Fungi and parasites

What is mycology

Mycology

-          The study of fungi

-          Traditional identification through phenotypic analysis is becoming more difficult

o   Culturing fungi poses a major health risk for laboratorians

-          Fungi cause a broad spectrum of disorders: infection of skin, hair, nails, and/or mucosal membranes, allergic diseases, and life-threating invasive diseases

-          Sequencing and real-time PCR have allowed for higher sensitivity, specificity, and the rapid detection and typing of fungi

Describe yeast and mold typing

Yeast and mold typing

-          Fungi can be typed by using amplicon-based sequencing

-          Perform PCR first, targeting a specific region (generate amplicons)

-          Then do sequencing of the amplicons to determine types

-          Common targets for mold typing = ITS regions or 28S rRNA

o   Targets are usually available as “multicopy targets” and enhance test performance and sensitivity

What are clinically relevant fungi

Clinically relevant fungi:

-          Candida, aspergillus, histoplasma, Rhizopus, blastomyces, cryptococcus

What are the characteristics and targets of Candida species 

Candida species

-          Invasive candidiasis is currently diagnosed by blood culture – slow turnaround time and poor sensitivity give the need for a better method

o   Some candida species will grow in 1 day while others take 3+ days

-          Can affect the oral cavity, vagina, penis, or other parts of the body

-          Panfungal PCR assays look for 5 different Candida species: C. albicans, C. parapsilosis, C. tropicalis, C. glabrata, and C. krusei

-          Its important to know which Candida species is present because treatment can differ between each of them

What are the characteristics and targets of Aspergillus fumigatus 

Aspergillus fumigatus

-          Aspergillus naturally exists in nature found in air, soil, and organic matter

o   Common both indoor and outdoor

-          We normally inhale the spore form of the fungi with no consequences

-          Immunocompromised patients are the individuals that suffer from the inhalation because their immune system cannot control growth of the mold

-          Infection can range from allergic sinusitis to invasive pulmonary aspergillosis

-          Aspergillus fumigatus is the cause of 90% of aspergillosis

What are the characteristics and targets of Histoplasmosis

Histoplasmosis

-          An infection caused by the fungus Histoplasma capsulatum

-          Histoplasma lives in the soil, particularly in areas with a high amount of bird and bat dropping (also humid environments)

-          People breathe in spores from the air which can cause fever, cough, fatigue, chills, headache, and body aches

o   Incubation is typically 3-17 days before symptoms start

-          If infection persists, the infection can spread from the lunds to other organs

For fungi, name the organisms that are common targets for molecular-based laboratory tests, and describe basic characteristics such as pathophysiology of infection

Fungi – found

Molecular tests commonly detect:

·       Histoplasma capsulatum

·       Blastomyces

·       Coccidioides immitis

·       Cryptococcus neoformans

·       Aspergillus spp.

·       Candida spp.

·       Rhizopus and other zygomycetes

·       Pneumocystis carinii (now P. jirovecii)

Characteristics mentioned:

·       Cause significant disease especially in immunocompromised hosts

·       Often require culture or staining

·       Some are dimorphic fungi

·       Thick cell walls hinder molecular lysis

For parasite, name the organisms that are common targets for molecular-based laboratory tests, and describe basic characteristics such as pathophysiology of infection

Parasites found

Organisms detected by NAAT include:

·       Trypanosomes

·       Plasmodia (malaria)

·       Toxoplasma

·       Entamoeba histolytica

·       Giardia lamblia / intestinalis

·       Cryptosporidium parvum / spp.

·       Babesia

·       Trichomonas vaginalis

·       Microsporidia (e.g., Encephalitozoon spp.)

Basic characteristics noted:

·       Protozoa often survive harsh conditions

·       Detection complicated by low organism load

·       Stool matrices inhibit PCR

·       Oocysts and spores resist lysis

Describe molecular fungal testing

Molecular fungal testing

-          Labs will typically use samples from affected areas: body fluids, tissues, swabs of the mouth or various body parts, and even nails and skin

-          “panfungal” tests are multiplex fungal assays/fungal panels

-          PCR tests can be presence/absence or quantitative

-          Amplicons of the ITS or 28S rRNA regions can be analyzed by sequencing, restriction enzyme analysis, or sequence-specific PCR

What is parasitology

Parasitology

-          The study of parasites

-          Parasites are typically identified by direct observations of various morphological stages of the parasite in clinical specimens

o   Subject to false-negatives in cases where there are low concentrations of parasite present

o   Also requires a trained eye to identify specimens correctly

-          Parasites are not a major cause of infection in the US, unless obtained while traveling to parasite-endemic countries

