Viral and Parasitic Diarrhoea

Viral and parasitic diarrhoea Introduction  Diarrhoea is defined by WHO as having 3 or more loose or liquid stools/day or as having more stools than normal for that person. - Increase in frequency, fluidity or amount. - Increase in daily stool weight >200 gm. Epidemiology  Worldwide, about 1.7 to 5 billion cases of diarrhoea occur per year, with 1.26 million deaths; accounting for the second leading cause of death globally.  More common in developing countries, where young children get diarrhoea on an average of three times a year.  Can be classified as -  On basis of duration- i. Acute - <14 days ii. Persistent- >14 days iii. Chronic- > 4 wks  On basis of pathogenicity- i. Non-inflammatory ii. Inflammatory iii. Penetrating Viral agents causing diarrhea • Rotavirus • Calciviridae: • Norovirus • Sapovirus • Adenovirus • Astrovirus • Other viruses Parasitic Diarrhoea Protozoa Cestodes Trematodes Nematodes Entamoeba histolytica Taenia solium Fasciolopsis buski Trichinella spiralis Giardia lamblia Taenia saginata Clonorchis sinensis Trichuris trichiura Cryptosporidium parvum Taenia saginata asiatica Paragonimus westermani Strongyloides stercoralis Cyclospora cayetanensis Metagonimus yokogawai Ankylostoma duodenale Isospora belli Gastrodiscoides hominis Necator americanus Balantidium coli Schistosoma japonicum Trichostrongylus spp. Agents and their underlying mechanism Mechanism Non-inflammatory Virus Rotavirus Norovirus Enteric adenovirus Inflammatory Parasite Giardia lamblia Cryptosporidium species Cyclospora species Microsporidia Entamoeba histolytica Balantidium coli Agents causing traveller’s diarrhoea- (50-75% bacterial) Viral (<20%) Norovirus(<10%) Rotavirus Parasites (0-10%) Giardia lamblia, Cryptosporidium , Entamoeba histolytica, Cyclospora VIRAL DIARRHOEA ROTAVIRUS  Reoviridae  60-80 nm in size, Icosahedral symmetry Epidemiology  Single most important cause of gastroenteritis in young children.  Worldwide, about 3-5 million cases/year; nearly 1 million deaths.  Most common group causing diarrhoea is group A and type is G1P[8]. Pathogenesis  Destroy the mature enterocytes in the villi of proximal small intestine.  Gastric and colonic mucosa are spared.  Multiply in cytoplasm and destroy their transport mechanism.  NSP4 act as enterotoxin and induces secretion by altering epithelial cell function and permeability.  Damaged cells may slough into intestinal lumen and release viruses in feces.  Viral excretion usually lasts 2-12 days, but may be prolonged in patient with poor nutrition or HIV. Transmission of rotavirus  Feco- oral  Poor hygiene Clinical presentation(winter vomiting disease)  IP- 1-3 days, abrupt onset.  Vomiting followed by watery diarrhoea.  Fever and abdominal pain. Adenovirus  2nd most common viral agents of endemic diarrheal illness of infants and young children.  Space vehicle shaped appearance.  Type 40 and 41 are associated with diarrhoea. Calciviridae  27-40 nm in size with icosahedral symmetry.  ss positive sense RNA  2 genus: Norovirus and Sapovirus Norovirus Sapovirus Norwalk virus Sapporo-like virus Most important cause of epidemic viral Sporadic cases and occasional outbreaks gastroenteritis in adults of diarrhea in infants, young children and elderly Food born illness: contaminated food and water, shellfish Milder disease than Norwalk virus Astroviruses  28-30 nm size,Icosahedral symmetry, ss positive sense RNA.  Serotype 1 is most common (7 serotype)  Causes sporadic cases and occasional outbreaks of diarrhoea in all age. Other viruses  Among respiratory viruses-SARS corona virus, Influenza A/H5N1, Influenza A/ H1N1( the 2009 pandemic strain)  Picornavirus: polio Laboratory diagnosis COLLECTION OF SPECIMEN Feces collected early in the illness is most ideal specimen. - Avoid using a container with preservatives, metal ions, detergents, or serum. Rectal swabs may be collected by the physician, nursing or laboratory staff.  Transport feces or swab to the Microbiology laboratory immediately.  