PARASITOLOGY

INTRODUCTION TO PARASITOLOGY

INTRODUCTION I. SYMBIOSIS (Sym = Together; Bio = Living  “Living Together”) -an association between two different species of organisms -primary aim: to obtain food and seek shelter and protection from the bigger organism -commonly known as Living Together -there are three types of Symbiotic Relationships: Commensalism + Mutualism + Parasitism ORGANISM Commensalism Mutualism Parasitism

Favorable Favorable Very Favorable

HOST Not Affected Favorable Not Favorable

II. PARASITOLOGY -the study of the parasites -the science that deals with a specialized group of animals which are physiologically dependent on other organisms for their survival -Neva&Brown: science that deals with organisms that take up their abodes, temporarily or permanently, on or within other living organisms for food, with the relationship of these organisms to their host **IMPORTANT NOTE: o The key in studying Parasitology is in the Life Cycle o If we know the life cycle and its important components, we would understand why it is the key III. DEFINITION OF TERMS A. Parasites -organisms ranging from microscopic to macroscopic which are known to inflict disease in man -exhibit great diversity in morphology and physiology -animals depending on bigger organisms for food and shelter -has intimate almost always obligatory relationship with the host (ALWAYS harms the host) **Ascaris lumbricoides -compete for available nutrients (sugars, proteins, fats) in the food we had already eaten -tangled worms may obstruct the gut -may damage vital organs during erratic migration **Description:  Harms its Host  Associated closely with host and derive nutrition from host  May kill the host B. Hosts -the bigger organisms in which the parasite lives -provides the nourishment and protection needed by the parasites **Description:

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 

Does not harm the parasites Provides shelter to its parasite (parasites need the host)

PARASITES I. CLASSIFICATION OF PARASITES -Parasites: organism or animal that depends upon another bigger organism for food -parasites can be classified according to location in their hosts, degree of association, and taxonomic groups A. According to Location 1. Ectoparasites -found outside or on the surface of the body of the host -may cause INFESTATION -ex) Scabies (Sarcoptes scabiei – human itch mite) 2. Endoparasites -found living within the host in any internal organs (GIT, Respiratory Tract, RBCs) -may cause INFECTION -examples of Endoparasites : • Ascaris (GIT Lumen) • Plasmodium (RBC) – Malarial Parasite • Schistosoma (Mesenteric Veins) B. According to Degree of Association 1. Obligate Parasite -those who cannot survive or reproduce outside the host -always parasitic (must always be in contact with a susceptible organism) -ex) Trichomonas vaginalis 2. Facultative Parasite -those that may exist in a free living state or as a commensal -if opportunity presents itself, they may become Parasitic -they can both live as free living or parasitic parasites -ex) Strongyloides stercoralis 3. Permanent Parasite -if the live continuously in the host, and remain in the body of the host – from the early stage until maturity -requires continuous contact with the host -ex) Pediculus, Trichomonas vaginalis 4. Intermittent (Temporary) Parasites -contact with the host is only temporarily – closely associated with its host temporarily to feed -ex) Anopheles mosquitoes (they only bite when they want your blood) 5. Accidental Parasites -enters the host that is not normally its host -does not develop to full maturity since the host is not physiologically compatible -association lasts only for a short time -ex) Echinococcus, Gnathostoma, Toxocara 6. Aberrant Parasite -moves out of the normal location within its host -its Migration to ectopic sites may lead to complications -also known as erratic

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-ex) Ascaris lumbricoides (intestines, then they migrate to bile duct, liver, or appendix) 7. Spurious Parasite -organisms taken in by accident and then passed out without causing any harm to unsuitable host -example of Commnesalism (ex- Fruit Fly larvae or Plant Nematode)

II. TYPES OF HOST -Host: organism in which a parasites live -classified according to the following: A. Definitive Host -harbors the sexual or the mature stage of the parasite (site where sexual reproduction occurs, if present) -ex) Man, Pig, Dog, Mosquitoes  Mosquitoes: definitive host in Malaria  Ascaris: man is the definitive host B. Intermediate Host -host that harbors the asexual or the immature / larval forms of the parasites -site where the asexual reproduction occurs -Paratenic Host: host in which the parasite does not undergo any development C. Reservoir Host -these are the animals that harbor the parasite that are also parasitic to man -site where the asexual reproduction occurs -ex) Dog may carry hookworm eggs from one place to another, but eggs do not hatch or pass through any development in these animals D. Accidental (Incidental) Host -these are the hosts that accidentally ingested the Parasites (that usually affect the animal kingdom) -infection occurs in a host other than the normal host species -Zoonosis: condition wherein animal parasites accidentally infect man -ex) Hyatid Tapeworms are usually found in dogs and sheep  Zoonosis E. Vector -an invertebrate animal that transmits a parasitic organism from one host to another -has two types: Mechanical Vector + Biological Host 1. Mechanical Vector

-an invertebrate animal which merely transfer parasitic organisms from one host to another (by carrying feces on their feet) -ex) Flies, Cockroaches

2. Biological Vector

-there is a development of the parasite inside the host -harbor the parasite internally in which it may undergo further development, multiplication, or both -ex) Mosquitoes in Malaria

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III. TAXONOMIC GROUPINGS OF PARASITES A. Protozoans (Unicellular Eukaryotic Parasites) o Phylum Sarcomastigophora o Phylum Apicomplexa o Phylum Microspora o Phylum Ciliophora PHYLUM REPRODUCTION Sarcomastigophora: Asexual Sarcodina Sarcomastigophora: Asexual Mastigophora

Apicomplexa

Asexual & Sexual

Ciliophora

LOCOMOTION Pseudopodia

SPECIES Entamoeba histolytica

Flagella

Giardia lamblia Dientamoeba fragilis Trypnasoma spp Loishmania spp Plasmodium spp Toxoplasma Sarcocystis Balantidium coli

None

Cilia

1. Phylum Sarcomastigophora -has Subphylum: Sarcodina and Mastigophora -both reproduce Asexually a. Subphylum Sarcodina

-move by Pseudopodia (can only move in one direction) -Pseudopodia: cytoplasmic protrusions -ex) Amoeba, Entamoeba (pathogen)

b. Subphylum Mastigophora

-move by flagella (Flagellates) -Flagellum: long threadlike extension of cytoplasm -ex) Giardia, Trypanosoma, Leishmania, Trichomonas

2. Phylum Apicomplexa -no locomatory organ -have a complex life cycle with alternating sexual and asexual generations -Blood Parasites that cause Malaria: Isospora, Cyclospora, Cryptosporidium, Sarcocystis -Other Parasites: Taxoplasma, Sarcocystis 3. Phylum Ciliophora -move by beating of many Cilia -mostly free living and symbiotic species -ex) Balantidium coli (the only pathogenic ciliate) 4. Phylum Microspora

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-no locomatory organ -intracellular parasite of many kinds of vertebrates and invertebrates -rarely cause disease in immunocompetent persons

B. Helminths (Metazoa; Wormlike Invertebrates) PHYLUM ASCHELMINTHES CLASS NEMATODA AKA Round worms Shape Elongated, Cylindrical Segmentation Unegmented Sexes Separate 1. Phylum Aschelminthes a. Class Nematoda

PHYLUM PLATYHELMINTHES CLASS CESTODA CLASS TREMATODA Flatworms Flukes Elongated, Ribbon Like Leaf-shaped, Elongated Segmented Unsegmented Hermaphrodite Hermaphrodite

