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Saturday, March 2, 2019

Study of Parasites ( Entamoeba, Plasmodium, Ascaris, Taenia)


Entamoeba histolytica


 Entamoeba histolytica is an invasive, pathogenic protozoan, causing amoebiasis, and an important cause of diarrhea in developing countries. Our understanding of its epidemiology has dramatically changed since this amoeba was distinguished from another morphologically similar one, Entamoeba dispar, a non pathogenic and commensal parasite. These two species can now be distinguished mainly through molecular and immunological proceduresis an invasive, pathogenic protozoan, causing amoebiasis, and an important cause of diarrhea in developing countries. Our understanding of its epidemiology has dramatically changed since this amoeba was distinguished from another morphologically similar one, Entamoeba dispar, a non pathogenic and commensal parasite. These two species can now be distinguished mainly through molecular and immunological procedures

 
The life cycle of the parasite is represented by two forms: the cyst and the trophozoite. The cyst is the infective and non motile form of the parasite.  It is excreted in the feces and can survive for weeks in the environment.  Mature cysts possess 4 nuclei and average 20 µm in diameter. The trophozoite is the motile form, with a size ranging from 10 to 60 µm. It colonizes the intestinal tract leading mainly to tissue destruction and secretory bloody diarrhea.
 
Amoebiasis is basically an acute disease acquired by: (i) ingestion of cysts present in contaminated food, water, or plants, (ii) through person to person contact, (iii) exposure in endemic areas, and (iv) swimming in contaminated water.  Clinical manifestations range from the asymptomatic carrier state to dysenteric symptoms represented by abdominal pain and bloody diarrhea.

The organism can be prevalent in cold regions as well as tropical and subtropical regions that have contaminated water. In fact, E. histolytica is an important cause of morbidity and/or mortality wherever sewage facilities are inadequate. As is the case for other intestinal protozoan pathogens, wastewater treatment techniques are reported not to be very efficient for E. histolytica elimination possibly because of their resistance to disinfectants and the small size of the cysts. Stabilization ponds have been reported to be more effective than activated sludge for their abatement.  Sedimentation and filtration can enhance the removal of cysts from wastewater. 









Plasmodium

Plasmodium, a genus of parasitic protozoans of the sporozoan subclass Coccidia that are the causative organisms of malariaPlasmodium, which infects red blood cells in mammals (including humans), birds, and reptiles, occurs worldwide, especially in tropical and temperate zones. The organism is transmitted by the bite of the female Anopheles mosquito. Other insects and some mites may also transmit forms of malaria to animals.

Five species cause human malaria: P. vivax (producing the most widespread form), P. ovale(relatively uncommon), P. falciparum (producing the most severe symptoms), P. malariae, and P. knowlesi. There are several species that have been isolated from chimpanzees, including P. reichenowi and P. gaboniP. falciparumP. gaboni, and other species have been isolated from gorillas. Examples of parasites found in reptiles include P. mexicanum and P. floridense, and those in birds include P. relictum and P. juxtanucleare.

Plasmodium species exhibit three life-cycle stages—gametocytessporozoites, and merozoites. Gametocytes within a mosquito develop into sporozoites. The sporozoites are transmitted via the saliva of a feeding mosquito to the human bloodstream. From there they enter liver parenchyma cells, where they divide and form merozoites. The merozoites are released into the bloodstream and infect red blood cells. Rapid division of the merozoites results in the destruction of the red blood cells, and the newly multiplied merozoites then infect new red blood cells. Some merozoites may develop into gametocytes, which can be ingested by a feeding mosquito, starting the life cycle over again. The red blood cells destroyed by the merozoites liberate toxins that cause the periodic chill-and-fever cycles that are the typical symptoms of malaria. P. vivaxP. ovale, and P. falciparum repeat this chill-fever cycle every 48 hours (tertian malaria), and P. malariae repeats it every 72 hours (quartan malaria). P. knowlesi has a 24-hour life cycle and thus can cause daily spikes in fever.













Ascaris lumbricoides


Ascaris lumbricoides is an intestinal round worm. It is the largest intestinal nematode to infect Human. The adult worm lives in small intestine and grow to a length of more than 30 cm. Human is only the natural host and reservoir of infection.
The round worm infection occurs worldwide. The number of infected persons is estimated to be more than 2 billion. 

Morphology:

Adult:
The round worm resembles to earthworm. It is elongated tapering to both end, anterior being thinner than posterior. Freshly excreted worm is yellowish pink in color, which gradually changes to white.

