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Viability assessment of Ascaris suum eggs stained with fluorescent dyes using digital colorimetric analysis
Experimental Parasitology 178 (2017) 7e13
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Viability assessment of Ascaris suum eggs stained with fluorescent dyes using digital colorimetric analysis chniak a, Zbigniew Osin ski b, Magdalena Włodarczyk a, Jolanta Zdybel a, *, Marek Pro c c, Tomasz Cencek a Jacek Karamon a, Teresa Kłape a b c
Department of Parasitology and Invasive Diseases, National Veterinary Research Institute in Pulawy, Al. Partyzantow 57, 24-100 Pulawy, Poland Department of Hygiene of Animal Feedingstuffs, National Veterinary Research Institute in Pulawy, Al. Partyzantow 57, 24-100 Pulawy, Poland Department of Biological Health Hazards and Parasitology, Institute of Rural Health, Jaczewskiego 2, 20-090 Lublin, Poland
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Fluorescent dyes are used for the differentiation of live and dead nematode eggs. In the study computer software was used for colour analysis of dyed eggs. This method gives repeatable results for the classification of dead and live eggs.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 October 2016 Received in revised form 28 March 2017 Accepted 23 April 2017 Available online 4 May 2017
The aim of the study was to develop a method for the colorimetric evaluation of nematode eggs using appropriate instruments. The materials for the study were live and dead (inactivated) eggs of the Ascaris suum. Viability of the eggs was assessed using four different kits for fluorescent staining (for each technique, a series of photos were taken). Images of stained eggs were analysed using graphic software with RGB (red-green-blue) function. The viability of the eggs was assessed according to the relative positions of the distributions of colour intensities of live or dead eggs - distributions area's overlap index (DAOI), and distributions area's separation index (DASI) were calculated. Computer analysis of the intensity of green colour was not satisfactory. However, analysis of images in the spectrum of red colour proved useful for the effective differentiation between live or dead eggs. The best parameters were observed using the Annexin V FITC Apoptosis Detection Kit (DASI ¼ 41 and 67). The investigation confirmed the usefulness of fluorescent dyes used in conjunction with digital analysis for the assessment of the viability of A. suum eggs. The use of computer software allowed a better objectivity of the assessment, especially in the case of doubtful staining. © 2017 Elsevier Inc. All rights reserved.
Keywords: Ascaris suum eggs Viability assessment Fluorescent dyes Digital colorimetric analysis
1. Introduction For the safety for humans and animals, it is a necessary to monitor the parasitological contamination of wastewater, sewage
* Corresponding author. E-mail address: [email protected] (J. Zdybel). http://dx.doi.org/10.1016/j.exppara.2017.04.012 0014-4894/© 2017 Elsevier Inc. All rights reserved.
sludge and organic fertilizers prior to their introduction into the environment The need to conduct parasitological studies of these substances results from legal regulations in effect in Poland, the EU and worldwide. In accordance with these regulations, sewage sludge or organic fertilizers introduced into the soil must not contain live eggs of the intestinal parasites of the genera Ascaris, Toxocara and Trichuris (Directive 86/278/EEC, 1986; US EPA, 1999;
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WHO, 2006; Final Report by Milieu Ltd, 2008; Gantzer et al., 2001, Regulation of the Minister of Environment, 2015). However, the differentiation of live eggs (invasive) and dead eggs (non-invasive) is an element of parasitological studies that causes many problems (Da˛ browska et al., 2014). At present, in Poland, similar to other countries worldwide, the method of viability assessment of nematode eggs recommended in the legal regulations in effect consists of evaluating for the appearance of larvae in eggs incubated in a moist chamber at approximately 27 C for a period of 2e3 weeks, and their periodical observation under a microscope (Polish Standard, 2001; Bazeli, 2007). However, this method is very time-consuming, and requires an experienced investigator. In microbiology, staining techniques with fluorescent dyes are used for the differentiation of live and dead cells of bacteria and fungi. These techniques are more often described in the literature, which results from an increased interest in quick and precise methods that are alternatives to traditional methods (Sadowska and Grajek, 2009). Fluorescent dyes contain mainly polycyclic aromatic compounds, which may absorb a specific wavelength, particularly with respect to ultraviolet radiation, and emit light of a lower energy in the visible spectrum (Haugland, 2001). The energies of emission of fluorochromes have different values; therefore, the visible radiation has colours from deep red to green and blue. Due to the high selectiveness, these substances enable the investigation of intracellular single cell parameters, such as membrane potential, membrane integrity, respiratory activity, content of intracellular enzymes and nucleic acids, intracellular pH, and metabolic activity (Shapiro, 2000; Nebe-von-Caron et al., 2000; Sadowska and Grajek, 2009). Each dye kit used for differentiation of cells contains solutions that stain live and dead cells, enabling their simultaneous identification. For example, in the FluoCell Double Staining Kit (Calcein AM/PI), one of the dyes (calcein) is highly lipophilic and therefore easily penetrates the intact structure of the egg sheath into its interior. In a live cell, the dye is subject to decomposition by cytoplasmic esterases into fluorescein (a substance with lipophilic properties) and emits strong, green fluorescence. Live cells show high esterase activity, resulting in considerable amounts of fluorescein, which consequently leads to intense green fluorescence. Dead cells are characterized by lower esterase activity and more poorly preserved integrity of the cell membrane; therefore, they accumulate less of the hydrolysis product, which results in lower values of green fluorescence (De˛ bowska et al., 2007). The second dye, propidium iodide, penetrates exclusively into the interior of eggs with a damaged sheath structure and reacts with nucleic acids to stain the egg red. Both dyes may be excited by a wavelength of 490 nm, which allows for the simultaneous monitoring (using a fluorescence microscope) of live and dead eggs. Fluorescent dyes are also used for the differentiation of live and dead eggs of intestinal parasites. However, the studies devoted to these methods are scarce, and the majority of researchers have noted their limited usefulness (Da˛ browska et al., 2014; Karkashan et al., 2015). The aim of the study was to develop a method that would allow for the colorimetric evaluation of nematode eggs using proper instruments, which would eliminate the stages based on subjective assessments by analysts. The elaborated method should enable more reliable and repeatable results for the classification of dead and live eggs stained using fluorescent dye kits and also allow for a reduction in the amount of time devoted to the observations and, at the same time, reduce the duration of the examination.
