A Guide to Utilization of the Microbiology Laboratory for Diagnosis of Infectious Diseases: 2018 Update by the Infectious Disease Society of America and the American Society of Microbiology1 includes guidance for infections of the gastrointestinal tract. The guide states for many gastrointestinal infections, particularly noninflammatory diarrhea and acute gastroenteritis of short duration, no laboratory testing is recommended. The specimen of choice to diagnose diarrheal illness is diarrheal stool, not a formed stool or a swab, except in pediatrics where a swab is acceptable when feces are noted on the swab. Multiple stool specimens are rarely indicated for the detection of stool pathogens. Toxin or nucleic acid amplification testing for Clostridium difficile (C. difficile) should only be performed on diarrheal stool, unless the patient has an ileus.
The guide states the appropriate approach to the diagnosis of diarrheal illness is determined by the patient’s age and status, severity of the disease, duration and type of illness, time of year, and geographic location. For severe, bloody, febrile, dysenteric, nosocomial, or persistent diarrhea, fecal testing using culture or culture-independent methods is indicated. To determine what organisms, methods, and screening parameters are included as part of the routine culture or culture-independent method, communication with the laboratory is required. Stool cultures often fail to detect the causative agent; when necessary, culture-independent methods are recommended as adjunct methods.
Culture-independent methods can detect pathogens in one to five hours compared to twenty-four to ninety-six hours required for cultures. The assays are reported to be more sensitive than culture and have much higher detection rates. Highly multiplexed assays allow for the detection of mixed infections. The importance of each pathogen is unclear and they may allow for the detection of pathogens where the indication for treatment is unclear. Culture-independent methods should not be used as a test of cure; they will detect both viable and nonviable organisms.
For C. difficile, toxin detection by either enzyme immunoassay (EIA) or immunochromatographic methods are widely used in clinical practice. They have a reported sensitivity of 70%-85% but are significantly faster than the toxigenic culture and the cytotoxin assay. Glutamine dehydrogenase (GDH) antigen assays are sensitive but not specific. NAATs for the detection of C. difficile have reported sensitivity of 93%-100%.1 To reduce turnaround time, costs, and improve accuracy, some laboratories utilize an algorithm which uses GDH as a rapid screening test with EIA for toxin A and toxin B detection with or without cytotoxin testing or NAAT to arbitrate discrepancies between GDH and EIA toxin results. NAAT detects viable and nonviable organisms. To reduce the identification of colonized patients, some laboratories are performing both NAAT and toxin production tests. When testing is limited to patients not receiving laxatives and with unexplained and new-onset diarrhea, NAAT alone, or toxin EIA as part of a multistep algorithm are the recommended test options.
For parasites, historically it was recommended that three specimens collected over a seven to ten day period be submitted for ova and parasite (O&P) examination. Options today include O&P examination of a second or third specimen if the previous was negative and the patient remains symptomatic. Targeted use of immunoassay testing or NAAT for the most common parasites based on geographic location, patient demographics, and physician request can be used as a screen, with only negative patients with continued symptoms or patients with specific risk factors requiring full O&P examination. Immunoassays for Giardia are sensitive enough that only one specimen may be needed. For NAAT, no data are available on the number of specimens required to rule out infection. In routine procedures, pathogenic Entamoeba histolytica cannot be differentiated from nonpathogenic Entamoeba dispar using morphologic criteria. Only an immunoassay or NAAT can differentiate these organisms.
Gastroenteritis caused by viruses is often of short duration and self-limited. After resolution of symptoms, viral shedding may persist. Although included in some multiplex NAATs, testing is not routinely performed except in immunocompromised patients, infection control purposes, or outbreak investigations. In immunocompromised patients, laboratory testing for CMV should be considered using a quantitative NAAT performed on plasma.
The Clinical Practice Guidelines for Clostridium difficile Infection in Adults and Children: 2017 Update by the Infectious Diseases Society of America (IDSA) and Society for Healthcare Epidemiology of America (SHEA)11 states recommendations are intended to optimize patient care based on a systematic review of the evidence and an assessment of the benefits and harms of alternative care options. The guidelines include an algorithm GDH plus toxin, GDH plus toxin arbitrated by NAAT, or NAAT plus toxin. The recommendations for diagnosis include the preferred population for C. difficile testing is patients with unexplained and new-onset three or greater unformed stools in twenty-four hours. For laboratory testing, the recommendation is use a stool toxin test as part of a multistep algorithm rather than a NAAT alone for all specimens received in the laboratory when there are no preagreed institutional criteria for patient stool submission. When there are preagreed institutional criteria for patient stool submission, use NAAT alone or a multistep algorithm for testing rather than a toxin test alone. Regarding repeat testing, the recommendation is to not perform repeat testing (within seven days) during the same episode of diarrhea and do not test stool from asymptomatic patients, except for epidemiological studies.
