Formal ethyl acetate concentration technique procedure

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Lifecycle and symptoms by hookworm

The hookworm eggs hatch into larvae, first stage larvae (L1), known as rhabithform within 2448 hours in the soil during optimal conditions with suitable warmth, humidity and shade. (3,14). The larvae feed on bacteria and organic matter in the surrounding environment (18) before they develop into the infective larval stage, known as third stage larvae (L3) (3) within 5-10 days. They survive in the soil for several weeks before penetrating the skin of the human host causing itch and erythema (6,18). The L3 stage, filariform is about 600 microns (μm) in length (3) and it burrows through tissue until it reaches the blood vessels. The larvae migrate through the venoules to superior- or inferior vena cava towards the lungs, causing pneumonitis and eosinophilia (15). Further, by entering the alveolus it migrates to the bronchial tree and trachea, via pharynx by the movement of microvilli (19). The host then cough up the larvae which in turn is swallowed and transported to the small intestine. In contrast to N.americanus, A.duodonale can directly be swallowed from poorly rinsed vegetables. Stage L3 larvae develops into a worm that attach themselves to the duodenal mucosa (6), damages the intestinal mucosa and sucks 0,03-0,3 ml of blood per worm and day (15). After 5-8 weeks the worm reach sexual maturity and mate, resulting in the female worm releasing thousands of eggs each day into the intestinal lumen that are passed through the feces (3,15). Microscopic examination of stools can establish the diagnosis (19). Normally the hookworm lives inside its human host for several years (3,15).
Infection by hookworm are often asymptomatic though a consequence of decreasing hemoglobin levels is iron deficiency anemia (1,6). Anemia is a serious health condition that mainly affect women of a reproductive age and young children (7,20). Anemia and low iron count carries risks that include intrauterine growth retardation, low birth weight, preterm birth, maternal and infant mortality (7,9). Other consequences may include allergic reactions at the entry site, nausea, vomiting and diarrhea (15). The morbidity is directly related to the number of worms harboured (21). If the infection become chronical, emaciation as well as mental and physical retardation may occur because of the great loss of blood and other nutrients. Secondary infection by bacteria in the intestinal mucosa may also occur (15).

Diagnosis and treatment of hookworm infection

Because hookworm cannot be cultured, microscopic examination is the pillar of diagnostic parasitology. Other methods for parasitic diagnosis are available, for example molecular analysis such as polymerase chain reaction (PCR) (3,15), that gives rapid, sensitive and specific diagnostic results. However, microscopy still remains the most common method of choice. This because of its low costs and simple techniques in combination with good results. The diagnosis of hookworm is established by first macroscopically examine the faecal samples for blood, mucus or other abnormalities (15). If dark stools are seen this indicate bleeding higher in gastrointestinal tract, while fresh blood indicate bleeding from the lower parts of the gastrointestinal tract. Presence of blood or mucus can come from a parasitic infection, but also from a number of different pathogenic conditions and should always be reported. Hookworm eggs are found in all types of faecal consistency; however, in very loose specimens’ eggs may be hard to find because of the dilution factor (6). Hookworm eggs do not appear in the stool in consistent numbers on a daily basis, this because the eggs are intermittently released, therefore a number of three specimens should be collected at three separate days within ten days for an accurate diagnosis. It’s recommended to collect in clean wide-mounted containers. Regularly a waxed cardboard or plastic container with a tight- fitting lid is used. The specimen should not be contaminated with water or urine because water contain free—living organisms that can be mistaken for human parasites and urine destroy motile organisms. In addition, urine also cause helminth eggs hatch (15). To prevent helminth eggs from developing to larvae and preserve the morphology of the eggs several fixatives can be used with the recommended ratio of stool and fixative. In order to ensure proper fixation 5% or 10 % buffered formalin, sodium acetate-acetic acid- formalin (SAF) or Schaudinns fluid polyvinyl alcohol can be used. The fixative kills any bacteria or viruses in the stools, which is an important safety aspect (6). The samples can be transported in a fixative SAF-tube and then directly analysed at the laboratory. In case the sample is transported unfixed the containers should be placed in plastic bags when transported to the laboratory and then fixed in a SAF-tube within 24 hours. In order to enable the microscopic analysis, the stools samples have to be prepared. This can be done with a variety of different methods including direct wet mount, Kato-Katz, concentration/sedimentation or flotation (6,22). Different methods are useful in detecting different range of parasites (15). The diagnosis can be based mainly on the presence of eggs, regardless of the amount found. In order to establish the intensity of an infection it is necessary to quantify the number, this also is important when treatment effects are studied (8).
Willis technique is a flotation method based on a solution containing saturated sodium chloride, with higher density than hookworm eggs. The gravity of hookworm eggs is 1.055 g/cm3. The difference in density makes the helminth eggs float to the surface and adhere to a coverslip (23).
The sedimentation technique, also known as Formal ethyl acetate concentration method is based on a solution that have lower density than hookworm eggs, making the eggs sediment. Ethyl acetate is used to remove debris (6) and together with formalin fixed material it is the most common sedimentation method in routine laboratories (23). The sedimentation method is useful in diagnosis and recovering all types of helminth eggs.