-Nematodes or Roundworms -elongated, cylindrical worms attenuated at both ends -unsegmented worms; sexes are separate -reproduction by: • Oviparous (ex. Ascaris, Enterobius) • Viviparous (ex. Trichinella, Filarial Worms) **Infection by: • Ingestion of Eggs (ex. Ascaris, Trichuris) • Skin penetration by Larvae • Vector Borne • Ingestion of Encysted Larvae

2. Phylum Platyhelminthes -also called Flatworms – multicellular animals characterized by flat, bilaterally symmetric body -most Flatworms are Hermaphroditic, having both male and female reproductive systems a. Class Trematoda

-majority are transmitted by ingestion (except – Schistosoma) -unsegmented -Hermaphroditic -flattened, leaf-shaped or cylindrical -Order Digenea: contains all species that are parasitic to humans -Infection by: Metacercariae ORSkin penetration by Cercariae

b. Class Cestoda

-segmented -Hermaphroditic, ribbon like -reproduction by: Oviparous (Ex. Taenia); multiplication of larval forms -Infection by: Ingestion of Encysted Larvae

C. Arthropods 1. Phylum Arthropoda -have segmented body parts: Head Thorax and Abdomen -have three to four pairs of jointed appendages and covered by hard chitinuous skeleton -bilaterally symmetrical body **Classes in Phylum Arthropoda

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CLASS Crustacea Chilopoda Arachnida Insecta

DESCRIPTION Some serve as intermediate hosts spiderlike Includes most important of arthropods

EXAMPLE Crabs, shrimps, copepods Centipedes Scorpions, spiders, ticks, mites

**Class Insecta • Order Anoplura • Order Hemiptera • Order Coleoptera • Order Hymenoptera • Order Siphonaptera • Order Diptera

LIFE CYCLES I. TWO TYPES OF LIFE CYCLES: -Parasites undergo a Life Cycle during its existence -it involves survival and development in the external environment and in one or more hosts -life cycle consists of the various morphologic forms and developmental stages -life cycle summarizes development of the parasite from the time it leaves the host to start a new infection in a new susceptible host -knowledge of life cycles = preventive measures A. Direct Life Cycle -there is no intermediate host required to transmit the parasite from one host to another -parasites are transmitted from an infected individual to a susceptible host without requiring an intermediate host **Ex1) Trichomonas vaginalis -direct cycle where the parasite is immediately infective when it leaves the host -Man  Man **Ex2) Ascaris lumbricoides -direct life cycle where the parasite develops into the Infective Stage in the external environment -Man  Development in External Environment  Vectors (optional)  Man

B. Indirect Life Cycle -at least one intermediate host / vector is required for the parasite to complete its transmission from the originally infected host to the susceptible potential new host -the infective stage of the parasite develops in the Intermediate Host and the Susceptible Host acquires the infection through contact or exposure to the infected intermediate host **Ex) Schistosoma japonicum, Capillaria phiippinensis -Man  Intermediate Host  Contact / Ingestion of Raw Intermediate Host  Man

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II. COMPONENTS OF THE LIFE CYCLE • Infective Stage • Portal of Entry • Mode of Transmission • Path or Route of Migration • Developmental Stages • Habitat • Portal of Exit • Diagnostic Stage A. Infective Stage -stage of development of the parasite which when ingested to the definitive host will result to infection -in the direct life cycle: Infective Stage comes from original infected host -in the indirect life cycle: it originates from the Intermediate Host or Vector -ex) Ascaris – Infected Stage is the Embryonated Egg B. Portal of Entry -refers to site of opening in the susceptible host through which the infective stage enters -most common portal of entry: Mouth, Skin, Nose -ex) Ascaris – Portal of Entry: Mouth C. Mode of Transmission -refers to the process on how a new host acquires the infective stage of the parasite -man could acquire it by: Ingestion, Inhalation, Infected Intermediate Host, Skin penetration, Inoculation -ex) in Ascaris – Ingestion of Embryonated Egg; Hookworm by Skin penetration of Larva D. Path or Route of Migration -when the infective stage of parasite enters the new host, it usually undergoes without maturation stages -different developmental stages of parasites may occur in different parts or organs E. Developmental Stages -the parasite undergoes different developmental stages as it moves from the original infected hosts to the environment, inside the intermediate hosts & even w/in the new susceptible host before settling down -ex) For Helminths, developmental stage is Egg, Larva, Immature Adult and Adult

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F. Habitat -refers to the specific organ or site in the definitive host’s body where the sexually mature parasite resides -ex) Habitat of Ascaris is in the Lumen of Small Intestines G. Portal of Exit -opening or site in body wherein parasites leave the infected hosts to find a new susceptible host -ex) Eggs of Ascaris leave the host through the Anus H. Diagnostic Stage -stage passed out by the host or taken up by the vector or intermediate host -serves as a basis for establishing presence of the parasite in host or diagnosis of infection

EXPERIMENT 24: LUNG FLUKES

INTRODUCTION I. SLIDES TO VIEW: • Paragonimus westermani o Adult o Egg II. PARAGONIMUS WESTERMANI • Paragonimus westermani = Affects the Human Lungs (Lung Fluke) • Transmission = Ingestion of Insufficiently cooked Crabs or Cray Fish infected with Metacercariae • Infective Stage = Metacercaria • Diagnosis = Finding Medium Sized Operculated Ova in the Sputum • Pathology = Acute, Subacute Inflammations of Lung Tissue; it may also wander erratically to Brain and Skin **IMPORTANT Note: o Clinically, it is usually Mistaken with Tuberculosis Symptoms due to Hemoptysis o TO Differentiate, we find Operculated Ova in the Sputum, instead of Acid Fast Bacilli III. LIFE CYCLE OF P. WESTERMANI • Type = Indirect Life Cycle (One or Two Intermediate Hosts and One Definitive Host) • Immature Larvae develops inside the Intermediate Host and Matures into Adult Stage inside Man • Mode of Transmission = Ingestion of Raw Infected Crabs / Crayfish • Diagnostic Stage = Eggs in Sputum / Stool • Infective Stage = Metacercariae from Crabs • Definitive Host = Man • 1st Intermediate Host = Snails • 2nd Intermediate Host = Crabs / Crayfish

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DEFINITIVE HOST = MAN

SNAILS

Ingestion of Raw Infected Crabs / Crayfish

Miracidium Hatches & Penetrates Snail

Immature Worm Migrate into Peritoneal Cavity, Diaphragm and Lungs Adult Worm Encysted in Lung Tissue (Lungs have Cavitary Lung Lesions, Cough, Pleuritic Pain, Hemoptysis)

Sporocyst

CRABS / CRAYFISH Cercariae Enter the Crabs Encysts to form Metacercaria

Radiae Cercariae

Metacercariae (Infective Stage)

Cercariae is released in the Water

Eggs in Sputum or Stool

III. DESCRIPTION OF THE SLIDES: A. Paragonimus westermani Adult -Plump, Ovoid -Reddish-Brown Fluke (8-20mm in length; 5-9mm in breadth) **Diagnostic Features of this Fluke  Scale-Like Integumental Spines  Two Equal-Sized Ventral & Oral Suckers located in the Anterior Half **Other Structures:  Two Lobate Testes situated Side-by-Side  Centrally Located Lobate Ovary Anterior to the Testes  Laterally, the entire body is occupied by the Highly Branched Vitellaria  Excretory Bladder  Coiled Uterus B. Paragonimus westermani Egg -Medium Sized, Oval Shaped Egg (85 x 55u) -Operculated: has a characteristic Broader Operculated Anterior End and a Thicker Posterior End -expectorated in Sputum or when swallowed, it can be detected in Feces in Unembryonated Stage

