Introduction to Parasitology

Introduction to Parasitology

• MICR5829, Christopher Peacock, Christopher.peacock@uwa.edu.au

Lecture Objectives

• Understand parasitism concepts and terminology.
• Recognize parasite diversity and life cycles.
• Define specific parasitology terms.
• Know diagnostic tests for identifying infections.
• Understand treatment-related issues.

What are Parasites?

• Organisms acquiring basic nutritional needs through close contact with another living organism.
• Eukaryotic organisms:
  • Protozoa: Unicellular (e.g., Plasmodium).
  • Metazoan: Multicellular (e.g., helminths).
• Endoparasite: Lives inside another organism.
• Ectoparasite: Lives on an organism's external surface.

Why Study Parasites?

• Major causes of human and animal diseases.
  • Many human pathogens.
  • Prevalent in tropical or less-developed countries, dangerous to immunocompromised individuals.
• Eukaryotic nature makes them difficult to treat due to distant relation to humans.

Health Burden

• Table outlining diseases caused by protozoa and helminths, their endemicity, infections (in millions), mode of transmission, site of parasitism, duration of infection (years), and immunopathology.
  • Protozoa
    • Malaria (Plasmodium falciparum, P. vivax, P. ovale, P. malariae): Worldwide in tropics and subtropics, 300 million infections via Anopheles mosquito bite, parasitizes erythrocytes, infection lasts 1-2 years, immunopathology (+).
    • Visceral Leishmaniasis (Leishmania donovani): India, China, Africa, <1 million infections via sand fly bite, parasitizes macrophages, lifelong infection, immunopathology (+).
    • Cutaneous Leishmaniasis (Leishmania major, others): Worldwide in tropics and subtropics, 12 million infections via sand fly bite, parasitizes macrophages, lifelong infection, immunopathology (-/+).
    • African Trypanosomiasis (sleeping sickness) (Trypanosoma brucei): Sub-Saharan Africa, <1 million infections via tsetse fly bite, parasitizes bloodstream, infection lasts months, immunopathology (++).
    • South American Trypanosomiasis (Chagas' disease) (Trypanosoma cruzi): Latin America, 20 million infections via Reduviid bug bite, parasitizes blood and muscle, lifelong infection, immunopathology (++).
    • Toxoplasmosis (Toxoplasma gondii): Worldwide, >100 million infections via infective stages in cat feces and undercooked meat, parasitizes many cell types, lifelong infection.
  • Helminths
    • Ascariasis (roundworms) (Ascaris lumbricoides): Worldwide in tropics and subtropics, 1,000 million infections via infective eggs in fecally contaminated soil, parasitizes small intestine, infection lasts 1-2 years, immunopathology (-/+).
    • Hookworm (Ancylostoma duodenale, Necator americanum): Worldwide in tropics, 900 million infections via infective larvae in fecally contaminated soil, parasitizes small intestine, infection lasts 2-3 years, immunopathology (-/+).
    • Trichuriasis (whipworm) (Trichuris trichiura): Worldwide in tropics and subtropics, 500 million infections via infective eggs in fecally contaminated soil, parasitizes colon and cecum, infection lasts 5 years, immunopathology (+).
    • Lymphatic filariasis (Wuchereria bancrofti): Worldwide in tropics and subtropics, >100 million infections via mosquito bite, parasitizes lymphatics, infection lasts >10 years, immunopathology (++).
    • Onchocerciasis (river blindness) (Onchocerca volvulus): Africa, Central and South America, 20 million infections via blackfly bite, parasitizes subcutaneous tissues, infection lasts >10 years, immunopathology (++).
    • Schistosomiasis (Schistosoma mansoni, S. japonicum, S. haematobium): Africa, Arabia, South America, East and Southeast Asia, >200 million infections via infective cercariae from freshwater snails, parasitizes mesenteric veins and vesical plexus veins, infection lasts >10 years, immunopathology (++).
    • Cysticercosis (tissue) (Taenia solium (pig tapeworm)): Worldwide, infection via infected pork, parasitizes subcutaneous tissues and brain, lifelong infection, immunopathology (+).