The American College of Gastroenterology (ACG) Clinical Guideline: Diagnosis, Treatment, and Prevention of Acute Diarrheal Infections in Adults5 focus is primarily on immune-competent adult individuals and does not consider C. difficile associated infections. For epidemiology and public health considerations, diagnostic evaluation using stool culture and culture-independent methods, if available, should be used when the individual patient is at high risk for spreading the disease and during known or suspected outbreaks. The recommendations for diagnosis include use of stool diagnostic studies, if available, in cases of dysentery, moderate-to-severe disease, and symptoms lasting greater than seven days to clarify the etiology of the illness and enable specific directed therapy. For the majority of cases of acute diarrheal illness, traditional methods of diagnosis fail to identify the etiology. If available, the use of Food and Drug Administration (FDA) approved culture-independent methods of diagnosis can be recommended at least as an adjunct to traditional methods. Antibiotic sensitivity testing for management of acute diarrheal infections is not recommended.
Serological and clinical lab testing in individuals with persistent diarrheal symptoms (between fourteen and thirty days) is not recommended.5 In the evaluation of persistent symptoms, a thorough and directed history is essential. The history may direct further investigations. The role of clinical laboratory studies and endoscopy is uncertain and should be dictated by clinical suspicion and disease severity, within the context of most likely etiologies. An initial diagnostic evaluation should include tests for microbial pathogens. Although stool culture and microscopy remain the initial diagnostic tests, they both suffer from limitations. Enzyme-linked immunoassays and direct immunofluorescence staining may increase sensitivity, but may not be able to distinguish between pathogenic and nonpathogenic organisms. Singleplex and multiplex polymerase chain reaction (PCR) assays for the detection of enteric microbial pathogens are more sensitive than culture, microscopy, or antigen detection. In certain clinical situations (for example, post-antibiotic or hospital-acquired), testing for C. difficile may be recommended, in addition to serological and clinical laboratory testing.
While not considered in these guidelines, the work-up for chronic diarrhea is briefly discussed and should include the differential diagnoses such as celiac disease, Crohn’s disease, eosinophilic gastroenteritis, and [or] Whipple’s disease. In the situation of chronic diarrhea and abdominal symptoms occurring after a bout of infectious diarrhea, a diagnosis of postinfectious irritable bowel syndrome must be considered.
The 2017 Infectious Diseases Society of America Clinical Practice Guidelines for the Diagnosis and Management of Infectious Diarrhea10 recommend a detailed clinical and exposure history should be obtained from people with diarrhea under any circumstances and jurisdictional recommendations for outbreak reporting and infection control should be followed. People with fever or bloody diarrhea should be evaluated for enteropathogens for which antimicrobial agents may confer clinical benefit. Enteric fever should be considered in a febrile person with exposure to Salmonella enterica subspecies Typhi or Paratyphi. People of all ages with acute diarrhea should be evaluated for dehydration. When a possible Shiga toxin-producing organism is suggested by clinical or epidemic history, diagnostic approaches should be applied that detect Shiga toxin and distinguish Escherichia coli (E. coli) O157:H7 from other Shiga toxin-producing E. coli (STEC) in stool. Diagnostic tests that can distinguish between more potent Shiga toxin 2 and less potent Shiga toxin 1 could be used. Shigella dysenteriae type 1 and rarely other pathogens may produce Shiga toxin and should be considered as a cause of hemolytic-uremic syndrome (HUS), especially in people with suggestive international travel or personal contact with a traveler. Clinicians should evaluate for postinfectious and extraintestinal manifestations of enteric infections.
Stool testing should be performed for Salmonella, Shigella, Campylobacter, Yersinia, C. difficile, and STEC in people with diarrhea who also have fever, bloody or mucoid stools, severe abdominal cramping or tenderness, or signs of sepsis. Bloody stools are not expected with C. difficile infections. STEC 0157 should be assessed by culture and non-0157 STEC should be detected by Shiga toxin or genomic assays. Detection of Shiga toxin is needed to detect other STEC serotype. Stool testing should be performed under clearly identified circumstances for Salmonella, Shigella, Campylobacter, Yersinia, C. difficile, and STEC. Specifically, test for Yersinia enterocolitica in people with persistent abdominal pain and in people with fever at epidemiologic risk for yersiniosis. Also, test stool specimens for Vibrio species in people with large volume rice water stools or either exposure to salty or brackish waters, consumption of raw or undercooked shellfish, or travel to cholera-endemic regions within three days prior to onset of diarrhea. A broader set of bacterial, viral, and parasitic agents should be considered in the context of a possible outbreak of diarrheal illness. Selection of agents for testing should be based on a combination of host and epidemiologic risk factors and ideally in coordination with public health authorities.