IV. ANSWERS TO QUESTIONS ❖ Discuss the Specimens of Choice in confirming the Diagnosis of Paragonmiasis o Stool

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o

Sputum

❖ Give the Characteristic Morphological Feature of Paragonimus westermani Ova o Broader Operculated Anterior End o Thickened Posterior End

EXPERIMENT 27: SPOROZOANS

INTRODUCTION • •

• •

Sporozoans = Group of Unicellular Parasites with a characteristic Apical Complex Has NO Locomotory Organelles (except for Flagellated Microgametes in some groups) Plasmodium = Commonly seen in Man which can cause Malaria Classification of Sporozoans (Based on Affected Organ) o Blood Sporozoans o Tissue Sporozoans

I. SLIDES TO VIEW: • P. falciparum = Trophozoites, Schizonts, Gametocytes • P. vivax = Trophozoites, Schizonts, Gametocytes • P. malariae = Trophozoites, Schizonts, Gametocytes • P. ovale = Trophozoites, Schizonts, Gametocytes

II. PROCEDURE • Stained Blood Smears are shown • Pay attention to the Size of the Normal and Parasitized RBC, number of Chromatin Dots, Amount of Hemozoin Pigments, Appearance of the Cytoplasm, and presence / absence of Stippling on the Infected RBC’s Cytoplasm

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III. STRUCTURE OF THE PARASITE (PLASMODIUM) Host RBC Stippling

Cytoplasm Chromatin

Vacuole

IV. LIFE CYCLE OF MALARIA PARASITES • Type of Life Cycle = Indirect Cycle • Infective Stage = Sporozoites • Diagnostic Stage = Gametocytes • Biologic Vector = Female Anopheles Mosquito • Definitive Host = Female Anopheles Mosquito • Intermediate Host = Human • Two Phases = Human Phase + Mosquito Phase V. OTHER IMPORTANT SPOROZOANS: • Cryptosporidium • Isospora • Blastocystis • Pneumocystis carinii • Toxoplasma gondii V. STAGES OF THE PARASITE • Asexual Stages = Trophozoites + Schizonts • Immature Sexual Stages = Gametocyte • NOTE: Man is just an Intermediate Host in Malarial Infections because they harbor only the Immature Forms of the Parasite (the Mature forms are found in the Mosquito) A. Trophozoites (Ring Form) -it is the earliest stage -they develop from Sporozoites in the Pre-Erythrocytic (Hepatic Schizogony) Cycle; in the Blood Cycle, it develops from Merozoites – therefore, we see Throphozoites in BOTH Cycles of Human Phases -with ONE or TWO Chromatin Dots -ring-like structures with Few Malarial Pigment **Chromatin Dots:  Usually only ONE!  P.falciparum (exception) = More than One in Trophozoite Stage (thin ring like cytoplasm) **Cytoplasms:  Compact Cytoplasm = P.ovale and P.malariae

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  

Thin Delicate Cytoplasm = P. falciparum Amoeboid Cytoplasm = P.vivax Band form Cytoplasm = P.malariae

B. Schizonts -once the Chromatin Dot divides by Binary Fission, Parasite is now a Schizont -with Two or More chromatin Dots (Chromatin Dots now develop to form Merozoites) -with plenty or increasing amount of Malarial Pigment (Hemozoin Pigment) in Cytoplasm -can be divided into Growing / Immature and Mature (Cryptozoites) in the Pre-Erythrocytic Cycle **Merozoites  P.falciparum = 12-32  P.vivax = 12-24  P.malariae = 6-12  P.ovale = 4-12 **Tissue Schizonts:  Primary Tissue Schizonts  Secondary Tissue Schizonts C. Gametocytes -it is only an Immature form of the Sexual Forms of the Parasite -Banana Shaped in P.falciparum or special with definite Chromatin material -develops from Merozoites

DECSRIPTION OF THE SPECIES I. MORPHOLOGY (From Lab Manual) Trophozoite

Schizont

P. falciparum Size: Small-Medium Number: Numerous Shape: Ring&Comma Form Chromatin: Two Dots Cytoplasm: Regular Mature Forms: Compact Pigment: Coarse Grains

P. vivax Size: Small-Large Number: Few-Moderate Shape: Broken Ring Chromatin: Single Cytoplasm: Irregular Mature Forms: Compact Pigment: Fine Scattered

P. malariae Size: Small Number: Few Shape: Ring to Round Chromatin: Single Cytoplasm: Regular Pigment: Scattered

P. ovale Size: Smaller than vivax Number: Few Shape: Ring to Round Chromatin: Single Cytoplasm: Regular Pigment: Course, Scatter

Associate w/ Ring Forms Size: Small, Compact Number: Few

Size: Large Number: Few-Moderate

Size: Small, Compact Number: Few

Size: like malariae Number: Few

Mature Form:12-24 Merozoite

Mature Form:6-12 Merozoite

Mature: 4-12 Merozoite

Mature Form:12-13 Merozoites

Pigment: Loose Mass

Pigment: Concentrated Daisy Flower Like

Pigment: Concentrated

Pigment: Single Dark Mass

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Gametocyte

Mature: Banana Shaped Chromatin: Single Pigment: Scattered

Mature: Round Pigment: Scattered

Mature: Round Chromatin: Single Pigment: Scattered Peripherally

Mature: Round Chromatin: Single Pigment: Scattered

II. GENERAL CHARACTERISTICS (From Notes) FORM Infected RBC Size

P. falciparum Not Enlarged

P. vivax Enlarged

P. malariae Not Enlarged

P. ovale Enlarged

Shape

Round but may be Crenated

Round

Round

Oval; Fimbriated

Stipplings

Maurer’s Dots

Shuffner’s Dots

Zieman’s or James Dots

Shuffner’s Dots

Trophozoites

Small Rings Multiple w/ Acole Forms

Small & Large Rings Amoeboid

Small Compact Rings Band Forms

Small; Compact Rings

Schizont

Rare (Seen in Severe Cases)

Irregular Contours

Compact; Round

Compact

# of Merozoites

12-32

12-24

6-12 Forms Regular RosetteLike Clusters

4-12 Forms Irregular RosetteLike Clusters

Gametocyte

Banana / Sausage Shape

Round-Large

Round, Compact

Round, Small

ANSWERS TO QUESTIONS ❖ Plasmodium falciparum Schizont VS Plasmodium vivax Schizont FEATURES P. falciparum Size Small & Compact Number Few, Uncommon Mature Forms 12-32 Merozoites Cluster Compact Clusters Pigment Single Dark Mass

P. vivax Large Few to Moderate 12-24 or More Merozoites Irregular Clusters Loose Mass

EXPERIMENT 28: BLOOD & TISSUE FLAGELLATES

INTRODUCTION • • •

Trypanosoma and Leishmania Flagellates belong to Order Kinetoplastida of Superclass Mastigophora They Multiply in the Blood (Hemoflagellates) or Tissue of Humans All Species require an Arthropod Intermediate Host

I. SLIDES TO VIEW • Trypanosoma brucei gambiense (Trypomastigote) • Leishmania tropica (in Culture) • Leishmania tropica (Hamster) II. MORPHOLOGIC DIFFERENCES AMASTIGOTE

