BIO 100.1 Lecture Notes - Cestodes PDF

# MODULE 3: CESTODES ## CESTODES - Hymenolepis nana (Dwarf tapeworm) - Hymenolepis diminuta (Rat tapeworm) - Taenia saginata (Beef tapeworm) - Taenia solium (Pork tapeworm) - Diphyllobothrium latum (Broad fish tapeworm) - Dipylidium caninum (Double pored tapeworm) - Echinococcus granulosus (Hydatid...

# MODULE 3: CESTODES ## CESTODES - Hymenolepis nana (Dwarf tapeworm) - Hymenolepis diminuta (Rat tapeworm) - Taenia saginata (Beef tapeworm) - Taenia solium (Pork tapeworm) - Diphyllobothrium latum (Broad fish tapeworm) - Dipylidium caninum (Double pored tapeworm) - Echinococcus granulosus (Hydatid tapeworm) ## GENERAL CHARACTERISTICS: TAPEWORMS - flat bilaterally symmetrical body - no body cavity - hermaphroditic - size range: 1 mm to 20 meters or longer - parasitic in humans - The tegument is well adapted for transport functions since it is covered with numerous microvilli resembling those lining the lumen of the mammalian intestine. - The outer membrane of the cestode tegument projects out toward the host as numerous finger-shaped tubes called microtriches. - Microtriches completely cover the worm's surface including its suckers ## ADULT WORMS - flat and ribbon-like - Body plan: consists of the anterior attachment organ (scolex and neck) and a body (strobila) consisting of a chain of segments called proglottids ## Proglottids (segments): - those closest to the neck are undifferentiated; as they move caudally, each develops hermaphroditic sex organs; distal proglottids are gravid and contain eggs in a uterus; mature proglottids contain a single ovary; eggs are noted because they are visible on microscopy. ## CESTODES: TAPEWORMS ### Scolex (head) - head (anterior end) of a tapeworm bearing suckers and hooks for attachment ### Rostellum - small button-like structure on the scolex of “armed” tapeworms from which the hooks protrude - It may be retractable. ## Development of proglottids - New proglottids bud from behind the scolex. - Gravid proglottids are filled with eggs. - Eggs are sometimes released in feces but often are retained within the segment - Proglottids a few are usually shed every 2-3 days. - Hexacanth embryos (onchospheres) develop within the eggs. ## HEXACANTH EMBRYO - Hexacanth: a six hooked larva ## CESTODES: TAPEWORMS ### Diagnosis - Identifying the proglottids, eggs, or scolex. - Eggs of some tapeworms can be differentiated but not the Taenia spp. eggs. - Perianal swabs: eggs are left in the perianal skin as the gravid segments squeeze out of the anal opening ### Hymenolepis nana - The Dwarf Tapeworm - most common tapeworm infection in humans - Mode of Infection Ingestion of eggs or insects (grain or flour beetles or fleas) containing larvae. - Autoinfection - eggs remain in the intestine - Definitive hosts - man, rodents ## Diagnosis - finding eggs in feces ### Morphology - small 2 to 4 cm in length. ### Scolex - 4 sucking disks and short rostellum with hooks. ### Proglottid - are broader than long; rarely seen in feces specimens (usually disintegrate in intestine). ### Eggs - measuring 45 to 50 microns in diameter - exhibit polar filaments lying between the egg shell and the hexacanth embryo ## TREATMENT - Praziquantel (25 mg/kg) single dose ## PREVENTION & CONTROL - Rodent control - Elimination of the insect intermediate hosts - Protection of food ## Taenia spp. - two species that infect humans - Infective eggs from human feces are ingested by the cow or pig. - The onchosphere hatches and penetrates the intestinal mucosa of cow and pig. - It is delivered to various parts of body via the circulatory or lymphatic systems. - Localize and encyst in muscle. ### Infective stage - cysticercus (encysted larva) in the muscle - Human acquire the infection when uncooked or undercooked meat containing larvae is ingested. ### Scolex - attaches to human intestine - unarmed (no hooks); 4 sucking disks ### Larva - Cysticercus ( fluid filled bladder) with an invaginated scolex ### Eggs of both species are identical. ### The Beef Tapeworm - Human infection - ingestion of cysticercus (larval cyst) in flesh of cow - Size - up to 10 meters in length ### Life expectancy - 25 