For immunocompromised people with diarrhea, a broad differential diagnosis is recommended for evaluation of stool specimens by culture, viral studies and examination for parasites. People with acquired immune deficiency syndrome (AIDS) with persistent diarrhea should undergo testing for additional organisms including, but not limited to, Cryptosporidium, Cyclospora, Cystoisospora, microsporidia, Mycobacterium avium complex, and cytomegalovirus (CMV).
Unless treatment is indicated, diagnostic testing is not recommended in most cases of uncomplicated traveler’s diarrhea.10 Travelers with diarrhea lasting fourteen days or longer should be evaluated for intestinal parasitic infections. Testing for C. difficile should be performed in travelers treated with antimicrobial agent(s) within the preceding eight to twelve weeks.
Clinical consideration should be included in the interpretation of multiplex NAAT because these assays detect deoxyribonucleic acid (DNA) and not necessarily viable organisms. All specimens that test positive for bacterial pathogens by culture-independent diagnostic testing such as antigen-based molecular assays (gastrointestinal tract panels) and where isolate submission is requested or required under public health reporting rules should be cultured in the clinical laboratory or at a public health laboratory to ensure that outbreaks of similar organisms are detected and investigated. A culture may also be required when antimicrobial susceptibility testing results would affect care or public health responses. Specimens from people involved in an outbreak should be tested per public health department guidance.
Culture independent, including panel-based multiplex molecular diagnostics from stool and blood specimens, and when indicated, culture-dependent diagnostic testing should be performed when there is a clinical suspicion of enteric fever or diarrhea with bacteremia.
Testing may be considered for C. difficile in people age two years old or greater that have a history of diarrhea following antimicrobial use and in people with healthcare associated diarrhea. A single diarrheal stool specimen is recommended for detection of toxin or a toxigenic C. difficile strain.
The optimal specimen for laboratory diagnosis of infectious diarrhea is a diarrheal stool specimen. Molecular techniques generally are more sensitive and less dependent on the quality of the specimen than culture. For identification of viral and protozoal agents, and C. difficile toxin, fresh stool is preferred. Follow-up testing is not recommended in most people following resolution of diarrhea. Collection and analysis of serial stool specimens using culture-dependent methods are recommended in certain situations by local health authorities.
A clinical and laboratory reevaluation may be indicated in people who do not respond to an initial course of treatment and should include consideration of noninfectious conditions. If clinical symptoms worsen and an antimicrobial agent has been given, antibiotic-associated diarrhea (non-C. difficile) should be considered.10 If the patient is hospitalized or has had healthcare exposure, C. difficile becomes an additional consideration, particularly if there is fever or leukocytosis. Stool should also be submitted for culture and susceptibility. If a bacterial etiology is confirmed, susceptibility testing may reveal whether worsening symptoms could be due to antimicrobial agent resistance. To reduce the identification of colonized patients, some laboratories are performing both NAAT and toxin production tests.1 The Clinical Practice Guidelines for Clostridium difficile Infection11 include an algorithm GDH plus toxin, GDH plus toxin arbitrated by NAAT, or NAAT plus toxin. Utilizing a multistep algorithm for testing for C. difficile is recommended unless there are preagreed institutional criteria for patient stool submission.11 When assessments for infectious agents do not reveal an etiology, consideration for noninfectious illnesses and inflammatory processes should occur. Inflammatory bowel disease and celiac disease are considerations. Postinfectious functional gastrointestinal disorders, including irritable bowel syndrome, may occur in 3% to 10% of adults following bacterial diarrhea. Post-infectious irritable bowel syndrome generally resolves within one year, but may persist for several years.
In the Guidelines for the prevention and treatment of travelers’ diarrhea: a graded expert panel report,14 microbiologic testing is recommended in returning travelers with severe or persistent symptoms or in those who fail empiric therapy. The authors also state that molecular testing, aimed at a broad range of clinically relevant pathogens, is preferred when rapid results are clinically important or non-molecular tests have failed to establish a diagnosis; however, the authors noted no studies have been published which show that using these tests improves patient outcomes.