PROMASTIGOTE

EPIMASTIGOTE

TRYPOMASTIGOTE

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AKA Morphology Flagellum Course of Flagellum Found in

Leishmania Ovoidal / Spherical Intracellular Axoneme Intracellular (Not Free)

Stage

Leishmania Diagnostic Stage

• •

Macrophages Reticuloendothelials

Leptomonas Spindle Shaped Free Flagellum Arise from Kinetoplast in the Anterior End Vector

Crithidia Spindle Shaped Free Flagellum Ends in Kinetoplast Anterior to Nucleus

Trypanosoma Spindle Shaped Free Flagellum Ends in Kinetoplast Posterior to the Nucleus

Leishmania Infective Stage

Typanosoma

Trypanosoma Infective Stage

Leishmania = Amastigote + Promastigote Trypanosoma = Trypomastigote + Epimastigote

III. TRYPANOSOMA SPECIES A. Comparison of Diseases and Vectors SPECIES DISEASE T. brucei gambiense West African Sleeping Sickness T. brucei rhodesiense East Sleeping Sickness T. cruzi Chaga’s Disease, Megacolon

VECTOR Riverine Tsetse Fly Woodland Tsetse Fly Reduviid Bug (Triatoma)

B. Life Cycles: 1. T. brucei gambiense  Infective Stage = Metacyclic Trypopmastigote  Diagnostic Stage = Trypomastigote  Stage that develops in the Salivary Glands of the Vector (Tsetse Fly) = Epimastigote  Mammalian Hosts = Humans, Pigs, Wild Animals  Vector = Riverine Tsetse fly (Glossina palpalis) = Day time biters  NOTE: Epimastigotes are NOT found in the Human’s Blood (it develops in the Tsetse Fly) 2. T. brucei rhodesiense  Infective Stage = Metacyclic Trypopmastigote  Diagnostic Stage = Trypomastigote  Stage that develops in the Salivary Glands of the Vector (Tsetse Fly) = Epimastigote  NOTE: Epimastigotes are NOT found in the Human’s Blood (it develops in the Tsetse Fly) 3. T. cruzi  Infective Stage = Metacyclic Trypomastigote  Epimastigote Develops inside the Reduviid Bug (Vector)  Diagnostic Phases: • Blood = Trypomastigote • Heart Tissues = Amastigote IV. LEISHMANIA SPECIES A. Comparison of the Diseases Caused: LEISHMANIA Leishmania tropica

DESCRIPTION Old World Cutaneous Leishmaniasis

DISEASES Dry, Chronic, Urban Cutaneous Leishmaniasis Oriental Sore Aleppo or Baghad Boil Recividans or Chronic Relapsing Cutaneous L.

Leishmania major

Old World Cutaneous Leishmaniasis

Wet or Acute Rural Cutaneous Leishmaniasis

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Leishmania aethiopica

Old World Cutaneous Leishmaniasis New World Cutaneous Leishmaniasis

Diffuse / Disseminated Cutaneous Leishmanisis

Leishmania braziliensis

Mucocutaneous Leishmaniasis

Mucocutaneous Leishmaniasis Espundia (disfigures face)

Leishmania donovani

Visceral Leishmaniasis

Visceral Leishmaniasis Kala-Azar or Black Disease (Zoonotic)

Leishmania mexicana

Diffuse / Disseminated Cutaneous Leishmaniasis Chiclero Ulcers

B. Life Cycle o Infective Stage = Metacyclic Promastigote o Diagnostic Stage = Amastigote (inside Macrophages) o Vector = Sandflies (Phlebotomus or Lutzomyia) o Reservoir Hosts = Humans, Dogs, Wild Animals o Habitat = Reticular Endothelial System

EXPERIMENT 29: FILARIAL WORMS

INTRODUCTION • • •

Filarial Worms = Slender Tissue dwelling Nematodes with Reduced Lips and Buccal Capsules All Species employ Arthropods as Intermediate Host Transmission = through Arthropod Vector Bite

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•

Infective Stage = Third Stage Larva (L3)

I. SLIDES TO VIEW (Blood Smears with): • Wuchereria bancrofti • Brugia malayi II. PROCEDURE • Blood Smears with Microfilariae • Take note of Morphological Differences between the Two Species and Tabulate based on the following: Shape & Size of the Larvae relative to the Host Cells, presence / absence of a Membrane Sheath and Number of Nuclei at the Posterior or Tail End III. FILARIAL WORMS OF MEDICAL IMPORTANCE FILARIASIS VECTOR Wuchureria bancrofti Lymphatic Mosquitoes Brugia malayi Lymphatic Mosquitoes Brugia timori Lymphatic Mosquitoes Onchoceria volvulus Subcutaneous Fly Loa loa Subcutaneous Fly Manzonella perstan Animal Midget Manzonella ozzardi Animal Midget

DEFINITIVE HOST Man Man Man Man Man Animal Animal

DISTRIBUTION Philippines Philippines Indonesia

IV. LIFE CYCLE • Infective Stage = L3 (Third Stage Larvae) • Diagnostic Stage = Microfilariae • Definitive Host = Man, Chimpanzee, Gorilla • Mosquito Borne L3 (Infective Stage)  Migrates to Lymph Gland  L1  L2  L3

Human Phase

Mosquito

EXAMINATION OF THE SLIDES I. STAGES OF THE PARASITE

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A. Adult Stage -found in Lymph Glands (they can create Obstruction, leading to Edema) -long slender, creamy white roundworm, smooth cuticle 1. Viviparous Female -Vulva opens to the Surface of the Worm near the middle of the Pharynx -gives birth to a Prelarval Form (Microfilariae) -6-10cm in length; 0.3cm wide 2. Males -possess Caudal Alae with two Spicules -40-50mm in length, 0.3wide B. Microfilariae -also known as the Pre-Larval Form (L1) -it is NOT an Infective Stage – it is a Diagnostic Stage -given birth by a Viviparous Female Worm (there is NO egg stage) -Body Nuclei = represents are Rudiments of Internal Organs **Morphology:  Morphology varies with Species  May contain sheath with or without striations  Body Nuclei represent Rudiment of Internal Organs  Stained with Giemsa **Comparison of Microfilariae between the Two Species: W. bancrofti Cuticular Sheath Present Cephalic Space Short Length = Width Body Curves Regular, Large Body Nuclei Round Medium-Size Well Separated Tail No Terminal Nuclei Pointed End

B. malayi Present Long Length > Width Irregular, Kinky, Small Small, Angular Overlapping (not separated) With Two Terminal Nuclei Blunt Tip

*NOTE: Microfilariae are found in the Blood only at Night Time II. COMPARISON OF LYMPHATIC AND SUBCUTANEOUS WORMS (BASED ON MICROFILARIAE) W. bancrofti B malayi Onchocerca volvulus Loa loa Sheath Present Present Absent Absent Cephalic Space Short Long Short Short Body Curves Regular Irregular Rigid Irregular Body Nuclei Separated Overlapping Separated Overlapping Tail No Nuclei With Terminal Nucleus No Nucleus With Terminal Nuclei III. ENDEMIC PLACES OF FILARIASIS IN THE PHILIPPINES • Luzon • Leyte • Marinduque • Mindoro • Palawan • Samar

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EXPERIMENT 30: SCHISTOSOMA (BLOOD FLUKES)

INTRODUCTION • • •

Schistosoma = Comprises the Blood Flukes or the Blood Trematodes They only require ONE Intermediate Host – have NO Metacercarial Stage Adults have Separate Sexes

