Specimen Collection and Diagnostic Accuracy

• Clinical Microbiology is taught at Ain Shams University, Faculty of Medicine, clinical pathology department, microbiology unit.

Diagnostic Errors

• Most diagnostic errors occur in the preanalytical phase, accounting for 68% of errors.
• Analytical errors account for 19% of errors.
• Post-analytical errors account for 13% of errors.
• Proper specimen collection in microbiology forms is the backbone of investigation procedures.

Biological Causes of Errors

• Errors can result due to specimens that naturally contain normal flora.
• Errors can result due to colonization of a sample.
• Colonization is a process by which sites normally inhabited by bacteria of the normal body flora have those bacteria but are not invading or causing damage. Sites may or may not be sterile.
• Infection occurs when organisms gain access to susceptible hosts and there is evidence of tissue invasion or damage.
• Opportunistic infections are caused by organisms causing infection of the host's defense mechanism is malfunctioning, and has low pathogenicity

Laboratory Diagnosis of Infectious Diseases - Direct and Indirect Methods

• Diagnostic methods can be either direct or indirect.
• Direct methods involve macroscopic and microscopic examination
• Macroscopic examination involves inspection of the specimen's gross morphology.
• Microscopic examination includes wet film examination and stained smear examination.
• Wet film: A part of the specimen is taken on a slide and examined directly, for detection of pus cells, red blood cells, fungi, and motile organisms like Trichomonas vaginalis and amoeba

Stained Smear and Gram's Stain

• Gram's stain is useful in identifying organisms as gram-positive, such as Staphylococci and Streptococci, or gram-negative, such as Neisseria, E. coli, and Klebsiella.
• Gram's stain is important for detecting the presence of organisms and differentiating according to gram reaction and morphology.
• Diagnostic accuracy is possible with Gram stains for meningitis (gram-negative diplococci intra and extracellular), throat swabs (Vincent angina), urethritis (gram-negative diplococci intra and extracellular), and vaginitis (Candida or anaerobic vaginosis).
• Gram's stain supports the diagnosis of suspected gas gangrene or tetanus cases and to check the quality of the specimen like sputum and wound swabs.

Ziehl Neelsen and Leishman Stains

• Ziehl Neelsen stain (ZN) is useful in detecting acid-fast bacilli, such as Mycobacteria, in clinical specimens.
• Leishman stain defines the type of cells in a smear.
• Leishman stain differentiates cells and identifies infections

Culture and Antimicrobial Susceptibility

• Cultivation is performed on conventional and/or selective culture media and incubation is performed for 24-48 hours or more aerobically.
• Facultative anaerobes will grow in the presence or absence of O2.
• Strictly aerobic organisms will grow only in the presence of O2.
• Anaerobic organisms cannot grow except in the absence of O2.
• Microaerophilic bacteria (Campylobacter) can grow only in an atmosphere with reduced O2 and increased CO2.
• Anaerobic cultures should be requested specifically when required.
• Identification of isolated organisms and determination of antimicrobial susceptibility of pathogenic organisms are performed on, blood, CSF, bile, fluids, throat swabs, skin swabs, wound swabs, ear swabs, nasal swabs, sputum sample, and faeces

Antigen and Nucleic Acid Detection

• Antigen detection is a rapid method for identifying organisms in a specimen Through surface antigens and soluble antigens produced by the organism in blood
• Nucleic Acid Detection(Molecular Diagnosis) is the use of molecular techniques in micro labs including nucleic acid probes, amplification systems e.g Polymerase chain reaction (PCR) for:
  • Rapid detection of microorganisms that non-cultivable on artificial culture media, e.g chlamydia and viruses.
  • Slow grower organisms e.g Mycobacterium tuberculosis.
  • Determination of genes responsible for antimicrobial resistance.
  • Bacterial typing in epidemiological investigations.

Indirect Methods using Serum

• Serum antibody detection is used for organisms non-cultivable on artificial culture media or difficult to be cultivated (e.g., Mycoplasma) and parasitic infections.
• Paired serum samples should be obtained from the patient, with the first collected during the acute stage and the second 10-14 days after the initial exposure to detect a 4-fold rise in antibodies.

Antibiotics

• Antibiotics is a type of chemical that is produced by microorganisms to kill or inhibit the growth of other organisms.
• Clinical Use of Antibiotics:
  • Prophylactic: Antibiotics used to prevent infection
  • Therapeutic-Empiric: Used when organism is unknown by syndrome known
  • Definitive: Using after Culture results

Antibiotic Combinations

• Antibiotic combinations can result in:
  • Synergism: Combination has a greater effect than the sum of the two individual drug effects.
  • Antagonism: Combination has less activity than that of an individual drug.
  • Indifference: The combined action equals the action of the effective one only.
• Antibiotic combinations are indicated for:
  • Mixed or severe infections, for a patient with E. faecalis endocarditis receiving penicillin + gentamycin.
  • Prevention or delay of development of drug resistance.
  • Empirical treatment for high-risk patients with serious infection.
• Failure of Antimicrobial Therapy factors
  • Improper drug choice, dose, route, timing, and duration.
  • Natural barriers (e.g., prostatic barrier in chronic prostatitis).
  • Antagonistic drug combinations.
  • Inadequate surgical drainage of abscesses.
  • Development of antimicrobial resistance.

