developed by Diatherix Laboratories (Huntsville, Ala.) provides simultaneous detection of the following pathogens: Clostridium difficile toxin B gene, Campylobacter jejuni, Escherichia coli strain O157, Listeria monocytogenes, Salmonella enterica, Shigella flexneri, Shigella sonnei, Vibrio parahaemolyticus, Giardia lamblia, Cryptosporidium parvum, adenovirus 40, and adenovirus 41. Target-enriched multiplex PCR (TEM-PCR) is the core molecular technology used for this panel. Nested gene-specific primers at extremely low concentrations are used to amplify targets during the first few cycles of PCR of the target-enrichment step. This is followed by exponential amplification using universal SuperPrimers. The Reverse SuperPrimer is labeled with biotin for subsequent detection of amplicons (Fig. 1A). The concentration of Forward and Reverse SuperPrimers facilitates asymmetric PCR producing biotin-labeled PCR products (Fig. 1B). These PCR products are hybridized to a complementary target-specific probe covalently coupled to a glass microarray (Microarrays Inc.) and detected with Streptavidin-labeled Phycoerythrin conjugate. Fluorescent signal corresponding to hybridized PCR products is detected on FLAIR reader (Sensovation, Germany), and results are reported as positive or negative for pathogens detected in the gastrointestinal panel.

Case. A seven-month-old female presented with a three-day history of diarrhea. The patient’s mother reported that the child was passing four to five bloody stools per day and experiencing low-grade fevers. The child’s oral intake remained normal and no abdominal pain was reported. The patient had been seen two weeks prior to the current visit with purulent nasal drainage and was treated with a seven-day course of Augmentin for sinusitis. Past medical history was unremarkable, and the patient was on no long-term medications. Physical exam revealed a well-nourished child in no apparent distress. Tympanic temperature was 99.2°F and heart rate was 124. Oral mucous membranes were moist and skin turgor was normal. The abdominal exam revealed normal active bowel sounds and no tenderness or mass.

A rectal swab stool specimen was obtained and submitted for laboratory testing with Diatherix’s gastrointestinal panel. Nucleic acid extractions, multiplex PCR amplification, and positive/negative signal detection were performed at the CLIA-certified Diatherix Laboratories. The results were reported to the physician’s office within seven hours of the sample having been received for laboratory testing. The patient’s rectal swab sample was positive for both C. difficile toxin B and Salmonella enterica. The patient was treated preferentially with a seven-day course of ampicillin for Salmonella enterica. Diarrhea subsequently resolved without recurrence.

Discussion. The significance of Clostridium difficile as a cause of gastroenteritis in the pediatric population has been a subject of debate for decades. Studies that have documented early colonization of the bowel flora in neonates and the apparent lack of symptoms in the face of positive cultures for the organism and the presence of toxin6 have cast doubt on the significance of Clostridium difficile as a cause of disease in this patient group. However, the emergence of a more virulent strain of Clostridium difficile that produces a binary toxin (B1/NAP1/O27) has led to a significant rise in the prevalence of the organism in hospitalized children who do not have comorbid factors.7 As a result, there has been a shift in the spectrum and prevalence of the disease in the pediatric population over the past decade.

Clinical presentation does not always provide direction for the diagnosis of gastroenteritis whether in the adult or pediatric patient. Early stages of Clostridium difficile infection are usually accompanied by mild diarrhea (five to 10 watery stools a day), low-grade fever, and mild abdominal cramping and tenderness. In the more severe forms of the disease, fever (usually 102° to 104°F), severe diarrhea (more than 10 watery stools a day) with blood, and marked abdominal pain and tenderness are present. These symptoms are not unique, however, as there is overlap with other intestinal pathogens that have both toxin and invasive components.

This case also exhibits co-detection of Clostridium difficile toxin B in association with a second pathogen, Salmonella enterica. In the new era of molecular diagnostics and specifically multiplex PCR in which multiple pathogens can be detected in a single specimen, clinicians are faced with a new level of information. The traditional paradigm wherein one pathogen causes clinical infection may be under challenge. Co-pathogens may work synergistically to cause clinical disease. Future molecular diagnostic testing will need to include, in addition to pathogen detection, other features that assist in determining pathogenesis and clinical illness.
We have presented this case report to draw attention to the fact that the spectrum and etiology of gastroenteritis in the pediatric population may be changing and molecular diagnostic tests are necessary to uncover complex etiology behind appropriate diagnosis and treatment of the pediatric population.

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Dr. Lazas is with Associates in Gastroenterology PLC, Nashville, Tenn., and Dr. Ber is with Sugarland Pediatrics LLC, Houma, La. Dr. Grigorenko is vice president of research and development, and Dr. Stalons is vice president of operations and clinical laboratory director—both at Diatherix Laboratories, Huntsville, Ala.