I. SLIDES TO VIEW: • Schistosoma japonicum = Egg, Cercaria, Adult • Schistosoma mansoni = Egg, Adult • Schistosoma mekongi = Egg, Adult

II. THREE MAJOR SPECIES SNAIL MAJOR INTERMEDIATE HOST SPECIES Schistosoma Oncomelania Hupensisjaponicum Quadrasi Schistosoma mansoni Schistosoma haematobium

Biomphalaria spp (Biomphalaria glabrata) Bulinus spp (Bulinus 18truncates)

HABITAT Veins of the Small Intestines Veins of the Large Intestines Veins of Genitourinary Tract

DISEASE Oriental Schistosomiasis Katayama Disease Schistosomiasis japonica Intestinal Bilharziasis Schistosomiasis mansoni Schistosomal Hematuria Vesical Schistosomiasis Urinary Bilharziasis

DISTRIBUTION Philippines (Misamis Oriental) China, Sulawesi in Indonesia, Japan, Taiwan Africa, South America, West Indies, Puerto Rico Middle East, Africa, Southern Tip of Europe

**NOTE: Schistosoma japonicum: produces the Most Number of the Eggs

III. LIFE CYCLE • Infective Stage = Cercaria • Diagnostic Stage = Eggs in Feces • Form which Enter the Snails = Miracidia • Intermediate Host = Snails (different species for specific species of parasites – see table) • Mode of Transmission = Skin Penetration

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EXAMINATION OF THE SLIDES I. PROCEDURE: • Note Morphologic Features of the Parasites and the Striking Differences between them II. MORPHOLOGY A. Adult Worms -0.6-2.5cm in length with Oral and Ventral Suckers -Narrow Elongated Shape; has separate Sexes; Integument is Smooth or Tuberculated depending upon species -number of Testes in Males and the length of the Uterus & Number of Eggs are Distinctive to the species -Excretory System = Flame Cells, Collecting Tubules leading into a Small Bladder with Terminal Excretory Pore MALE ADULT WORM Shorter by more Sturdy; Grayish Has a Cylindrical Anterior End Body Folded to form a Gynecophoral Canal

FEMALE ADULT WORM Longer More Slender Darker

**Gynecophoral Canal = Found in Males in which the Female is embraced during copulation B. Ova / Egg -with Spine or Knob-Like Projections -depending on the Stage of Development at the time they are passed out with the Feces, one may find them from Multicellular Stage to full Embryonation with a developed Miracidium within the Shell S. japonicum Egg S. mansoni Egg S. haematobium Egg *Developed Miracidium *Developed Miracidium *Developed Miracidium *Lateral Knob *Large Lateral Spine *Large Terminal Spine *Small Lateral Spine C. Miracidium -ciliated, pyriform in shape with a Primitive Gut D. Cercariae -forked-tailed when Discharged from the Snail -fork situated at the Posterior-Third of the Tail -Oral and Ventral Suckers are developed

III. ANSWERS TO QUESTIONS ❖ Differentiate the Appearance of the Ova of the Different Species of Schistosoma S. japonicum S. mansoni S. haematobium Oval Light Yellow=Brown Terminal Spine With minute Spine or Elongate Knob Lateral Spine Acid Fast

S. mekongi Oval Lateral Spine or Knob

❖ What Specimen are you going to collect to demonstrate the Ova of Schistotoma japonicum?

19

o o

Feces are Collected When eggs cannot be found in Feces, a Rectal Biopsy may be done

EXPERIMENT 36: INTESTINAL AMOEBA

INTRODUCTION •

• •

Protozoans in the Intestines obtain nutrients from the Parasitized Host or Predigested Food in the GIT Trophozoite = Active or Feeding Stage Cysts = form when the Environment is Unfavorable for Survival (Not Active, Non-Feeding)

I. SLIDES TO VIEW: • Entamoeba histolytica = Cyst, Trophozoite • Entamoeba coli = Cyst, Trophozoite • Entamoeba nana = Cyst, Trophozoite II. ENTAMOEBA HISTOLYTICA • The ONLY Pathogen (others are Commensals) • It has the Potential to Invade the Intestinal Wall & other Organs • Entamoeba dispar = another Intestinal Commensal which is morphologically similar w/ E. histolytica • They could be differentiated using Specific Tests (Isoenzyme Analysis, Immunoassays, PCR) **NOTE: LIFE CYCLE Discussed Below

EXAMINATION OF THE SLIDES I. TROPHOZOITES AND CYSTS:

Entamoeba histolytica

Entamoeba coli

TROPHOZOITES *Karyosome = Center *Nucleus = Eccentric w/ Fine Chromatin Granules *Directional Movements (Active Progressive) *Defined Ectoplasm *Blade-Like Hyaline Pseudopodia *Indistinct Nucleus *Ingested RBC

CYSTS *1-4 Nuclei (Mature Cyst) *Diffuse Glycogen Mass *Cigar-Shaped Chromatoidal Bars

*Karyosome = Eccentric Karyosome in Nucleus *Nucleus = w/ Course Chromatin Granules *More Sluggish Non-Directional Movements *More Granular Cytoplasm *Contain Ingested Bacteria *Narrower; Less Differentiated Ectoplasm *Blunter and Broader Pseudopodia *Heavier *Irregular Peripheral Chromatin *NO RBC inside

*As many as 8-Nuclei (Mature Cyst) *Larger; Slender *More Granular Cytoplasm *Splinter-Like Chromatoidal Bodies

20

Endolimax nana

*Karyosome = Eccentric, Large Irregular *No Peripheral Chromatin Granules

*Has 4-Nuclei

II. ANSWERS TO QUESTIONS ❖ What is the Morphologic Feature of Entamoeba histolytica that signifies Pathogenicity? o Trophozoite of E. histolytica is Active, Motile Feeding Stage that causes Pathology in the Colon o It is Anaerobic with no mitochondria o Erythrophagocytosis

EXPERIMENT 37: INTESTINAL FLAGELLATES & CILIATE

INTRODUCTION I. SLIDES TO VIEW: • Giardia lamblia • Dientamoeba fragilis • Chilomastix mesnili • Balantidium coli II. FLAGELLATES VS CILIATES A. Flagellates o Possesses one or more Flagella – Long Thread-Like Extrusions of the Cytoplasm o The Pathogenic Intestinal Flagellate of Man is Giardia lamblia o Classified into Two Groups (According to Habitat):  Intestinal, Oral, and Genital Flagellates  Blood and Tissue Flagellates **NOTE: ALL have Trophozoite and Cystic Stage (EXCEPT Trichomonas)  Dientamoeba fragilis was originally grouped under Amoebas (now it is a Flagellate)  Now, it is in the Order Trichomonadida  Dientamoeba fragilis has Two Nuclei and NO Cystic Stage B. Ciliates o Move by means of Cilia – Threads of Cytoplasm (shorter and more numerous than Flagella) o The only Pathogenic Ciliate of Man is Balantidium coli

III. REVIEW OF PROTOZOANS

Infective Stage Habitat Diagnosis Pathology

Entamoeba histolytica Amoebiasis Amoebic Dysentery Amoebic Hepatitis Mature Cyst Large Intestine (Cecum) Parasite in Feces Flask Shaped Ulcer

Transmission Drugs of Choice

Ingestion Metronidazole

Disease

Giardia lamblia Giardiasis Lambiasis Traveller’s Diarrhea Mature Cyst Small Intestines Cyst & Trophozoites Malabsorption Syndrome Steatorrheac Stool Ingestion Metronidazole