years or more ### Cysticercus - Ovoidal, milky white in color about 10 mm in diameter - Single scolex invaginated into a fluid-filled bladder. ### Gravid proglottids - one inch or slightly longer; uterus contains 15 to 20 primary branches. - The uterine pore is located in a lateral position. - Eggs are identical to T. solium measuring about 40 microns in diameter. ## PATHOLOGY AND CLINICAL MANIFESTATION - Common chief complaint: passage of proglottids or segments in the stool. - Non-specific symptoms: epigastric pain, weakness, weight loss, loss of appetite etc. ## TREATMENT - Praziquantel (25mg/kg) single dose ## PREVENTION & CONTROL - Treatment of infected cases - Rodent control - Food must be properly stored and protected from grain beetles. ## Hymenolepis diminuta - The Rat Tapeworm - a common parasite of rats, mice and other rodents - Mode of infection - ingestion of intermediate host (grain weevil) containing larval stages. - Grain weevils - most important intermediate host. - Infection is often due to accidental ingestion of grain beetles infesting dried grains, dried fruits, flour, and cereals. ### Morphology - larger than H. nana (adult as well as egg). - Size - adults measure 20 to 60 cm in length. ### Scolex - exhibits no hooks; larger than that of H. nana. - Eggs measure 60 to 80 microns in diameter and do not exhibit polar filaments. ## PATHOLOGY AND CLINICAL MANIFESTATION - Minimal and non-specific ## Diagnosis - finding eggs in feces ### Morphology - small 2 to 4 cm in length. ### Scolex - 4 sucking disks and short rostellum with hooks. ### Proglottid - are broader than long; rarely seen in feces specimens (usually disintegrate in intestine). ### Eggs - measuring 45 to 50 microns in diameter - exhibit polar filaments lying between the egg shell and the hexacanth embryo ## TREATMENT - Praziquantel (25 mg/kg) single dose ## PREVENTION & CONTROL - Rodent control - Elimination of the insect intermediate hosts - Protection of food ## Taenia spp. - two species that infect humans - Infective eggs from human feces are ingested by the cow or pig. - The onchosphere hatches and penetrates the intestinal mucosa of cow and pig. - It is delivered to various parts of body via the circulatory or lymphatic systems. - Localize and encyst in muscle. ### Infective stage - cysticercus (encysted larva) in the muscle - Human acquire the infection when uncooked or undercooked meat containing larvae is ingested. ### Scolex - attaches to human intestine - unarmed (no hooks); 4 sucking disks ### Larva - Cysticercus ( fluid filled bladder) with an invaginated scolex ### Eggs of both species are identical. ### The Beef Tapeworm - Human infection - ingestion of cysticercus (larval cyst) in flesh of cow - Size - up to 10 meters in length ### Life expectancy - 25 years or more ### Cysticercus - - Ovoidal, milky white in color about 10 mm in diameter - Single scolex invaginated into a fluid-filled bladder. ### Gravid proglottids - one inch or slightly longer; uterus contains 15 to 20 primary branches. - The uterine pore is located in a lateral position. - Eggs are identical to T. solium measuring about 40 microns in diameter. ## PATHOLOGY AND CLINICAL MANIFESTATION - Common chief complaint: _passage of proglottids or segments in the stool._ - Non-specific symptoms: _epigastric pain, weakness, weight loss, loss of appetite etc._ ## TREATMENT - The first line of choice is _praziquantel (5 to 10 mg per kg)_ single dose for both adults and children. - Criteria for cure include the ff: - recovery of the scolex - negative stool examination 3 months after treatment ## PREVENTION & CONTROL - Consumption of thoroughly cooked beef. ## Taenia solium - The Pork Tapeworm - Man is the only definitive host. - Infection - ingestion of cysticercus in flesh of swine - Size - up to 7 meters in length. - Life expectancy - 25 years or more. - Scolex - armed, with 4 sucking disks. - Gravid proglottid has 7-13 primary uterine branches. - Eggs are identical to T. saginata measuring about 40 microns in diameter. ## PATHOLOGY AND CLINICAL MANIFESTATION - Intestinal infection results in mild and non-specific abdominal complaints. - Most serious manifestation