Examples of Medical Importance

• Trichomoniasis: Commonest STD worldwide.
• Malnutrition and Parasites: ~15 million children die annually due to this combination.
• Malaria: ~350,000 African children die each year from malaria's effects.
• Cryptosporidia: The largest waterborne disease outbreak in the USA occurred in 1993 in Milwaukee, with 403,000 cases.

Opportunistic Pathogens

• Organisms generally non-pathogenic or reoccurring from a quiescent stage.
• The AIDS epidemic highlights that pathogenicity depends on the host.
• Harmless organisms to immunocompetent individuals can be lethal to immunocompromised individuals.
• Immunosuppression causes:
  • Other infections (AIDS).
  • Cancer.
  • Drug or radiotherapy (organ transplant or cancer treatment).
  • Age or pregnancy.
• Manifested through reactivation of latent/silent infections.

Parasite Life Cycles

• Parasites pass through developmental stages to complete life cycles.
  • Direct: One host in the life cycle.
  • Indirect: Two or more hosts required.
• Host Types:
  • Definitive/Primary Host: Parasite reaches maturity and undergoes sexual reproduction.
  • Reservoir Host: Harbors pathogen, often with minimal effect.
  • Secondary/Intermediate Host: Parasite undergoes asexual reproduction.
• Reproduction:
  • Both asexual and sexual reproduction occur (sexual normally in definitive host).

Transmission

• Direct: Passed from one infected host to another via physical means or direct invasion.
• Food or waterborne.
• Via an intermediate host or vector:
  • Very common among parasites.
  • Definitive host: Site where sexual stages of life cycle take place. May or may not be human.
  • Mechanical or essential life-cycle stages occur.
  • Distinction between intermediate host and vector is arbitrary, depends on size, speed, and active involvement (e.g., pig vs. mosquito).

Parasites and Health

• Helminth infections' association with protection against asthma and atopy.
  • 1913 Herrick noted eosinophil increase in asthma and roundworm infestation.
  • 1986 Godfrey's hygiene hypothesis in the Lancet revisited this.
• Developed nations show high asthma/allergy rates, low helminth infection rates, and vice versa in developing countries.
• Successful helminth therapy for asthma.
• Lack of helminth exposure in the developed world has increased the incidence of IBD, Crohn’s, ulcerative colitis, and other autoimmune-like disorders.
• IBD is more common in people with high socioeconomic status.
• Use of Trichuris suis and Necator americanus improves IBD.
• Search for non-live worm antigens to treat these diseases.
• Review – Elliot DE, Weinstock JV. Curr Opin Gastroenterol 2012 nov 28 Helminth infections and IBD (Crohn’s and UC)

Blame an Animal – or another human?

• Zoonosis: Disease transmitted to humans from animals (e.g., East African sleeping sickness). Most pathogens are zoonotic.
• Anthroponosis: Disease spread from humans to humans (e.g., smallpox, malaria). Usually implies a pathogen normally limited to infecting humans. Excludes rare/unusual cases.

Taxonomic Context

• Five Kingdom System (phenetic, based on phenotype):
  • Plantae, Animalia, Fungi, Protista, Monera.
  • Kingdom Protista served as a “dumping ground” for most single-celled eukaryotic microbes.
• The Domain System (phylogenetic) is superseding the Kingdom System, especially for the classification of microorganisms.
• Eukaryotic organisms form one domain (Eukarya), with Protista spread among supergroups.
• No current evidence suggests the order of divergence from the eukaryotic line.
• Probably occurred at least 1 billion years ago.