2. Materials and methods 2.1. Preparation of material for the studies: eggs of Ascaris suum The materials for the study were eggs of the intestinal parasites Ascaris suum (large pig roundworm). Parasites were collected from the intestinal tract content of pigs slaughtered in the registered slaughterhouse. The eggs were isolated directly from the uterus of the roundworm and resuspended in PBS buffer. A part of the material was subjected to thermal inactivation in a water bath at 70 C for 1 h. Two groups of eggs were produced, live eggs (non-inactivated) and dead eggs (inactivated). 2.2. Methods used for fluorescent staining Assessment of egg viability was performed using kits for fluorescent staining. The colouring of live and dead eggs was compared. Four kits of stain were used in the study as follows: a commercial FluoCell Double Staining Kit (Calcein AM/PI) (MoBi Tec, Germany), Annexin V FITC Apoptosis Detection Kit (Sigma-Aldrich, USA), LIVE/ DEAD BacLight Bacterial Viability Kit (Molecular Probes Inc., USA), and a kit composed of the dyes:fluorescein diacetate (SigmaAldrich, USA), and propidium iodide (MILLIPORE, USA). The study was conducted in two repetitions for each kit. 2.2.1. FluoCell Double Staining Kit (calcein AM/PI) The dying solution was prepared as follows: 20 ml of calcein and 10 ml of propidium iodide added to 5 ml of PBS. Subsequently, 200 ml containing about 150 of live eggs suspension and 200 ml containing about 150 of dead eggs were collected, and then 100 ml of previously prepared staining solution was added to each. After mixing, the samples were incubated for 15 min at 37 C. 2.2.2. Annexin V FITC Apoptosis Detection Kit A volume of 200 ml containing about 150 of A. suum egg suspension of was centrifuged. The supernatant was poured off and the sediment was resuspended in 200 ml 2 concentrate binding buffer. Then, 5 ml of annexin and 10 ml of propidium iodide were added to the prepared egg suspension. The samples were incubated for 15 min at room temperature. After incubation, 400 ml of binding solution was added to the samples at a working dilution. 2.2.3. LIVE/DEAD BacLight Bacterial Viability Kit A mixture was prepared of components A and B included in the kit, at the proportion 1:1. Then, 0.66 ml of the dye mixture was added per each 200-ml containing about 150 of A. suum egg suspension. The samples were mixed and incubated at room temperature (approx. 22 C) for 15 min protected from light. 2.2.4. Fluorescein diacetate and propidium iodide The initial solution of fluorescein diacetate was prepared at a concentration of 0.5 mg/ml acetone. Subsequently, the A dye was prepared, which constituted 0.4 ml/ml of the initial solution in PBS. The B dye was a solution of propidium iodide at a concentration of 20 mg/ml PBS. To perform the test, 200 ml containing about 150 of egg solution was sampled from both groups. To each group was added 100 ml of dye A and 30 ml of dye B. Previously mixed samples were incubated at room temperature for 3 min. 2.3. Assessment of stained eggs viability by means of fluorescence microscope After staining, the egg suspension was filtered using a subpressure filtering device with polycarbonate filters with a pore
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diameter of 12 mm (Whatman, USA). The filters were transferred to glass slides. Subsequently, several drops of glycerine were added and spread equally over the entire surface of the filter. The specimens were observed under a fluorescence microscope with the appropriate filter set with a wavelength of 490 nm and with a magnification of 400. The colouring of live and dead eggs was assessed for all dye kits.
2.4. Analysis of images of Ascaris suum stained eggs using graphic software with image analysis function RGB (red-green-blue) For colour analysis 25 live and 25 dead eggs were randomly selected from each group. For each of the staining techniques, a Canon EOS 700D camera was used, and two series were obtained (one series for each repetition), with 25 photos of inactivated eggs (dead) and 25 photos of non-inactivated (live) eggs each. The camera used was equipped with an 18-megapixel Hybrid CMOS AF matrix. For computer analysis of the image parameters in the RGB (RedGreen-Blue) mode, CorelDRAW software, version Graphic Suite 7, Corel Photo Paint module was used. Using this software, images of the stained eggs were analysed such that in a photo, a surface was determined from the middle of the egg with a size of 340 340 pixels. Subsequently, the parameters of the RGB histogram of the determined area of the image were analysed (mean value and standard deviation of saturation of two colours of the image, green and red). This analysis was based on the assumption that each colour can be compared using a parametric description of the intensity of each colour or its individual components using numbers. Such a system is commonly used in digital systems for the description of colours. In the RGB system applied, each colour is in the form of a mixture of pure colours, red, green and blue. The contribution of each component is expressed by the intensity according to the scale of 0e255 for each of the three basic colours (Fig. 1). To assess the differentiation of eggs (for each type of staining), the relative positions of the distributions of the obtained intensities of colours for live and dead eggs were considered. The assessment was performed using the distributions area's overlap index (DAOI), and the distributions area's separation index (DASI). The DAOI expressed the so-called common area of colour intensity intervals obtained for live and dead eggs, and it was an arithmetic difference between the maximum intensity values for stained live eggs and the minimum values for the computer analysed specimens of images of dead eggs (Maxlive - Mindeath). The DASI expressed the difference between the minimum intensity for dead eggs and maximum intensity for live eggs (Mindeath - Maxlive).
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2.5. Statistical methods To evaluate the techniques of staining and to select the most favourable for the developed method, the following parameters were analysed: specificity (Sp), sensitivity (Se) and accuracy (Ac). These parameters were determined using the receiver-operating characteristic analysis for diagnostic tests (ROC curve) with Youden correction (Greiner et al., 2000). The objective of the statistical techniques was selection of the cut point and its optimization by obtaining the best parameters of the analytical method developed. Statistical calculations were performed using the Epitools calculators (Sergeant, 2015). 3. Results Based on the results of these studies, all fluorescent dye kits used enabled a visual (microscopic) identification of live eggs (noninactivated) and dead eggs (inactivated). All kits applied in the study coloured the live (non-inactivated) A. suum eggs green (Fig. 2A and B, C, D). The egg sheaths were particularly intensely coloured. The dead eggs (inactivated) were coloured orange using three of the kits. The exception was the LIVE/DEAD BacLight
Fig. 2. Ascaris suum eggs: live (non-inactivated) and dead (inactivated) stained with the dye kits. A) FluoCell Double Staining Kit. B) Annexin V FITC Apoptosis Detection Kit. C) LIVE/DEAD BacLight Bacterial Viability Kit. D) Fluorescein diacetate and propidium iodide.