is neurocysticercosis. - Most serious zoonotic disease worldwide - cysticerci containing scolex can be found in the brain ## TREATMENT - The drugs of choice are praziquantel and niclosamide. - Cysticercosis (praziquantel 50 to 75 mg/kg) ## PREVENTION & CONTROL - Thorough cooking of meat - Freezing at -200C for 10 days kills the cysticerci Sanitary inspection of slaughtered pigs, cows and cattle. ## Diphyllobothrium latum - The Broad Fish Tapeworm - Two intermediate hosts are required: copepods (water flea) and fish - The infective stage is the plerocercoid larva which is ingested in raw or undercooked fish. - Larvae attach to the mucosa lining of the small intestine and develop into adults. ## Diagnosis - usually based on identification of gravid proglottids - Gravid proglottids are the size of rice grains and exhibit a genital pore on each side. - Eggs - contained within packets (up to 12 eggs/packet). ## Pathogenesis and Clinical Manifestations - Slight intestinal discomfort - Epigastric pain - Diarrhea ## Treatment - Praziquantel (5 to 10 mg/kg) single dose ## Echinococcus granulosus - The Hydatid Tapeworm - Adults are only in canines. Eggs are shed in the feces of infected animals. - Humans accidentally ingest eggs from close contact with an infected animal or from canine feces. - Hexacanth embryo penetrates the intestinal mucosa and migrates to tissues. - The hexacanth develops into the larval stage (hydatid cyst) in the tissue (liver, lung or brain, most often). - Adult worm - small, consists of only 3 segments. ### Hydatid cyst - a thin walled larva containing many thousands of invaginated scoleces. ## Major pathology - hydatid cysts in the brain and other organs (liver, lungs) ## Diagnosis - Radiographic findings - ELISA ## Treatment - Surgical management of the hydatid cyst (scolicidal agent - hibitane, 95% ethanol or 30% hypertonic solution to kill the cysts) - Albendazole (400 mg twice daily for one to six months) # MODULE 4 - HELMINTH INFECTION DIAGNOSIS ## PART I. ROUTINE TECHNIQUES ## LABORATORY DIAGNOSIS - Accurate diagnosis of parasitic infections can help decrease the prevalence and incidence of a parasitic infection. - A parasitology laboratory should be able to: - confirm a clinical impression that the condition has a parasitic nature - rule out a diagnosis - aid a clinician in the choice of proper medication - help in monitoring the effect of a treatment regimen - The ability of a parasitology laboratory to generate reliable results is dependent on the following: - Proper collection, handling and processing of the specimen - Skill of the laboratory analyst - Quality of the equipment ## Presumptive diagnosis - identifies the likely condition of a patient ## Definitive diagnosis - final diagnosis that is made after getting the results of tests ## Parasite Diagnosis - Demonstration of parasites - Adults - Eggs - Larvae - Cysts - Oocysts - Trophozoites - Detection of host immune response (antibodies and antigens) ## Specimens for parasitic examination - Stool - Blood - Sputum - Cerebrospinal fluid -Tissue aspirate - Tissue biopsies - Orifice swabs ## Stoolor Fecal Sample - Best collected in clean, wide-mouthed containers made of waxed cardboard or plastic with a tight-fitting lid to ensure retention of moisture and to prevent accidental spillage. - The stool specimen should be submitted with the following information: - patient's name - age - sex - date/time of collection - requesting physician ## ROUTINE TECHNIQUES ## DFS (Direct Fecal Smear) - Routine method of stool examination which uses an approximately 2 mg of stool. - Primarily useful in the detection of motile protozoan trophozoites - Trophozoites can be stained to demonstrate the nuclear morphology using the Nair's buffered methylene blue solution (BMB). - Entamoeba cytoplasm will stain pale blue and the nucleus darker blue. - Protozoan cysts can also be observed. - A weak iodine solution can be used as a temporary stain to demonstrate nuclei. - The cytoplasm will stain golden yellow, the nucleus will be pale and the glycogen will be deep brown. - Helminth eggs and larvae can also be detected using this preparation. ## DFS: SALINE