Gastrointestinal Infections PDF

Preview

Summary

Lab Diagnosis of Gastrointestinal Pathogens- Cary-Blair Medium, transport liquid media with a low nutrient concentration. Must be filled properly. Overfilling will overwhelm the buffers in the media and underfilling will not allow bacterial recovery. - Stools in clean dry containers are acceptable a...

Description

MLSC-3131U, Clinical Microbiology II Lab Diagnosis of Gastrointestinal Pathogens -

-

Cary-Blair Medium, transport liquid media with a low nutrient concentration. Must be filled properly. Overfilling will overwhelm the buffers in the media and underfilling will not allow bacterial recovery. Stools in clean dry containers are acceptable as long as they are set up within 2 hours of collection. Rectal swabs are acceptable for stool pathogens, typically from infants and small children. Blood cultures are occasionally worked on in the case of Typhoid fever. More common in undeveloped countries. Specimen Rejection, occurs when o Information is missing o Sample is preserved incorrectly (stool sample in formalin, except for parasitology). o Unpreserved stool outside of 2 hours of collection. o Duplicate specimens (1-2 stools per day are set up per patient) o In-patients that are hospitalized for more than three days should not be cultured for routine pathogens (GI pathogens are typically infections that occur outside of the hospital). If they do develop symptoms, check for Clostridium difficile. o Leaky containers or containers with stool on the outside of the container contaminate the sample.

Routine Examination -

-

Stools are investigated for o Salmonella spp. o Shigella spp. o Campylobacter spp. o E. coli O157:H7 o Yersinia enterocolitica (depending on patient age, children) Pathogens like Aeromonas and Vibrio are only investigated if specifically requested based on patient exposure. Stool samples are set up on Mac and either XLD or HEK agar. These plates allow us to look for Salmonella and Shigella. CAMPY media is also set up to look for Campylobacter along with Sorbitol MAC media to look for E. coli O157:H7. Selenite broth is also set up to look for Salmonella. CIN plates are set up for specimens from children to look for Yersinia.

MLSC-3131U, Clinical Microbiology II

Yersinia enterocolitica -

GNB, ferments mannitol and shows bulls-eye red pigment on CIN. Explosively catalase positive. Incubated on CIN at 30-32 degrees for 24 hours. A/A sick on TSI. Urea positive (75%) and ONPG-PA-M is +/-/- (motility is positive at RT). Causes pseudo-appendicitis in children 1 month – 10 years old.

Campylobacter -

Usually treated with the patient’s own self defense. Most common cause of bacterial gastroenteritis. Incubated on CAMPY agar at 42 degrees for 48 hours. GNB (gull winged), oxidase positive, catalase weak positive and motile.

Vibrio -

Eye-brow shaped GNBs, live in salt water environments. Isolated in Thiosulfate citrate bile salts sucrose. Causes Cholera, which results in diarrhea and severe dehydration. Very mucoid organism and oxidase positive.

Clostridium difficile -

-

-

-

Hospital acquired infection. Infection occurs when people are placed on broad spectrum antibiotics leading to its overgrowth. Causes pseudomembranous colitis. Has NG on most media. Produces enterotoxins but does not cause symptoms if there is an adequate amount of normal flora. Seen as normal flora in children. Antibiotics will make the infection worse since the antibiotics caused this issue in the first place (loss of more normal flora). Instead, the patient’s normal flora is replenished through stool transplants along with lab generated probiotics. Samples for this are collected in dry sterile containers since we will not look for it in a normal culture and screen. It is still a stool sample but does not use special transport media. We look for the toxins it produces (A and B) to ID it. Testing Methodologies o Glutamate Dehydrogenase, looks for this enzyme, which is made in high amounts by C. difficile, for ID. Cheap but only used as a screen since the enzyme is only present when C. difficile is present. Does not tell us if it is producing toxins leading to pseudomembranous colitis (not specific). o Toxin Production, done after a positive screen and looks for the actual toxins being produced through immunoassays. Tend to lack sensitivity.

MLSC-3131U, Clinical Microbiology II

-

o PCR, the genes of the toxins are detected. Specific, sensitive but very expensive. Done when the previous two tests give mixed answers. These methodologies are done to limit cost and maximize efficiency. Patient must have extreme diarrhea for a valid test.

Helicobacter pylori -

Lives in the stomach and associated with stomach ulcers and cancer. Detected by testing patient blood sample for antibodies against Helicobacter pylori.

Double Stranded RNA Viruses -

Rotavirus, causes the stomach flu. Vaccine exists, but not all children are vaccinated. Suspected infection is tested for with ELISA, PCR or Latex Agglutination.

Single Strand RNA Viruses -

Calciviridae, include Noroviruses. Usually causes stomach flu in the winter and last for 24 hours. People with school aged children or work in a nursing home are typically at risk. Also hits cruise ships. Highly infectious and identified through PCR.

