CHAPTER II

REVIEW OF RELATED LITERATURE

This part presents the theoretical writing, related studies, theories and principle

that would serve as the theoretical basis for the researchers study and would offer

important learning for the completion of the study.

2.1 Conceptual Literature

2.1.1 Fecalysis

Fecalysis is a series of tests done on a stool through chemical,

microbiological and microscopic view. It diagnoses certain conditions that affects

digestive tract. Among these conditions are the infection from parasites, viruses,

bacteria and deprived nutrient absorption or cancer.

2.1.1.1 Manual Process of Fecalysis

In manual implementation of fecalysis, a secure amount of stool that

is placed in a sterile container is needed. Its color and consistency must be

observed microscopically. Then, one drop of normal saline solution should

be place into slides, by using applicator stick, the sample is spread in the

slide and mixed. It is again examined microscopically by observing for any

RBC, puss cell and parasites. Microscope is adjusted to its high power field

to distinguish the parasites such as entamoebas.

2.1.1.2 Automated Fecalysis

Various laboratory equipment is now present as technology

evolved. One of this equipment is the Belson LXL1000, an automatic stool

analyzer that can examine multiple stool samples. Its features include

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automatic dilution, high speed mechanical mixing, automatic sample

loading and it continuously processed stool samples for about 120 samples

testing per hour.

The machine is efficient and provides fast service to medical

laboratories. Though, it is costly and its availability is rare.

Figure 2.1 Belson LXL1000

Belson LXL 1000 operates by putting the sample in the cup which

is well sealed and extremely sanitary and put it on the 1st slot for the sample

cups. There are 24 slots for sample cups which makes it possible to test

24 samples at the same time. There is a test card, which is used for the

examination of OBT, gut microbes, virus and etc. There are two needles

here. These needles add reagent to the sample cup and mix them

automatically. Another needle picks up samples to the counting pool

through the pipeline and put samples on the test card. And the pipeline

would be cleaned automatically afterwards. There three cameras on the

equipment. And they can display the result of the test on the computer

screen. There is an external camera which is used for capturing the test

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 result of the test card and the result is displayed on the screen. There is

also internal camera which is used to capture the shape, color and

characteristic of the sample and displayed on the screen. There is an

internal microscope which transmits data to the computer through the

electronic eyepiece. And they are displayed on the screen.

2.1.2 Description and Morphology of Entamoeba histolytica

Figure 2.2 Sample image of the Morphological Structure of Entamoeba histolytica Cyst

Entamoeba histolytica is an anaerobic parasitic protozoan, part of genus

Entamoeba which is predominantly infecting humans and primates. Its

morphological characteristic includes the number of nucleus. This parasite is

consisted of two stages, the trophozoite and cyst. The trophozoites may range in

size from 10-60 um and have irregular circular shape and a single nucleus with a

prominent nuclear border, central karysome and vacuole. Trophozoites ingest red

blood cells and absorbs their nutrients to survive. Some trophozoites transform

into cysts.

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 On the other hand, Figure 2.2 shows that Entamoeba histolytica cyst is

made up of a circular shape body which consist of crystalline ribosomes. It includes

a number of nuclei which ranges from one nucleus in early stage and four nuclei

when mature. The nucleus itself can be describe as the ring and dot portion of the

given protozoa.

2.1.3 Algorithm

An algorithm refers to a specific set of instructions for carrying out

procedure or solve a problem. Given some set of initial conditions, it can be

performed in a prescribed sequence to achieve a certain goal and has a

recognizable set of end conditions.

Basically, algorithms tell exactly how to do something to achieved the

required output or to accomplished well-defined task. One of the most important

aspects of algorithm. It is often easy to come up with algorithm to solve a problem,

however if the algorithm is slow, it is not effective.

Image processing served as the proposed process in this particular study.

There are different algorithms and techniques developed in image processing.

From these techniques, the algorithm that will be used in this study is selected.

2.1.3.1 Some Fundamental Steps involve in Image Processing

2.1.3.1a Image Enhancement

Image enhancement technique brings out the detail in an

image that is obscured or highlight certain features of interest in an

image. Enhancement techniques include contrast adjustment,

filtering, morphological filtering and equalization. Image

enhancement operations typically return a modified version of the

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original image and are frequently used as a preprocessing step to

improve the results of image analysis techniques.

