Automatic Cryptosporidium and Giardia viability detection in treated water
© Badsha et al.; licensee Springer. 2013
Received: 17 May 2013
Accepted: 10 October 2013
Published: 31 October 2013
In the automatic detection of Cryptosporidium and Giardia (oo)cysts in water samples, low contrast and noise in the microscopic images can adversely affect the accuracy of the segmentation results. An improved partial differential equation (PDE) filtering that achieves a better trade-off between noise removal and edge preservation is introduced where the compass operator is utilized to attenuate noise while retaining edge information at the cytoplasm wall and around the nuclei of the (oo)cysts. Then the anatomically important information is separated from the unwanted background noise using the Otsu method to improve the detection accuracy. Once the (oo)cysts are located, a simple technique to classify the two types of protozoans using area, roundness metric and eccentricity is implemented. Finally, the number of nuclei in the cytoplasm of each (oo)cyst is counted to check the viability of individual parasite. The proposed system is tested on 40 microscopic images obtained from treated water samples, and it gives excellent detection and viability rates of 97% and 98%, respectively.
KeywordsParasite calculation Parasite differentiation Nucleus calculation Automatic viability confirmation
Cryptosporidium parvum and Giardia lamblia are two common waterborne parasites infecting humans worldwide . In infected humans, the organisms normally attack the small intestine after being ingested [2, 3]. They are found mostly in surface waters, where their concentration is related to the level of faecal pollution or human use of the water [4–6]. Cryptosporidium and Giardia parasites can also be found in infected sheep, cattle and many other animal species such as cat, beaver, deer and flies. Besides diarrhoea, other symptoms of giardiasis and cryptosporidiosis are slight fever, fatigue and myalgia. In rare cases, these parasites may infect the lungs and trachea, resulting in cough, dehydration and extreme weight loss [7–9].
The impact of epidemic Cryptosporidium and Giardia outbreaks has motivated many researchers to find measures to prevent them from recurring worldwide. As a result, studies related to the transmission, detection and life cycle of the parasites have proliferated. The process of detecting the presence of Cryptosporidium and Giardia (oo)cysts in water and wastewater samples involves filtration, isolation, staining and microscopy. In microscopy, direct visual inspection under a microscope is performed by an expert to detect the presence of Cryptosporidium and Giardia (oo)cysts. This manual inspection is tedious and time-consuming. Attempts have been made to implement automatic detection of (oo)cysts in microscopic images to increase the efficiency of the process. Widmer et al. used an artificial neural network (ANN) algorithm to accurately identify Cryptosporidium and Giardia (oo)cyst images. Back-propagation technique was employed in the training process, and adjustment of weights was made within the neuron layers. The aim was to minimize the error between the predicted and the correct solution . Fernandez-Canque et al. implemented an algorithm that detected Cryptosporidium oocysts using shape and colour features in fluorescein isothiocyanate (FITC)-stained images .
In this paper, an automation detection system is developed to detect the presence of Cryptosporidium and Giardia (oo)cysts in samples of treated water concentrate. The system employs a modified partial differential equation (PDE) filter to denoise FITC- and 4′,6-diamidino-2-phenylindole (DAPI)-stained images. The modified fourth-order PDE filter uses the compass operator instead of the common Laplacian operator so that more edge information is preserved. In addition, roundness metric, nucleus counting and viability measure are also incorporated as additional features. This paper is organized as follows. Section 2 contains details of the steps involved in the system, and Section 3 describes the experimental results of implementing it on 40 stained images. Finally, conclusion is presented in Section 4.
2. Proposed system
After greyscale conversion, it is observed that the greyscale images contain significant noise. As a result, small nuclei appear almost similar to background noise in the image. Therefore, the noise in the DAPI images needs to be filtered while important edge information must be preserved. Preserving the orientation of anatomically significant edges while detecting the nuclei is a challenging task. For this purpose, a modified fourth-order PDE diffusion filter is used.
where E(I) is the energy function, |∇2 I| is the absolute value of the Laplacian of I and Ω is the support domain in the image (I). Since f(|∇2 I|) is an increasing function of |∇2 I|, its global minimum is at |∇2 I| = 0. The Laplacian of an image at a pixel is zero if the image is planar in its neighbourhood. Therefore, the minimization of the functional is equivalent to smoothing the image to piecewise planar.
The coefficient of the Laplacian operator is c(|∇2 I |). Dissipation can be measured by this coefficient. The Laplacian operator is isotropic or uniform to the orientations; therefore, the compass operator has been used as it is free from the orientations. So the coefficient of the new PDE will be c(|O k I|).
Using the Otsu thresholding, the greyscale image is converted into a binary image . The process is described as follows:
where A and P are the area and perimeter of the object, respectively.
An (oo)cyst is considered viable if there are nuclei inside its body. The positions of the (oo)cysts are highlighted in the FITC, but their nuclei can only be seen clearly in the DAPI image. Therefore, the FITC and DAPI binary images have to be merged to see whether the nuclei lie within the (oo)cysts. Since the area and perimeter of each (oo)cyst are already known, if there are no nuclei within the area of the (oo)cyst in the merged image, it is regarded as nonviable.
From the resultant image, nucleus counting is done for viability confirmation. Counting nuclei is similar to counting objects in the image. The number of objects/nuclei can be counted by the following steps .
Set the number of nuclei to 0.
- 2.Scan the image until we locate a pixel (x,y) belonging to a nucleus, i.e. f(x,y = 1).
Increase the number of nuclei by 1.
Find all pixels connected to pixel (x,y), i.e. find the entire object.
Remove the object from the image by setting all its pixel values to 0.
Continue scanning (back to step 2) until we reach the end of the image.
4. Experimental results
Decision system to identify the Cryptosporidium and Giardia
Roundness ≥ 0.9 ∩ area ≤ 1.0e + 003 × 1.24
Eccentricity > 1 ∩ area ≥ 1.0e + 003 × 0.24
Performance evaluation in terms of detection and checking the viability of Cryptosporidium
Result (average, %)
Performance evaluation in terms of detection and checking the viability of Giardia
Result (average, %)
Figure 8a,b displays the FITC and DAPI images which contain both Cryptosporidium and Giardia (oo)cysts, respectively. Figure 8c shows the position of the detected Cryptosporidium oocyst, and Figure 8d shows the value of its roundness metric. Figure 8e confirms the viability of the oocyst and the number of nuclei residing in it. Similarly, Figure 9a,b shows the DAPI and FITC images containing both types of (oo)cysts. Figure 9c shows the location of the detected Giardia cyst, and Figure 9d shows the value of its eccentricity. Finally, Figure 9e confirms the viability of the cyst and the number of nuclei within it.
In this work, a robust technique is presented for image filtering of DAPI- and FITC-stained images for Cryptosporidium and Giardia (oo)cyst detection using compass operator with fourth-order PDE. Roundness metric and eccentricity are utilized to differentiate Cryptosporidium and Giardia (oo)cysts in 40 images. The performance of the system is measured in terms of detection, viability and nucleus counting rates and compared against those of the human observer. Excellent rates of more than 97% are achieved for both parasites on all measures. In the future, segmentation and identification of overlapping and occluded nuclei and (oo)cysts in clean and dirty images will be considered.
This research is fully supported by Exploratory Research Grant Scheme (ER031-2011A) under the Ministry of Higher Education, Malaysia.
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