Lecture Notes on Septicemia and Gastroenteritis 5

Diagnosing Infections

Comparing and Contrasting Symptoms

• Initial clues come from differing symptoms, but crossover can occur (e.g., Salmonella septicaemia).

• Patients can have multiple conditions simultaneously, an important lesson for practicals.

• The way infections are handled in the lab varies significantly based on the type of infection.

Handling Blood Samples: Dilution Effect

• Blood volume in humans averages 7-9 liters, diluting microbes entering the bloodstream.

• Small sample volumes (5-10 mL per bottle, up to 40 mL total) are tested, assuming circulation distributes organisms.

• Septicaemia assumes even distribution, but other bloodstream infections may not exhibit this pattern (e.g., infected endocarditis).

• Infected endocarditis: Microbes grow in clumps, potentially leading to missed detection in samples.

Blood Culture Media

• Blood culture bottles contain highly nutritious enrichment media to maximize microbial growth.

• Two bottles are used: one for aerobic and one for anaerobic conditions, indicated by cap colors.

• Samples are directly inoculated into bottles via syringe to prevent clotting and sent to clinical labs urgently. The porters then ship it urgently to the clinical labs and place the blood cultures on the machines.

Incubation and Monitoring

• Bottles incubated on continuous monitoring machines that scan for color changes indicating carbon dioxide levels.

• Colorimetric reaction on a disk at the bottle's base detects $CO_2$ changes.

• Machines scan every 30 seconds and produce a graph. A threshold level triggers flagging.

• Slow rises in $CO_2$ may indicate white blood cell death instead of bacterial growth.

• Most bacterial cultures flag positive within 2-3 days, reflecting the dilution effect.

Follow-Up Procedures

• Positive flags trigger immediate Gram staining and subculturing of the sample.

• Gram stains should be prepared carefully to preserve natural growth characteristics (e.g., staphylococci in grape-like bunches, streptococci in pearl necklace chains).

• Gram stain results inform antibiotic choices, especially if initial antibiotics are ineffective.

• Cultures are grown on nutritious, selective media to identify any present organisms.

• Columbia blood agar, chocolate agar (heated to lyse cells and release nutrients), and anaerobic blood agar are commonly used.

• Growth patterns in aerobic vs. anaerobic bottles offer clues: facultative bacteria grow in both, strict aerobes in aerobic only, and anaerobes in anaerobic only.

• Blood is normally sterile, resulting in pure growth of a single organism in most cases.

Rapid Identification Techniques

• Direct identification from the blood clot is possible using MALDI-TOF mass spectrometry or API tests.

• Results must be interpreted cautiously due to potential interference from other components in the clot.

Clinical Microbiologist Review

• Clinical microbiologists review positive blood cultures daily, checking patient records for existing infections (e.g., UTIs).

• Matching Gram stain results between blood and urine cultures suggests the same organism, informing antibiotic selection.

• Full identification to species level is crucial, with potential for further subtyping.

Contamination Considerations

• Blood cultures taken from a vein through the skin can be contaminated by normal skin flora (e.g., coagulase-negative staphylococci, diphtheroids).

• False positive rates increase with inexperienced staff.

• Assess patient illness signs and match with existing infections.

• Avoid drawing blood through existing lines or catheters due to biofilm contamination.

Gastroenteritis Diagnosis

Selective and Differential Media for Fecal Samples

• Fecal samples require selective differential media due to diverse normal flora (35,000+ organisms).

• Salmonella-Shigella (SS) agar targets non-lactose fermenters with hydrogen sulfide ($H_2S$) production, indicating Salmonella.

• Differentiating pathogens from normal flora among Enterobacteriaceae is challenging as its presnt in the gut in high numbers.

Molecular Tests

• Molecular tests for Salmonella and Shigella are used because they are always considered pathogenic.

• Molecular methods were initially limited by substances in feces inhibiting PCR, but advances have mitigated this.

• Culture testing may continue alongside molecular methods for antibiotic sensitivity testing.

Sorbitol Testing

• Sorbitol is used as a different agent instead of lactose in media for E. coli O157 detection.

• Tellurite and cefixime are present as selective agents added to suppress other organisms' growth.

Campylobacter Detection

• Campylobacter detection uses a selective charcoal-based medium with antibiotics and modified incubation.

• Plates are incubated at 42°C in reduced oxygen and raised carbon dioxide ($CO_2$) for 48 hours.

• Microaerophilic (requires reduced oxygen) and capnophilic (requires increased carbon dioxide). The extended incubation is to make data skewed more for Campylobacter.

• While Campylobacter prefers 37°C, 42°C suppresses E. coli growth.

• Emphasis on Campylobacter jejuni may lead to underreporting of other Campylobacter species due to incubation conditions.