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Microscopic appearance of hookworm eggs

The eggs of the two species A.duodenale and N.americanus cannot be differentiated by basic light microscopy and is counted as hookworm combined. The eggs measure 55-75 μm x 36-40 μm and have a thin wall, is oval with rounded ends and contains an undeveloped embryo with 8-32 cells (22). A clean space between the embryo and shell is a distinct feature (19). A common similar specie, Trichostrongylus spp is often mistaken for hookworm eggs by unexperienced laboratory scientists. To separate the species, the size can be used as the Trichostrongylus spp is greater measuring 73-95 μm x 40-50 μm. Trichostrongylus spp also have thicker walls and are tapered at one end (6).

Formal ethyl acetate concentration technique procedure

A 3ml amount of solvent ethyl acetate, CH3COOC2H5 (Xinlong Chemical CO., Ltd. Guangang Province China) were added using 3ml Pasteur pipette and the solution was shaken vigorously for one minute after a lid was putted on. After the centrifugation (Eppendorf centrifuge 5702, Eppendorf Nordic Aps, Horsholm, Denmark) at 1500 x g for three minutes, a sediment was obtained, three different layers consisting of formalin, plug of debris and ethyl acetate that was decanted to a slush. The pellet suspends in residual water and a 76 x 26mm slide were prepare for examination. A drop of the mixed pellet was placed on slides until all of the pellet was used. This was done with disposable pipette (Labdesign, Täby, Sweden) and one drop of 0,9% sodium chloride was added. Finally, the slide was mounted with a 21 x 26mm coverslip and then examined. The slide was placed in a Petri dish with a wet filter paper until it was analysed under a light microscope. First a 10 X objective was used in combination with a 10X ocular. If a suspected hookworm egg was seen the objective was changed to 40X.

Willis flotation and Ethyl acetate concentration

All 101 collected sample were analysed with Willis flotation technique and Formal ethyl acetate concentration. By both methods a total of 17 positive samples were found making the overall prevalence 16,8%. No significant quantitate differences between the two methods were establish and the independent analyse was interpret equivalent, the image quality was judged to be different between the both methods. In formal ethyl acetate concentration the image showed more debris in contrast to a cleaner image found in Willis flotation method, see figure 2a and 2b respectively.

Table of contents :

Introduction
Background
Prevalence
Lifecycle and symptoms by hookworm
Diagnosis and treatment of hookworm infection
Risk factors and prevention
Microscopic appearance of hookworm eggs
Purpose
Materials and methods
Study population
Stool sample collection and preparation
Willis technique procedure
Formal ethyl acetate concentration technique procedure
Quality Control
Health care education
Ethical considerations
Statistical analysis
Results
Willis flotation and Ethyl acetate concentration
Gender
Age
Prevalence of hookworm infection
Discussion
Methodological issues
Result discussion
Conclusion
Acknowledgments
References

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