2.1.3.1a.1 Some general methods involved in Image

Enhancement

a. Histogram Equalization

The gray level values of the pixels within an image is

uniformly redistributed so that the number of pixels at any one gray

level is equal. Moreover, the contrast of images is enhanced by

transforming the values in an intensity image so that the histogram

of the output image approximately matches a specified histogram.

b. Median Filtering

A median filter operation on an image removes long tailed

noise such as negative exponential and salt and pepper type noise

from an image with a minimum blurring of the image. The median

filter is defined as the median of all pixels within a local region of an

image. Pixels that are included in the median calculation are

specified by a mask. The median filter performs much better than

the arithmetic mean filter in removing salt and pepper noise from an

image and in preserving the spatial details contained within the

image. The median filter can easily remove outlier noise from image

that contain less than 50% of its pixels as outliers.

c. Contrast Correction

Contrast correction is used of enhance the visual

appearance of an image. Contrast modification of an image is

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 defined as stored in the structured image. The program then

computes the average of the image and removes this value from

the original pixel. Next, the averaged removed pixel is multiplied by

specified contrast parameter. The last step implements a program

to add back the average to the contrast corrected pixel and store

the result in the structure.

2.1.3.1b Image Segmentation

Image segmentation is the process of driving an image into

multiple parts. This is typically used to identify objects or other

relevant information in digital images. A lot of image segmentation

methods have been already used in medical applications to

segment tissues and body organs.

One big application area of image segmentation is the

object detection. Its results consist of transforming an image into

different phases and keeping track of the important characteristics

of each phase. Example of image segmentation result is the

segmentation of the MR-image of the brain which is segmented into

four different phases.

2.1.3.1b Some general methods involved in Image Segmentation

a. Edge Detection

Edge detection finds the edges in an input image by

approximating the gradient magnitude of an image. It convolves the

input matrix with the Sobel or Roberts kernel. It outputs two gradient

components of an image, which are the results of this convolution

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 operation. It can perform thresholding operation on the gradient

magnitudes and output a binary image, which is a matrix of Boolean

value. If a pixel value is 1, it is an edge.

b. Thresholding

Thresholding is used in image processing to separate an

objects pixels from the background pixels. Thresholding converts a

multi-gray level image into binary image containing two gray level

values.

c. Clustering

Clustering is the process of counting and labeling of

objects within an image. Clusters consists of a pixel groupings that

are related to one another by a predetermined measure. This

measure can be defined as a distance between clusters or a

similarity measure such as pixel value or it may be a complex set of

identifiers and constraints that define membership inn a cluster. The

cluster may ultimately be mapped into a binary image as a unique

image.

2.2 Related Studies

2.2.1 A Computer Vision Approach to Micro-Nucleus Automatic Detection for

Protozoan Parasites Segmentation

This paper by Hsiao-Yu Wang and Hung-Yuan Chung presents a novel

multiple detection schemes to segment protozoan parasites and their nucleuses

from microscopic images. The methods used for this paper are different protozoan

parasites segmentation scheme. First, the image was first transformed into gray-

level ones and equalized color spaces using gamma equalization. Then, median-

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mean filter and two-class edge enhancement algorithm were used to erase noises

and enhance the boundaries of protozoan parasite. K-mean algorithm was used

to cluster the pixels of each image into two major clusters which are the protozoan

parasite and the background. The largest clustered or connected pixels are

considered as the protozoan parasite and morphological closing operation is used

to determine a more complete region of the it.

Second, after detecting the region of the protozoan parasite, multiple

nucleuses inside it are needed to be detected. The detection scheme that they

used are composed of different methods. This includes the adaptive boundary

erasure, iterative gamma equalization, two-means, modified connected

component detection and circular mask scoring methods. They are performed

iteratively to detect the first most significant nucleus inside the protozoan parasite.

Lastly, the height and the width of the nucleus were measured and considered as

the conditions for further detection of other nucleus. The scheme gained 96.64%

accuracy for 112 experimental protozoan parasite microscopic images but the

software that they used was not stated on the paper.