Vibrio Detection

• TCBS (thiosulfate-citrate-bile salts-sucrose) agar detects Vibrio, especially after travel or seafood consumption.

• Differential reactions based on sugar fermentation and selective inhibition (bile salts, alkaline pH) favor Vibrio growth.

Clostridium difficile Testing

• C. difficile is detected by toxin testing (ELISA) rather than culture due to unpleasant odor and focus on toxin presence.

• Glutamate dehydrogenase (GDH) screen followed by C. difficile toxin-specific test and you do a bacterial glutamate dehydrogenase.

Viral Gastroenteritis Diagnosis

• Traditionally diagnosed by electron microscopy, now largely replaced by ELISA-based antigen detection or molecular methods.

• Data skewed toward children due to testing guidelines and parental behavior.

• Children are more likely to take a child to the doctor rather than an adult.

Parasite Detection in Fecal Samples

• Fecal parasites are detected via iodine wet preps and modified Ziehl-Neelsen/oil immersion staining.

• Iodine highlights internal cell structures, while Ziehl-Neelsen detects Cryptosporidium.

• Molecular methods (PCR) are increasingly used for Cryptosporidium and Giardia.

Antibiotic Sensitivity Testing

Testing for Sensitivities

• Antibiotic sensitivity profiles are tested for septicaemia and gastroenteritis but not always released for the latter.

• For simple infections, a limited set of antibiotics is tested, and only those recommended by national guidelines are reported.

• Gentamicin results are withheld from general practice samples due to IV administration.

No antibiotics for gastroenteritis

• Antibiotics are generally avoided in gastroenteritis to prevent diarrhea, C. difficile infection, and antimicrobial resistance.

• Treatment focuses on hydration and electrolyte balance.

• For C. difficile, the first step is to stop the causative antibiotics.

• If that is not possible, then treat with vancomycin or metronidazole.

Treating Septicaemia

• Septicaemia requires immediate broad-spectrum antibiotics before sensitivity results are available.

• Antibiotic therapy is then tailored based on the identified agent and any secondary infection sites.

• Supportive care includes maintaining renal function, blood pressure, homeostasis, and potentially mechanical ventilation. while dealing with infection

• Protect the organs and potentially administer steroids.

• Long-term damage prevention and management of autoimmune follow-up are essential.

• Immunocompromised patients with septicaemia require bactericidal antibiotics.

• Must also treat patients that are immunocompromised for cryptosporidium.

Epidemiology of Bloodstream Infections and Diarrheal Diseases

Bloodstream Infections Monitoring

• Bloodstream infections, while numerous, are not all routinely reported.

• Methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE) from blood cultures are exceptions.

• Most septicaemias are not transmissible, focusing on individual treatment rather than public health.

• Sentinel surveys periodically monitor specific organisms to assess threats and patterns.

  identifying key risk factors for sepsis

• major trauma

• medical intervention that circumvents patients normal defences.

• existing defects of either innate or acquired immune system

• splenectomy, The surgical removal of the spleen, which can significantly impair the immune response and increase susceptibility to infections.

• age (Immunosenescence) Immunosenescence refers to the age-related decline and dysregulation of the immune system, leading to a reduced ability to fight infections, respond to vaccines, and combat cancer, as well as an increased risk of autoimmune diseases.

Diarrheal Diseases Monitoring

• Diarrheal diseases are monitored globally by the WHO.

• Diarrhea is the second most common cause of death in children under five globally, though rare in the UK.

• Maintaining hydration is crucial, but contaminated water sources pose a challenge.

• Malnutrition exacerbates (to cause (a disease or its symptoms) to become more severe.) recovery challenges in developing countries.

• Clean water helps prevent and treat diarrheal diseases.

• Death from infection (Shigella and Cholera) can be caused from dehydration leading to kidney failure or heart failure. Also from salt imbalance leading to heart failure.

Epidemiology in the UK

• Data is skewed towards bacterial infections due to higher testing rates which are under-reported.

• Data in fecal microbiology is skewed towards children due to testing of children.

• Campylobacter is the most common, salmonella is the second most common, Shigella/Colo is third.

• Shigella from overseas and E. coli is from your home.

• Campylobacter is linked to migratory patterns of Canada geese and improper cooking methods.

• C. diff infections are hospital-associated, with higher rates in those over 65.

Risk Factors

• Double cut-off with risk in Under 1-2 and Over 80.

• Over 65 more likely to acquire disease and be unwell but over 70 more likely to die.

• Contaminated food, especially at home (50% of cases are sporadic), is a major risk factor.

• Foreign travel exposes individuals to untreated water, potentially contaminated with parasites and to contaminated Ice cubes.

• Age, reduced stomach acidity (due to antacids), broad-spectrum antibiotics, and hospitalization increase risk.