L6 Diagnostics and Infection Control

Why Diagnosing Infection is Challenging

• Symptoms often identify a syndrome- not a specific pathogen

• Normal microbiota and contaminants can complicate interpretation

  • A positive result does not automatically establish causation!

• Some pathogens are difficult, slow, or unsafe to culture

• Best diagnostic method depends on disease stage and specimen source

• Therefore, a laboratory result must be interpreted in the clinical context

• Interesting point: The same pathogen may require different diagnostic strategies depending on whether the question is acute infection, past exposure, treatment selection, or outbreak surveillance

Diagnosing Infectious Diseases… not always that simple

• 42-year-old patient with:

  • Persistent cough

  • Fatigue and weight loss

  • Fever and night sweats

• Chest X-ray: Suggests chronic lung infection

  • TB is suspected

• How would you diagnose this patient?

• What specimen would you collect?

• What are you trying to detect?

  • The pathogen?

  • The host immune response?

• Which diagnostic methods could you use?

  • Microscopy

  • Culture

  • Molecular testing (PCR)

  • Antigen/antibody testing

• Would one test be enough?

• How do speed, sensitivity, and specificity affect your choice?

• Tests most useful for diagnosis: PCR

Diagnosing Infectious Diseases

What do we know about TB anyway?

• Mycobacterium tuberculosis causes tuberculosis (TB) and has caused enormous human mortality

• M. tuberculosis grows slowly, making traditional laboratory diagnosis time-consuming

• TB symptoms can develop gradually, delaying recognition of active disease

• Diagnosis is especially challenging where laboratory resources and supplies are limited

• Delayed diagnosis can worsen patient outcomes and allowed continued transmission

The Clinical Microbiology Laboratory

Major functions

• Receive and assess clinical specimens

• Detect and isolate infectious agents

• Identify organisms using phenotypic, immunologic, or molecular methods

• Perform antimicrobial susceptibility testing when appropriate

• Communicate clinically important results rapidly

Laboratory safety is risk-based: Containment, PPE, engineering controls, and work practices are matched to the organism and procedure

Healthcare-Associated Infections (HAIs): A Diagnostic Problem

• Up to 2 million HAIs occur annually in the United States

• Healthcare settings concentrate susceptible hosts, invasive devices, antibiotics, and opportunities for transmission

• Laboratory diagnostics helps distinguish infection from colonization and supports outbreak recognition

• Strain and resistance patterns can reveal shared sources or transmission chains

• Infection prevention depends on timely detection and communication

Diagnostic microbiology serves both the individual patient and the healthcare population

Factors found to contribute to HAIs

• Bed space

• Low compliance with hand hygiene practices

• Lack of resources including rooms for isolation or cohorting (grouping together patients with the same infection)

• Lack of trained IPC practitioners and limited opportunities for staff training, High patient-to-nurse ratio

• Increasing use of complex and medical surgical procedures

• Increasing use of invasive medical devices (e.g., mechanical ventilators, urinary catheters, central intravenous lines) without proper IPC training or laboratory support

• Inadvertent contamination of prepared supplies/pharmaceuticals (e.g., IV fluid, infant formula, general medications)

• Suboptimal cleaning, disinfection, and sterilization practices

• Antibiotic resistance due to overuse of broad-spectrum antibiotics

The Diagnostic Workflow

• Laboratory identification of microbial pathogens

• Flowchart shows alternative paths for identifying pathogens or pathogen exposure in the clinical laboratory

Critical principle: Errors made BEFORE testing- wrong site, poor collection, delayed transport- can invalidate everything downstream

Specimen Selection, Collection & Transport

A diagnostic test is only as good as its specimen!