If transport is to be delayed, the specimen may be refrigerated up to 24 hours at 2-8°C. If the delay is greater than 24 hours, the specimen must be frozen at -20°C or colder until tested. Direct detection of virus  Electron microscopy(EM): • Rota virus- wheel shaped • Adeno virus- space vehicle shaped • Astrovirus-star shaped peplomers  Immunoelectron microscopy(IEM): after adding specific antiviral antibodies. Virus isolation- for adeno virus primary human embryonic kidney cell line and A 549 cell line are most susceptible. Isolation of other viruses are difficult. Detection of viral antigen: ELISA and Latex agglutination RT-PCR: most sensitive Antibody detection: ELISA Typing methods: RNA sequence typing and neutralization test Vaccination for rotavirus  Rotashield vaccine –(1999 )Withdrawn because of its association with intussusception .  Current regimen: ROTARIX ROTATEQ ROTAVAC Monovalent Pentavalent Monovalent 2 doses, 6 and 10 weeks 3 doses, 6,10 and 14 weeks 3 doses, 6 ,10 and 14 weeks Live attenuated Live attenuated Live attenuated oral oral oral Parasitic diarrhoea Entamoeba histolytica • 3rd most common parasitic cause of death worldwide. • In India prevalence rate is around 15%. • 3 stages-trophozoite, precyst and cyst. • Intestinal amoebiasis may manifest as amoebic dysentery, appendicitis, amoeboma and fulminant colitis. Giardia lamblia • Intestinal flagellate. • In India prevalence rate ranges from 3-32% • 2 stages-trophozoite and cyst. • Diarrhoea is often foul smelling with fat and mucus but no blood. • Chronicity leads to recurrent episodes of foul smelling diarrhoea, foul smelling flatus, sulfurous belching with rotten egg taste and weight loss. Cryptosporidium parvum, Cyclospora cayetanensis, Isospora belli • Intestinal coccidian parasites. • Causes self limiting diarrhoea in immunocompetent healthy individuals • Opportunistic pathogen in immunocompromised patients causing chronic persistent life threatening diarrhoea. • Other less common protozoans are Dientamoeba fragilis, Sarcocystis, Microsporidia, Blastocystis hominis, Balantidium coli etc. Tinea solium and saginata • Definitive host- man • Intermediate host- pig/cattle • Caused by ingestion of contaminated pork/beef. • Intestinal taeniasis cause change in bowel habit (constipation/diarrhoea) • Obstruction by migrating proglottids can lead to appendicitis or cholangitis. Fasciolopsis buski • Largest and most common intestinal fluke. • Light infection cause dysentery while severe infection may result in obstruction of intestinal tract causing malabsorption and protein losing enteropathy with profuse yellowish green stool. Trichuris trichiura (whip worm) • Large intestinal nematode. • Causes Trichuris dysentery syndrome. Small intestinal nematodes  Hookworm • A.duodenale - old world hook worm • N.americanus- new world hook worm • Cause inflammatory diarrhoea, chronic infection may lead to iron deficiency anemia and PEM. Other less common helminths are Diphyllobothrium latum, Clonorchis sinensis, Heterophyes heterophyes, Mentagonimus yokogawai, Gastrodiscoides hominis,Trichinella spiralis, Strongyloides stercoralis etc. Laboratory Diagnosis of Parasitic Diarrhoea  Sample collection  Macroscopic examination  Microscopic examination  Concentration techniques  Culture techniques  Immunodiagnostic methods  Molecular methods Sample Collection  STOOL • Wide mouthed, clean, leak-proof, screw capped containers. • TIMING- before starting antiparasitic drugs. • FREQUENCY- At-least 3 stool specimens collected on alternate days(3rd specimen after purgatives). • WHEN TO EXAMINE- Liquid stool between 15 to 30 minutes, semisolid within 1 hour and formed stool upto 24 hrs after collection • Several preservatives like 10% formalin or poly vinyl alcohol can be used to maintain morphology of cysts and eggs. Macroscopic Examination  Mucoid bloody stool: Acute amoebic dysentery, Intestinal schistosomiasis, Invasive balantidiasis.  Frothy pale offensive stool: Giardiasis  Dark red stool: upper GIT bleeding  Bright red stool: lower GIT bleeding  Presence of worm Microscopic Examination  DirectWet Mount Preparation • Saline Preparation • Lugol’s Iodine Preparation • Buffered Methylene Blue Preparation • Eosin solution Preparation  Staining Preparations • Trichrome Staining • Iron hematoxylin stain • Modified ZN Staining Direct wet mount • Examined under low power objective (10X), followed by high power objective (40X). • Following structures are visualised Normal constituents- plant fiber, starch cells, animal hair, pollen grain, fat globules, air bubbles etc.  