Fig. 1. Analysis of stained egg images using graphic software: a) determination of the analysed surface from inside the stained egg and b) reading based on the histogram for the green channel of the mean value of colour saturation. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Bacterial Viability Kit, in which the dead eggs (inactivated) were yellow (Fig. 3 C). For all staining methods, there was a low percentage of inactivated eggs coloured yellow to yellow-green (Fig. 3). The images show that an unequivocal classification of these eggs into live and dead based on their colour was difficult. Such an analysis is characterized by the high subjectivity of evaluations by those who perform the analysis. Therefore, the images were subjected to colour analysis using the graphic software CorelDRAW Graphic Suite 7. Tables 1 and 2 demonstrate the results (for two repetitions) of the analysis of intensity of components of the colours, green (G) and red (R) of the RGB image using the graphic software. Within the range of the green colour, regardless of the method of staining, live eggs of A. suum yielded lower mean values of colour intensity (114.8e183.2) than dead eggs (146.1e205.6). The lowest mean intensity of green colour was obtained for staining with fluorescein diacetate þ propidium iodine (dead: 146.1 and 160.5, and live: 131.3 and 138.9, respectively). The highest mean intensities of green colour were obtained for staining with the LIVE/ DEAD BacLight Bacterial Viability Kit (dead: 205.6 and 192.0, and live: 182.0 and 183.2, respectively). Within the range of green colour, a common area was observed for all four types of staining. For the FluoCell Double Staining Kit (Calcein AM/PI), the common value for the range of results for analysis of image of live and dead eggs was 74.2 in the first repetition, and 32.9 in the second repetition, compared to the scale of 255 possible levels of colour intensity constituting 29% and 13% of the common area, respectively. However, for fluorescein diacetate þ propidium iodine, the values were 122.2 and 112.7, which in this scale was as much as 48% and 44% of the common area, respectively. Such a large common area made it impossible to determine threshold values for the colour of live and dead eggs. Therefore, for green, statistical calculations were discontinued. At the same time, the values for the intensity of the red colour were subjected to the same assessment for the images of live and dead eggs. Table 2 presents the results obtained. For the red colour, live eggs of Ascaris suum yielded mean values of colour intensity within the range of 48.9e100, and the values for dead eggs were in the range of 188e228. The lowest mean intensity for the red colour was obtained using the LIVE/DEAD BacLight, (live, 48.9 and 52.9, and dead, 188 and 203. The highest mean intensities for red colour were obtained for fluorescein diacetate þ propidium iodine (live, 86.8 and 100, and dead, 228 and 204). For one case of staining with fluorescein diacetate þ propidium iodine, a small degree of mutual overlapping for red colour intensity was observed for live and dead eggs. For the remaining staining methods, the red colour intensity intervals measured in
live and dead eggs were separable. Using the Annexin V FITC Apoptosis Detection Kit, the distribution area separation index of results for analysis of the red colour of live and dead eggs assumed the highest values, equal to 41 and 67, in the second repetition of the study series. This constituted, with the total range of values possible of 255, the area of separation of two groups within the range of 16%e26%. The subsequent staining with FluoCell Double Staining Kit (Calcein AM/PI) resulted in an area characterizing the difference between live and dead eggs within the range 13.3% (value 34) - 18.4% (value 47). For fluorescein diacetate þ propidium iodine, the least favourable results were obtained for characterizing dead and live eggs of A. suum using the analysis of red colour, from the mutual overlapping of 0.8% of the range to 10.2% (value 26), the area of which is the difference between extreme results, Mindeath and Maxlive. Distributions of the analysed results in the majority of cases were not close to the normal distribution (Gauss), which excluded the application of statistical parametric methods for the assessment of staining methods. Therefore, the distribution area separation index of the results of red colour intensity for live and dead eggs (DASIred) was evaluated. Arithmetically, this was the difference between the minimum values of intensities of stained dead eggs and the maximum values for computer analysed sections of live eggs images. For further analysis, the receiver-operating characteristic analysis for diagnostic tests (ROC) was applied using Youden's J index. The calculated Youden's J index was used to determine the most beneficial cut-off point for the qualification of results characterizing live and dead eggs and was most stable for repetitions of studies using the Annexin V FITC Apoptosis Detection Kit. The Youden's J differences obtained between repetitions were only 0.5. The FluoCell Double Staining Kit (Calcein AM/PI) method differed in the Youden's J value by 1.8. Within the range of 255 possible results, the differences were very small, which indicates high stability of the results obtained using these staining methods. With the remaining two staining methods assessed, the differences were larger, with values of 7.9 for the LIVE/DEAD BacLight Bacterial Viability Kit and 20.1 for fluorescein diacetate þ propidium iodine, which indicates lower stability compared to the other two methods. The determination of cut-off points using Youden's statistics gave the following parameters of analytical methods for the first three staining methods: sensitivity SE ¼ 1.00 (CL95% ¼ 0.867e1.00) and specificity SP ¼ 1.00 (CL95% ¼ 0.862e1.00). Considering the last evaluated staining method, in the second repetition, the difficulty of differentiating a low percentage of live from dead eggs using fluorescein diacetate þ propidium iodine, assuming a lower value of cut-off point using Youden's statistics of 155.5, yielded a sensitivity of SE ¼ 0.96 (CL95% ¼ 0.805e0.993). However, this indicates that dead eggs will not be detected by 4%, on average, and considering the confidence interval, in the worst case, by as much as 19.5%. 4. Discussion
Fig. 3. Dead Ascaris suum eggs coloured yellow or yellow-green stained with the dye kit Fluorescein diacetate þ propidium iodide. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Attention is frequently paid to limits of traditional methods for the determination of parasite eggs vitality, in which the only differentiation criterion is the visual method (Błaszczyk and KrzyskoŁupicka, 2014; Kalisz et al., 2003). These methods are imprecise and burdened with high error due to the subjectivity of assessment of the person performing the test. The use of fluorescent dyes for the specific simultaneous staining of live and dead eggs would be beneficial. Such methods are often used in microbiology for the differentiation of bacteria and fungi. In our own studies, three commercial kits of fluorescent dyes were used. FluoCell Double Staining Kit (Calcein AM/PI) (Mo BiTec, Germany), Annexin V FITC Apoptosis Detection Kit (Sigma-Aldrich, USA), LIVE/DEAD BacLight
