Introduction to Parasitology & Lab Diagnosis of Parasitic

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Introduction to Parasitology

&

Lab Diagnosis of Parasitic DiseasesDr. Sudheer Kher


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Why study Parasitology?

Many of these parasites are causative agents of majorpublic health problems of the world.Recent estimates of prevalence of parasites in the worldare:

Ascaris 1.5 billion

Hookworms 1.3 billionWhipworms 1 billionFilarial worms 657 millionMalaria 500 millionSchistosomes 270 million

Amebiasis 50 millionTaenia tapeworms 50 millionClonorchis 20 millionChagas’ Disease 15 million

These parasites cause untold suffering and death in theworld today.


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The burden of some major parasitic infectionsParasite Diseases No. people infected Deaths/yr

Plasmodium malaria 273 million 1.12 million

Soil transmitted helminths:

Roundworm ( Ascaris)

Whipworm (Trichuris)

Hookworm ( Ancylostoma and

Necator )

Pnemonitis, intestinal obstruction

Bloody diarrhoea, rectal prolapse

Coughing, wheezing, abdominal pain

and anaemia

2 billion 200,000

Schistosoma Renal tract and intestinal disease 200 million 15,000

Filariae Lymphatic filariasis and

elephantiasis

120 million Not fatal but 40

million disfigured or

incapacitated

Trypanasoma cruzi Chagas disease (cardiovascular) 13 million 14,000

African trypanosomes African sleeping sickness 0.3 – 0.5 million 48,000

Leishamania Cutaneous, mucocutaneous and

visceral leishmaniasis

12 million; 2 million new

cases/yr

50,000


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Key definitions: What is ….?

Medical parasitology: “the study and medical

implications of parasites that infect humans”

A parasite: “a living organism that acquires someof its basic nutritional requirements through its

intimate contact with another living organism”.Parasites may be simple unicellular protozoa orcomplex multicellular metazoa

Eukaryote: a cell with a well-definedchromosome in a membrane-bound nucleus. Allparasitic organisms are eukaryotes


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K d fi iti Wh t i ?


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Key definitions: What is ….?

Host: “the organism in, or on, which the parasite lives and causes

harm”

Definitive host: “the organism in which the adult or sexuallymature stage of the parasite lives”

Intermediate host: “the organism in which the parasite livesduring a period of its development only”

Zoonosis: “a parasitic disease in which an animal is normally thehost - but which also infects man”

Vector: “a living carrier (e.g.an arthropod) that transports apathogenic organism from an infected to a non-infected host”. Atypical example is the female Anopheles mosquito that transmitsmalaria


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Taxonomic classification of protozoa

Sub

kingdom

Phylum Sub-phylum Genus-

examples

Species-

examples

Protozoa Sarcomastig-

ophorafurther divided into

Sarcodina-- - moveby pseudopodia

Entamoeba E. histolytica

Mastigophoramove by flagella

Giardia G. lamblia

Apicomplexano organelle of

locomotion

Plasmodium P. falciparum,

P. vivax,

P. malariae,

P. ovale

Ciliophoramove by cillia

Balantidium B. coli

MicrosporaSpore-forming

Enterocyto-zoa E. bienusi


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Examples of important intestinal protozoaTransmitted by the faecal-oral route and

cause diarrhoea

Giardia lamblia: world-wide distribution,lives in the small intestine and results inmalabsorption

Entamoeba histolytica: may invade thecolon and cause bloody diarrhoea –amoebic dysentery. Also causes ameobicliver abscess.

Cryptosporidium parvum: more prevalentin the immunocompromised

Cyclospora cyatenensis - parasitises thesmall intestinal mucosa and may causediarrhoea for several weeks

Balantidium coli: a large motile ciliatedparasite that lives in the colon of pigs,humans and rodents and can lead tocolonic ulceration

Enterocytozoon bienusi: a microsporidianthat parasitises the small intestine. Alsomore common in theimmunocompromised.


