Interpreting the GI Pathogen Plus Profile (GPP) Report. Bernadette M. Mandes Wildemore, MD Medical and Laboratory Director DRG Laboratory

Transcription

1 Interpreting the GI Pathogen Plus Profile (GPP) Report Bernadette M. Mandes Wildemore, MD Medical and Laboratory Director DRG Laboratory

2 All tests were developed and their performance characteristics determined by DRG Laboratory Diagnosis and treatment are the responsibility of the ordering physician

3 All testing at DRG is performed via molecular methods Polymerase Chain Reaction (PCR) Enzyme Linked Immuno-sorbent Assay (ELISA)

4 Sections of the GPP Report Bacteria and Toxins by PCR Antibiotic Resistance by PCR GI Health Markers Parasites by PCR Fungi by PCR

5 Each of the analytes are listed by name on the left of the report

6 The quantitative value is listed for each analyte in the column labeled Quantitation

7 The result is listed for each analyte in the column labeled Result

8 It is imperative that the ordering clinician work in concert with the laboratory values as provided by DRG. As our laboratorians do not actually SEE the patient, it is critical for the treating physician to be familiar and comfortable with the lab values we provide. Consultation with our laboratory medicine specialists are always available if questions arise

9 Result interpretation for Bacteria and Toxins section of GPP For most of the values in the Bacteria and Toxins by PCR section of the report, DRG has determined the cut-off or nominal value as to 350 In other words, 350 is where the value turns from negative to positive The exceptions include the Helicobacter pylori (HP) markers and the HP stool antigen (please see later slides)

10 Interpreting the GPP line by line First, let s start with the Bacteria and Toxins by PCR section

11 Interpreting the GPP Clostridium difficile (toxins A and B) Bacterium that can cause significant diarrhea and colitis ( 3 loose stools in 24 hours) Complaints usually associated with current or recent antibiotic therapy Usually accompany treatment with Fluoroquinolone Clindamycin Cephalosporin Penicillin BUT can be tied to any antibiotic use Particularly of concern in very young or very old, or immunocompromised patients

12 Interpreting the GPP C. difficile is a critical infection to identify and treat aggressively (i.e., antibiotics) Suggested treatment protocol is the responsibility of the ordering clinician; however, treatment generally includes Stop ALL non essential antibiotics Confirm that C. difficile toxin is present via confirmatory method (DRG assay works for this) AVOID empiric therapy until diagnosis is confirmed Treat

13 Interpreting the GPP Campylobacter Campylobacter enteritis due to C. jejuni, C. coli, and C. lari are clinically indistinguishable Complaints include belly pain, cramping, and diarrhea Treatment is generally supportive (except for immunocompromised patients)

14 Interpreting the GPP Escherichia coli (E. coli) 0:157 While E. coli may be considered normal flora in some humans, significant pathogenic strains exist Pathogenic E. coli strains are categorized into pathotypes Six pathotypes are associated with diarrhea and collectively are referred to as diarrheagenic E. coli.

15 Interpreting the GPP Diarrheagenic Escherichia coli types Shiga toxin-producing E. coli (STEC). Often times, this is the specific pathotype involved in the more noteworthy outbreaks The Shiga toxin producing E. coli strains include two major strains: Stx1 and Stx2 Enterotoxigenic E. coli (ETEC) Suggested treatment protocol is the responsibility of the ordering clinician; however, treatment generally includes Supportive therapy (hydration) Antibiotics should be avoided Antibiotics and Imodium may increase the risk of hemolytic uremic syndrome (HUS), a potentially fatal complication

16 Interpreting the Helicobacter pylori (HP) values on the GPP The HP values are one of the exceptions to the 350 rule for many of the analytes. For these, the cut off values are below: Helicobacter pylori (H. pylori) caga vaca icea oipa 1500