Dientamoeba fragilis

Trophozoite Large Intestines (Cecum) Binucleate Trophozoites

Balantidium coli Balantidiasis Balantidosis Balantidial Dysentery Mature Cyst Large Intestines Cysts & Trophozoites

Uncertain Iodoquinol

Ingestion Tetracycline

21

Trophozoite Nucleus Karyosome Movement

Single & Eccentric Center Unidirectional

Others

Easily Destroyed

Cyst Description

Cigar-Chromatoid Bodies 1-4 Nuclei

Two Nuclei (Binucleate) Center 4-Flagella Twisting Movements Sucking Disk Ventrally Pear Shaped (Old Man) 2-Parabasal Bodies 2-Axonemes

Two Nuclei (Binucleate)

Ellipsoidal 2-4 Nuclei

NONE

Pseudopods Non-Directional No Flagella

Macro / Micro

Surrounded by Cili Cytosome & Cytopyge

Thick Cyst Wall Round or Oval

EXAMINATION OF THE SLIDES I. TROPHOZOITES AND CYSTS Giardia lamblia

TROPHOZOITE *Sucking Disk on Ventral Surface *Pear Shaped *Old Man’s Face *Two Nuclei with Large Central Karyosomes *Two Axonemes *Two Blepharoplasts *Two Deeply Staining Bars (Parabasal Bodies) *4-Pairs of Flagella

CYST *2-4 Nuclei *Surrounded by a Cyst Wall *Ellipsoidal (9-12um)

Dientamoeba fragilis

*Sluggish Non-Directional Motility *Two Nuclei *NO Flagella!!! *May Ingest RBC

NONE

Chilomastix mesnili

*One Nucleus *Four Anterior Flagella *Cytosomal Groove *Curved Posteriorly

*One Nucleus *Lemon-Pear-Shaped *Clear Knob *Cytosome

Balantidium coli

*Surrounded by Cili *Has Vacuoles and Inclusions in the Cytoplasm *Some unique structures: • Cytostome = Primitive Mouth • Cytopyge = Excretory Pore • Macronucleus = Bean / Kidney Shaped • Micronucleus = within the Macronucleus

*Almost similar: Round or Oval *Thick Cyst Wall

II. ANSWERS TO QUESTIONS

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❖ Give the Characteristic Morphologic Features FLAGELLATE / CILIATE LOCOMOTORY ORGANELLE Giardia lamblia 1-4 Flagella

DISTINCT FEATURES Two-Nuclei Large Central Karyosome

Dientamoeba fragilis

Amoebaflagellate

Two-Nuclei (Binucleate) No Cysts!

Chilomastix mesnii

Flagella

Trophozoite = Tear Drop Shape Evident Nucleus Has a Cystosome (Cell Mouth) Cysts = Lemon Shaped

Balantidium coli

Cilia

Trophozoites: Large & Oval

EXPERIMENT 38: INTESTINAL NEMATODES

INTRODUCTION I. SLIDES TO VIEW A. Adult Forms: o Ascaris lumbricoides o Trichuris trichiura o Enterobius vermicularis o Hookworms (Necator americanus, Ancylostoma ceylanicum, Ancylostoma caninum) o Capillaria philippinensis o Trichinella spiralis B. Larva o Strongyloides stercoralis o Hookworms (Necator americanus, Ancylostoma ceylanicum) o Trichinella spiralis C. Ova o o o o o

Ascaris lumbricoides (Fertilized, Unfertilized, and Decorticated Ova) Trichuris trichiura Hookworms (Necator americanus, Ancylostoma ceylanicum) Capillaria philippinensis Enterobius vermicularis

II. NEMATODES • Nematodes / Roundworms = comprise a Large Group of Helminths of simple structure • Elongated, Unsegmented, Bilaterally Symmetrical • Separate Sexes • Males < Females • Males have one or two Copulatory Spicules

23

EXAMINATION OF THE SLIDES I. OVERALL CHARACTERISTICS Ascaris Trichuris lumbricoi trichiur des a General Characteristics Common Giant Round Whipworm Name Worm Diseases Ascariasis Trichuriasis Ascaris Infection Trichocephaliasis

Enterobius vermic ularis

Hookworms

Strongyloides stercoralis

Capillaria philippinensis

Pinworm / Seatworm Enterobiasis Oxyuriasis

Hookworms

Threadworm

Ancylostomiasis Uncinariasis Necatoriasis

Strongyloides Strongyloidosis Cochin-China

Capillariasis Pudoc’s Disease

Habitat

Small Intestines Heart-Lung Route Filariform Larva

Small Intestines Heart-Lung Route Filariform Larva

Small Intestines

Eggs in Feces

Small Intestines Heart-Lung Route Embryonated Egg

Large Intestines

Large Intestines

Embryonated Egg

Embryonated Egg

Eggs in Stool

Eggs in Stool

Eggs in Feces

Transmission Definitive Host Intermediate Host Pathology

Ingestion Man

Ingestion Man (Monkeys)

Eggs in Perianal Region Ingested Man ONLY

Skin Penetration Man

Rhabditiform Larva Skin Penetration Man

NONE

NONE

NONE

NONE

NONE

Freshwater Fish

Loeffler’s Syndrome

Rectal Prolapse

Pruritus Ani

Creeping Eruptions MH-Anemia Pneumonia-Like

Larva Curens

Borborygmi Gurgling Stomach

Treatment

Mebendazole Pyrantel Pamoate Albendazole

Mebendazole Albendazole

Mebendazole Pyrantel Pamoate Albendazole

Mebendazole Pyrantel Pamoate Albendazole

Albendazole Thiabendazole

Mebendazole Albendazol Thiabendazole

Morphology Female Adult

Pointed Ends

Blunt Posterior

Sharp Pointed End

Parasitic Free-Living

Male Adult

Curved Poserior

Curved Posterior

Curved Posterior

Atypical = 2-3 Rows Typical = 1-Row Long Spicular

Infective Stage Diagnosis

Bursa to hold

No Parasitic Male

Third Stage Larva

Ingestion Man

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Eggs

Rhabditiform Larva Filariform Larva Distinct Features

End

Single Spicule Retractile Sheet

Single Spicule

female

Fertilized = Embryo Unfertilized = Granules --

Lemon /Barrel Shaped Bipolar Mucus Plug --

D-Shaped With Embryo

2-8 Cell Stage

-3-Buccal Lips

-Anterior Whip like Posterior Round

--Cephalic Alae

Long Buccal Capsule Pointed Tail

Free-Living

Sheath

Peanut Shaped

Short Buccal Capsule Forked Tail

Rounded Ends

II. RHABDITIFORM & FILARIFORM LARVAE HOOKWORMS Rhabditiform Larva Buccal Cavity Genital Primordium Filariform Larva Tail Esophagus

Long Buccal Cavity Small Genital Primordium

STRONGYLOIDES

Short Buccal Cavity Large Genital Primodrium

Pointed Tail Notched / Forked Tail Shorter Esophagus Longer Esophagus EXPERIMENT 39: DIRECT FECAL SMEAR