The worm is sexually diamorphic.
§  Adult male: 15-30 cm in length, 3-4 mm in diameter, tail curved
§  Adult female; 20-40 cm length, 2-6mm diameter, tail straight
Egg:
Ascaris egg is round or oval, 60*40 µm size, thick brown shell and have rough surface. It is the infective form of parasite.
§  i) Un fertilized egg; large, more elongated (38-55*78-105) µm
§  ii) fertilized egg; ovoid (35-50*50-70)µm, golden brown color

 Life cycle:
The life cycle of Ascaris completes in single host. Human.

§  Adult worm lives in small intestine
§  Stages in life cycle:
Stage I: Eggs in faeces
§  Sexually mature female produces as many as 200,000 eggs per day, which are shed along with faeces in unembryonated form. They are non infective.
Stage II: Development in soil
§  Embryonation occurs in soil as optimum temperature of 20-25C with sufficient moisture and O2
§  Infective larva develops within egg in about 3-6 weeks.
Stage III: Human infection and liberation of larvae
§  Human get infection with ingestion of embryonated egg contaminated food and water
§  Within embryonated state inside egg, first stage larvae develops into second stage larvae. This second stage larvae is known as Rhabtitiform larvae
§  Second stage larve is stimulated to hatch out by the presence of alkaline pH in small intestine and solubilization of its outer layer by bile.
Stage IV: migration of larvae through lungs
§  Hatched out larvae penetrates the intestinal wall and carried to liver through portal circulation
§  It then travels via blood to heart and to lungs by pulmonary circulation within 4-7 days of infection.
§  The larvae in lungs molds twice, enlarge and breaks into alveoli.
Stage V: Re-entry to stomach and small intestine
§  From alveoli, the Larvae then pass up through bronchi and into trachea and then swallowed.
§  The larvae passes down the oesophagus to the stomach and reached into small intestine once again.
§  Small intestine is the normal habitat of Ascaris and it colonises here.
§  Within intestine parasite molds twice and mature into adult worm.
§  Sexual maturation occurs with 6-10 weeks and the mature female discharges its eggs in intestinal lumen and excreted along with faeces, continuing the life cycle.
§  The life span of parasite is 12-18 months

Pathogenesis:

1. Mode of transmission:

§  faeco-oral route, by contaminated vegetables or water.

2. Pathogenesis:

Infection of A. lumbricoides in man is known as Ascariasis. There are two phase in ascariasis.
Phase I: migrating larvae
§  The migrating larvae causes pathological lesions. The severity of lesions depends upon the sensitivity of host, nutritional status of host and number of migrating larvae.
§  During migration and molding through lungs, larvae may causes pneumonia with low grade fever, cough and other allergic symptoms.
Phase II: Adult worm
§  Few worm in intestine produce no major symptoms and but some time give abdominal pain especially in children.
§  The adult worm produce trauma in host tissue and the wandering adults may block the appendical lumen or common bile duct and even small intestine.
§  Large number of adult worms affects the nutritional status of host by robbing the nutrition leading to malnutrition and growth retardation in children.
§  The metabolites of living or dead worm are toxic and immunogenic.
§  lumbricoides also produces various allergic toxin, which manifests fever, conjunctivitis and irritation.












Taenia
Taenia is a flat worm that is commonly called tapeworm because of its flat and ribbon-like body. Taenia solium is the pork tapeworm(hookworm) and the secondary host is pig, while Tsaginata is the beef tapeworm whose secondary hosts are cows, buffalos, sheep and goat.
Adult tapeworm lives in the small intestine of man attached to the intestinal mucosa by its scolex. The larval stage occurs in the tissues of the secondary host which is usually pig but sometimes other animals also.
Morphology
T. solium is long, whitish, dorsoventrally flattened and ribbon-like worm that can reach a length of 2-3 meters. Body is divided into hundreds of segments called proglottids.
There are three regions in the body, the scolex, unsegmented neck and the segmented posterior region called strobila.
Scolex is the anterior end of the body; about 1mm in diameter with 4 cup-like suckers and an anterior rounded portion called the rostellum having 20-30 curved chitinous hooks, which are used for attachment to the intestinal wall of the host.
Neck is short, narrow and unsegmented area behind the scolex. This is the growth zone or area of proliferation where new segments or proglottids are added to the body.
Strobila forms the bulk of body and consists of a series of proglottids arranged in a linear fashion. The strobila may contain as many as 800-900 proglottids. Proglottids are self-contained units, each with a complete set of both male and female reproductive organs and a part of excretory and nervous systems. The youngest proglottid is just behind the neck and the oldest is at the posterior end of strobila.