Treatment -

-

GI infection causes diarrhea which results in dehydration. Dehydration will lead to a loss of electrolytes. Therefore, treatment is mainly focused on rehydration and electrolyte replenishment. Antibiotics are only used in severe infections since these infections are self-limiting. The patients will get over the infection on their own. The use of drugs that will stop diarrhea will increase the contact time of the pathogen to the intestinal wall. The diarrhea will stop; however, the pathogen will not be flushed out of the body. May result in the pathogen crossing into the blood stream and cause septicemia. Many cases of diarrhea are caused by viruses, so antibiotics are often rendered useless anyways.

Reporting -

Always list the organisms we look for and whether we found them or not. Do not list “no pathogens present” as we are only sure that we did not find what we were looking for. All isolates of enteric pathogens, except for Campylobacter, should be sent to PHL for surveillance purposes.

MLSC-3131U, Clinical Microbiology II Susceptibility Testing -

-

Salmonella, most cases resolve without antibiotic treatment and routinely do not require AST. Shigella, isolates should be run for AST, with a report given after. This is because it causes very bloody diarrhea, giving the organism a very high chance of dissemination into the blood. Yersinia, Campylobacter and Vibrio, most cases resolve without antibiotic treatment and routinely do not require AST. E. Coli O157:H7, AST is not done since there is no evidence that antibiotics are effective against this organism strain. Salmonella typhi, isolates should have AST run and reported.

Media -

-

-

-

-

-

-

SMAC, E. coli O157:H7 ferments lactose but not sorbitol. If it is an NSF, run latex agglutination. If the bacteria is positive for latex agglutination, run API 20E with a SMAC purity plate. If it is E. coli O157:H7, report to doctor, infection control and send the isolate to PHL. Colorless colonies on XLD, Sorbitol MAC (positive for E. Coli O157:H7) and HEK mean that the bacteria have not used the sugars provided (non-fermenters). Non-fermenters with or without H2S production require a full work up. CAMPY, isolates campylobacter when incubated at 42 degrees Celsius. Contains antibiotics that inhibit other organisms so any growth on this media requires a full work up. Colourless, pinpoint and flat to convex (may also spread across the plate). If there is growth, confirm with a gram stain of gull-wing shaped GNB. Selenite Broth, contains selenium which is a selective agent and very toxic to normal flora. Also contains Cysteine which acts as a reducing agent and provides nitrogen sources to enrich the media. The broth is also toxic to the pathogens, so incubation time in selenite is kept to a minimum, 12-18 hours, and then subcultured onto HEK. Do not mix the broth and only inoculate it on the top of the broth. CIN, differential and selective media that isolates Yersinia enterocolitica. Has mannitol which Yersinia enterocolitica will ferment to produce red precipitate (bulls-eye colonies). Yersinia enterocolitica is mesophilic and grows best at lower temperatures (30-32 degrees Celsius). If there are bulls-eye colonies, run catalase. All enterobacterales are catalase positive, but Yersinia is explosively catalase positive. If catalase positive, run API 20E. MAC, contains lactose to differentiate LFs from NLFs. For stool samples, we only work on NLFs because LFs do not cause GI infections. Yersinia enterocolitica also grows on MAC and will look the same as they do on CIN. If your MAC/HEK organisms look like Yersinia enterocolitica, a catalase of the Yersinia enterocolitica from the MAC or HEK plates will tell you if it really is Yersinia enterocolitica. If it is explosively catalase positive, you know

MLSC-3131U, Clinical Microbiology II that all three plates have Yersinia enterocolitica. Stop using the MAC/HEK plates for work up at this point, since CIN is better for Yersinia enterocolitica. Salmonella -

-

NLF with or without H2S (only some strains are H2S negative). ONPG-Phenylalanine-Motility is -/-/+. If isolated, set up API 20E or Vitek. If it is positive for Salmonella, perform a multi-valent serotype. If the serotype is positive, send out a preliminary for Salmonella and send the isolate to the PHL for serotyping. Serology, looks for the O (cell membrane), H (flagella) and Vi (capsular) antigens. The Vi antigen can mask the O antigen, so the isolate is heated to break down the heat-labile Vi antigen. This reveals the heat-stabile O antigen while preserving the heat-stabile H antigen.

Shigella -

NLF with an ONPG-PA-M that is +/-/-. Urea positive. Requires three days for incubation. Has four serogroups.

Biochemicals -

TSI, ALK/A is very suspicious of GI pathogens. A/A and A/A with H2S are not suspect of pathogens. ALK/A with H2S may be salmonella. ONPG and Motility, non-motile organisms are suspicious of stool pathogens. Phenylalanine, all Phenylalanine positive bacteria are not stool pathogens....