Coinfection of the Intestinal Tract with Aeromonas hydrophila, Clostridium difficile and Rotavirus – a Case Report

Most cases of acute diarrhea in adults are of infectious etiology, likely viral and self-limited. Among those with severe diarrhea, however, bacterial causes are responsible for most cases. Apart from the standard stool cultures, to increase the positive yield a novel multiplex molecular test can be performed simultaneously. The authors present a patient with recurrent diarrhea and detection of Aeromonas hydrophila by culturing and Rotavirus and Clostridioides difficile by multiplex molecular test. They discuss and justify which is the most likely actionable pathogen. Good communication between the physicians and interpretation on the multiple positive results in the context of clinical picture and the test employed were important for a better management and favourable outcome of the patient.


INTRODUCTION
Diarrheal disease is one of the top ten leading causes of death worldwide and is a particular concern for children younger than five years old in resource-limited settings. 1 Among adults in resource-rich settings, diarrhea is often a "nuisance disease" in the healthy individual. Most cases of acute diarrhea are of infectious etiology, commonly viral and self-limited. Noroviruses have surpassed rotaviruses as the most common pathogens in regions where rotavirus vaccine has become routine. Among those with severe diarrhea, however, bacterial causes are responsible for most cases. 2 Nosocomial Clostridioides (formerly Clostridium) difficile (Cl. difficile) infection is one of the most common hospital-acquired (nosocomial) infections and is an increasingly frequent cause of morbidity and mortality among older adult hospitalized patient. 3 Stool cultures have been the standard diagnostic tool for determining the microbial etiology of suspected bacterial infectious diarrhea although time consuming, costly, and with low positive yield. Rapid and accurate diagnostic tools are needed for appropriate management of infectious diarrhea. Commercially available multiplex polymerase reaction (PCR) may improve patient care by allowing clinicians to choose the appropriate antimicrobials or to avoid them if not indicated. 4 However, their potential for yielding results of unclear medical significance, including false positives and multiple positives per sample has been extensively documented. 5 The authors aimed to evaluate a patient with recurrent diarrhea and possible coinfection with enteric pathogens. Methods, characteristic of clinical and epidemiological research, were applied. Methods, implemented in the establishment of the etiological diagnosis, include standard stool culturing (Salmonella spp., Shigella spp., Campylobacter spp.), automatic identification techniques and antibiotic susceptibility testing; for Cl. difficile Toxins A and B by enzyme-linked immunosorbent assay (ELISA) (Savyon Diagnostics) and stool sample through multiplex polymerase chain reaction (mPCR) FilmArray (Biofire ® ) (a gastrointestinal panel for 22 pathogens causing diarrhea). Detection of Aeromonas: Bacterial pure culture was detected, with lactose-negative colonies lacking dark center, normally associated with the production of hydrogen sulfide. Microscopy revealed gram-negative rods. After positive oxidase test the diagnostic thinking shifted to Pseudomonas, Aeromonas or NAG Vibrio as possible causative agents of infectious diarrhea. Further cultivation on Kligler-iron agar proved glucose fermentation, lack of lactose fermentation and lack of H2S production. The indole test was positive and urease production negative. The final identification was performed by VITEK-2 automated system (BioMerieux, France), which proved the microorganism to be Aeromonas hydrophila (97%). The antimicrobial sensitivity was determined by disk-diffusion test (DDT) of Bauer-Cirby on Mueller-Hinton agar and the minimal inhibitory concentrations (MIC) were evaluated by VITEK-2. The interpretation was made with regard to the criteria of EUCAST 2019. The isolate was susceptible to all antibiotics recommended by the standard (cefepime, ceftazidime, ciprofloxacin, levofloxacin, trimethoprim/ sulfamethoxazole, aztreonam). Serum and fecal cytokine concentrations on IL-1b (15.6-500 pg/ml), IL-6 (6.25-200 pg/ml), IL-8 (62.5-2000 pg/ml), IL-10 (12.5-400 pg/ ml), TNF-l (25-800 pg/ml) were determined via ELISA with Diaclone SAS, France kits and reported using spectrophotometric techniques (TECAN sunrise reader, Tecan Trading AG, Switzerland). The research was carried out in the Department of Microbiology and Immunology and the Research Institute of the Medical University of Plovdiv. The present scientific report has been realized thanks to the scientific research project DPDP -02/2018 of the Medical University of Plovdiv.