Cellular elements like pus cells (inflammatory diarrhoea),RBC (dysentery)  Charcoat leyden crystals :They are breakdown products of eosinophils. seen in amoebic dysentery, Isospora belli etc.  Trophozoites and cyst of protozoa and eggs and larvae of helminths.  Saline mount: useful in detection of trophozoites and cysts of protozoa, and eggs and larvae of helminths • Motility of trophozoites and larvae can be demonstratred. • Bile staining property can be appreciated.  Iodine Mount: nuclear details of cysts, helminthic eggs and larvae are visualized, helps in species identification. • Disadvantage- motility of trophozoites and larvae is lost and bile staining property cannot be appreciated. Parasitic forms seen in fecal sample Parasite and its form Features Entamoeba histolyticatrophozoite 15-20 µm, motile, single spherical nucleus Entamoeba histolytica- cyst Spherical, 12-15µm, 4 nuclei with compact centrally located karyosome. Immature cyst have chromatid bars and glycogen vacoule. Giardia lamblia- trophozoite 10-20 µm in length and 5-15 µm in width, pear shaped with tapering ends, falling leaf like motility, 2 nuclei, 2 adhesive discs,4 pairs flagella Giardia lamblia- cyst Oval,11-14 µm in length and 7-10 µm in width, 4 nuclei and remnants of axonemes present Image Parasite & its form Features Hookworm egg Oval, 60×40 µm, non bile stained, segmented ovum, empty space b/w shell and blastomeres. Trichuris trichiura egg Elongated,barrel shaped, 50×20 µm, mucus plug present at poles, contains unembryonated egg. Taenia egg Spherical, 30-40µm, thick shell lined by prominent radial striations, embryonated oncosphere possess 3 pairs of hooklets. Fasciolopsis buski egg Large (130-140 µm×80-85 µm) Operculated bile stained eggs Diphyllobothrium latum egg Oval,70×50 µm, operculated Image Relative sizes of parasitic eggs  Buffered Methylene Blue Preparation : • Stains only trophozoites of amoeba. • It does not stain amoebic cyst or trophozoites and cyst of flagellates. • Nucleus and the inclusions such as RBC or yeast cells stain dark blue. • Cytoplasm stains light blue.  Eosin Wet Preparation : • Detection of trophozoites and cyst. • They can be much more easily detected against the pink-red background of eosin preparation. Permanent stained smear  Accurate diagnosis of cysts and trophozoites by staining internal structures. This stain is not recommended for the detection or visualization of helminth eggs and larvae. 1. Iron hematoxylin stain: Organisms - bluish or greyish with black nuclear structures. Background - pale blue-grey colour. 2. Trichrome stain: Cytoplasm of protozoan trophozoites - blue green colour. Cysts - purple. Nuclei and Charcot-Leyden crystals- red colour. Background - green.  Modified acid fast stain: used for identification of oocyst of Cryptosporidium parvum, Cyclospora caytenensis and Isospora belli. Oocyst will stain pink to deep red colour against a blue background.  Direct fluorescent antibody staining: gold standard test for cryptosporidiosis. Oocyst are detected using fluorescent labelled monoclonal antibody directed against cyst wall antigen. • Oocyst of Cyclospora have property of Auto- fluorescence • Oocyst of Isospora belli are detected using fluorescent stained smear by auramine-rhodamine stain. Concentration technique • Used to increase detection rate when parasite concentration is low in stool specimen. • Mainly performed to differentiate parasitic eggs from debris. • It makes eggs more visible by removing organic and inorganic elements of stool. • Eggs, cyst and larvae are recovered but trophozoites get destroyed. • Commonly used techniques are 1. Sedimentation techniques 2. Flotation techniques SEDIMENTATION TECHNIQUE • Principle: stool is concentrated by centrifugation. Eggs and cyst are concentrated at the bottom of the tube as they have greater density than suspending medium.  