Pathogenesis and Diagnosis of Fungal Infections​-66

What is pathogenicity?

Pathogenicity is the ability of an organism to cause disease (i.e., harm the host). This ability is a genetic trait of the pathogen. The damage done to the host depends on host-pathogen interactions. Commensals and opportunistic pathogens lack this inherent ability to cause disease.

What is virulence?

Virulence refers to the degree of pathology caused by the organism. It is correlated with the ability to replicate in the host and may be affected by other factors.ability of microorganism to cause damage

What is a pathogen?

A pathogen is an agent that causes disease (damage to the host)

What is microbial pathogenesis?

Microbial pathogenesis refers to the biochemical mechanisms whereby microbes (bacteria, fungi, parasites, viruses) cause disease.

How has the demonstration that a pathogen causes disease evolved over time?

• 19th century: Koch’s postulates

• 20th century: Molecular Koch’s postulates (Stanley Falkow, 1988)

• 21st century: Koch’s postulates adapted for genomics, metagenomics, proteomics

What are modern considerations and limitations when demonstrating that a microbe causes disease?

• Microbe is found in diseased tissue but not normal tissue

• Some pathogens (e.g., Staphylococcus aureus, Helicobacter pylori, Candida albicans) may colonize without always causing disease

• Microbe can be isolated from diseased tissue as a pure culture

• Non-culturable organisms (e.g., Treponema pallidum) must be identified by PCR

• Some diseases are caused by multiple organisms (e.g., periodontal disease)

• Microbe can cause disease when inoculated into an animal or human

• Some microbes (e.g., gonococcus) have no animal model

• Microbe must be re-isolated in pure culture from the host

• Disease may require multiple organisms to develop

• Therapeutic or preventative measures should eliminate the disease

What are Molecular Koch’s Postulates, and what do they demonstrate?

• The virulence gene is associated with microbes that cause disease and is absent or inactive in non-virulent strains.

• Disrupting the gene in a virulent strain results in loss of virulence (avirulence).

• Introducing the cloned gene into an avirulent strain confers virulence.

• The gene must be expressed during infection.

• Redundant virulence factors may exist.

• Virulence may depend on the host organism and immune response.

• The gene may not have evolved strictly to cause disease but may provide general fitness advantages in microbial populations.

• Requires microbial culture and genetic tools.

• Developed by Stanley Falkow in 1988.

What are the 21st Century Koch’s Postulates, and what modern tools are used?

• Use of metagenomics (bulk sequencing or microarrays, including 16S rRNA or random sequencing) to define new species, genes, or pathways

• Supported by initiatives like the Human Microbiome Project

• Microarray identification is a key tool

• The nucleic acid sequence of the putative pathogen must be present during disease and at sites of disease

• The nucleic acid sequence should be absent or reduced in healthy controls

• There should be a dose response relationship between nucleic acid quantity and disease severity

What are key virulence factors of opportunistic fungal pathogens?

Candida albicans:

• Yeast to hyphae transition

• Phenotypic switching

• Secreted proteinases and phospholipases

Cryptococcus neoformans:

• Polysaccharide capsule

• Melanin production

Aspergillus spp.:

• Small aerosolized conidia

• Secreted enzymes

• Gliotoxin?

What are the virulence factors of Candida albicans?

Adhesins, biofilm formation, dimorphic switching (yeast to hyphae), secreted hydrolytic enzymes (proteases, lipases, phospholipases), phenotypic switching

How do fungi evade the host immune system?

• Fungi hide by masking antigens

• Some fungi are too large to be phagocytosed

• Fungi avoid lysis by escaping the phagolysosome

• Fungi can germinate and replicate within macrophages

• Fungi interfere with immune activation (e.g., Coccidioides metalloprotease hydrolyzes the immunodominant antigen SOWgp during endospore development)

What is the morphogenesis of Candida albicans within macrophages?

Candida albicans undergoes morphogenesis within macrophages, transitioning from yeast to hyphal forms, which contributes to its escape from the phagocyte and enhances virulence.

What are mycotoxins and what characterizes mycotoxicoses?

Mycotoxins are secondary fungal metabolites. Mycotoxicoses can be acute or chronic. Optimal temperature for mycotoxin biosynthesis is 20°C to 30°C. Their role during active mycotic infections is unclear. Diagnosis is often based on epidemiological evidence.

What are key mycotoxins, their effects, and the fungi that produce trichothecenes?

• Aflatoxin B1: Causes hepatitis and hepatic cancer

• Ochratoxin A: Nephrotoxic

• T-2 toxin (a Trichothecene): Causes tremors, loss of vision, gastrointestinal and respiratory symptoms

What histopathological stains are used in diagnosing fungal infections?

• Routine: H&E (hematoxylin and eosin)

• Special: GMS (Gomori methenamine silver), PAS (Periodic acid-Schiff), Mucicarmine, India Ink

What microbiological methods are used to diagnose fungal infections?

• Direct microscopy of clinical specimens using KOH wet mount, Gram stain, Giemsa stain, and calcofluor white

• Culture and identification of clinical isolates

• Antifungal susceptibility testing

What biochemical methods are used to diagnose fungal infections?

• Detection of fungal metabolites (e.g., D-arabinitol, D-mannitol)

• Identification of cell wall and/or capsular components

• Detection of fungal enzymes

What molecular methods are used to diagnose fungal infections?

• Direct detection using nucleic acid amplification

• Identification of fungi after culture

• Strain typing methods such as karyotyping, RFLP (Restriction Fragment Length Polymorphism), and MLST (Multilocus Sequence Typing)

What immunologic methods are used to diagnose fungal infections?

• Serology: Includes antibody screening and complement fixation to detect patient antibodies

• Fungal antigen detection in CSF or serum, using methods such as latex agglutination, enzyme immunoassays, or lateral flow immunoassays

What is 16S rRNA gene sequencing used for, and what are key features of bacterial 16S rRNA?

16S rRNA gene sequencing is mostly used as a nonculture-based method, such as in microbiome research.
Bacterial 16S ribosomal RNA has a conserved secondary structure with variable regions that allow for species identification.

What are the characteristics of the ITS1, ITS2, and 5.8S rRNA regions in fungal identification?

• ITS1 and ITS2 regions are highly variable, which allows for species-level identification of fungi.

• The 5.8S rRNA gene region is conserved and provides a stable reference point.

In practice:
The variability in ITS1 and ITS2 makes them ideal for distinguishing fungal species, while the conserved 5.8S region helps align and compare sequences across samples. Together, they enable accurate identification and phylogenetic analysis (e.g., building dendrograms).