Some concerns regarding molecular testing addressed in the guidelines include the detection of colonization rather than infection and the use of molecular diagnostic methods does not allow for resistance characterization. Thus, the clinician may be in a position of ordering both culture-based and molecular tests to fully evaluate the patient’s illness and identify the best therapy. Further studies are needed to evaluate the utility of these assays in a clinical setting of the returning traveler with diarrhea.
The increase in worldwide tourism has led to an increase in imported diseases in patients returning to their countries.13 Gastrointestinal problems remain the first complaint among travelers with health problems. Although most episodes of acute traveler’s diarrhea may resolve without the need for antimicrobial treatment and without identification of the causative agent(s), antibiotic treatment reduces the duration of diarrhea as well as related symptoms and time spent incapacitated. In the author’s experience, 35% of the patients attending the Tropical Medicine Unit of the hospital with traveler’s diarrhea caused by enteroaggregative E. coli (EAEC) or enterotoxigenic E. coli (ETEC) need antimicrobial therapy due to the severity or persistence of the symptoms. Bloody diarrhea and persistent diarrhea always require further investigation. Early diagnosis allows an appropriate antibiotic prescription, if needed, and helps to decrease unnecessary prescriptions. Antibiotic treatment can increase the risk of HUS in cases of STEC; therefore, rapid detection can have a significant impact on patient outcome. An additional advantage of rapid diagnosis is the potential to improve public health response during outbreaks.
Molecular techniques have been widely introduced in the routine diagnosis of diarrhea in several microbiological laboratories worldwide. Multiplex molecular assays offer the possibility of syndromic diagnosis as they usually detect the most common microorganisms causing diarrhea. An important advantage is the automation of the laboratory workflow. Additional advantages include speed, no need for trained personnel, minimal handling time, and reduced risk of contamination. Previous studies have shown the sensitivity of molecular assays is higher than culture and microscopy based methods. The improvement in sensitivity implies advantages as well as some difficulties in interpreting the results. The specificity of molecular assays should be improved. One of the main limitations is the failure to distinguish between infection and colonization or the detection of insignificant pathogen load that might not be involved in any clinical feature. A more accurate method to quantify the enteropathogens present in the stool samples is needed. All microbiological results should be interpreted within a clinical context. In addition to the detection of low load of enteropathogens, false positive results can be obtained due to detection of nonviable microorganisms, free DNA or ribonucleic acid (RNA), or nonspecific amplification within the multiplex reaction. In addition, not having live organisms from culture does not allow for typing studies to assess for an outbreak. The detection of antibiotic resistance by molecular methods is another problem. The last limitation is the fixed composition of the majority of the molecular panels commercially available. There is a need for open multiplex molecular platforms where the consumer can combine different pathogens to be detected based on the local epidemiology, patient characteristics, and origin of the diarrhea.
The health technology assessment for multiplex tests to identify gastrointestinal bacteria, viruses and parasites in people with suspected infectious gastroenteritis15 concluded gastrointestinal pathogen panel testing will generally correctly identify pathogens identified by conventional testing. However, these tests also generate considerable additional positive results of uncertain clinical importance with potential workplace implications for those diagnosed. Early diagnosis may change the management of patients positively or negatively. Negative results could lead to an earlier discharge from the hospital. Positive findings could result in unnecessary treatment when symptoms would spontaneously resolve with watchful waiting. Further research is needed.
Multiple studies3,4,16-21 evaluated the FilmArray GIP panel. In most of the studies, the FilmArray specimens were submitted in Cary-Blair enteric transport medium. A few studies used frozen specimens. The studies used several different versions of the test, including version investigational use only (IUO), version 1.7, and version 2.0. The test consists of automated nucleic acid extraction, reverse transcription, amplification, and analysis, with the results available in approximately one hour per run per specimen. The test detects twenty-two agents, including bacteria, viruses, and parasites. The results of the multiplex panel were compared to conventional laboratory techniques.
In the Buss et al3 clinical trial, the test had 100% sensitivity/positive percent agreement (PPA) for twelve of the twenty-two targets and had greater than or equal to 94.5% sensitivity/PPA for an additional seven of twenty-two targets. It was not possible to assess the sensitivity/PPA for Vibrio spp., Vibrio cholerae, and Entamoeba histolytica, as the organisms were not detected by comparator methods, or at all in the case of Entamoeba histolytica. The specificity/negative percent agreement (NPA) was greater than or equal to 97.1% for all targets. In the Piralla et al17 trial, the PPA was 87.5% and the NPA was 77.1%.