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Table 1 Parameters of the intensity of green colour for images of live and dead Ascaris suum eggs using various staining techniques. Analysis within the range of basic colour
Repetition
FluoCell Double Staining Kit (Calcein AM/PI)
I
I II
LIVE/DEAD BacLight Bacterial Viability Kit
I II
Fluorescein diacetate þ propidium iodide
Intensity values for green colour
Dead Live Dead Live Dead Live Dead Live Dead Live Dead Live Dead Live Dead Live
II Annexin V FITC Apoptosis Detection Kit
Type of egg
I II
Min
Mean
Max
Std. deviation
DAOI
116 88.7 119.9 94.2 121.3 101.6 123.8 115.9 119.5 136.8 119.5 138.1 73.3 80.7 82.8 108.5
174.1 119.6 188.2 114.8 179.3 151.3 179.5 149.3 205.6 182 192 183.2 146.1 131.3 160.5 138.9
225.5 190.2 225.5 152.8 254.9 209.7 218.7 185.9 255 218.4 246 216 212.3 195.5 208.4 195.5
28.9 18.9 29.6 13.2 33.1 22.7 28.5 19.2 37.4 23 43.2 19.8 41.5 25.1 39.1 23.3
74.2 32.9 88.4 62.1 98.9 96.5 122.2 112,7
Table 2 Parameters of the intensity of red colour for images of live and dead eggs of Ascaris suum using various staining techniques. Staining technique
Repetition Type of egg Intensity values for red colour Min Mean Max Std. deviation DASI (difference between Mindeath and Maxlive)
FluoCell Double Staining Kit (Calcein AM/PI) I
Dead
163 217
240 17.4
Live
33.1 58.9
129 20.5
Dead
161 223
250 24.9
Live
34.4 62.2
114 19.3
Dead
175 221
251 20.7
Live
55.5 84.9
134 20.6
Dead
175 225
255 25.4
Live
37.3 73.6
108 17
Dead
109 203
252 40.8
Live
24.4 52.9
80.8 13.4
II
Dead Live
117 188 20.8 48.9
I
Dead
II
Annexin V FITC Apoptosis Detection Kit
I
II
LIVE/DEAD BacLight Bacterial Viability Kit
Fluorescein diacetate þ propidium iodide
I
II
Youden's J Method parameters obtained (cut-point) (95% CL)
34
162.9
47
161.1
41
175.1
67
174.6
28.2
108.9
239 40.2 79.2 17.2
37.8
116.8
156 204
255 31.9
26
155.5
Live
53.7 86.8
130 19.4
Dead
164 228
254 26.8
2
175.6
Live
65.2 100
166 25.8
Bacterial Viability Kit (Molecular Probes Inc., USA) and a kit using fluorescein diacetate (Sigma-Aldrich, USA) and propidium iodine (MILLIPORE, USA). The majority of the dyes stained live eggs of A. suum green, whereas dead eggs were orange. A slightly different colouring of eggs was obtained with the kit LIVE/DEAD BacLight, consisting of two components, SYTO 9 and PI. According to the manufacturer, the SYTO 9 component penetrates into bacterial cells that are live and those with intact cellular structure, whereas the PI component stains dead cells exclusively. In our studies, live cells (not exposed
SE ¼ 1.00 (0.867e1.00) SP ¼ 1.00 (0.862e1.00) SE ¼ 1.00 (0.867e1.00) SP ¼ 1.00 (0.862e1.00) SE ¼ 1.00 (0.867e1.00) SP ¼ 1.00 (0.862e1.00) SE ¼ 1.00 (0.867e1.00) SP ¼ 1.00 (0.862e1.00) SE ¼ 1.00 (0.867e1.00) SP ¼ 1.00 (0.862e1.00) SE ¼ 1.00 (0.867e1.00) SP ¼ 1.00 (0.862e1.00) SE ¼ 1.00 (0.867e1.00) SP ¼ 1.00 (0.862e1.00) SE ¼ 0.96 (0.805e0.993) SP ¼ 1.00 (0.862e1.00)
to a high temperature) were stained yellow, consistent with the description, although a particularly clear colouring was obtained for egg sheaths. This is consistent with the results of previous studies conducted by Da˛ browska et al. (2014) and Karkashan et al. (2015). In the experiment performed by Karkashan, live eggs with intact sheath structure absorbed neither SYTO 9 nor PI. To facilitate the absorption of dyes, the researcher used sodium hypochlorite, which removed the outer protein layer of the egg sheath. However, this allowed only the staining of the inner lipoprotein layer by both dye A (SYTO 9), and dye B (PI). In our experiment, dye absorption
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occurred without removal of the outer protein sheath. The differences between own results and those obtained by Karkashan et al. (2015) may result from the use of eggs prepared differently for the experiment (e.g., obtaining mature eggs from the final section of the roundworm uterus or obtaining them from faeces, or the ‘whitening’ of eggs using sodium hypochlorite). It may also be a result of the observation method because Karkashan observed egg fluorescence using a confocal microscope, whereas in our studies, a traditional fluorescence microscope was used, in which individual layers emitting light mutually overlap. Nevertheless, this does not explain the relatively low intensity of red colour in live eggs in our experiment. For dead eggs, Karkashan et al. (2015) observed that both dyes penetrated into their interiors. Similarly, in our studies, this was observed for dead eggs stained an intermediate colour between green and red fluorescence, which indicates the presence of both dyes inside the eggs and, at the same time, hindered interpretation of the results. Gasol et al. (1999) encountered similar difficulties in the determination of bacterial cells with the Live/Dead BacLight kit using flow cytometry. To obtain a greater differentiation of colours, the researchers washed the filter with stained bacteria with isopropyl alcohol. Because both dyes components of the kit react with nucleic acids during competition for the binding site of one component, isopropanol removed the dye that was more weakly bound. An uneven absorption and binding of dyes to eggs of A. suum was also observed for the remaining dye kits, although to a lower degree. Some of the eggs showed yellow fluorescence caused by the penetration of both dyes into the inside of the eggs. Such eggs caused difficulties in interpretation of results and their classification as live or dead eggs. Difficulties with obtaining a uniform colour were also reported by de Victorica and Galvan (2003), and Jensen et al. (2009). These researchers found that some dyes may be toxic to live eggs and larvae and cause the leakage of components directly into the interior of dying eggs, thus making observation difficult. Therefore, according to the WHO (WHO, 2004), it is recommended to examine fluorescence dyes within several minutes after their application. An uneven intensity of colours may also be noted inside dead eggs, as seen on images in the report by Karkashan et al. (2015). This was likely due to various amounts of DNA, which both dyes bind (after penetration through intact structures of cells), emitting fluorescence. The eggs of A. suum used by these researchers, at the moment of inactivation with high temperature, were at various stages of development. Some contained developed larvae, which may have resulted in their more intense colour, in contrast to those containing an embryo at the initial stages of development. As a result of the these studies, the staining that would allow for the differentiation of eggs into live or dead has risks of a difficult and often faulty image for visual assessment. Therefore, for the determination of egg viability, additional instruments were used, which allowed the elimination of the risk of erroneous assessment. For this purpose, computer software was used to compare the mean intensity of colours, with red and green for live and dead eggs, respectively. The results obtained for the intensity of basic green colour were not satisfactory. A considerable common area described by the DAOI value between the results for live and dead eggs, particularly for fluorescein diacetate þ propidium iodide. This made it impossible to use this approach for the development of a reliable method for the identification of live and dead eggs. This partially results from properties of the digital description of colours, in which the yellow and orange colour indicated dead eggs in the microscopic image and is composed of the basic colours RGB, to which the basic green colour has a great contribution. The use of images within the basic red colour showed its usefulness for the effective