PREPARATION - a. Saline Preparation. - Place one drop of physiological saline on a microscope slide. - Take a small quantity of feces on one end of a toothpick and stir into the saline to produce a translucent suspension. - Apply a cover slip and examine under a microscope. - b. Iodine Preparation. - Place one drop of Lugol's iodine solution instead of saline solution on a slide. - Take up a bit of feces on the end of a toothpick and stir into the iodine to form a thin suspension. - Apply a cover slip and examine. Observe the portions that are colored by the iodine. - Or, a drop of iodine can also be added to the edge of the coverslip of a previously examined saline mount. - The iodine will diffuse into the stool-saline mixture, kill the organisms, and stain the cellular elements. ## KATO THICK SMEAR - About 50 to 60 mg of stool is placed over a glass slide and covered with cut cellophane soaked in a mixture of glycerine and malachite green solution. - Glycerine is a clearing solution. - Malachite green is used to give color to the cellophane in order to give a pale green background to the eggs and to minimize the brightness of the microscopic field. - Green cellophane may be used if malachite green is not available. - The preparation is best examined within 10 to 20 minutes. - Useful in mass stool examinations (detecting STH eggs) - If the preparation is kept too long before examination, hookworm eggs are distorted making identification very difficult. - Usefulness is limited if stools are diarrheic or watery. - It is not able to detect protozoan cysts and trophozoites. - Uses a measured amount of stool which has been sieved through a wire mesh and pressed under cellophane paper soaked in glycerine- malachite green solution. - Uniform amount of stool is examined through the use of a template with a uniform-sized hole in the middle. - All eggs are counted from the whole preparation. - The total egg count is multiplied with a factor depending on the amount of stool used. - Consistency of the stool-main determinant for the sensitivity of this technique. - Drier stools yield higher egg counts than moist ones. - The technique can only be done on fresh formed stools and not on liquid and preserved samples. ## Kato-Katz Method or the Cellophane Covered Thick Smear - Useful for assessing the intensity of infection in schistosomiasis and common soil-transmitted helminthiasis like ascariasis, trichuriasis and hookworm infection. ## Ascaris lumbricoides fertile egg - Characteristics of fertile egg: - Round - thick shell and outer mammilated albuminous layer (corticated egg) - Size: length: 45-60 µm width: 35-50 μm - Characteristics of unfertile egg: - longer and narrower - thin shell - irregular mammilated coating - Size: length: 88-94 µm width: 39- 44 μm with refractile granules ## CONCENTRATION - Applicable in cases of light infections and if there is a need to recover more parasites - Based either on sedimentation or flotation techniques. - Sedimentation: The parasite has a higher specific gravity than the reagent. - Flotation: The parasite has a lower specific gravity and will float to the surface of the preparation. ## Sedimentation Procedures ### ACID ETHER CONCENTRATION TECHNIQUE (AECT) - Main reagents: - 40% HCI dissolve albuminous material - Ether- dissolve neutral fats in stool - Recommended for the recovery of Trichuris, Capillaria, Schistosoma, other trematode eggs - Can be used in processing stools from cats and dogs - Drawback: loss of parasite to the plug of debris and possible destruction of protozoan cysts ### FORMALIN ETHER CONCENTRATION TECHNIQUE - Main reagents: - 10% formalin: all purpose fixative - Ether: dissolve neutral fats in stool - Useful in the recovery of both helminth eggs and protozoan cysts - Parasite morphology is preserved in formalin. - Sediments from FECT can be stored for a long period of time. - Ether: flammable compound - Ethyl acetate: may be used in place of ether. However, it is not as efficient as ether in the extraction of fat or mucoidal material from the stool. ## FLOATATION PROCEDURES ### Zinc Sulfate (ZnSO4) Flotation - Main reagent: 33% ZnSO4 - Ideal specific gravity: 1.18 to 