INTRODUCTION I. PRINCIPLES • Direct Fecal Smear (DFS) = Simplest and most Rapid of all Fecal Examination Techniques • Recommended for Identification of Protozoan Trophozoites and Detection of Helminthic Infection • One Direct Fecal Smear Preparation contains approximately 2mg of Feces • Saline and / or Lugol’s Iodine Solution can be used to Emulsify the Fecal Material A. Unstained Film -useful for the Study of Living Parasite Objects -ex) Motile Protozoan Trophozoites, Helminth Eggs, and Nematode Larva B. Iodine Film -employed to study the Diagnostic Features of Protozoan Cysts

II. PROCEDURE • Place 1-2 Drops Saline at the Center of a Glass Slide • With an Applicator Stick, poke at various portions of the Specimen (it with blood streaks, make sure you touch your applicator at this portion) • Make a smooth uniform emulsion in the drop of saline by Rotatory Motion starting from the Center • Place the edge of Cover Slip in the slide so that it touches the edge of the water • Slowly lower the coverslip to prevent Air Bubbles • Examine slide (first with LPO, then with High Power)

25

ANSWERS TO QUESTIONS ❖ Give the Advantage of using Logol’s Iodine over Saline Solution in Direct Fecal Smear o Lugol’s Iodine or D’Antoni’s Iodine helps visualize internal structures of Trophozoites and Cysts o Because Stains will kill Motile Trophozoites, it is recommended that a Saline Smear and an Iodine Smear be prepared Separately ❖ Briefly Discuss the Advantages and Disadvantages of a Direct Fecal Smear o Advantages  Allows the viewer to Detect Motile Protozoa  Can view Helminth Eggs / Larvae, Protozoan Cysts, WBCs, Some Yeast o

Disadvantages  Should NOT be performed on Preserved Specimens  Should be used for Fresh Stool Specimens that are very Soft or Liquid  Cannot be examined using Oil Immersion

EXPERIMENT 40: CELLULOSE TAPE PERIANAL SWAB

INTRODUCTION I. PRINCIPLES • Pinworm Infection (Enterobius vermicularis) is suspected in children with Perianal Itching, Insomnia, and Restlessness • Evidence depends on recovery of Adult Worms, Eggs, or Both • They are RARELY Found in Stool Examination • Pinworm Infection can be best diagnosed by Swabbing Perianal Area using Graham’s Cellophane (Cellulose) Tape Method II. GRAHAM’S CELLOPHANE TAPE METHOD • Highest Sensitivity and Specificity Results • Best to Collect Specimens in the Morning before the Patient bathes or defecates

III. PROCEDURE • Place a Strip of Cellulose Tape, Sticky Side Down, on a slide and on one end a Small Strip of Paper • Hold a Slide Against a Tongue Depressor one inch from the end and lift the tape away from the Slide • Loop the Tape over the end of the Tongue Depressor to expose the Gummed Surface (Hold the Tape by the Paper

26

• • •

Tab attached to its End Portion Hold the Tape and Slide against the Tongue Depressor Press the gummed surface of the tape against several areas of the Perianal Area Replace the tape on the slide and examine directly under the microscope for Pinworm Eggs or Taenia Eggs

ANSWERS TO QUESTIONS ❖ Why is Early Morning the BEST Time to collect Specimens? o The highest Positive Results and Greatest Number of Eggs can be detected in the morning o Specimens are best obtained a few hours after the Person has retired (10-11pm) or the first thing in the morning before bowel movement or bath

EXPERIMENT 41: KATO THICK SMEAR AND KATO-KATZ THICK SMEAR

KATO THICK SMEAR (CELLOPHANE THICK SMEAR) I. PRINCIPLES • Kato Thick Smear = Qualitative Method to detect Helminthic Infections (better than Direct Fecal Smear) • Requires 50-60mg Fresh Fecal Material so that even Light Infection can be detected • Cellophane Strips used is Soaked in Glycerine-Malachite Green Solution for 24 hours before use • This method is very useful in Mass Examination of Common Soil-Transmitted Helminthic Infecitons **Glycerine-Malachite Green Solution o Glycerine = clears the Fecal Film to visualize the Helminth Eggs o Malachite Green = Dye used to protect the eyes from Intense Light Needed in examining Thick Smear o Preparation: 500ml Distilled Water + 500ml Glycerine + 5ml 3% Malachite Green Solution in H2O **NOTE: This Method is NOT Suitable for Diarrheic Stool and CANT Detect Protozoan Cysts and Trophozoites

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II. PROCEDURE • Place 50-60mg of Stool at the Center of a Glass Slide and Cover with a Square Piece of Pre-Treated Cellophane • Using a Rubber Stopper, press the Cellophane gently to spread the Stool Specimen evenly, approximating the Circumference of the Rubber Stopper • Leave the prepared slide at room temperature for 10-20minutes – during this time, the Microscopic Field becomes clear due to the action of Glycerine on the Stool Constituents • Examine the Slide (The slide should be examined after 10-20 minutes or within 1 hour after preparation • Allowing the slide to stand for Long Periods of time will cause Drying and Shells of Hookworm Ova will become transparent and difficult to see III. ADVANTAGES VS DISADVANTAGES: A. Advantage o Time Saving, Simple, Economical o Useful in Mass Examination of Common Soil-Transmitted Helminth Infections B. Disadvantages: o Not Suitable for Diarrheic Stool o Cannot be used to detect Protozoan Cysts & Trophozoites

KATO-KATZ THICK SMEAR (MODIFIED THICK SMEAR) I. PRINCIPLES • Kato-Katz Thick Smear is a Modification of Kato-Thick Smear Method • It is a Quantitative Method for counting Helminthic Eggs • The Number of Eggs per gram (NPEG) of Feces can be computed by Multiplying the Number of Eggs Observed per Thick Smear by 24 II. PROCEDURE • Place about Half a Gram of Fecal Sample on Filter Paper • Place the wire net or screen on top of the fecal sample • Using a Flat-Sided Applicator Stick, scrape across the upper surface of the screen to sieve the Fecal Material • Place a Template on a Clean Microscope Slide

28

• • • • • • •

Transfer a small amount of Sieved Fecal Material into the hole of the Template and carefully fill the hole Remove the Template carefully so that all the Fecal Material is left on the slide Cover the Fecal Sample on the slide with a Glycerine-Malachite Soaked Cellophane Strip Invert the Slide and Press the Fecal Sample against the Cellophane gently on a Smooth Surface to spread evenly Let stand for about 20 minutes and examine Count all eggs seen in the whole preparation Multiply the Total Eggs counted by Factor 24 to express the Count as Eggs per Gram Feces (EPG) **IMPORTANT Notes: o The Wire-Mesh Net is used to Separate Fecal Material from the Large Debris which may affect the Approximate Fecal Weight and Clearing of Fecal Film by Glycerol

ANSWERS TO QUESTIONS ❖ Discuss the Advantages of Thick Smear over the Direct Fecal Smear o Thick Smear can be used to Obtain an Accurate measure of the Number of Eggs in a given amount of Sample so that the Worm Burden can be inferred o However, Adult Schistosomes take weeks to months to pass eggs into the Feces ❖ Give the Rationale of using Glycerine-Malachite Green Solution in these two procedures o It is a dye used to Dye the Smear and Protect the Eyes from the Intense Light that is required for the Microscopic Examination of the Specimen

EXERCISE 42: INTESTINAL TAPEWORMS AND FLUKES

INTRODUCTION •

Cestoda (Tapeworms) & Trematoda (Flukes) of Phylum Platyhelminthes (or Flatworms) are Exclusively Parasitic

I. SLIDES TO VIEW A. Trematodes 1. Adult, Egg  Fasciolopsis buski

29

   