differentiation between live and dead eggs of A. suum. Additionally, selection of the staining method is of important for obtaining good parameters of the analytical method. The Annexin V FITC Apoptosis Detection Kit was the most beneficial. Good parameters of detection were obtained (DASI ¼ 41 and 67), with a small probability of obtaining false positive and false negative results, with a diagnostic sensitivity SE ¼ 1.00 (0.867e1.00) and specificity SP ¼ 1.00 (0.862e1.00). Analysis of parameters obtained while preserving the intra-laboratory repeatability conditions with a series of specimens in two repetitions, also confirmed the superiority of staining with the Annexin V FITC Apoptosis Detection Kit. The Youden J index for these staining series was nearly identical. However, for fluorescein diacetate þ propidium iodide, even the most favourable cut-off point for the differentiation between live and dead eggs of A. suum, may cause the occurrence of a certain percentage of wrongly classified eggs. The estimated parameter of diagnostic sensitivity of the method is SE ¼ 0.96 (0.805e0.993). It should be emphasized, however, that the presented study was conducted under experimental conditions. This is the first description of such a method for the estimation of colorimetric viability of stained eggs, which is the basis for further, more practical application. Therefore, there is a need of further investigations with the use of samples from naturally contaminated environmental samples. 5. Conclusion These studies confirm the usefulness of fluorescent dyes for the viability assessment of the eggs of intestinal parasites of A. suum. The staining methods may be an alternative to time-consuming traditional methods based on subjective evaluation by a researcher. The additional use of computer software for the analysis of images within the range of red colour allows the avoidance of erroneous assessment in the case of doubtful staining. The advantage of this method is also obtaining more repeatable and reliable results for the classification of live and dead eggs. Acknowledgments Funding: This work was supported by the Polish Ministry of Science and Higher Education [grant number NN 305 6013 39]. References Bazeli, M., 2007. Biological Analysis of Sewage Sludge. BIOM Biological Laboratory (In Polish). Pracownia Biologiczna BIOM. Wyd. PPR STUDIO K2. Piła. Błaszczyk, K., Krzysko-Łupicka, T., 2014. Overview of the research methods for _ sewage sludge used in Poland. Inzynieria i Ochr. Srodowiska 17 (1), 117e133. Da˛ browska, J., Zdybel, J., Karamon, J., Kochanowski, M., Stojecki, K., Cencek, T., 2014. Assessment of viability of the nematode eggs (Ascaris, Toxocara, Trichuris) in sewage sludge with the use of LIVE/DEAD Bacterial Viability Kit. Ann. Agric. Environ. Med. 21 (1), 35e41. De˛ bowska, R., Bazela, K., Eris, I., 2007. Apoptoza i ochronne działanie kwasu foliowego. Dermatol. Estet. 9 (2), 83e90. Directive 86/278/EEC, 1986. Council directive on the protection of the environment, and in particular of the soil, when sewage sludge is used in agriculture. Off. J. Eur. Communities L 181, 6e12. de Victorica, J., Galvan, M., 2003. Preliminary testing of a rapid coupled methodology for quantitation/viability determination of helminth eggs in raw and treated wastewater. Water Res. 37, 1278e1287. Final Report by Milieu Ltd, 2008. WRc and RPA for the European Commission DG Environment under Study Contract DG ENV.G.4/ETU/2008/0076 R. Environmental, economic and social impacts of the use of sewage sludge on land. Milieu Ltd, (Belgium), Brussels. Gantzer, C., Gaspard, P., Galvez, L., Huyard, A., Dumouthier, N., Schwartzbrod, J., 2001. Monitoring of bacterial and parasitological contamination during various treatment of sludge. Water Res. 35 (16), 3763e3770. Gasol, J.M., Zweifel, U.L., Peters, F., Furhman, J.A., Hagstrom, A., 1999. Significance of size and nucleic acid content heterogeneity as measured by flow cytometry in natural planktonic bacteria. Appl. Environ. Microbiol. 65, 4475e4483. Greiner, M., Pfeiffer, D., Smith, R.D., 2000. Principles and practical application of the
M. Włodarczyk et al. / Experimental Parasitology 178 (2017) 7e13 receiver-operating characteristic analysis for diagnostic tests. Prev. Veterinary Med. 45, 23e41. Haugland, R.P., 2001. Handbook of Fluorescent Probes and Research Chemicals, eighth ed. Molecular Probes. Jensen, P.K.M., Phuc, P.D., Konradsen, F., Klank, L.T., Dalsgaard, A., 2009. Survival of Ascaris eggs and hygienic quality of human excreta in Vietnamese composting latrines. Environ. Health 8, 57. Kalisz, L., Nechay, A., Ka zmierczuk, M., Sałbut, J., Szyprowska, E., Gierczak, A., Kostrzewa-Szulc, J., 2003. Physico-chemical and biological referential methods of investigating municipal sewage sludge. In: The Main Inspectorate for Environment Protection. Environmental Monitoring Library, Warsaw. Karkashan, A., Khallaf, B., Morris, J., Thurbon, N., Rouch, D., Smith, S.R., Deighton, M., 2015. Comparison of methodologies for enumerating and detecting the viability of Ascaris eggs in sewage sludge by standard incubation - microscopy, the BacLight Live/Dead viability assay and Rother vital dyes. Water Res. 68, 533e544. Nebe-von-Caron, G., Stephens, P.J., Hewitt, C.J., Powell, J.R., Badley, R.A., 2000. Analysis of bacterial function by multi-colour fluorescence flow cytometry and single cell sorting. J. Microbiol. Methods 42, 97e114.
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Polish Standard PN-Z-19000-4, 2001. Soil Quality e Assessment of the Soil Sanitary Conditions e Detection of Eggs of the Intestinal Parasites Ascaris lumbricoides and Trichuris trichiura (In Polish). Regulation of the Minister of the Environment of 6 February 2015 on municipal sewage sludge, 2015. J. Laws volume 257. Sadowska, J., Grajek, W., 2009. Analysis of physiological state of single bacterial cell using fluorescent staining methods. Biotechnologia 4 (87), 102e114. Sergeant, E., 2015. Epitools Epidemiological calculators., AusVet Animal Health Services and Australian Biosecurity Cooperative Research Centre for Emerging Infectious Disease. Shapiro, H.M., 2000. Microbial analysis at the single-cell level: tasks and techniques. J. Microbiol. Methods 42, 3e16. US. Environmental Protection Agency, 1999. Environmental Regulations and Technology: Control of Pathogens and Vector Attraction in Sewage Sludge. EPA/625/ R-92/013. Revised edition. U.S. EPA, Washington, D.C. WHO, 2004. Integrated Guide to Sanitary Parasitology. World Health Organization, Geneva, Switzerland. WHO-EM/CEH/121/E. WHO, 2006. Guidelines for the safe use of wastewater, excreta and greywater. In: Policy and Regulatory Aspect, 1. World Helth Organization, Geneva.