Formalin ether concentration technique  Formalin-ethyl acetate concentration technique  Formalin-acetone concentration technique • Advantage: sensitivity of detecting ova and cyst increases. • Size and shape of parasite is maintained. • Easy and inexpensive and remove fecal odour. • Disadvantage: trophozoites are killed. Formalin ether Concentration technique Thoroughly mix 4 gm of stool specimen into 10mL 5-10% formalin- stand for 30 minutes. Filter through double layered gauze piece into a conical centrifuge tube. Centrifuge the suspension at about 500× g for 10 minutes. Discard supernatant, resuspend sediment with formalin. Centrifuge again and repeat washing until supernatant is clear. After the last wash, decant the supernatant and add 3 to 4 ml of 10% formalin to the sediment. Add equal amount of solvent ether, Stopper the tube and shake vigorously. Centrifuge at 500× g for 2 minutes. Four layers should result as follows – a top layer of ether; – plug of debris; – layer of formalin; and – sediment Free the plug of debris from the side of the tube with an applicator stick. Carefully decant the top three layers.Mix the remaining sediment. Transfer one drop each to a drop of saline and iodine on a glass slide. Cover with a coverslip and examine microscopically. FLOTATION TECHNIQUE • Principle: suspending the specimen in medium of greater density than that of helminthic eggs and protozoan cysts, so eggs and cyst float at the top and are collected.  Saturated salt solution flotation technique  Zinc sulphate flotation technique  Sheather’s sugar flotation technique • Disadvantage: can’t use for heavy eggs like unfertilized egg of A.lumbricoides, larvae of Strongyloides, Taenia eggs, operculated eggs of trematodes. • walls of eggs and cyst will often collapse, hindering identification. Saturated salt flotation technique Place 4 gm of fresh stool in a flat bottomed container. Add few drops of saturated salt solution (specific gravity 1.20), stir to make a fine emulsion. Fill container with salt solution until a convex meniscus is formed. Lay a glass slide over the top, avoiding any bubbles being trapped. Leave for 20 min, quickly invert the slide. Examine under microscope after putting a coverslip. Entero test(String test) • Done for the detection of trophozoites of Giardia lamblia and larvae of Strongyloides stercoralis . • It uses a gelatin capsule attached to the thread. • One end of capsule is attached to the patient’s cheek and then capsule is swallowed. • Capsule get dissolved in intestine releasing the thread which is kept there for 4 to 6 hours to take duodenal fluid. • Later the thread is withdrawn and shake in saline to release trophozoites/larvae of parasites which can be detected microscopically. String test for Giardiasis Egg quantification Methods  Direct smear counting method of Beaver  Kato’s cellophane tape  Stoll’s method or dilution egg counting method- Mix 4 g of feces with 56 ml of N/10 NaOH in Stoll’s flask. 0.15 ml is transferred to slide and total number of egg is counted (n) with low power objective. Number of egg per gram of feces N= n × 100. Culture Techniques Culture methods for Protozoa • Four types: 1. Axenic culture: parasites are grown on pure culture without any bacterial associate. USE- when bacterial flora interfere with the result like preparation of antigen for serology, drug susceptibility testing. 2. Xenic culture: parasites grown in association with an unknown microbe. 3. Monoxenic culture: parasites are grown with single known bacterium. 4. Polyxenic culture: contains multiple bacterial supplements , starch and serum. Culture media for intestinal protozoans Culture medium Type Important ingredients Use for Boeck and Dr bohlav’s medium Polyxenic Solidified egg or blood, locke’s solution, inactivated bovine or calf serum E. histolytica Balamuth’s medium Polyxenic Egg-yolk-liver concentrate infusion medium E. histolytica Robinson’s medium Polyxenic Erythromycin, Bacto peptone, phthalate solution, bovine serum, E.coli strain 0111 and R-medium E. histolytica Jones medium Polyxenic Horse serum and yeast autolysate in phosphate buffered saline E. histolytica Diamond’s (TYM) medium Axenic Trypticase-yeast extractmaltose E. histolytica Giardia lamblia Culture Techniques for Larval-stage Nematode • Copro culture methods are especially useful for specific identification of hookworm, Strongyloides stercoralis, and Trichostrongylus species.. • In the culture medium eggs hatch out into rhabtidiform larvae which is used to differentiate between hookworm, Strongyloides stercoralis and Trichostrongulus species. • Further transformation into filariform larvae differentiate into A.duodenale and N.americanus • Various techniques can be used  Harada Mori filter paper strip culture.  