Taenia solium  (Pig wom)           Taenia Saginata (Beef worm)







Isolation and Identification of Clinically Important Aspergillus sp.,



Aim:
            To isolate and identify the Aspergillus sp., from clinical samples.

Introduction:

            Aspergillus species are ubiquitous fungi, commonly occurring in soil, water, and decaying vegetation. Route of acquiring infection being inhalation of fungal spores. From lungs, dissemination takes place leading to systemic aspergillosis. Occurrence of Aspergillus infection in association with chronic lung disease like pulmonary TB, lung abscess, bronchopneumonia, with residual lung cavity, asthma and lung malignancy has been documented. Out of 185 species of genus Aspergillus, only 20 can cause human infection, Some examples are Aspergillus flavus, Aspergillus fumigates, Aspergillus niger and Aspergillus nidulans. Fumigatus is the most common species found in human infection all over the world.

Materials Required:

Clinical sample
Sabouraud dextrose agar
Lacto phenol cotton blue staining

Procedure:

1. Clinical specimens were streaked on SDA plate and it was incubated at 37 °C for 5-7 days.
2. Fungal growth was identified by colony morphology on plate.
3. Microscopic observations:

Lacto phenol cotton blue staining:

Growth of Fungus were taken from culture medium on clean grease free sterile microscopic slide and mixed with 1-2 drops of LPCB stain. Focused the slide first under 10x to find the area and then under 40x for identification of fungus.

Slide culture technique.

            10 mm square block from Potato dextrose agar (PDA) was cut using sterile coverslip or scalpel. The growth of Fungus were taken from culture medium i.e. Sabouraud’s dextrose agar (SDA) with the help of L shaped wire and inoculated into four corner of the block and then a sterile coverslip was kept over the block. The inoculated side culture was incubated in a wet Petri dish at 25 ±5°C in Biological Oxygen Demand incubator for 48 to 72 hours. Then the coverslip was removed and placed over a drop of Lactophenol Cotton Blue (LPCB) stain in clean sterile glass slide and then focused under 10x to find the area and then under 40x for identification of fungus.
Result and Interpretation:
            Obtained fungal culture from clinical sample was identified as Aspergillus niger based on Colony morphology and Microscopic characterization.








Colony Morphology of Aspergillus species
A. niger:
Colonies consist of a compact white or yellow basal felt covered by a dense layer of dark-brown to black conidial heads
A.  flavus:

Colonies are granular, flat, often with radial grooves, yellow at first but quickly becoming bright to dark yellow-green with age.
A. fumigates :
Colonies are typically blue-green with a suede-like surface consisting of a dense felt of conidiophores. 
A. nidulans :  
Colonies are typically plain green in colour with dark red-brown cleistothecia developing within and upon the conidial layer.

 Microscopic markers of selected Aspergillus species fungal pathogens :

Organism (s) 

Hyphae 
Other features 
A. fumigatus 
2.5–8 µm wide, septate, hyaline, acute angle branching, tree- or fan-like branching. Stipes may resemble hyphae of zygomycetes 
Conidial head uniseriate, columnar, conidia in chains or detached and dispersed. Single or paired conidia may resemble yeast cells 
A. niger 
See A. fumigatus 
Conidial head biseriate, radiate, conidia in chains or detached and dispersed. Single or paired conidia may resemble yeast cells 
A. terreus 
See A. fumigatus 
Small, round, hyaline conidia (‘accessory’ conidia) attached to the vegetative hyphae 





Ref:

N. McClenny, Medical Mycology, Volume 43, Issue Supplement_1, 1 January 2005, Pages S125–S128, https://doi.org/10.1080/13693780500052222.

Raksha et al., Pilot study on identification of Aspergillus species and its antifungal drug sensitivity testing by disc diffusion method, Int.J.Curr.Microbiol.App.Sci (2014) 3(12): 555-562.

Shrimal et al., ISOLATION OF ASPERGILLUS SPECIES FROM SPUTUM SAMPLES: A STUDY CONDUCTED IN A TERTIARY CARE HOSPITAL, AHMEDABAD, NATIONAL JOURNAL OF MEDICAL RESEARCH, eISSN: 2277 8810, Volume 3│Issue 3│ 2013, 289 – 291.

https://mycology.adelaide.edu.au/descriptions/hyphomycetes/aspergillus/