Taxonomic Information

• The NLM website (http://www.ncbi.nlm.nih.gov/Taxonomy) is useful for discovering these arcane terms
• Plasmodium falciparum:
  • Eukaryota
  • Alveolata
  • Apicomplexa
  • Aconoidasida
  • Haemosporida
  • Plasmodium
  • Plasmodium (Laverania)
  • Plasmodium falciparum

Human Parasitic Disease - Overview

• Protozoa
  • Unicellular and small organisms (2um up to 80ums).
  • ~25 are common infections in humans, 50 less common
  • Cause of major mortality events (malaria, leishmaniasis, Chagas disease, cryptosporidiosis, amoebiasis).
  • Often more severe in children
• Helminths
  • Multicellular, 15um up to several meters.
  • 30 are common with about 250 rare infections.
  • Very common in rural and tropical regions, less so in developed countries.
  • Often cause chronic debilitating diseases

Protozoa Parasites

• Can be intracellular/extracellular parasites.
• Transmission: vectors (malaria, leishmaniasis, Chagas disease), food/water (Giardiasis, amoebiasis, toxoplasmosis), or directly (trichomoniasis).
• Often difficult to diagnose directly.
• Contain organelles common to all eukaryotes (nuclei, cell membrane, ER, mitochondria, Golgi bodies, lysosomes, food vacuoles).

Protozoan parasites of humans

Map of the human body and location of protozoan parasites. Listing:

• Brain:
  • P. falciparum
  • T. cruzi
  • T.brucei sub-gp
• Lymph nodes: T. gambiense.
• Bone marrow: L. donovani
• Heart muscle: T. cruzi
• Spleen:
  • L. donovani
  • P. falciparum
• Large intestine
  • B. coli
  • E. histolytica
  • G. lamblia
• Skin:
  • L. major, L. mexicana & L tropica
• Blood:
  • Species of Trypanosoma and Plasmodium
• Liver
  • E. histolytica
  • L donovani
  • P. falciparum
• Lungs
  • E. histolytica
• Oro-nasal mucous membrane
  • L brasiliensis

Protozoa Classification by Locomotion

• With pseudopodia – Amoeboids
  • Entamoeba histolytica, Naegleria fowleri
• With flagella – Flagellates
  • Giardia lamblia, Trichomonas vaginalis
• With cilia – Ciliates
  • Balantidium coli
• Immobile stage – Sporozoans
  • Plasmodium, Cryptosporidia

Helminth Parasites

• General Categories by Phylum: Metazoa

Helminths Classification

• Nematodes (Roundworms)
  • Round in cross-section, have body cavities, a straight alimentary canal, and an anus.
  • Examples: Ascaris (roundworm), Trichuris (whipworm), Ancylostoma (hookworm), Necator (hookworm), Enterobius (pinworm or threadworm), Filarial worms
• Platyhelminthes (Flatworms)
  • Dorsoventrally flattened, no body cavity, and if present, the alimentary canal is blind-ending.
  • Cestodes (Tapeworms)
    • Adult tapeworms are found in the intestine of their host. Have a head (scolex) with sucking organs and a segmented body but no alimentary canal. Each body segment is hermaphrodite.
    • Examples: Taenia (tapeworm), Echinococcus
  • Trematodes
    • Non-segmented, usually leaf-shaped, with two suckers but no distinct head. They have an alimentary canal and are usually hermaphrodite and leaf-shaped. Schistosomes are the exception; they are thread-like and have separate sexes.
    • Examples: Fasciolopsis (liver fluke), Schistosoma (not leaf-shaped!), Paragonimus

Helminths - Health Burden

Global Prevalence and Distribution of Helminth Infections

Ectoparasites

• Insects and arachnids that feed on human skin or tissues.
• Impact on human disease:
  • Transmit other diseases.
  • Cause skin irritation.
  • Cause allergic reaction.
  • Bites or to faeces.