For the parasites discussed in lecture, identify the infectious stage responsible for transmission and is capable of infecting new hosts

Parasite	Infectious Stage	Notes
Giardia lamblia	Cyst	Ingested from contaminated water/food; cysts survive in environment.
Entamoeba histolytica	Cyst	Ingested; trophozoites are not infectious.
Cryptosporidium parvum	Sporulated oocyst	Immediately infectious when excreted; resistant to chlorine.
Trichomonas vaginalis	Trophozoite	Transmitted sexually; does NOT form cysts.
Toxoplasma gondii	Tissue cysts, oocysts, tachyzoites	Oocysts from cat feces; tissue cysts in meat; tachyzoites via congenital transmission.
Plasmodium spp.	Sporozoite	Injected by Anopheles mosquito during blood meal.
Babesia microti	Sporozoite (in tick saliva)	Transmitted by Ixodes tick; resembles malaria.
Trypanosoma brucei	Metacyclic trypomastigote	Transmitted by Tsetse fly.
Trypanosoma cruzi	Metacyclic trypomastigote	Deposited in reduviid bug feces → enters through mucosa/broken skin.
Leishmania spp.	Promastigote	Injected by sandfly.

What is Entamoeba histolytica

Entamoeba histolytica

-          E. histolytica is the causative agent of amoebic dysentery and can penetrate through the gut wall and reach the liver

-          Transmission can occur through contaminated food, water, or hands

o   Both cysts and trophozoites are passed in feces, but trophozoites do not survive outside of the host

-          PCR can detect E. histoltica in stool, abscess aspirates, and tissue biopsies

What is Giardia lamblia

Giardia lamblia

-          A protozoan flagellate that causes diarrhea and other intestinal symptoms

o   Originally named G. duodenalis but later renamed; can also be referred to as G. intestinalis

-          Transmission occurs through contaminated food, water, or by swallowing recreational water

-          Giardia cysts can be ingested and once at the small intestines, each cyst releases two trophozoites

-          Cysts shed in feces can survive several months in cold water or soil

-          There are different types of Giardia, with types A and B occuring in humans

What is cryptosporidium species

Cryptosporidium species

-          Cryptosporidium is a protozoan parasite that has emerged as a cause of diarrheal illness worldwide

o   Outbreaks during 2009-20017 in the US were linked to pools, cattle, and childcare settings

-          Transmission is fecal-oral by ingestion of oocysts containing infectious sporozoites

-          C. parvum and C. hominis account for most human cases

-          Species specific primers/probes can be used to detect Cryptosporidium species

-          PCR has resulted in a 22-fold increase in the detection of Cryptosporidium and Giardia compared to conventional microscopy

What is cryptosporidium infections

Cryptosporidium infection

-          Sporozoites parasitize the epithelial cells of the GI tract

-          The parasites undergo asexual and sexual multiplication

-          Asexual multiplication results in oocysts

o   Thin-walled oocysts can auto-infect the patient

o   Thick-walled oocysts can be shed in feces

Describe complications that are encountered when isolating nucleic acids from protozoan parasites

·       Oocysts/spores have tough walls, resistant to lysis

·       Stool contains PCR inhibitors

·       Low organism load causes false negatives

·       Complex sample matrices require modified extraction methods

Describe protozoan parasite DNA isolation

 

Protozoan parasite DNA isolation

-          Nucleic acid isolation from oocysts and spores of protozoan parasites is complicated

-          The fecal matrix presents its own difficulty with potential inhibitors; you must be able to isolate nucleic acids from all parasitic stages present in the sample

-          Preserving clinical specimens in formalin may inhibit amplification

-          Cary blair media has been an acceptable storage media for transport

What are advantages of molecular tests for detection of intestinal parasites

Advantages of molecular tests for detection of intestinal parasites

-          Multiplex real-time PCR for the detection of several different parasites at one time

-          MDx outperforms microscopy in multiple areas, including diagnostic sensitivity and standardizability

-          Lower DNA load found in microscopy-negative samples (more sensitive)

What is a GI syndromic panel

GI syndromic panel

-          When do you use a syndromic GI panel?

o   Use it when there are individuals at high risk of spreading disease or during an outbreak

o   Use it on individuals presenting with symptoms of:

§  Dysentery

§  Diarrhea with fever, severe abdominal cramps, or signs of sepsis

§  Moderate to severe disease

§  Symptoms lasting more than 7 days

§  Immunocompromised patients with diarrhea