17 Interpreting the GPP report What are the virulence factors (VF) as reported on the GPP? These factors give additional information to the treating physician regarding the potential for the development of gastric cancer (GC) The development of GC involves the interplay among three important factors The agent (generally, H. pylori) and its pathogenicity Host (patient) characteristics Environment Additional information regarding the individual VF is available on our website for the DRG HP panel (HPP) report

18 Interpreting the GPP report The HP stool antigen value is the other analyte that differs from the 350 rule. The cut off value for the stool antigen is 3 ng/ml H. pylori stool antigen ¹ : Negative or positive The superscript one refers to the area of the report that reads: Please note: The Helicobacter pylori (H. pylori) value can remain positive even in the absence of an active infection. The H. pylori stool antigen functions to determine if the infection is active or current; therefore, favoring treatment. The cut off value for HP stool antigen is 3ng/mL

19 Critical numbers in the Bacteria and Toxins by PCR section of GPP Report Below are key values that should alert the clinician to a significant issue Clostridium difficile (C. difficile) toxin A and/or toxin B C. difficile is a potentially noteworthy infection that generally requires antibiotic treatment If symptoms warrant, ALL non-essential antibiotics should be stopped Treat case with appropriate antibiotic H. pylori stool antigen A positive value indicates an active or current infection, and usually suggests treatment This is even more critical when the patient is positive for one or more of the virulent factors

20 Reliable methods to determine eradication of HP infection Noninvasive testing options for H. pylori include serology, urea breath testing (UBT), and stool antigen Serologic testing has poor sensitivity and specificity values The sensitivities and specificities of UBT and stool antigen are essentially equivalent However, UBT requires the patient make significant dietary and medical changes

21 Interpreting the GPP report Salmonella spp. Significant diarrheal illness Most patients develop diarrhea, fever, and abdominal cramps within 12 and 72 hours after infection Usually self-limited, but may be of special concern for immunocompromised patients Severe illness may require hospitalization for blood borne illness

22 Interpreting the GPP report Shigella spp. Significant diarrheal illness Symptoms include diarrhea (sometimes bloody), fever, abdominal pain, and tenesmus Symptoms usually self limited except in immunocompromised Bismuth subsalicylate (e.g., Pepto-Bismol ) may be helpful Antibiotics should be avoided

23 If any of the bacteria or toxins, or stool antigen are determined positive by DRG, AND the antibiotic resistance box is checked on the TRF, you will see the report below

24 Each of the values listed in the Antibiotic Resistance by PCR section of the report are commonly used antibiotics for the treatment of H. pylori However, the antibiotics are not specific for H. pylori The result will be either sensitive or resistant Resistant values indicate antibiotics that likely won t work for this patient It is important to note that the results in this section are not organism specific; rather, they are patient specific

25 Resistance traits are genetically encoded, so at DRG we test for the specific genes that confer antibiotic resistance (AR) Although nucleic acid-based detections systems are generally rapid and sensitive, it is important to remember that the presence of a resistance gene does not necessarily equate to treatment failure Resistance is an ever evolving field as bacteria continue to evolve with the transfer of resistance one to another

26 Beta lactam antibiotics target the penicillin-binding proteins (PBPs). The beta-lactam ring portion of this group of antibiotics binds to these different PBPs, rendering them unable to perform their role in cell wall synthesis This then leads to death of the bacterial cell due to osmotic instability or autolysis Specific examples include penicillin, carbapenam, and cephalosporin

27 Nitroimidazole antibiotics have been used to combat anaerobic bacterial and parasitic infections The most common example is metronidazole (Flagyl ).

28 Fluoroquinolones belong to a family of broad spectrum, systemic antibacterial agents Fluoroquinolones are active against a wide range of aerobic gram-positive and gram-negative organisms Examples include ciprofloxacin (Cipro ) and lexofloxacin (Levaquin ).