Ectoparasites examples

• Mostly due to ticks and fleas
• Myiasis: Bot fly, Tumbu fly

Parasites - Direct Transmission

• Trichomonas vaginalis – very common, commonest STI
• Naegleria fowleri – very uncommon
• Hookworms (Anclyostoma and Necator)
• Strongyloides stercoralis
• Few pathogens in humans are spread by direct contact, however, there are several important infections spread this way

Parasites – Food and Waterborne

• Includes a majority of parasitic infections
• Very effective mechanisms of transmission
• Often leads to outbreaks of infection
• Most of the global parasites are transmitted this way – Giardia, toxoplasma, amoebiasis, pin worm, tapeworms.
• Higher prevalence in tropical countries due to contaminated food and water
• Parasites have environmentally resistant stage
• Most inhabit the gut

Parasites – Food or Water Borne

• Enteric protozoa – Entamoeba histolytica, Cryptosporidium spp, Giardia intestinalis – very common
• Enteric helminths – most tapeworms and some flukes, Strongyloides, Trichuris and Enterobius– common
• Systemic parasites – Toxoplasma, Trichinella, liver flukes, Taenia solium
• Majority of intestinal parasites and some invasive parasites.
• Potential for large outbreaks to occur
• Tend to have global occurrence though more prevalent in the tropics
• Prevention is often down to hygiene and cultural differences

Water Outbreak Examples

• Sydney Water Scare, 1998: Parasites in the city's supply led to warnings for 1.5 million residents to boil tap water.
• Giardiasis Tests in Scranton, 1984: Families warned about hygiene and illness signs due to serious stomach infection outbreak.
• Milwaukee Outbreak: Massive cryptosporidiosis outbreak after spring thaw affected >400,000 people due to high turbidity in treated water and oocysts identified in ice.
• Upper-Hill School amoebiasis outbreak

Vector-Borne Parasitic Diseases

Restrictions placed on vector-borne diseases

• Parasite that depend on a vector for part of the life cycle will be restricted to the range of the vector.
• The parasite will have to adapt to at least two very different environments (37/25^oC, gut/other sites), usually requires significant morphological change.
• Parasite will have to adapt to vector behaviour or change vector behaviour.
• Most feed on blood (haematophagous) – some obligate, others only females
• Benefits: Have a good delivery system, rely on vector for source of energy, in many vector was original host.

World's Deadliest Animals (by number of people killed per year)

• Mosquito: 725,000
• Human: 475,000
• Snake: 50,000
• Dog (Rabies): 25,000
• Tsetse Fly (Sleeping Sickness): 10,000
• Assassin Bug (Chagas Disease): 10,000
• Freshwater Snails (Schistosomiasis): 10,000
• Ascaris Roundworm: 2,500
• Tapeworm: 2,000
• Crocodile: 1,000
• Hippopotamus: 500
• Elephant: 100
• Lion: 100
• Wolf: 10
• Shark: 10

Parasites - Diagnosis

• What tests can be used?
  1. Fecal (stool examination) – For ova, cysts, trophozoites, adults – intestinal parasites
  2. Urine sampling – ova
  3. CSF – protozoan parasites
  4. Blood tests
     • Blood smear – direct observation of parasites
     • Serology – look for antibodies or antigens
  5. Invasive aspirates or biopsy – risks involved, specialist medical staff needed
  6. MRI imaging, X-ray, CAT scan
  7. Post mortem – tissue parasites
  8. PCR based tests
• Gold Standard in diagnosis - See the parasite!!

Direct Microscopy examples

• Visualization of parasite - cysts

Parasites - Diagnosis

• Fecal or urine examination
  1. Fecal stool examination directly for enteric protozoan trophozoites (needs to be fresh), cysts, trophozoites, adults – intestinal parasites
  2. Can also use PCR, multiplex or real time quantitative PCR or serological antigen detection for evidence of parasites
  3. Some rapid chromotographic tests available
  4. Can use culture on fecal material
• Entamoeba histolytica cyst and trophozoite
• Coccidian parasites such as Cryptosporidium spp and Isospora belli
• Schistosome eggs from feces and urine