In all of the studies, the multiplex test was positive for more pathogens than the conventional laboratory techniques and detected multiple pathogens in a single specimen at a higher rate than the conventional laboratory techniques. Limitations to the studies included a low number of positive specimens for some of the test targets, the test cannot differentiate between live and dead organisms, the test can detect organisms at non-pathogenic levels, the significance and clinical implications of detected coinfections is unclear, many gastrointestinal pathogens can be shed asymptomatically (colonization) or for prolonged periods of time after symptoms subside, and the results of the studies could be influenced by geographic location, season of sampling and the patient population analyzed.
In the Beal et al16 trial, the goal was assessing the impact of the test. The study found patients who had multiplex panel testing had fewer additional stool tests, fewer imaging studies, and fewer days on antibiotics. The overall length of stay was decreased by 0.5 days. Some limitations of the study include the investigators did not confirm the results when the multiplex panel results and the conventional testing did not agree and a historical cohort of patients was used for the control group.
In the Cybulski et al19 trial, the clinical impact of the multiplex panel test was assessed. The investigators found patients with infections detected by the multiplex panel test were more likely to receive targeted rather than empirical therapy. Reduced time to treatment and an impact on antibiotic prescribing were also observed in STEC infections. Patients infected with pathogens identified both by culture and the multiplex panel test (concordant) tended to have greater symptom severity than those patients with positive multiplex panel tests and negative cultures (discordant). The investigators stated these observations were consistent with a higher organism burden required for positive cultures and the relationship of disease severity to the level of organism burden. Patients with coinfections were generally younger and were more likely to have recent international travel. Limitations to the study included an insufficient size of certain subgroups to draw definitive conclusions, the retrospective medical record review is subject to confounding factors and biases, and the study was limited to two hospitals within a single healthcare system.
The Khare et al8 trial compared two commercial multiplex panels for detection of gastrointestinal pathogens. Conventional laboratory techniques, testing on the FilmArray platform, and testing on the MagPix platform (xTag gastrointestinal pathogens panel) were performed on each specimen. The investigators followed the manufacturer’s instructions for the FilmArray platform; however, they modified the manufacturer’s FDA cleared protocol for the xTag panel. They used Cary-Blair stool instead of raw stool. The investigators found that the majority of the targets represented on the panels showed high sensitivity and specificity; however, there were analytes on both panels that showed poorer performance. The FilmArray Aeromonas assay had low sensitivity and the xTag assay for Yersinia enterocolitica had low sensitivity. The xTag assay for norovirus had low specificity. This study also found a high percentage of stool samples positive for two or more pathogens. Limitations of the study included different volumes of stool were tested by the FilmArray assay (200 microliters) and the xTag assay (100 microliters), the xTag sample was performed on more dilute Cary-Blair specimens rather than the FDA cleared raw stool, the investigators were unable to correlate the results of testing with clinical information and treatment decisions, the study identified a relatively low number of positive results for many of the targets and did not identify any samples testing positive for several analytes, and the multiplex assays detected a number of pathogens that were not confirmed.
Multiple studies6,9,22 compared Verigene, FilmArray, and xTag assays. Huang et al22 found that each of the assays demonstrated good clinical performance in their patient population. The Verigene and FilmArray assays provided rapid, on-demand testing for individual specimens in a moderate complexity environment. The xTag assay was higher complexity but allowed greater throughput in a single batch.
The Binnicker9 study noted challenges associated with interpretation of the results of the multiplex panels. First, the multiplex panels target microbial nucleic acid and cannot distinguish between viable, replicating organisms responsible for disease and nonviable pathogens or remnant nucleic acid. The second challenge identified is the use of multiplex panels may increase the detection of C. difficile and potentially impact isolation and treatment decisions. The third challenge relates to how health care providers will use and interpret the large amount of data that will be made available with the broad implementation of the multiplex panels. In the conclusions, the author found each of the multiplex panels had unique advantages and limitations that a clinical microbiology lab must consider. Although the panels increased both the positivity rate and the number of coinfections detected, cultures will continue to be needed for antibiotic susceptibility and epidemiologic investigations. Future studies need to address clinically related issues, including the impact of the panels on antibiotic use and the influence the results have on management decisions and patient outcomes. In the author’s opinion, reserving the highly multiplexed panels to immunocompromised patients, the critically ill, or patients with prolonged diarrhea may improve test utilization and reduce inappropriate antibiotic use.
The Ramanan et al6 study found that in addition to broad coverage and the ability to identify a higher rate of coinfections, the multiplex panels offered reduced turnaround time. It is important for clinical laboratories to be actively involved in the development of test utilization strategies focusing on the use and interpretation of the results of the tests. Each of the panels had unique advantages and limitations. Conventional methods are still needed to detect pathogens not covered by the panels and provide antimicrobial susceptibility information.