Microbiology and Meningitis Quiz

Bacteremia and Sepsis

• Bacteremia is bacteria in the bloodstream
• Septicemia is bacteremia with clinical signs and symptoms like fever, chills, hypothermia, hyperventilation, and septic shock.
• Exotoxins and/or endotoxins can evoke a massive immune response
• Mortality rate is greater than 50%

Types of Bacteremia

• Primary bacteremia: No other known infected site
• Secondary bacteremia: Associated with an infected body site
• Occult bacteremia: No known cause, often in children
• Pseudobacteremia: False bacteremia, contaminated materials are the source of organisms in blood culture media

Bacteremia Patterns

• Transient: Bacteria are in the bloodstream for minutes to hours, often from a traumatized body site (e.g., mucous membrane, skin, minor surgery) or early stages of some diseases (e.g., meningitis, osteomyelitis)
• Intermittent: Bacteria are periodically released into the bloodstream (e.g., caused by pneumonia, meningitis, and abscess)
• Continuous: Bacteria are present constantly in the bloodstream; often in individuals with infected intravascular sites

Bacteremia Sources

• Intravascular: Associated with the vascular system (e.g., infected heart valves, catheters, veins)
• Extravascular: Outside the vascular system; lymphatic vessels carry organisms to the bloodstream (e.g., urinary tract infections, respiratory tract infections, abscesses)

Intravascular Bacteremia Causes

• Bacteria infection of the endocardium (e.g., viridans streptococci - normal oral flora, Enterococci, coagulase-negative staphylococci (e.g. S. epidermidis), S. aureus, Corynebacterium - normal skin flora)

Extravascular Bacteremia Causes

• Bacteria from a localized site of infection (e.g., Meningitis caused by N. meningitidis, H. influenzae type B, and S. pneumoniae. Other sites are enterics, anaerobes, NFGR, and other GNR.)

Bacteremia Detection

• Mortality rates are high, so prompt detection is crucial
• Obtain blood via an aseptic venipuncture
• Isolate, identify, and test for the organism's susceptibility to different antibiotics/antimicrobials
• Common causes of nosocomial bacteremia: coagulase-negative staphylococci, enterococcus, and Candida albicans; Enterobacteriaceae (E. coli., K. pneumonia, Enterobacter spp.), Serratia spp. Nonfermenting GNR (e.g., P. aeruginosa, A. baumannii)

Bacteremia Collection Sites and Methods

• Venipuncture: Peripheral aseptic venipuncture is preferred; indwelling intravascular catheters aren't recommended due to potential contamination; only use if no other option.
• Methods: Needle and syringe (preferred method), butterfly, tube method via lysis centrifugation system

Bacteremia Collection Procedure

• Specimen container preparation (disinfect tops of culture bottles & tubes with 70% alcohol or iodine)
• Site preparation (clean skin with alcohol to remove debris and oil, swab with iodine in concentric circles, let iodine dry for at least 1 minute)
• Specimen collection
• Site care (remove iodine with alcohol)

Bacteremia Collection Difficult Cases

• Difficult to recover organisms in clotted specimens
• Recommended anticoagulant is SPS

Sodium Polyanethol Sulfonate (SPS)

• Prevents clotting
• Inactivates complement
• Inhibits phagocytosis
• Neutralizes some antimicrobial agents
• Inhibits N. gonorrhoeae and N. meningitidis
• 0.025% concentration in blood culture media
• SPS blood collection tubes available but not always recommended.

Blood Volume

• Larger Volume = More positive results
• Recommended amount varies with patient age; children have high levels of bacteremia and require 100-1,000 organisms/mL; infants and children need 1-5 mL; and adults need 10 mL minimum

Blood-to-Broth Ratio

• 1:5 to 1:10
• Prevention of clots, and dilution of inhibitory factors

Timing and Number of Cultures

• Usually 2-3 sets of aerobic and anaerobic blood culture sets
• Getting 1 set is 80% detection rate
• 2 sets = 90% detection
• 3 sets = 99% detection
• Take cultures from different sites
• Collect before antimicrobial therapy

Newer Recommendation

• Three blood cultures collected simultaneously or one right after the other

Transport

• High priority transportation (STAT)
• Store at 37°C if transport is delayed

Culture Media - Broth

• Includes BHI, Brucella, Columbia, Thioglycollate, Trypticase Soy, and special formulations
• Key components: SPS, nutrients, headspace with CO2, antibiotic removal device, and osmotic stabilizers

Culture Media - Agar

• Lysis-centrifugation method uses agar media
• Uses biphasic culture bottles (agar and broth)

Incubation Conditions

• 35-37°C
• Time varies according to the organism; some require longer incubation

Manual Culture Methods – Conventional Broth

• Broth bottles examined daily for growth, turbidity, gas production, discoloration
• Blind subcultures

Manual Culture Methods – Biphasic

• Castaneda bottle: tipped to cover agar and allow inoculation; colonies appear after incubation
• Newer versions use blood added and use a paddle device attached to aerobic bottle with media containing CHOC or MAC; paddle bathed, and paddle/broth is examined daily for growth

Manual Culture Methods – Lysis-Centrifugation

• Collect blood in a special isolator tube; anticoagulate and lyse the blood; centrifuge the tube
• Inoculate onto agar plates; incubate and check daily for appearance of colonies

Instrumentation

• BACTEC systems: Measure CO2 production by metabolizing organisms
• Semiautomated system: BACTEC 460
• Continuous-Monitoring Systems: BacT/Alert, BACTEC 9000, ESP

BACTEC 460

• First automated system
• Uses radioactive carbon; microorganisms metabolize carbon and produce radioactive CO2; amount of radioactive CO2 in headspace is determined