Echinostoma ilocanum Clonorchis sinensis Opisthorchis sp Fasciola hepatica / gigantica

2. Adult  Fasciola hepatica  Metagonimus yokogaeai  Haplorchis taichui 3. Metacercaria B. Cestodes 1. Adult  Taenia solium  Taenia saginata 2. Egg, Scolex, Mature / Gravid Proglottid of:  Taenia solium  Taenia saginata  Diphyllobothrium latum  Hymenolepis nana  Hymenolepis diminuta  Dipylidium caninum 3. Cysticercus cellulosae II. TREMATODES (Flukes) • Have Conspicuous Suckers • Leaf-Shaped, Unsegmented, and Dorsoventrally Flattened • External Cuticle which most species may be covered with Scales, Spines, Tubercles, Ridges • They have Complex Life Cycles, involving one or more Intermediate Hosts • Transmission (Majority) = Eating Inadequately Cooked Second Intermediate Host containing the Infective Metacercariae

III. CESTODES (Tapeworms) • Endoparasitic Worms with Elongated, Segmented, and Flat Body devoid of a Body Cavity • Adult Tapeworm = Scolex (Head) + Neck + Proglottids that comprise the Strobila or Body of the Tapeworm • Each proglottid contains one or two sets of Male and Female Reproductive Organs

EXAMINATION OF SLIDES: I. TREMATODES (FLUKES)

30

A. Intestinal Flukes (Trematodes) Disease Common Name Life Cycle Definitive Host

Fasciolopsis buski Fasciolopsiasis Giant Intestinal Fluke Indirect Humans, Hogs, Dogs

1st Intermediate 2nd Intermediate Habitat Infective Stage Diagnostic Stage Morphology Testes Suckers Egg

Unique Char.

Planorbid Snails Water Plants Small Intestines Metacercaria Eggs in Feces

Echinostoma ilocanum Echinostomiasis Garrison’s Fluke Indirect Humans, Mammals, Birds Planorbid Snails Snails (Pila luzonica) Small Intestines Metacercaria Eggs in Feces

Heterophyes heterophyes Heterophyiasis None Indirect Humans, Fish-Eating Mammals Brakish-Water Snails Freshwater Fish Small Intestines Metacercaria Eggs in Feces

Metagonimus yokogawai Mentagonimiasis None Indirect Humans, Fish-Eating Mammals Snails Freshwater Fish Small Intestines Metacercaria Eggs in Feces

Dendritic Tandem Oral + Ventral Unembryonated Operculated

Lobed Tandem Oral + Ventral Unembryonated Operculated

Lobate Side by Side (Opposite) Oral + Ventral + Genital Embryonated Operculated

Oval Obliquely Side by Side Oral + Ventral + Genital Embryonated Operculated

Vitellaria Lateral to the Ceca

Surrounded by Crown of Spines

Fan-Shaped Vitellaria

B. Liver Flukes (Trematodes) Fasciola spp General Characteristics Disease Fascioliasis Liver Rot Pharyngeal Fascioliasis Halzoun

Clonorchis sinensis

Opisthorchis spp

Clonorchiasis

Opisthorchiasis

Common Name

Sheep Liver Fluke (F. hepatica) Liver Fluke (F. gigantica)

Chinese / Oriental Liver Fluke

Cat Liver Fluke

Life Cycle Infective Stage Definitive Host Diagnostic Stage 1st Intermediate 2nd Intermediate Reservoir Host Habitat

Indirect Metacercariae Man, Sheep, Cow, Deer, etc Ova in Feces Lymneid Snail Water Plants Bile Ducts & Biliary Passages

Indirect Metaceracariae Man and Dog Ova in Feces Planorbid (Operculate) Snails Freshwater Fish (Cyprinoid) None Bile Ducts & Biliary Passages

Indirect Metaceracariae Man and Dog Ova in Feces Planorbid Snails Freshwater Fish (Cyprinoid) None Bile Ducts & Biliary Passages

Leaf Shape Oral < Ventral Dendritic; Tandem Fan-Shaped Unembryonated Branched Branched Cephalic Cone / Shoulder

Oblong, Lanceolate Shaped Oral > Ventral Branched; Tandem Oval Embryonated Simple Diffuse, Irregularly Distributed Eggs smaller than C. sinensis

Oblong, Lanceolate Shaped Oral = Ventral Lobate; Oblique Oval Embryonated Simple Cluster / Compressed Follicles

Morphology Shape Suckers Testes Ovary Egg Intestinal Ceca Vitellaria Unique Feature

II. CESTODES

31

Taenia solium

Taenia saginata

Diphyllobothrium latum

Hymenolepis nana

Hymenolepis diminu ta

Dipylidium caninum

General Characteristics AKA Pork Tapeworm

Beef Tapeworm

Broad-FishTapeworm

Dwarf Tapeworm

Rat Tapeworm

Dog Tapeworm

Disease

Taeniasis

Diphyllobothriasis (Adult)

Hymenolepiasis

Hymenoleipasis

Dipylidiasis

Taeniasis (Adult)

Sparganosis (Larva)

Cysticercosis (Larva)

Habitat Larval Form

Small Intestines Cysticercus cellulosae Cysticercus Egg

Small Intestines Cysticercus bovis

Small Intestines Plerocercoid Larva

Small Intestines Cysticercoid

Small Intestines Cysticercoid

Small Intestines Cysticercoid

Cysticercus

Embryonated Egg

Cysticercoid

Cysticercoid

Gravid Proglottids Embryonated Egg

Gravid Proglottids Embryonated Egg

Plerocercoid Larva Procercoid Larva Unembryonated Egg

Embryonated Egg

Embryonated Egg

Man Only

Man Only

Man, Dog, Cat, etc

Man, Mice, Rats

Rat, Mouse, Man

Embryonated (Packets) Dogs, Cats, Man

Pig

Cattle

Fleas, Louse

Ingestion

NONE Required (Rat Fleas) Ingestion

Fleas, Beetle

Ingestion

First: Copepod Second: Fish Ingestion

Ingestion

Ingestion

Globular Armed Rostellum Four Suckers

Pyriform No Rostellum Four Suckers

Spatulate / Almond Bothria (Grooves) Grooves

Trilobed Ovary

Bilobed Ovary More Testes

Bilobed Ovary Broader > Long Rosette Like Uterus

Club-Shaped Unarmed / Rudiment Rostellum Bilobed Ovary 3-Testes

Rows of Spine Four Suckers

Mature Segment

Globular Retractile.Rostellu m Four Suckers Bilobed Ovary 3-Testes

Gravid Segment Egg

13 Branches

Embryonated

Embryonated

Embryonated

Saccular Uterus Egg Masses Embryonated

Infective Stage Diagnostic Stage Definitive Host Intermediate Host Transmission Morphology Scolex

Distinct Feature

Unembryonated Longest Tapeworm

Egg Bigger than H.nana

Pumpkin-Seed Like 2-Sets of Repro. Organ Membranous Capsules Embryonated (Packets) Rice Grain Appearance

32

ANSWERS TO QUESTIONS I. CLASSIFICATION OF FLUKES A. Classification of Flukes According to Size (Those in Bold Letters are the Intestinal Flukes) 1. Large Flukes (2-8cm)  Fasciolopsis buski (largest)  Fasciola spp 2. Medium Fluke (1-2cm)  Echinostoma ilocanum  Clonorchis sinensis  Opisthorchis spp  Paragonimus westermanii 3. Small Flukes (