1.20 - If parasites are exposed to high specific gravity, distortion and shrinkage of protozoan cysts and thin-walled nematode eggs may occur ### Brine Flotation - Makes use of a saturated table salt solution - Stools are directly mixed with the brine solution - There is no need for centrifugation since helminth eggs rise to the surface of the solution. - Low cost and simple - Hookworm eggs and Schistosoma become shrunken - Not useful for operculated eggs like Clonorchis, Opisthorchis, and heterophyids because these do not float in brine solution ### Sheather's Sugar Flotation - Boiled sugar solution preserved with phenol is used in this method. - Recovery of coccidian oocysts (Cryptosporidium, Cyclospora and Isospora) # MODULE 4 - HELMINTH INFECTION DIAGNOSIS ## PART I. SPECIAL TECHNIQUES ## Stool Culture Method - nematode species identification ## HARADA-MORI CULTURE (Test Tube Culture Method) - purpose of culturing and recovering nematode larvae (Strongyloides stercoralis, hookworm) - Stools for culture should not be refrigerated. ## Strongyloides stercoralis - nematode (roundworm) causing strongyloidiasis - Males grow only to about 0.9 mm (0.04 in) in length. - Females can grow from 2.0 to 2.5 mm (0.08 to 0.10 in) in length. - free-living rhabditiform and parasitic filariform stages ## Autoinfection - Rhabditiform pass down the large intestine and develop into filariform. - Filariform invade the mucosa and enter the circulation to start another parasitic cycle without leaving the body of the host. - Explains how some people remain infected for more than 30 years (Vietnam war veterans who returned to US). ## Perianal Swab - used to recover eggs of Enterobius vermicularis and Taenia spp. ## Enterobius vermicularis - human pinworm - causes enterobiasis or oxyuriasis (familial disease) - characterized by perianal itching or pruritus ani ## EPIDEMIOLOGY ### Philippines - PR 29% among schoolchildren from exclusive private schools and 56% among those from public schools - eggs collected from fingertips and under fingernails of school children ## MODE OF TRANSMISSION - Route of infection is through the mouth - Respiratory system (inhalation of dust containing Enterobius eggs) - Anus - hatched larvae enter the anus and cause retroinfection when they go back into the large intestine - Eggs contaminate underwear and beddings. ## EGG - Elongated, ovoid, flattened on the ventral side giving an appearance similar to a letter "D". - Size: length: 50-60 µm width: 20-30 µm - Translucent shell - consists of outer triple covering - albuminous (mechanical protection) - inner embryonic lipoidal membrane (chemical protection) ## ADULT WORMS - found in the lower ileum and cecum - Gravid female worms migrate down the intestinal tract - Exit through the anus to deposit eggs on the perianal skin - Single female lays eggs per day with an average of 11, 105 eggs - migrate to the perianal area even during daytime but more migration occurs in the evening hours ## CLINICAL MANIFESTATION - Intense itching of the perianal region leads to scratching and secondary bacterial infection. - Migration of egg-laying females to the anus causes irritation of the perianal region. ## DIAGNOSIS - Graham's scotch adhesive tape swab - Eggs are collected from the perianal area instead of from the feces. - Finding adult worms or eggs on microscopic examinations. ## Adult worms may be seen in the feces or in the perianal region. ## TREATMENT - Pyrantel pamoate (10 mg/kg) - Albendazole (400 mg) - Mebendazole (500 mg) - Cure may be considered only after seven perianal smears using scotch tape method are found negative. ## PREVENTION & CONTROL - Personal cleanliness and personal hygiene. - Fingernails should be cut. - The use of showers rather than bathtubs is suggested and infected persons should sleep alone. - Underwear, night clothes etc. should be boiled - Familial disease (chemotheraphy of the entire family) ## Perianal Swab - Enterobius gravid female migrates out through the anus at night time, deposits eggs on the perianal skin - Taenia spp. gravid segments can crawl out of the anus and in the process, ova are squeezed out of the segment and are deposited