Parasites - Diagnosis

• Blood collection
  1. Thin and thick film examination directly for parasites like Plasmodium, trypanosomes and filarial worms. Can also use PCR, multiplex or real time quantitative PCR or
  2. Can use serological antigen/antibody detection for evidence of parasites not directly detectable
  3. Can use rapid chromatographic tests for malaria
  4. Can use culture on blood for parasites that are hard to find
• Plamodium falciparum in blood
• Trypanosoma gambiensie (African trypanosomiasis)
• Lymphatic filiriasis – microfilaria in the blood

Importance of Serological Diagnosis

• Diagnosis of systemic parasites – Trypanosoma cruzi – Chagas disease
• Advantages – do not need presence of parasite, easy to do, sensitive, can be used to monitor progress of treatment
• Disadvantages – not good for acute infection, may not differentiate past and present infections, can get cross reactivity and incorrect result

Parasites - Diagnosis

• Tissue biopsies, aspirates or PM
  1. Tissue biopsies can be used to identify parasites that invade the body. Provides information on the damage done to the host but can have low sensitivity in detecting some parasites
  2. Can use PCR or immunocytochemistry on tissue sections
  3. Is invasive but can be used to detect systemic parasites
• Cutaneous leishmaniasis
• Onchocerca volvulus in lymph node
• Primary amoebic encephalitis – Naegleria fowlerii

Issues Related to Diagnosis of Neglected Diseases

• Giardia, Cryptosporidium, Entamoeba, Cyclospora, Dientamoeba detection methods
  • Microscopy: Low cost, point-of-care (POC) detection
  • Immunodetection: Antibody or parasite detection
  • DNA detection: Enteric panel detection

Infections in the Field (Concept of Operation)

Flow chart including:

• Patent blood work
• Positive field test
• Transport sample to Remote Army Medical Lab or Naval Hospital Ship
• Patent medical history to outside experts (Comm Link)
• Blood from finger to confirm result
• Both tests are used to estimate parasite density (severity of infection)
• Confirmatory Microscopic Analysis, PCR Analysis, MAGIChip
• Malaria species ID and drug sensitivity
• Malaria strain and therapeutics to field

Cell Free DNA - Extending Use of PCR

• Possible mechanisms for release of parasite cfDNA into different fluid compartments of the host:
  • Passive release: parasite degradation, cellular apoptosis/necrosis
  • Active secretion: direct release from parasite or through excretory secretory products
• Possible variations in diagnostic accuracy of direct parasitological methods and NAATS in relation to the presence of intact parasite stages and parasite cfDNA in clinical samples
  • CSF / Urine
  • Saliva / Sputum
  • Lymph
  • Peritoneal fluid / Amniotic fluid
  • Stool
  • Plasma / Serum
• cfDNA detection techniques (NAATS):
  • conventional PCR
  • nested PCR
  • real-time/quantitative PCR
  • multiplex PCR
  • digital PCR
  • LAMP

PCR: A New Dimension

• Simultaneous detection of leishmania, dog, and human DNA in sand flies informs on outbreaks of leishmaniasis.

Parasite Identification using sequencing to target numerous parasites

Using the Universal Parasite Diagnostic to testing animal tissues by:

• Extract DNA
• Host-specific restriction digestions, separating Host DNA and Parasite DNA
• 18S rDNA PCR and Illumina sequencing
• Results for: Trypanosoma cruzi, Tapeworms, Babesia sp., Hepatozoon sp., Dracunculus sp., Filarial nematodes, Ascarids

Multiplex PCR with Fluorescent Probes for Common and Pathogenic Intestinal Parasites

• BD MAX™ Enteric Parasite Panel
• Achieves CE Mark to Detect Common and Pathogenic Intestinal Parasites
• Fully automated molecular platform: pathogen extraction, amplification & detection
• Supports IVD assays & Open System Reagents (OSR)
• Compliant to FDA's Guidance Molecular Diagnostic Instruments with Combined Functions (OSR)
• Variable run size (1 to 24 samples) with multiple assays
• Simplified and Efficient Workflow (less than one minute of hands-on time per sample)