• Collect from the site most likely to contain the pathogen

• Collect before antimicrobial therapy when feasible

• Use aseptic technique to reduce contamination

• Obtain adequate volume- especially for blood cultures

• Transport promptly under conditions that preserve the target organism

Direct Examination: Rapid First Clues

• Microscopy can reveal cell morphology and abundance, arrangement, and host inflammatory cells

• Differential stains can rapidly narrow the possibilities

• Negative microscopy does not rule out infection when organism burden is low

• Direct results often guide immediate culture choices and empiric therapy

Culture, Isolation & Colony Morphology

• Isolation of a pure culture permits definitive phenotypic testing

• Diagnostic clues: Colony size, shape, texture, pigment, hemolysis, and growth conditions

• Failure to grow may reflect the organism- or the culture conditions

• Culture is powerful:

  • Adv: Produces a living isolate

  • Disadv: Can be slow and selective

Phenotypic Identification: Biochemical Profiles

• Biochemical tests ask what metabolic reactions an isolate can perform

• Identification is based on a pattern of positive and negative reactions; i.e., metabolic “fingerprint”

• Tests may detect substrate use, enzyme activity, fermentation products, or other metabolic traits

• Miniaturized and automated systems speed testing and standardize interpretation

• Warning: Phenotypic expression can vary with growth conditions and organisms state

Choosing the Right Treatment: Antimicrobial Susceptibility Testing - 1

Choosing the Right Treatment: Antimicrobial Susceptibility Testing - 2

Goals

• Determine which antimicrobial is most likely to inhibit the identified pathogen

• Susceptibility results support de-escalation from broad empiric therapy to a narrower targeted drug when clinically appropriate

Interpreting Susceptibility Results

Laboratory result ≠ treatment decision by itself

• MICs and inhibition zones are compared with organism-specific breakpoints

• “Susceptible” predicts likely success only with appropriate dosing and drug exposure

• Anatomical site, host factors, toxicity, route, and drug penetration matter

• Resistance mechanisms can be spread within and between microbial populations

• Accurate identification is essential before interpreting many susceptibility results

Immunological Diagnosis: Antigen or Antibody?

Detect the pathogen (antigen)

• Can provide evidence of current infection

• Useful when a characteristic microbial antigen is present

• May be rapid and adaptable to point-of-care formats

• Sensitivity depends on antigen abundance and sampling

Detect the host response (antibody)

• Useful when direct pathogen detection is difficult

• Response may take days to weeks to become detectable

• IgM/IgG patterns and rising titer can inform timing

• Past infection or vaccination can complicate interpretation

Diagnostic antibodies are often monoclonal reagents selected for highly specific recognition of a microbial antigen

Serology & Antibody Titer

• Antibody titer = reciprocal of the highest serum dilution that remains positive

• Paired acute and convalescent sera can demonstrate a rising antibody response

• IgM often appears earlier; IgG becomes prominent later and may persist

• Timing of a sample collection is central to interpretation

Pattern of infection and immunity in untreated typhoid fever patients

• Body temperature indicates acute disease progression over time

• Antibody titer is shown as the reciprocal of the highest serial dilution causing agglutination of Salmonella enterica (typhi)

• Presence of bacteria in blood, feces, and urine determined from cultures

• Bacteria clear from the blood as the antibody titer rises, whereas clearance from feces and urine requires more time

• Body temperature drops to normal as the antibody titer rises

Antigen- & Antibody-Based Diagnostic Assays - 1

Core idea: Specific antigen-antibody binding is converted into a visible or measurable signal

Antigen- & Antibody-Based Diagnostic Assays - 2

Antigen- & Antibody-Based Diagnostic Assays - 3

Antigen- & Antibody-Based Diagnostic Assays - 4

Nucleic-Acid-Based Diagnosis: Polymerase chain reaction (PCR)

• PCR selectively amplifies a defined DNA target through repeated thermal cycles

• Primers provide sequence specificity

• Each cycle can approximately double to target, creating exponential amplification

• RNA targets can be detected after reverse transcription to DNA

• PCR can detect organisms that are slow-growing, nonculturable, or present at low abundance

Adv: Speed and sensitivity

Disadv: Contamination risk, dependence on known target sequences, and the possibility of detecting DNA from nonviable organisms

Real-Time PCR (qPCR): Detect While You Amplify

• Fluorescence is measured during each amplification cycle

• The cycle at which fluorescence crosses a threshold reflects starting target abundance

• (a) Earlier threshold crossing generally indicated more starting template

• qPCR reduces post-amplification handling and can be highly sensitive

• Multiplex formats can detect several targets in the same reaction

Choosing the Right Diagnostic Tool

No single method is best for every infectious disease (for example: PCR for rapid detection plus culture for antimicrobial susceptibility testing).

Best practice: Match the test to the biological question, specimen, disease stage, and clinical decision