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Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr
Viability assessment of Ascaris suum eggs stained with fluorescent dyes using digital colorimetric analysis chniak a, Zbigniew Osin ski b, Magdalena Włodarczyk a, Jolanta Zdybel a, *, Marek Pro c c, Tomasz Cencek a Jacek Karamon a, Teresa Kłape a b c
Department of Parasitology and Invasive Diseases, National Veterinary Research Institute in Pulawy, Al. Partyzantow 57, 24-100 Pulawy, Poland Department of Hygiene of Animal Feedingstuffs, National Veterinary Research Institute in Pulawy, Al. Partyzantow 57, 24-100 Pulawy, Poland Department of Biological Health Hazards and Parasitology, Institute of Rural Health, Jaczewskiego 2, 20-090 Lublin, Poland
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Fluorescent dyes are used for the differentiation of live and dead nematode eggs. In the study computer software was used for colour analysis of dyed eggs. This method gives repeatable results for the classification of dead and live eggs.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 October 2016 Received in revised form 28 March 2017 Accepted 23 April 2017 Available online 4 May 2017
The aim of the study was to develop a method for the colorimetric evaluation of nematode eggs using appropriate instruments. The materials for the study were live and dead (inactivated) eggs of the Ascaris suum. Viability of the eggs was assessed using four different kits for fluorescent staining (for each technique, a series of photos were taken). Images of stained eggs were analysed using graphic software with RGB (red-green-blue) function. The viability of the eggs was assessed according to the relative positions of the distributions of colour intensities of live or dead eggs - distributions area's overlap index (DAOI), and distributions area's separation index (DASI) were calculated. Computer analysis of the intensity of green colour was not satisfactory. However, analysis of images in the spectrum of red colour proved useful for the effective differentiation between live or dead eggs. The best parameters were observed using the Annexin V FITC Apoptosis Detection Kit (DASI ¼ 41 and 67). The investigation confirmed the usefulness of fluorescent dyes used in conjunction with digital analysis for the assessment of the viability of A. suum eggs. The use of computer software allowed a better objectivity of the assessment, especially in the case of doubtful staining. © 2017 Elsevier Inc. All rights reserved.
Keywords: Ascaris suum eggs Viability assessment Fluorescent dyes Digital colorimetric analysis
1. Introduction For the safety for humans and animals, it is a necessary to monitor the parasitological contamination of wastewater, sewage
* Corresponding author. E-mail address: [email protected] (J. Zdybel). http://dx.doi.org/10.1016/j.exppara.2017.04.012 0014-4894/© 2017 Elsevier Inc. All rights reserved.
sludge and organic fertilizers prior to their introduction into the environment The need to conduct parasitological studies of these substances results from legal regulations in effect in Poland, the EU and worldwide. In accordance with these regulations, sewage sludge or organic fertilizers introduced into the soil must not contain live eggs of the intestinal parasites of the genera Ascaris, Toxocara and Trichuris (Directive 86/278/EEC, 1986; US EPA, 1999;
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WHO, 2006; Final Report by Milieu Ltd, 2008; Gantzer et al., 2001, Regulation of the Minister of Environment, 2015). However, the differentiation of live eggs (invasive) and dead eggs (non-invasive) is an element of parasitological studies that causes many problems (Da˛ browska et al., 2014). At present, in Poland, similar to other countries worldwide, the method of viability assessment of nematode eggs recommended in the legal regulations in effect consists of evaluating for the appearance of larvae in eggs incubated in a moist chamber at approximately 27 C for a period of 2e3 weeks, and their periodical observation under a microscope (Polish Standard, 2001; Bazeli, 2007). However, this method is very time-consuming, and requires an experienced investigator. In microbiology, staining techniques with fluorescent dyes are used for the differentiation of live and dead cells of bacteria and fungi. These techniques are more often described in the literature, which results from an increased interest in quick and precise methods that are alternatives to traditional methods (Sadowska and Grajek, 2009). Fluorescent dyes contain mainly polycyclic aromatic compounds, which may absorb a specific wavelength, particularly with respect to ultraviolet radiation, and emit light of a lower energy in the visible spectrum (Haugland, 2001). The energies of emission of fluorochromes have different values; therefore, the visible radiation has colours from deep red to green and blue. Due to the high selectiveness, these substances enable the investigation of intracellular single cell parameters, such as membrane potential, membrane integrity, respiratory activity, content of intracellular enzymes and nucleic acids, intracellular pH, and metabolic activity (Shapiro, 2000; Nebe-von-Caron et al., 2000; Sadowska and Grajek, 2009). Each dye kit used for differentiation of cells contains solutions that stain live and dead cells, enabling their simultaneous identification. For example, in the FluoCell Double Staining Kit (Calcein AM/PI), one of the dyes (calcein) is highly lipophilic and therefore easily penetrates the intact structure of the egg sheath into its interior. In a live cell, the dye is subject to decomposition by cytoplasmic esterases into fluorescein (a substance with lipophilic properties) and emits strong, green fluorescence. Live cells show high esterase activity, resulting in considerable amounts of fluorescein, which consequently leads to intense green fluorescence. Dead cells are characterized by lower esterase activity and more poorly preserved integrity of the cell membrane; therefore, they accumulate less of the hydrolysis product, which results in lower values of green fluorescence (De˛ bowska et al., 2007). The second dye, propidium iodide, penetrates exclusively into the interior of eggs with a damaged sheath structure and reacts with nucleic acids to stain the egg red. Both dyes may be excited by a wavelength of 490 nm, which allows for the simultaneous monitoring (using a fluorescence microscope) of live and dead eggs. Fluorescent dyes are also used for the differentiation of live and dead eggs of intestinal parasites. However, the studies devoted to these methods are scarce, and the majority of researchers have noted their limited usefulness (Da˛ browska et al., 2014; Karkashan et al., 2015). The aim of the study was to develop a method that would allow for the colorimetric evaluation of nematode eggs using proper instruments, which would eliminate the stages based on subjective assessments by analysts. The elaborated method should enable more reliable and repeatable results for the classification of dead and live eggs stained using fluorescent dye kits and also allow for a reduction in the amount of time devoted to the observations and, at the same time, reduce the duration of the examination.