2.2.2 An Image-Based Entamoeba Automatic Detecting System

Recently, Lai et al. proposed an extraction scheme for protozoan parasite

images. The main focus is to determine whether there exists amoeba in the image

through image processing techniques. The methods used in the study were: (1)

preprocessing stage (2) image enhancement and (3) image segmentation. In order

to prevent problems to the segmentation results, the researcher process the pre-

processing steps which includes color images convert to grayscale images,

histogram equalization and power law transformation. Then, image enhancement

which includes two steps, run length enhancement and bi-group enhancement

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was used in order to gain the complement contours. Lastly, the image

segmentation technique which includes automatic threshold selection and closing

operation was applied to separate the edges of the objects and the background in

the image. The results obtained 98.73% accuracy from 75 experimental images

that was tested.

2.2.3 Automatic Recognition of Human Parasite Cysts on Microscopic Stools

Images using Principal Component Analysis and Probabilistic Neural

Network

The study performed by Beaudelaire Saha et.al, took consideration on

having digital microscopic cyst images for the detection of Entamoeba species

through PCC (Principal Component Analysis) and PNN (Probabilistic Neural

Network) methods. Indeed, image segmentation turned out to be their fundamental

method for the detection of several species. An algorithm was clearly presented

on how the detection from digital images could be obtained. First, a pre-processing

stage was performed. It was made possible through edge detection using multi-

scale wavelet transform, segmentation through circular hough transform and

parasite extraction through computation of active contour based on gradient vector

flow model. A feature extraction and dimensionality reduction was also executed

through PCA and parasite image resizing in order for an easier differentiation of

one specie to another. Lastly, the classification stage was implemented through

PNN training in order to categorize and name a specie from one another. Indeed,

the algorithm presented through the Probabilistic Neural Network Toolbox from

MatLab resulted to 100% accuracy in which nine kinds of intestinal parasites were

obtained from 540 samples of digital protozoan cysts images.

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2.2.4 - Red Blood Cells, White Blood Cells and Epithelial Cells Detection and

Counting Using Image Segmentation On Matlab

This study of Caringal, Glenn A. De Lunas, Caroline E. and Endaya, John

Kenneth M. aimed to develop a Matlab program that will detect and count the Red

Blood Cells, White Blood Cells and Epithelial Cells that are present on urine. This

study is developed for the purpose of bringing an automated approach of a

microscopic urine test. This research is limited with detecting the RBC, WBC, and

Epithelial Cells. The researchers limited their study with the following cells

because of their significance to a persons health.

The program was developed using MatLab. Researches were done about

image segmentation and its applications, the microscopic characteristics of red

and white blood cells and epithelial cells to be able to made this study possible.

The RBC detection simply used circular though transform in imfindcircles function

because of its circular property. On the other hand, the process of detecting WBC

and epithelial cells both started by converting the image to grayscale using the

srgb to cmyk function. The cyan color space was extracted and image thresholding

were done. Image segmentation was implemented using edge detection and blob

detection and the binary image was generated. In filtering the detected blobs, WBC

and epithelial cells were differentiated by their shape and size. Their respective

counts were saved and displayed on the graphical user interface.

RBC counting, the result of evaluation gave an average percent error is

31.8%. While WBC counting resulted to an average percent error 39%. Lastly,

Epithelial Cells Counting had an average percent error of 24.13% which is not that

large.

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2.2.5 Microscopy Image Processing Analysis for Automatic Detection of

Human Intestinal Parasites ALO and TTO

K.H. Ghazali, R.S. Hadi and M. Zeehaida published a study about an

automatic system to diagnose human intestinal parasites using image processing

technique. The overall research methodology is divided into two-part decision

system and interfacing system. The first part of research methodology was the

image acquisition of Ascaris Lumbricoides Ova (ALO) and Trichuris Trichiura Ova

(TTO) parasites using a microscope camera. Next part is the image processing

analysis that consists of pre-processing, image processing technique, feature

extraction and classification. The best pre-processing approach has been explored

that focused on the segmentation of target object parasite cell. The captured

images have been analyzed by performing image enhancement using contrast

enhancement, edge enhancement using unsharp filtering, noise reduction using

median filter and edge detection using canny filter. The final stage in the

development of the decision is classification method which have been already

obtained from the results of the image analysis and pre-processing stage. For the

interfacing system, a complete automatic glass slider system was developed.