on the perianal skin. ## Adult worms - flat and ribbon-like - Body plan: consists of the anterior attachment organ (scolex) and a body (strobila) consisting of a chain of segments called proglottids ## CELLULOSE TAPE OR SCOTCH ΤΑΡΕ ΜΕΤHOD - performed by sampling the perianal skin using a strip of cellulose tape adapted over a glass slide. - Sticky side is applied to the skin - Specimen collection: early in the morning before the patient has taken a bath or before the patient has washed the perineum - Collected specimens are examined under the microscope for the presence of eggs or the adult Enterobius. - A drop of toluene or xylene helps in the visualization of eggs. ## STAINING OF STOOL SPECIMEN - Examination of the nuclear characteristics of amoebae: - Useful in the identification of other intestinal protozoans like Balantidium and Giardia. - Stains used: - Trichome - Iron-Hematoxylin - Chlorazol Black E ## Acid-fast staining - Coccidian oocyst: Cryptosporidium - Oocysts in clinical specimens may be difficult to detect without special staining - requires spreading of a thin layer of stool on a glass slide either from fresh or concentrated specimens - Parasite stains stain pink to red with blue or green background - Materials: - Reagents may be purchased commercially or prepared in the laboratory. ## Cryptosporidiosis: Cryptosporidium spp. - Cryptosporidium parvum and C. hominis - leading causes of human cryptosporidiosis - cause of severe diarrhea in immunocompromised individuals ## Examination of sputum - First morning specimen is considered the best specimen to examine - 10% sodium chloride or hydrogen peroxide (inductants) may increase the amount of sputum collection - Parasites recovered from sputum: - Migrating larvae of Ascaris lumbricoides, Strongyloides stercoralis, hookworms ## Sputum examination: - oval in shape - yellowish to brown in color - thick- shelled (80 to 118 µm by 48 to 60 μm) - Flattened but with prominent operculum. - Opposite to the operculum is a thickened abopercular portion. ## Examination of sputum - Echinococcus granulosus - affects the liver and lungs of infected host - Hydatid Tapeworm - Humans accidentally ingest eggs from close contact with an infected animal or from canine feces. - Microscopic examination - wet mount using saline or iodine is useful when searching for protozoan trophozoites - Sputume concentration - 3% NaOH added, centrifuge Supernate is discarded and the sediment is examined. ## Examination of urine - Parasite recovery: collected first thing in the morning - Specimen for diagnosis of Trichomonas vaginalis, Schistosoma haematobium - Sample is centrifuged and the sediment is studied under the microscope. ## Examination of Tissue Biopsy Material ### Muscle Biopsy - diagnosis of Trichinella spiralis infection - small pieces of muscles are pressed between two glass slides and the preparation is examined under the microscope ## Life cycle ### Encysted larvae in the muscle fibers - infective stage - Humans acquire the infection through ingestion or raw or insufficiently cooked meat (pork). - Cysts are digested in the stomach. - Larvae excyst either in the stomach or small intestine. - Larvae penetrate the mucosa, pass through the lymphatic system into the circulation and into the striated muscles. - The larvae grow and develop in the muscles. - After three weeks, the larvae start to coil into individual cysts. # MODULE 5 - ACANTHOCEPHALANS ## Acanthocephalans - Acanthocephalas (also known as spiny- or thorny-headed worms) are common parasites of wildlife and some domestic animal species, but they rarely infect humans. - Species recovered from humans include Macracanthorynchus hirudinaceus, Macracanthorynchus ingens, Moniliformis moniliformis, Acanthocephalus rauschi, Pseudoacanthocephalus bufonis, Corynosoma strumosum, and Bolbosoma sp. - M. hirudinaceus and M. moniliformis are the most common species implicated in human infections. ## Natural definitive hosts - include rats (Moniliformis moniliformis), swine (Macracanthorynchus hirudinaceus), and raccoons (Macracanthorynchus ingens) - The insect intermediate host varies by species but is usually scarabaeoid or