2. Materials and methods 2.1. Preparation of material for the studies: eggs of Ascaris suum The materials for the study were eggs of the intestinal parasites Ascaris suum (large pig roundworm). Parasites were collected from the intestinal tract content of pigs slaughtered in the registered slaughterhouse. The eggs were isolated directly from the uterus of the roundworm and resuspended in PBS buffer. A part of the material was subjected to thermal inactivation in a water bath at 70 C for 1 h. Two groups of eggs were produced, live eggs (non-inactivated) and dead eggs (inactivated). 2.2. Methods used for fluorescent staining Assessment of egg viability was performed using kits for fluorescent staining. The colouring of live and dead eggs was compared. Four kits of stain were used in the study as follows: a commercial FluoCell Double Staining Kit (Calcein AM/PI) (MoBi Tec, Germany), Annexin V FITC Apoptosis Detection Kit (Sigma-Aldrich, USA), LIVE/ DEAD BacLight Bacterial Viability Kit (Molecular Probes Inc., USA), and a kit composed of the dyes:fluorescein diacetate (SigmaAldrich, USA), and propidium iodide (MILLIPORE, USA). The study was conducted in two repetitions for each kit. 2.2.1. FluoCell Double Staining Kit (calcein AM/PI) The dying solution was prepared as follows: 20 ml of calcein and 10 ml of propidium iodide added to 5 ml of PBS. Subsequently, 200 ml containing about 150 of live eggs suspension and 200 ml containing about 150 of dead eggs were collected, and then 100 ml of previously prepared staining solution was added to each. After mixing, the samples were incubated for 15 min at 37 C. 2.2.2. Annexin V FITC Apoptosis Detection Kit A volume of 200 ml containing about 150 of A. suum egg suspension of was centrifuged. The supernatant was poured off and the sediment was resuspended in 200 ml 2 concentrate binding buffer. Then, 5 ml of annexin and 10 ml of propidium iodide were added to the prepared egg suspension. The samples were incubated for 15 min at room temperature. After incubation, 400 ml of binding solution was added to the samples at a working dilution. 2.2.3. LIVE/DEAD BacLight Bacterial Viability Kit A mixture was prepared of components A and B included in the kit, at the proportion 1:1. Then, 0.66 ml of the dye mixture was added per each 200-ml containing about 150 of A. suum egg suspension. The samples were mixed and incubated at room temperature (approx. 22 C) for 15 min protected from light. 2.2.4. Fluorescein diacetate and propidium iodide The initial solution of fluorescein diacetate was prepared at a concentration of 0.5 mg/ml acetone. Subsequently, the A dye was prepared, which constituted 0.4 ml/ml of the initial solution in PBS. The B dye was a solution of propidium iodide at a concentration of 20 mg/ml PBS. To perform the test, 200 ml containing about 150 of egg solution was sampled from both groups. To each group was added 100 ml of dye A and 30 ml of dye B. Previously mixed samples were incubated at room temperature for 3 min. 2.3. Assessment of stained eggs viability by means of fluorescence microscope After staining, the egg suspension was filtered using a subpressure filtering device with polycarbonate filters with a pore
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diameter of 12 mm (Whatman, USA). The filters were transferred to glass slides. Subsequently, several drops of glycerine were added and spread equally over the entire surface of the filter. The specimens were observed under a fluorescence microscope with the appropriate filter set with a wavelength of 490 nm and with a magnification of 400. The colouring of live and dead eggs was assessed for all dye kits.
2.4. Analysis of images of Ascaris suum stained eggs using graphic software with image analysis function RGB (red-green-blue) For colour analysis 25 live and 25 dead eggs were randomly selected from each group. For each of the staining techniques, a Canon EOS 700D camera was used, and two series were obtained (one series for each repetition), with 25 photos of inactivated eggs (dead) and 25 photos of non-inactivated (live) eggs each. The camera used was equipped with an 18-megapixel Hybrid CMOS AF matrix. For computer analysis of the image parameters in the RGB (RedGreen-Blue) mode, CorelDRAW software, version Graphic Suite 7, Corel Photo Paint module was used. Using this software, images of the stained eggs were analysed such that in a photo, a surface was determined from the middle of the egg with a size of 340 340 pixels. Subsequently, the parameters of the RGB histogram of the determined area of the image were analysed (mean value and standard deviation of saturation of two colours of the image, green and red). This analysis was based on the assumption that each colour can be compared using a parametric description of the intensity of each colour or its individual components using numbers. Such a system is commonly used in digital systems for the description of colours. In the RGB system applied, each colour is in the form of a mixture of pure colours, red, green and blue. The contribution of each component is expressed by the intensity according to the scale of 0e255 for each of the three basic colours (Fig. 1). To assess the differentiation of eggs (for each type of staining), the relative positions of the distributions of the obtained intensities of colours for live and dead eggs were considered. The assessment was performed using the distributions area's overlap index (DAOI), and the distributions area's separation index (DASI). The DAOI expressed the so-called common area of colour intensity intervals obtained for live and dead eggs, and it was an arithmetic difference between the maximum intensity values for stained live eggs and the minimum values for the computer analysed specimens of images of dead eggs (Maxlive - Mindeath). The DASI expressed the difference between the minimum intensity for dead eggs and maximum intensity for live eggs (Mindeath - Maxlive).
9
2.5. Statistical methods To evaluate the techniques of staining and to select the most favourable for the developed method, the following parameters were analysed: specificity (Sp), sensitivity (Se) and accuracy (Ac). These parameters were determined using the receiver-operating characteristic analysis for diagnostic tests (ROC curve) with Youden correction (Greiner et al., 2000). The objective of the statistical techniques was selection of the cut point and its optimization by obtaining the best parameters of the analytical method developed. Statistical calculations were performed using the Epitools calculators (Sergeant, 2015). 3. Results Based on the results of these studies, all fluorescent dye kits used enabled a visual (microscopic) identification of live eggs (noninactivated) and dead eggs (inactivated). All kits applied in the study coloured the live (non-inactivated) A. suum eggs green (Fig. 2A and B, C, D). The egg sheaths were particularly intensely coloured. The dead eggs (inactivated) were coloured orange using three of the kits. The exception was the LIVE/DEAD BacLight
Fig. 2. Ascaris suum eggs: live (non-inactivated) and dead (inactivated) stained with the dye kits. A) FluoCell Double Staining Kit. B) Annexin V FITC Apoptosis Detection Kit. C) LIVE/DEAD BacLight Bacterial Viability Kit. D) Fluorescein diacetate and propidium iodide.