Emerging Antimicrobial Resistance

• "ESKAPE" pathogens cause the majority of hospital-acquired infections that escape the effects of antibacterial drugs.
  • Enterococcus faecium
  • Staphylococcus aureus
  • Klebsiella pneumoniae
  • Acinetobacter baumanii
  • Pseudomonas aeruginos
  • Enterobacter species.

Gram-Positive Cocci and Gram-Negative Bacilli

• Gram positive cocci:
  • Vancomycin-resistant Enterococci (VRE) Vancomycin is a drug reserved as last resort for treating life-threatening infections caused by gram-positive organisms that are resistant to all beta-lactam drugs.
  • Methicillin-resistant Staphylococcus aureus (MRSA)=resistant to methicillin as well as all other beta-lactam drugs. Infections caused by these strains are generally treated with vancomycin.
• Gram negative bacilli:
  • Extended spectrum beta-lactmases (ESBLs) are enzymes that give resistance. This is common mainly among Enterobacteriaceae.
  • Carbapenamases Resistance to most types of antibiotics.

Infection Control and Prevention (HAI)

• Health care-associated infection: An infection acquired during hospitalization and which was not present or incubating at the time of admission.
• Types of infections:
  • Bloodstream infection (BSI)
  • Surgical site infection (SSI)
  • Hospital-acquired UTI
  • Hospital-acquired pneumonia
  • Catheter-related infections.
• Sources of infection:
  • Autogenous (self-infection from the patient's own flora).
  • Cross-infection via hands --Contamination occurs upon contact with
• Microbial Flora of the Hand:
  • Resident flora: skin antiseptic solutions (e.g., Diphtheroids, Coagulase-negative Staphylococci).
  • Transient flora: Organisms which colonizes the superficial layers of the skin and are often acquired by healthcare contact.

The effect of infections:

• Prolonged hospitalization.
• Increased hospital morbidity and mortality.
• Elevated healthcare costs.
• Emergence of resistant strains and antibiotic resistance.

Precautions to prevent transmission of infectious agents

• Standard precautions designed to protect and prevent infectious agents from patients, staff and visistors.
• Used hand hygiene.
• Use of personal protective equipment
• Respiratory hygiene/cough etiquette.
• Safe injection practices.
• Safe handling of potentially contaminated equipment or surfaces.
• Transmission based isolation precautions: -Contact isolation = gloves + Gown -Droplet isolation= Mask room Separate rooms
  • Air-borne isolation= N95 mask and Private room with negative pressure

Meningitis specifics

• 30 year old has fever and neck stiffness with altered blood glucose
• Caused from: Coliforms, S. agalactiae , Listeria monocytogenes, Viral: Herpes simplex virus 2.
• Sample for blood culture from lumbar puncture. Collected using aspetic means
• Subject to microbiological exam, WBC count and chemical exam.
• All done aseptically using clear tubing for collection and transportation
• Rapid diagnostic tests include the N species detection tests and molecular diagnostics.

Body fluid effusion

• Abnormal collection of fluid in the pleural space, pericardial sac, abdominal cavity (ascites) or intra-articular joints is abnormal can be caused from inflammatory or malignant diseases

A Sore throat

• Sore throats can be caused by bacteria, viruses, and fungal infections.
• Swabs of throat can determine the cause of acute streptococcal pharyngitis

Group A beta-hemolytic sequelae

• Diagnostic markers for strep can indicate an issue with heart conditions in the future

Pneumonia infections

• Community acquired pneumonia.Typical pathogens for bacterial or viral pneumonia in children and from chronic bronchitis
• Causative pathogens: S pneumoniae, Haemophilus influenza, S. aureus ,Moraxella catarrhalis. Bacterial,Viral,Mycoplasma and fungal detection

Tuberculosis

• Tuberculosis is caused my Mycobacterium
• Pulmoary infection will require sucessive sputum samples or bronchoaveolar samples
• Samples are detected using stain with ZN and detection with acid-fast bacili
• TB detection happens on solid media, fluid media and molecular diagnosies.

Blood stream

• the presence of bacteremia or fungemia causes SIRS
• Blood will then need to be cultured and detected.
• Blood culture is used when there are suspicions of infective endocarditis or the possibility of early meningitis and pneumonia.

Wounds

• Absesses can be caused by surgery and trauma and interrupts the skin surface
• Specimens are looked at using a stained film to detect bacteria such as E. colli.

Anaerboic detection

• Most are classified as non-spore and can appear on a variety of infections and wounds
• Detection happens with Gram Stains such as those in Clostridium infections
• Diagnostic methods focus on testing for A& B toxins