Petri dish (slant culture ) technique  Baerman funnel technique  Charcoal culture method  Agar plate technique Harada Mori filter paper strip culture Smear is made with 0.5-1 g of feces in the center of a narrow strip of filter paper. Filter paper is placed in a conical centrifuge tube with sterile water in such a way that lower end dip in water. Seal the mouth of tube and Incubate for 7-10 days at room temperature. Larvae develop on filter paper and migrate to water which is examined under microscope. Baermann technique  Useful in examining containing small number of Strongyloides larva.  Exploits property of Strongyloides larva to migrate from cooler to warmer area. A glass funnel is used fitted with a rubber tubing and clamp. A round wire screen with a gauze is placed on the funnel. 5 g feces placed on gauze. Funnel is filled with warm water, left for 1-2 hr. Clamp is opened ,710 ml fluid is collected in a beaker. Centrifuge and examine microscop pically. Baermann technique IMMUNODIAGNOSTIC METHODS 1. Detection of parasite specific antibody in serum 2. Detection of circulating parasitic antigen in the serum  Methods are useful when  Parasites occur in very small number  Parasite reside in internal organs and morphological identification is not possible.  Parasites can be detected only during early course of disease. Antibody detection tests • Recent techniques like IFA, ELISA, rapid immunochromatographic test(ICT) and Immunoblot (western blot) are sensitive and specific methods for antibody detection. • Antigens used are Crude antigens obtained from cultured parasites, animal inoculation or by natural infection to human.  Recombinant or synthetic antigens.  From related parasites or related bacteria. • Limitations: cannot distinguish between acute and chronic infection. • Less specific- cross reactive antibodies (heterogenicity of parasite) • Speciation is not possible ELISA for antibody detection Disease Antibody against Comments Amoebiasis Lectin antigen of Entamoeba histolytica Sensitivity 75-85% Specificity >85% Cryptosporidiosis Cryptosporidium parvum oocyst antigen Useful for seroepidemiology Strongyloidiasis Crude larval antigens Sensitivity (95%) useful when stool microscopy is negative Antigen detection tests • Most commonly used tests are  ELISA  Direct fluorescent antibody assay(DFA)  ICT • Advantages: provide information about acute and chronic infection. • Used to monitor the response to the treatment. • Useful when microscopy is negative. • To diagnose congenital infections. • Assesing the severity of infections. ELISA for antigen detection Disease Antigen detected Comments Amoebiasis 170 kDa of Lectin antigen of Entamoeba histolytica in stool or serum Stool- Sensi & Speci >95% Serum-Sensitivity >65% Specificity>90% Giardiasis Cyst wall protein antigen in stool Sensitivity 90-100% Specificity 90-100% Cryptosporidiosis Cryptosporidium parvum oocyst antigen in stool Sensitivity 66-100% Specificity >90% Intestinal taeniasis Taenia specific antigen in stool by using polyclonal Taenia antibodies. More sensitive than stool examination but can’t differentite between T.saginata & T.solium Molecular Methods  Polymerase Chain Reaction (PCR): in-vitro procedure for DNA amplification  Advantage- more sensitive and specific, rapid, used for speciation, drug resistance detection Disease Target Comments Nested PCR targeting small subunit rRNA genes Can differentiate subspecies E.histolytica/dispar/ moshkovskii Real time PCR More sensitive and take less time Cryptosporidiosis C.parvum gene from both clinical and environmental sample Can differentiate various genotypes, useful in outbreak Strongyloidiasis Real time PCR to detect Strongyloides DNA in stool Nealy 100%specificity and high sensitivity, research purpose Amoebiasis Treatment  Rehydration– replace the loss of fluid and electrolytes  Zinc therapy, Probiotics  Anti parasitic drugs Parasite Drug Amoebic colitis Metronidazole, Tinidazole, Ornidazole Giardiasis Metronidazole, Tinidazole, Furazolidone in children Cryptosporidium Nitazoxanide Cyclospora and Isospora Cotrimoxazole Cestodes and Trematodes Praziquental and Niclosamide Nematodes Mebendazole, Albendazole, Pyrantel pamoate