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Examples of important systemic protozoa

Detected in the blood

Plasmodium: the cause of malaria.There are 4 species that infect man: P.falciparum, P. vivax, P. ovale and P.malariae

Toxoplasma gondi: transmitted by theingestion of oocysts from cat faeces.

Infection can lead to ocular problemsand is also a cause of neonataltoxoplasmosis

Leishmania: transmitted by sand flies,can lead to visceral, cutaneous andmucocutaneous leishmaniasis

Trypanosoma: haemoflagellates whichcause

In Africa - sleeping sickness(transmitted by the Tsetse fly)

In South America - Chagasdisease (transmitted by theReduviid bug)

Typical lesion of cutaneous leishmaniasis

Tsetse fly – the vector of African

trypanosomiasis It has a painful bite!


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Taxonomic classification of helminthsSub

kingdom

Phylum Class Genus – examples

Metazoa NematodesRound worms; appear round incross section, they have body

cavities, a straight alimentary

canal and an anus

Ascaris (roundworm)

Trichuris (whipworm)

Ancylostoma (hookworm)

Necator (hookworm)

Enterobius (pinworm or

threadworm)

Strongyloides

PlatyhelminthesFlat worms; dorsoventrally

flattened, no body cavity and, if

present, the alimentary canal is

blind ending

Cestodes Adult tapeworms are found in the

intestine of their host

They have a head (scolex) with

sucking organs, a segmented

body but no alimentary canal

Each body segment is

hermaphrodite

Taenia (tapeworm)

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

Fasciolopsis (liver fluke)

Schistosoma (not leaf

shaped!)

E l f i t t t i t ti l


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Examples of important metazoa – intestinal

nematodes

Trichuris (whipworm)

A soil transmitted helminthprevalent in warm, humid conditions

Can cause diarrhoea, rectal prolapseand anaemia in heavily-infected people

Ancylostoma and Necator (hookworms)

A major cause of anaemia in the tropics

Strongyloidesinhabits the small bowel

infection more severe inimmunospressed people (e.g.HIV/AIDS, malnutrition, intercurrentdisease)

Enterobius (pinworm orthreadworm)

prevalent in cold and temperate climatesbut rare in the tropics

found mainly in children

Ascaris (roundworm)

Found world-wide in conditions of poorhygiene, transmitted by the faecal- oral

route

Adult worms lives in the small intestine

Causes eosinophilia

Heavy intestinal infections may occur with Ascar is .

Adult worms can be several cms long.

Examples of important metazoa


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Examples of important metazoa –

systemic nematodesFilaria including:

Onchocerca volvulus –Transmitted by thesimulium black fly, thismicrofilarial parasite cancause visual impairment,

blindness and severeitching of the skin in thoseinfected

Wuchereria bancrofti – Themajor causative agent oflymphatic filariasis

Brugia malayi – Anothermicrofilarial parasite thatcauses lymphatic filariasis

Toxocara

A world-wide infection of

dogs and cats

Human infection occurs

when embryonated eggs

are ingested from dog orcat faeces

It is common in children

and can cause visceral

larva migrans (VLM)

Examples of important flatworms


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Examples of important flatworms -

cestodesIntestinal - (“tapeworms”)

Taenia saginataworldwide

acquired by ingestion ofcontaminated, uncooked beef

a common infection but causesminimal symptoms

Taenia soliumworldwide

acquired by ingestion ofcontaminated, uncooked porkthat contains cystercerci

Less common, but causes

cystercicosis – a systemicdisease where cysticerci encystin muscles and in the brain –may lead to epilepsy

SystemicEchinococcus granulosus (dogtapeworm) and Echinicoccus

multilocularis (rodent tapeworm)

Hydatid disease occurs when

the larval stages of these

organisms are ingested

The larvae may develop in the

human host and cause space-

occupying lesions in severalorgans, e.g. liver, brain

Examples of important metazoa trematodes


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Examples of important metazoa –trematodes

(flukes)

Intestinal

Fasciolopsis buski - A commonparasite of humans and pigs inSouth- east Asia. This parasite isone of the largest trematodes toinfect man (8cm in length) andlives in the upper intestine.Chronic infection leads toinflammation, ulceration andhaemorrhage of the smallintestine

Fasciola hepatica (liver fluke)- Primarily, a

parasite of sheep, humans become infectedwhen they ingest metacercariae that have

encysted on watercress. The adult trematode

lives in the intra-hepatic bile ducts of the liver.