Based on the result, more than 95% of accuracy was obtained for the classification

of both parasites ALO and TTO.

2.2.6 Detection of presence of Parasites in Human RBC in Case of

Diagnosing Malaria Using Image Processing

Pranati Rakshit and Kriti Bhowmik presented a piece of work which is

innovative of its kind where some infected blood samples are analyzed to

determine stage of the disease malaria. For pre-processing, the method that they

used is the converting of the colored blood smear image into binary image. Further

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processes includes edge detection Harris corner detection and region selection.

Under edge detection, sobel operator was used since it is best for biomedical

images. It executes a 2-D spatial gradient measurement on an image and

emphasizes on regions of high spatial frequency that are related to the edges, it is

used to find the approximate absolute gradient magnitude at each point of an input

gray scale image. Harris corner detection was needed in order to find out corner

points. Region selection is then used to calculate some properties of image

regions. Overall calculation says that in 97.73% cases the diagnosis of malaria is

correct detected by pathologists. They considered several samples of infected

blood with different stages of Malaria and used Matlab 7.14 software to study the

detection. Apart from detecting malaria, severity of the disease in human body can

also be diagnosed in an efficient way through this technique.

2.2.7 - Prevalence of Entamoeba species in captive primates in zoological

gardens in the UK.

Carl S. Regan, Lisa Yon, Maqsud Hossain and Hany M. Elsheikha

underwent a research regarding the occurrence of amoeba in animal feces among

the six zoos in United Kingdom. The purposed of the study is to distinguish the

different kinds of Entamoeba specie such as Entamoeba histolytica and

Entamoeba dispar, and Entamoeba coli that were present in animal stool. It also

aimed to identify which kind of parasite is commonly existing in myriad number of

animals. The study used PCR in examining the samples. Initially, the samples were

preserved via formalin and prepared using a Ridleys formol-ether concentration

technique to improve the samples appearance in a microscope. The study also

used Minitab 15 for the evaluation of the data. It also used the QlAamp DNA stool

Mini Kit to directly extract parasite DNA from stool. The study concluded that the

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 presence of Entamoeba at multiple zoos in United Kingdom had the percentage of

28.9% based on the tested samples. The most common Entamoeba specie that

was detected is the Entamoeba coli. Through this study the detection and clear

differentiation of Entamoeba histolytica and Entamoeba dispar were successfully

recognized.

2.2.8 Detection and Differentiation of Entamoeba Histolytica and Entamoeba

Dispar Isolates in Clinical Samples by PCR and Enzyme-Linked

Immunosorbent Assay

This study performed by Patrick Gonin and Louise Trudel presented a

different technique for the detection and differentiation of Entamoeba histolytica

and Dispar species. In this study the use of PCR (Polymer Chain Reaction) and

ELISA (Enzyme-Linked Immunosorbent Assay) was used and compared. Since

the two target species were considered to be morphologically identical from one

another, PCR used a genetic method of approach wherein samples taken

undergone DNA processing. On the other hand, ELISA offered serological method

in which the detection of the two species can be identify from the serum which was

the watery portion obtained from the stool samples. These two techniques resulted

to 85% accuracy from the several tests performed in the laboratory. Equipment

used were purely obtained from the laboratory and no programming tool was used.

2.3 Synthesis

The related literatures included in this study serves as references for the

researchers in developing their proposed program. The concepts and theories from the

cited studies contribute a lot in making this research possible.

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 The related studies give ideas for further improvement and development of the

proposed project. Through the review of related studies, the researchers found out the

differences and similarities between the other researchers work and the proposed study

The study of by Hsiao-Yu Wang and Hung-Yuan Chung, no image processing

software has been stated on their paper but the algorithms used were completely detailed.

The processing were basically divided into two parts. The first part focuses on the

detection of the protozoan parasite. It includes several algorithms to determine the region

of it. The second part is more on the nucleus detection inside the protozoan parasite. The

experiment shows that the proposed scheme can detect the nucleuses with indistinct

boundaries effectively.

For the study conducted by Lai et al, the researchers focus on developing an

amoeba automatic detection system based on the image processing techniques. It

focuses on whether there exists an amoeba in the image. Preprocessing stage, image

enhancement and image segmentation are the methods used. This study concentrates

only on amoeba detection only.