hydrophilid beetles for M. hirudinaceus and likely M. ingens, and beetles or cockroaches for M. moniliformis. ## Geographic Distribution - Cases of acanthocephaliasis more commonly occur in areas where insects are eaten for dietary or medicinal purposes or in children who consume insects. - Macracanthorynchus hirudinaceus is found wherever wild or domestic swine occur. - Macracanthorynchus ingens is highly endemic in raccoons from the southeastern United States, and the recorded human cases originate from Texas and Florida. - The distribution of Moniliformis moniliformis is not known but is likely cosmopolitan. ## Clinical Presentation - Clinical symptoms of acanthocephaliasis are often severe, due in part to the mechanical damage caused by the insertion of the armed proboscis into the lumen of the host's intestine. - Symptoms generally include abdominal pain and related digestive complaints. However, low-intensity or early infections may be asymptomatic. ## Acanthocephalans - parasitic, spiny or thorny headed worms - necrotrophic - prominent characteristic of acanthocephalans is a protrusible proboscis armed with recurved hooks - proboscis, neck, trunk ### Proboscis - armed with hooks - worm attaches to the wall of the intestine - The proboscis is hollow and fluid-filled. - Attached to its inner apex is a pair of muscles, called proboscis retractor muscles, which extend the length of the proboscis. - From the circular canal two sac-like projections called the lemnisci run into the cavity of the body. ### Life cycle - - Each species of Acanthocephala uses at least two hosts in its life cycle. - The eggs contain a fully developed acanthor and ingested by an insect intermediate host. - The acanthor molts into a second larval stage, called an acanthella inside the body cavity of an insect until it reaches the infective stage called a cystacanth. ## Research work - A case of Moniliformis moniliformis (Acanthocephala) infection in Iran - A case of Moniliformis moniliformis (Acanthocephala) infection in Iran - Objective of the study: To present a report on human infection of M. moniliformis in 2 year old girl in Iran. ## Clinical symptoms and treatment - Symptoms: abdominal pain, diarrhea, vomiting, facial edema - Treatment: Levamisole, clinical symptoms reduced in 2 weeks # MODULE 6 - DIAGNOSTIC ASSAYS AND ADVANCES IN HELMINTHOLOGY ## Helminth Diagnostic Assays - foundation of a successful health care system, providing critical information that health care providers and patients need to make the right medical conditions - Diagnostic Assays - Immunological Diagnostic Procedure ELISA (Enzyme-linked immunosorbent Assay) - Nucleic Acid Diagnostic Systems Types of polymerase chain reaction ## Immunodiagnosis - These methods either detect antigen or antibodies in clinical specimens. - The use of Enzyme-Linked Immunosorbent Assay (ELISA) can supplement the microscopic examination. - The antibody detection assays use either whole helminths from in vivo and in vitro cultures or recombinant and soluble crude extracts as antigens. - Using a mixture of antigens increases the detection of helminth antibodies such as in Cocktail ELISA. ## Development of Diagnostic Tests - Prevention, control, treatment of helminth infection - early detection and accurate identification of the causative helminth organism - Pathogenicity of helminth cannot be determined by microscopy - limits the detection of parasitic disease-causing organism - Molecular diagnostic procedures using either immunological or DNA detection methodologies have been devised ## Molecular diagnostics in Helminthology - detect and measure the presence of genetic material or proteins associated with a specific health condition or disease - foundation of a successful health care system providing information that health care providers and patients need - provide objective, quantitative measurements- prevention, detection, diagnosis, treatment, and successful management of health conditions ## Molecular diagnostic: Detection strategy ### Specific - assay must yield a positive response for only the target organism or molecule <h3> Sensitive - Diagnostic test must identify very small