29 Tetracycline is generally used in the treatment of infections of the urinary tract, respiratory tract, and the intestines The use of tetracycline is becoming more and more limited due to widespread development of resistance in the causative organisms

30 Genetic resistance is associated with presence of the nitroimidazole resistance genes nima-h and nimj The affected drugs include metronidazol, tinidazol, and ornidazol

31 Vancomycin is indicated for the treatment of serious, life -threatening infections by gram-positive bacteria unresponsive to other antibiotics

32 Next, let s move on to the GI Health Markers section of the GPP report

33 Anti-Gliadin IgA This first analyte is to determine if a patient has a sensitivity to gluten This assay does NOT confirm celiac disease, as that is only possible through an invasive small intestinal biopsy The anti-gliadin assay is a superlative non invasive alternative to detect the antibodies commonly found in patients that have celiac disease Our assay detects circulating IgA antibodies to gliadin, an antibody found in ~80% of patients with celiac disease It is directed against the alpha/beta and gamma (α,β,γ) gliadins, and may also found in a number of patients who are not enteropathic Patients with values over 100 U/L should be considered for a gluten free diet to relieve GI symptoms such as bloating, diarrhea, constipation, and irritable bowel

34 Calprotectin and Lactoferrin Elevated levels of calprotectin and lactoferrin indicate that neutrophils (PMNs) have migrated in to the lumen of the intestine These PMNs are markers of inflammation within the gut High calprotectin and lactoferrin levels are indicative of inflammatory bowel disease (IBD); however, the assay does not differentiate ulcerated colitis from Crohn disease This test may be useful for monitoring disease activity The test is also positive in patients with bacterial infection

35 Fecal fat Values over 400 are indicative of fat malabsorption syndromes that lead to steatorrhea Disorders of exocrine pancreatic function Celiac disease Short bowel syndrome (in which much of the small intestine has had to be surgically removed and the remaining portion cannot completely absorb all of the fat) Small bowel bacterial overgrowth syndrome (SIBO)

36 Fecal occult blood (FOBT) This is the only non quantitative assay we perform at DRG Laboratory This assay looks for microscopic (occult) blood in the stool secondary to upper or lower intestinal bleeding; however, false positives are possible The value with be negative or positive A positive value should prompt a search for the cause of the bleeding

37 siga 1 and siga 2 These markers are indicative of secretory IgA This is the antibody that lines the interior of the intestines Cut off value 1000; however much higher values are possible Anything that comes in to contact with the mucosa of the gut may be seen as a foreigner by the immune system siga 1 > siga 2 IBD

38 Next, let s move to the Parasites by PCR section of the GPP report

39 Blastocystis hominis Gastrointestinal parasite found in ~23% of US population (in 2000) Four commonly described forms are the vacuolar, granular, amoeboid, and cyst forms Fecal oral transmission within humans has only been identified for the cyst form Symptoms include diarrhea, nausea, abdominal cramps, bloating, excessive gas, and anal itching May also be associated with irritable bowel syndrome (IBS) Suggested treatment includes nitazoxanide

40 Entamoeba histolytica This parasite may infect up to 10% of the world s population Symptoms can include fulminating dysentery, bloody diarrhea, weight loss, fatigue, abdominal pain, and amoeboma Cysts survive outside the host in water, in soils, and on foods

41 Giardia spp. Intestinal parasite of infected humans Infection occurs by ingesting or coming into contact with contaminated food, soil, or water (seasonal infection from fresh water lakes is common) Symptoms include violent diarrhea, excess gas, stomach or abdominal cramps, upset stomach, and nausea

42 Trichomonas spp. Intestinal parasite of infected humans The trichomonad species include T. vaginalis, T. tenax, T. hominis T. hominis is also known as Pentatrichomonas hominis This third organism inhabits the intestinal tract in the area of the cecum (portion of the intestines between the small and large bowel) Diagnosis of this organism as pathogenic is currently controversial, but is believed to be a marker for other, clearly pathogenic organisms in the stool Furthermore, this organism is also believed to be associated with diarrhea, in addition to being found in some liver abscesses