The study performed by Beaudelaire Saha et.al, employs several techniques which

involves image edge detection, segmentation and parasite extraction with the application

of Neural Network Toolbox from MatLab. On the other hand, this study involves the same

approach of employing techniques including image edge detection and circular Hough

Transform but instead of using a toolbox from MatLab, this study focusses only upon the

implementation of several codes for the detection process. Both studies are concerned for

the protozoan cysts since its the most common stage of a protozoa that can be easily

identify and can be found from stool samples. This study focusses only upon the detection

of Entamoeba histolytica cyst and no any other species are concerned. This is because

other species are rarely found on stool samples. Lastly, both studies use MatLab as a

computer programming software tool as a platform where processing of digital images

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could be done. Indeed, this study offered a GUI (Graphical User Interface) for an easier

interaction and processing of results for the users which is not yet done by the previous

study.

The study of Caringal, Glenn A. De Lunas, Caroline E. and Endaya, John Kenneth

M is about the identification and counting of RBC, WBC and Epithelial cells in urine

samples. It used image processing using a computer programming software which is the

Matlab. Graphic User Interface Program is also implemented. Different codes are applied

such as codes for detection of circles, for reading and showing of the test image. There is

a certain algorithm used in order to attain the desired result: Image segmentation, Edge

Detection using Sobel Operator, Dilating Image using line Structured Element, Filling

Holes in the Image, Removing the Objects in the Border, and Eroding an Image.

In comparison the previous study focused on urinalysis and detection of RBC,

WBC and Epithelial cells while the current study focuses in fecalysis and detection of

E.histolytica only. In contrast, both used and will use the same kind of method which is

image processing specifically image segmentation through the aid of MATLAB software.

Next, the study presented by K.H. Ghazali, R.S. Hadi and M. Zeehaida which is an

automatic system to diagnose human intestinal parasites. The overall research

methodology of the study is divided into two parts decision system and the interfacing

system. The decision system was composed of image acquisition of ALO and TTO using

a microscope camera, image processing stage, image analysis and the classification

method. On the other hand, interfacing system was composed of complete automatic

glass slider system consisting of a microscope, an automatic glass slider handler, a

camera for image acquisition, an arduino interfacing card and a high-end PC to process

and analyze the parasite images. The system capture the images of the sample using a

digital camera attached to the microscope and then directly process the detection of

parasites using the desktop computer. The samples were handled by the automatic

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handler system through the interface with the desktop computer. The system was provided

result directly to the medical technician.

With regards to the study of Pranati Rakshit and Kriti Bhowmik, the stages of

malaria are analyzed and can be detected. They used Matlab as their tool for processing

the sample images that they have collected. Earlier stages of malaria has been seen and

as it matures within RBC. Apart from, detecting malaria, their study can help other severe

diseases in human body and make way to diagnosis by efficient techniques of digital

image processing.

Then, for the research of Carl S. Regan, Lisa Yon, Maqsud Hossain and Hany M.

Elsheikha, the differentiation and recognition of Entamoebas in non-human primates were

conducted through the feces as the sample and PCR as the method. The study focused

on the examination of stool in different zoological gardens to identify the existence of

pathogenic and non-pathogenic parasites. Compared the difference of each species of

Entamoeba through DNA extraction which is molecularly examined in PCR method.

In comparison with the current study, this study tackles broad identification of

Entamoeba since it used a DNA extraction and differentiate various types of parasite.

Meanwhile the current researchers limit the scope in recognizing specifically the

E.hystolica through image segmentation using MATLAB, a computer programming

software.

Lastly, the study of Patrick Gonin and Louise Trudel, detection and differentiation

of Entamoeba histolytica and Entamoeba dispar is made possible through the aid of an

in-depth specific biological method, PCR (Polymer Chain Reaction) and ELISA (Enzyme-

Linked Immunosorbent Assay). This is a comparative study that relates the efficiency of

each mentioned method. Researchers concluded that PCR is the most useful in

differentiating the specific kind of specie.

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 In comparison, the said previous study focusses primarily on the use of biological

method while this study uses image segmentation method through a computer

programming software which is the MatLab. This study focusses only on the detection of

Entamoeba histolytica cyst and there is no other related specie concerned.

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