Continuous Monitoring Systems (BacT/Alert, BACTEC 9000, ESP)

• Uses special bottles, incubator, agitator, and detection system monitoring for growth
• Computer monitors the bottles

BacT/Alert

• Colorimetric method
• Monitors CO2 production
• Bottles with gas-permeable sensors; sensor changes from green to yellow as gas is generated

BACTEC 9000

• Uses fluorescent sensors in the base of the bottles
• Fluorescence increases as CO2 is generated

ESP/VersaTREK

• Manometric system
• Bottles attached to a pressure transducer; measures gas pressure inside the bottles

Positive Blood Cultures

• Critical values detected by appearance of turbidity, hemolysis, gas production; colonies on subculture plates
• Instrumentation-assisted identification and antimicrobial susceptibility testing

Gram-Stained Smears

• First step in work-up
• Determine morphology and gram stain reaction

Subculture

• Positive cultures subcultured onto agar media to isolate colonies
• Media selection depends on gram stain reaction; aerobic: BAP, CHOC, or both in CO2; MAC, EMB added for GNR; mixed cultures using PEA or CNA; anaerobic: anaBAP

Reporting Results

• Notify physician immediately upon detection of critical values
• Document report with time, date, performed tests, and number of positive cultures; including gram-stained smear results
• Preliminary report after 48 hours; final report if no growth after 7 days of incubation

Contaminant

• Determine clinical significance of isolate
• Probable contaminant: Skin flora (e.g., Corynebacterium spp., Propionbacterium acnes, S. epidermidis, Micrococcus); growth of multiple organisms from one culture; Growth of the same organism in repeated cultures. Growth of same organism in blood and from a sterile body site

Special Cultures

• Brucella spp.: Prolonged incubation (30 days); Nutritionally deficient streptococci; Suspected GPC if no growth on subcultured plates; overlaid culture with a streak of S. aureus
• Campylobacter: Will grow in blood culture, but subculture must be in microaerophilic conditions (e.g., CAMPY agar)

Antimicrobial Therapy

• Empiric initially
• Narrow spectrum after ID and susceptibility testing is completed

Central Nervous System

• Introduction: CNS includes the brain, spinal cord, and meninges.
• Term: CSF is clear and colorless liquid bathing the brain and spinal cord. It's tested for culture, cell count, glucose, and protein levels. Meningitis is inflammation of the meninges, and purulent meningitis is also known as pyogenic meningitis (pus in the meninges), often caused by bacteria. Aseptic meningitis is typically caused by a virus. Encephalitis is inflammation of the brain, and meningoencephalitis is inflammation of the brain and meninges.

Routes of Infection

• Hematogenous: Most common route where organisms from a site of infection enter the bloodstream and travel to the CNS. N. meningitidis is an example.
• Contiguous spread: Organisms spread from an infected adjacent site (like sinusitis) to the meninges.
• Trauma: Breach of CNS protective barrier (e.g., skull fracture) allows microbial contamination.
• Surgery: Microbial contamination can occur during surgical procedures.
• Shunts: Placed to drain fluid from the CNS, these can serve as a portal of entry for microbes

Acute Bacterial Meningitis Symptoms

• Flu-like symptoms
• Headache
• Fever
• Nausea
• Vomiting
• Nuchal rigidity (stiff neck)
• Photophobia (sensitivity to light)
• Mental status changes

Causative Agents

• Neonates: E. coli; other GNRs (Klebsiella, Enterobacter); S. agalactiae; Listeria monocytogenes
• Young children (6 months-5 years): H. influenzae type b; N. meningitidis; S. pneumoniae
• Older Children/Adults: Meningococci; Pneumococci; GNRs; L. monocytogenes (immunocompromised individuals)
• Elderly: Same as above but with possible shunt

Specimen Collection

• Lumbar puncture: Spinal needle inserted between the 3rd and 4th lumbar vertebrae to collect CSF
• Specimen transported at room temperature (RT) within 15 minutes of collection; STAT processing; and stored in incubator or at RT for pathogens that are not sensitive to refrigeration (e.g., meningococci, pneumococci, H. influenzae)
• 3-4 tubes are collected; Tube #1 (protein and glucose); Tube #2 (gram stain and culture); Tube #3 (cell count and differential).

Microbiology Processing

• STAT processing
• 1 mL CSF → centrifuge and collect sediment for smear and culture
• Gram stain sensitivity: 75-90%
• Thick smears

Culture Media

• BAP, CHOC media are standard/most common
• MAC or EMB if GNR is suspected from gram stained smears
• anaBAP requested for anaerobic bacteria
• Anaerobes rarely isolated
• Incubate at 35-37°C with CO2

Antigen Detection Tests

• Available for group B streptococci, Hib, meningococci, pneumococci
• Should supplement but not replace smear and culture tests

Other Types of Meningitis

• Spirochetes: T. pallidum, B. burgdorferi
• Viral: Aseptic meningitis
• Mycobacterial: M. tuberculosis
• Fungal: Rare, chronic meningitis in immunocompromised individuals
• Paraistic: Rare

CSF Findings

• Table describing organisms, cells (e.g., neutrophils, lymphocytes, or eosinophils), and diagnostic tests for various types of meningitis

Brain Abscesses

• Caused by N.F. organisms (anaerobic bacteria, staphylococci, viridans, other streptococci)
• Aspiration and biopsy material transported in anaerobic conditions
• Examined microscopically; cultured aerobically and anaerobically

Encephalitis/Meningoencephalitis

• Most common: Viruses
• Bacteria: L. monocytogenes, Rickettsia, and Mycoplasma
• Parasites: Naegleria and Acanthamoeba