amounts of the target organism even in the presence of other potential interfering potential organism <h3> Simple - simplicity required for the test to run efficiently, effectively, and inexpensively on a routine basis - can be used in the field ## Molecular Diagnosis - Polymerase Chain Reaction (PCR) based assays are available in helminth infection diagnosis. - This tool was developed to detect nucleic-acid sequences specific to the helminth in question. - This molecular assay can detect and measure the presence of genetic material or proteins associated with specific helminth infections. ## PCR-based assay - uses two oligonucleotide primers which flank the helminth target sequence - Taq polymerase which amplifies the target gene of interest - successive cycles of DNA denaturation and annealing of primers - DNA sequencing follows to analyze the genetic composition of the helminth gene. ## Comparison of some of the methods used to diagnose helminth infection - Microscopic examination - Advantages - Simple - direct detection of helminth - differentiates morphologically distinct organisms - Disadvantages - slow, laborious and tedious - low sensitivity - cannot discriminate between similar organisms - requires a high skill level ## Microscopy - Microscopy as a tool to diagnose helminth infections remains the gold standard in diagnosis. - It is simple, informative, and can be rapid for a definitive diagnosis of helminth infections. - However, microscopic detection of helminths may be limited by the low density in the clinical specimen examined. It may also be affected by the stage of the disease and the biology of the helminth. - Helminth concentration techniques and culture methods are useful alternatives in helminth infection diagnosis. - Consequently, these techniques are highly dependent on an experienced microscopist for accurate identification of the helminth agent. - Microscopic examination on the part of a well-trained technician can also be labor intensive when a lot of samples need to be examined in a given period of time such as during epidemiologic investigations. ## Detection of antibodies in serum - Advantages - simple and fast - can be used to screen a large number of samples - Disadvantages - not always specific - does not distinguish between past and current infections ## PCR - Advantages - fast, sensitive, and specific - detects helminth directly - can distinguish different species - Disadvantages - expensive and multistep - possible false positives and false negatives ## ELISA (Enzyme-linked Immunosorbent Assay) - immunological test used in detecting helminth infection - Example: NTD's (Neglected Tropical Diseases) Schistosomiasis endemic in the Philippines. - procedure may be either indirect or direct ## Example application: - Development and optimization of cocktail-ELISA for a unified surveillance of zoonotic schistosomiasis in multiple host species ## Schistosoma japonicum recombinant antigens - A recombinant antigen is an antigen that has been artificially reproduced. - The most common method to make a recombinant antigen is to insert the genetic code for the antigen into E. coli; the bacteria produce the foreign protein which is then purified from the culture broth. ## Nucleic Acid-Based Diagnostic Techniques - Detect specific nucleic acid (DNA or RNA) - In the case of infectious diseases, nucleic acid-based diagnostics detect DNA or RNA from the infecting organism - Advantages - High sensitivity - Earlier detection of infections - Quantification - Species differentiation - Disadvantages - Can be time consuming - Expensive - Extensive training required - Requires cross contamination provisions - ## Nucleic acid based diagnostic test: ### Polymerase Chain Reaction - Types of PCR - Singleplex PCR - Multiple PCR - Advantages - Quantitative - Single-Step PCR versus Multiplex - Singleplex PCR - require a single primer in the amplification of target DNA - Multiplex PCR - require many primers to amply multiple targets in the DNA - Real-time (qPCR) - Quantitative qPCR - Quantification of target DNA using fluorescent dyes - Visualization and simultaneous detection of DNA - more precise than the normal