“Fascioliasis” can lead to severe anaemia in

humans

Clonorchis sinensis (liver fluke)- Widespreadin China, Japan, Korea and Taiwan, this

parasite is acquired by ingestion of infective

metacercariae in raw or pickled fish

Paragonimus westermani ( lung fluke)-

Widespread in the Far East and South east

Asia, the parasite is acquired by ingestion ofinfective metacercariae in raw or pickled

crustaceans

Schistosoma haematobium, S. mansoni and

S. japonicum – see below


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Schistosomiasis (bilharzia)


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Hookworm (1)

Epidemiology

>1200m infections each year of which

100m are symptomatic

It is due to 2 parasites both of which

occur worldwide:

Necator americanus - predominant

species in sub-Saharan Africa,

south Asia and the Pacific

Ancylostoma duodenale –

predominant in S. Europe, N. Africa,

western Asia, northern China,

Japan and the west coast of

America

Hookworm is a major cause of anaemia


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Hookworm (2)Life cycle

Adult worms live in the intestine and excrete eggs in the faeces

In the absence of latrines, eggs contaminate soil and develop in warm, damp conditions

eggs hatch and infective filariform larvae develop in about one week and remain infective in soil for

many weeks

filariform larvae penetrate the skin when a person walks barefoot in the soil

larva migrate from the skin to the lungs via the lymphatic and blood systems

larvae penetrate the capillary wall to enter the alveolus

Larvae are propelled up the respiratory tree to the epiglottis where they are swallowed

Develops to adult stage in upper intestine; adult worms are fully mature after about 5 weeks

Eggs are excreted in the faeces

Note: eating soil (pica) is a common practice. Ingested filariform larvae of A. duodenale can pass

directly to the gut mucosa

Egg of A. duodenale in faecal smear (size 57-76 µm

by 35-47 µm) Filariform larvae


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Hookworm (3)

Pathology

Hookworms move several times a dayto different attachment sites in the upperintestinal mucosa to ingest blood

They secrete an anticoagulant whichcauses the old attachment sites tocontinue to bleed

Heavy hookworm infection results in

chronic haemorrhage from the duodenaland jejunal mucosa

The combination of constant blood lossdue to hookworm infection and poor ironintake in the diet results in irondeficiency anaemia

A. duodenale ingests 4-5 times more

blood each day than N. americanusIn a child, the continued daily loss of10ml of blood can lead to severeanaemia Adult male and female worms of A.

duodenale


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L h ti fil i i (1)


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Lymphatic filariasis (1)

Epidemiology

120m people infected in >80

countries in Africa, Asia, the

Pacific islands and South and

Central America

40m of those infected aredisfigured or severely

incapacitated

95% cases due to Wuchereria

bancrofti , other species include

Brugia malayi and Brugia timori

A female Anopheles mosquito taking

a blood meal

L h ti fil i i (2)


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Life cycle

Wuchereria bancrofti is mainlytransmitted by

Culex mosquitoes in India

Anopheline mosquitoes in AfricaB. malayi and B. timori are transmittedmainly by Mansonia mosquitoes

Larval forms of the parasite

(microfilariae) are taken up by a femalemosquito when it takes a blood mealfrom a human infected with adult worms

The microfilariae develop inside themosquito

When the mosquito takes another bloodmeal the infective filariform larvae enterthe bite wound

Filariform larvae migrate to thelymphatics and lymph glands

Larvae develop into sexually matureadult worms over 3-12 monthsdepending on the species of filarialworm

Microfilaria of B. malayi in thickblood film (H&E stain; source: CDC)