Rapid Diagnostic Tests

• Easy to run, no special technical skills required
• Relatively cheap
• Varying sensitivity and specificity
• Stable in poor environments
• May need confirmation test
• Can use a range of patient samples

Anti-Protozoal Drugs

• Malaria:
  • Quinine/chloroquine – 1638 or 1820/1934
  • Hydroxychloroquine - 1955
  • Mefloquine – 1984
  • Atovaquone/proguanil – 1961
  • Doxycycline, Clindamycin, Tetracycline –
  • Primaquine - 1946
  • Pyrimethamine/sulfadoxine 1953
  • Artesunate derivatives -1977
• Intestinal parasites:
  • Metranidazole/tinidazole – 1959
  • Iodoquinol – 1953
  • Nifurtimox -1940
• Others:
  • Trimethroprim-sulfamethoxazole - 1932
  • Range of antbiotics like azithromycin, Clindamycin
  • Albendazole
  • Paromomycin
  • Pentavalent antimony

Anti-Protozoal Drugs

• African trypansomiasis (sleeping sickness (HAT))
  • Trypan blue 1906 – turned people blue!
  • Suramin – 1917, used only for the acute stage of T.b. rhodesiensie
  • Pentamidine – 1941 – acute stage T.b. gambiensie
  • Melarsoprol – 1949 until 1990 the only treatment for tertiary stage disease, arsenical, kills 5% of patients (encephalopathy).
  • Eflornithine – 1970, licensed in 1990 for T.b. gambiensie
• Chagas disease - Only 2 drugs neither cures chronic disease, neither drug FDA approved
  • Benznidazole – 1974, treats acute stage only, manages chronic infections, toxicity increases with age of patient
  • Nifurtimox – 1940, acute stage only, manages chronic infections
• Leishmaniasis:
  • Pentavalent antimony – 1929
  • Paromomycin – 1960s, aminoglycoside antibiotic
  • Amphoteracin B – 1960s, severe side effects, liposomal
  • Miltefosine – 1980s, as a anticancer drug, only drug taken orally
• All drugs toxic and hard to administer

Anti Helminthic Drugs

• Benzimidazole family- 1962- triclabendazole, albendazole, mebendazole, thiabendazole, flubendazole – Prevents polymerisation of tubulin in parasite cytoskeleton
• Rafoxanide – Nicolsamide 1960s – Interferes with oxidative phosphorylation in worms
• Praziquantal – 1970 (not licensed for humans in UK) – Poorly understood, increases membrane permeability to ions
• Imidazothiazoles – 1966 –Levamisole, thiazole – Nicotinic acetylcholine receptor agonist – overstimulates parasite muscles leading to paralysis
• Macrocyclic lactones – (1978) Avermectin, Ivermectin, Abamectin – Block electrical activity of nerves leading to influx of Cl ions
• Diethylcarbamazine - 1947 – Inhibits arachidonic acid metabolism in microfilaria

References

• Manson’s Tropical Diseases 21st edition
• Diagnostic Medical Parasitology Lynn Shore Garcia
• Wikipedia
• Animal Planet Videos – Monsters inside me
• Atlas of Medical Protozoology P.I. Chiodini, A. H. Moody
• Atlas of Human Parasitology Lawrence Ash, Thomas Orithel
• Atlas of medical Parasitology Vigar Zamen

Practice Questions

• Which of these parasites is transmitted directly from one person to another
  • A. Leishmania
  • B. Trichomonas vaginalis
  • C. Giardia Intestinalis
  • D. Cryptosporidium
• Which of these diseases is best diagnosed using serology
  • A. Naegleriasis (Primary ameobic encephalitis)
  • B. Malaria
  • C. Giardiasis
  • D. Chronic Chagas disease (Trypanosoma cruzi)
• SAQ. Define what cell free DNA is and how it can be used in diagnosis of parasites