DISCUSSION
In this study we presented a patient with recurrent diarrhea and evidence of 3 enteric pathogens: A. hydrophila, Cl. difficile and rotavirus.
It is not easy to decide which of the verified pathogens has a leading role in the disease.
Rotavirus gastroenteritis has a pronounced seasonality, occurring mainly during the cold months as epidemics or nosocomial infections in young children. Rotavirus antigen may be identified by several means, ELISA assay being the most common. Only the first encounter with rotavirus can lead to more severe diarrhea, and subsequent infections are mild or unapparent. Rotavirus asymptomatic carriage occurs in about 3% of adults. However, this patient we present had recurrent diarrhea and abdominal pain. 1,2 The discrepancy between the severe clinical presentation and molecular result made rotavirus unlikely reason. It is quite difficult to evaluate the significance of toxigenic Cl. difficile. In our case it has not been detected by ELISA, but by multiplex PCR. ELISA for Cl. difficile toxin A/B is the most commonly used diagnostic test. 6 ELISA is not a reliable method when used alone, due to the wide range of specificity (from 75% to >99% 7 ) and sensitivity (from 32% to 73% 8 ) that depend on the reference standard used 9 . It is preferable to use a method that detects toxin A and B, as strains that only produce toxin B and not toxin A are reported. 10 The Infectious Diseases Society of America, the Society for Healthcare Epidemiology of America and the European Society of Clinical Microbiology and Infectious Diseases guidelines recommend Cl. difficile toxin detection and nucleic acid amplification test (NAAT) as the most effective and sensitive diagnostic tests. To optimize Cl. difficile diagnosis two-step algorithms are currently recommended: e.g. glutamate dehydrogenase test (GDH) test plus toxin detection (ELISA); GDH test plus evidence of toxin arbitrated by NAAT; or NAAT plus toxin detection test. 11 No single test can be recommended as a stand-alone test for diagnosing Cl. difficile. 1 Although re-examination of feces in patients with negative tests for Cl. difficile is a common procedure, there is little evidence to support this practice. This is due to the low sensitivity of ELISA and studies show that 10% of patients who initially test negative for Cl. difficile toxin will test positive in the second testing. [12][13][14][15][16] The inability to verify toxigenic Cl. difficile by other means in the case presented made us doubt this diagnosis. Moreover, previous antibiotic use may shift the likelihood that the detected Cl. difficile are more than colonizers.
Low concentrations of IL-6 in feces can be regarded as another finding, which is difficult to be explained. We would rather expect elevated values of the aforementioned as a result, additionally supported by data from other authors in various intestinal infections with diarrheal syndrome. Moreover, according to other studies, the level of IL-6 in feces correlates with the severity of the disease development. 17,18 Culturing of A. hydrophila from the stool of the patient is the only direct evidence of the presence of an enteric pathogen. The genus Aeromonas consists of gram-negative rods widely distributed in freshwater, estuarine, and marine environments. The genus Aeromonas was re-categorized from the family Vibrionaceae to the family Aeromonadaceae in the mid-1980s. 19 About 95.4% of the strains associated with humans correspond to 4 species: A. caviae, A. dhakensis, A. veronii, and A. hydrophila. Aeromonas species are considered emerging pathogens that cause a wide spectrum of diseases in humans, mainly diarrhea, wound infection and bacteriemia. 20 Aeromonas organisms grow in routine culture but are frequently overlooked unless their isolation is specified; thus, when Aeromonas is suspected, the laboratory must be advised to look for this organism. 21 Clinical studies have demonstrated differences in antimicrobial susceptibility between species, highlighting the importance of both species identification and susceptibility testing for all isolates, particularly in the setting of serious infection. Most cases of Aeromonas-associated diarrhea are self-limited and can be managed with supportive therapy, including oral and intravenous rehydration. Based on anecdotal data, antibiotics may be of value in patients with severe diarrhea and/or a history of immunosuppression. 22 Antibiotic therapy is always indicated in the setting of wound infection and bacteremia. Empiric therapy of suspected Aeromonas infections (severe diarrhea, wound infections, bacteremia) with a fluoroquinolone, third generation cephalosporin, or trimethoprim/sul-famethoxazole is recommended pending species identification and susceptibility testing. 23 While initially missed to be cultured, A. hydrophila was detected during the second admission of our patient. Despite this unexpected finding, her recent exposure to seaweed food supplement might be the explanation. Appropriate therapy led to her uneventful recovery.
In summary, we were able to detect simultaneously A. hydrophila by culturing the stool sample and rotavirus and Cl. difficile by applying mPCR. Mixed positive finding per sample by mPCR is not uncommon. It is a result of its reliance on amplification of genetic loci which may persist in the gut during asymptomatic carriage or be transferred horizontally between enteric bacteria. 24,25 Therefore, all identified bacteria in the stool should be confirmed in conventional culture methods. Clinical judgment combined with mPCR and confirmatory stool cultures can provide an approach to infectious gastroenteritis that is both rapid and accurate. Finally, we considered that A. hydrophila played a major role as a possible pathogen in the clinical presentation of the patient described herein while positive mPCR results were accepted as non-contributing accidental findings.

CONCLUSIONS
In case of co-detected pathogens the clinician has to decide which is the more (or the most) actionable agent. Good communication between physicians and always interpretation of the results in the context of clinical finding are crucial for favourable outcome of the patient.