Adult worms of B. malayi in section in a

lymph node (source: Univ South Carolina)

L h ti fil i i (3)


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Pathology

Adult worms live in the afferentlymphatic vessels and cause severe

disruption to the lymphatic system

Scrotal damage and massive swelling

may occur when adult Wuchereria

bancrofti lodge in the lymphatics of

the spermatic cord

Late stage disease is typified by

elephantiasis – painful and disfiguring

swelling of the limbs

Trauma and secondary bacterial

infection of affected tissues is

common

Elephantiasis of the leg

(source: WHO/TDR/Crump)

L h ti fil i i (4)


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Symptoms and signs – 3 stages

1. Asymptomatic stage

There is internal damage to the lymphaticsand kidneys

2. Acute stage – Filarial lymphangitis

Characterised by bouts of fever

heat, redness, pain, swelling andtenderness of the lymph nodes and ducts

3. Chronic stage

Usually results in elephantiasis as a resultof chronic lymphoedema

There is a massive overgrowth of tissueresulting in severe deformities

The legs are often affected and result ininability to walk

The scrotum is often affected in men andthe breasts and vulva in women

Elderly male with massive hydrocoele, and

elephantiasis of the leg. Also has nodules in the groin

due to onchocerciasis (source: WHO/TDR/Crump)

L h ti fil i i (5)


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Diagnosis

Microscopic examination of Giemsastained thick blood films for the presenceof microfilariae

W. bancrofti shows marked nocturnalperiodicity, so it’s best to collect bloodsamples between 10pm and 1 am

Serology

TreatmentDiethylcarbamazine (DEC) rapidly killsmicrofilariae and will kill adult worms ifgiven in full dosage over 3 weeks

Release of antigens from dying microfilariacauses allergic-type reactions – add anantihistamine and aspirin to treatmentregimen

Other treatment options areivermectin

combination of DEC and albendazole

Prevention and control

Rapid diagnosis and treatment of infectedindividualsMass drug administration to at riskcommunities

Vector control: eliminate mosquitobreeding sites through improved sanitationand enviromental management

Personal protection against mosquito bites

by insecticides, bednets and repellants

L b t Di i f P iti I f ti


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Laboratory Diagnosis of Parasitic Infections

Purpose –Confirmation of clinical suspicion

Identification of unsuspected infection

Methods same as used in Bacteriology &

Virology but significance of different methods

varies.

Isolation least important, morphological

identification very important.Serology relatively less important

Morphological identification


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Examination of faeces –Gross

MicroscopySaline mount

Iodine Mount

Thick smears – not commonly used

Permanent stained smears

Iron hematoxylene

Whearley’s trichrome stain

Concentration methodsFloatation techniques

Sedimentation techniques



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Examination of BloodThin Smear

Thick smear

Wet mount for microfilaria

Stains used

Cultivation of parasites


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Cultivation of parasites

Culture methods – Amoeba

Leishmania & Trypanosoma

Malarial parasite

Animal inoculation – Not practical

Xenodiagnosis – Vectors infected

experimentally

Immunological diagnosis



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Serology – All tests

availableIHA

ELISA

CIEP

IF

CFTMore useful in

Amoebiasis

Leishmaniasis

Malaria

ToxoplasmosisTrichinosis

Filariasis

Echinococcosis

Skin Tests – Specificity low,

cross reactions commonCasoni’s test

Leishmanin test

Sources of information


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Sources of information

The Special Programme for Research and Training

in Tropical Diseases (TDR UNICEF, UNDP, World

Bank, WHO) website:

ww.who.int/tdr/media/image.html

University of South Carolina School of Medicine:http://pathmicro.med.sc.edu/book/parasit-sta.htm

Lecture notes on Tropical Medicine, Dion R Bell,

Fourth edition, 1996, Blackwell Science.

Parasites and human disease, W. Crewe and

D.R.W. Haddock, 1985, First edition, Edward Arnold

Documents

Introduction to Parasitology & Lab Diagnosis of Parasitic