43 Please keep in mind that values in the range of MFI (particularly among the parasites) are molecularly positive, but may not be clinically significant Treatment based on these results is recommended only if clinical symptoms and appropriate assessment of the patient warrant

44 Lastly, let s move to the Fungi by PCR section of the report

45 Candida spp. This yeast is an opportunistic infection commonly found on skin Gastrointestinal infection with candida species is usually a symptom of dysbiosis Return to a healthy gut microbiome is most often suggested Candida infection also accompanies overzealous use of antibiotics or antacids, alcohol overuse, or poor diet

46 The results indicated on this report represent organisms that may be commensal and normally found in a given patient, particularly given his or her environmental exposures. A positive value does not necessarily indicate the need of treatment. Values given here should always be interpreted in concert with the greater clinical picture for an individual patient. Any laboratory value should never be taken in isolation without careful clinical judgement. DRG Laboratory

47 References Kostic, Aleksandar D; Dirk Gevers, Chandra Sekhar Pedamallu, Monia Michaud, Fujiko Duke, Ashlee M Earl, Akinyemi I Ojesina, Joonil Jung, Adam J Bass, Josep Tabernero, José Baselga, Chen Liu, Ramesh A Shivdasani, Shuji Ogino, Bruce W Birren, Curtis Huttenhower, Wendy S Garrett, Matthew Meyerson ( ). "Genomic analysis identifies association of Fusobacterium with colorectal carcinoma". Genome Research 22 (2): Yoshikawa H, Wu Z, Kimata I; et al. (January 2004). "Polymerase chain reaction-based genotype classification among human Blastocystis hominis populations isolated from differrent countries". Parasitol. Res. 92 (1): Blastocystis hominis infection. Diseases and Infections. Mayo Foundation for Medical Education and Research. Last accessed October 7, Bonner A, et. al. The Nonplanar Secretory IgA2 and Near Planar Secretory IgA1 Solution Structures Rationalize Their DifferentMucosal Immune Responses. J Biol Chem Feb 20; 284(8): Entamoeba histolytica Infection. General Information. Centers for Disease Control and Prevention. Last accessed October 7, 2015 Giardia infection (giardiasis). Diseases and Infections. Mayo Foundation for Medical Education and Research. Last accessed October 7, Glocker E. The Need for Resistance Surveillance and Antimicrobial Susceptibility Testing of Helicobacter pylori. Digestion Sep 16;92(3): Haley K, Gaddy J. Metalloregulation of Helicobacter pylori physiology and pathogenesis. Front Microbiol Sep 2;6:911. ecollection Kane SV, Sandborn WJ, Rufo PA, Zholudev A, Boone J, Lyerly D, Camilleri M, Hanauer SB. Fecal lactoferrin is a sensitive and specific marker in identifying intestinal inflammation. Am J Gastroenterol Jun;98(6): Navidad J. Evaluation of Luminex xtag gastrointestinal pathogen analyte-specific reagents for high-throughput, simultaneous detection of bacteria, viruses, and parasites of clinical and public health importance. J Clin Microbiol Sep;51(9): doi: /JCM Epub 2013 Jul 12. Sayehmiri F. Prevalence of caga and vaca among Helicobacter pylori-infected patients in Iran: a systematic review and meta-analysis. J Infect Dev Ctries Jul 30;9(7): Warner RH, Stevens FM, McCarthy CF. Salivary SIgA and SIgA 1 in coeliac disease, inflammatory bowel disease and controls. Ir J Med Sci Jan-Mar;168(1):33-5. Hemmatinezhad, B. et. al. (2016) VacA, caga, icea and oipa genotypes status and antimicrobial resistance properties of Helicobacter pylori isolated from various types of ready to eat foods. Ann Clin Microbiol Antimicrob Jan 20;15(1):2.

48 Thank you!