Fig. 1. Analysis of stained egg images using graphic software: a) determination of the analysed surface from inside the stained egg and b) reading based on the histogram for the green channel of the mean value of colour saturation. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Bacterial Viability Kit, in which the dead eggs (inactivated) were yellow (Fig. 3 C). For all staining methods, there was a low percentage of inactivated eggs coloured yellow to yellow-green (Fig. 3). The images show that an unequivocal classification of these eggs into live and dead based on their colour was difficult. Such an analysis is characterized by the high subjectivity of evaluations by those who perform the analysis. Therefore, the images were subjected to colour analysis using the graphic software CorelDRAW Graphic Suite 7. Tables 1 and 2 demonstrate the results (for two repetitions) of the analysis of intensity of components of the colours, green (G) and red (R) of the RGB image using the graphic software. Within the range of the green colour, regardless of the method of staining, live eggs of A. suum yielded lower mean values of colour intensity (114.8e183.2) than dead eggs (146.1e205.6). The lowest mean intensity of green colour was obtained for staining with fluorescein diacetate þ propidium iodine (dead: 146.1 and 160.5, and live: 131.3 and 138.9, respectively). The highest mean intensities of green colour were obtained for staining with the LIVE/ DEAD BacLight Bacterial Viability Kit (dead: 205.6 and 192.0, and live: 182.0 and 183.2, respectively). Within the range of green colour, a common area was observed for all four types of staining. For the FluoCell Double Staining Kit (Calcein AM/PI), the common value for the range of results for analysis of image of live and dead eggs was 74.2 in the first repetition, and 32.9 in the second repetition, compared to the scale of 255 possible levels of colour intensity constituting 29% and 13% of the common area, respectively. However, for fluorescein diacetate þ propidium iodine, the values were 122.2 and 112.7, which in this scale was as much as 48% and 44% of the common area, respectively. Such a large common area made it impossible to determine threshold values for the colour of live and dead eggs. Therefore, for green, statistical calculations were discontinued. At the same time, the values for the intensity of the red colour were subjected to the same assessment for the images of live and dead eggs. Table 2 presents the results obtained. For the red colour, live eggs of Ascaris suum yielded mean values of colour intensity within the range of 48.9e100, and the values for dead eggs were in the range of 188e228. The lowest mean intensity for the red colour was obtained using the LIVE/DEAD BacLight, (live, 48.9 and 52.9, and dead, 188 and 203. The highest mean intensities for red colour were obtained for fluorescein diacetate þ propidium iodine (live, 86.8 and 100, and dead, 228 and 204). For one case of staining with fluorescein diacetate þ propidium iodine, a small degree of mutual overlapping for red colour intensity was observed for live and dead eggs. For the remaining staining methods, the red colour intensity intervals measured in
live and dead eggs were separable. Using the Annexin V FITC Apoptosis Detection Kit, the distribution area separation index of results for analysis of the red colour of live and dead eggs assumed the highest values, equal to 41 and 67, in the second repetition of the study series. This constituted, with the total range of values possible of 255, the area of separation of two groups within the range of 16%e26%. The subsequent staining with FluoCell Double Staining Kit (Calcein AM/PI) resulted in an area characterizing the difference between live and dead eggs within the range 13.3% (value 34) - 18.4% (value 47). For fluorescein diacetate þ propidium iodine, the least favourable results were obtained for characterizing dead and live eggs of A. suum using the analysis of red colour, from the mutual overlapping of 0.8% of the range to 10.2% (value 26), the area of which is the difference between extreme results, Mindeath and Maxlive. Distributions of the analysed results in the majority of cases were not close to the normal distribution (Gauss), which excluded the application of statistical parametric methods for the assessment of staining methods. Therefore, the distribution area separation index of the results of red colour intensity for live and dead eggs (DASIred) was evaluated. Arithmetically, this was the difference between the minimum values of intensities of stained dead eggs and the maximum values for computer analysed sections of live eggs images. For further analysis, the receiver-operating characteristic analysis for diagnostic tests (ROC) was applied using Youden's J index. The calculated Youden's J index was used to determine the most beneficial cut-off point for the qualification of results characterizing live and dead eggs and was most stable for repetitions of studies using the Annexin V FITC Apoptosis Detection Kit. The Youden's J differences obtained between repetitions were only 0.5. The FluoCell Double Staining Kit (Calcein AM/PI) method differed in the Youden's J value by 1.8. Within the range of 255 possible results, the differences were very small, which indicates high stability of the results obtained using these staining methods. With the remaining two staining methods assessed, the differences were larger, with values of 7.9 for the LIVE/DEAD BacLight Bacterial Viability Kit and 20.1 for fluorescein diacetate þ propidium iodine, which indicates lower stability compared to the other two methods. The determination of cut-off points using Youden's statistics gave the following parameters of analytical methods for the first three staining methods: sensitivity SE ¼ 1.00 (CL95% ¼ 0.867e1.00) and specificity SP ¼ 1.00 (CL95% ¼ 0.862e1.00). Considering the last evaluated staining method, in the second repetition, the difficulty of differentiating a low percentage of live from dead eggs using fluorescein diacetate þ propidium iodine, assuming a lower value of cut-off point using Youden's statistics of 155.5, yielded a sensitivity of SE ¼ 0.96 (CL95% ¼ 0.805e0.993). However, this indicates that dead eggs will not be detected by 4%, on average, and considering the confidence interval, in the worst case, by as much as 19.5%. 4. Discussion
Fig. 3. Dead Ascaris suum eggs coloured yellow or yellow-green stained with the dye kit Fluorescein diacetate þ propidium iodide. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Attention is frequently paid to limits of traditional methods for the determination of parasite eggs vitality, in which the only differentiation criterion is the visual method (Błaszczyk and KrzyskoŁupicka, 2014; Kalisz et al., 2003). These methods are imprecise and burdened with high error due to the subjectivity of assessment of the person performing the test. The use of fluorescent dyes for the specific simultaneous staining of live and dead eggs would be beneficial. Such methods are often used in microbiology for the differentiation of bacteria and fungi. In our own studies, three commercial kits of fluorescent dyes were used. FluoCell Double Staining Kit (Calcein AM/PI) (Mo BiTec, Germany), Annexin V FITC Apoptosis Detection Kit (Sigma-Aldrich, USA), LIVE/DEAD BacLight
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Table 1 Parameters of the intensity of green colour for images of live and dead Ascaris suum eggs using various staining techniques. Analysis within the range of basic colour
Repetition
FluoCell Double Staining Kit (Calcein AM/PI)
I
I II
LIVE/DEAD BacLight Bacterial Viability Kit
I II
Fluorescein diacetate þ propidium iodide
Intensity values for green colour
Dead Live Dead Live Dead Live Dead Live Dead Live Dead Live Dead Live Dead Live
II Annexin V FITC Apoptosis Detection Kit
Type of egg
I II
Min
Mean
Max
Std. deviation
DAOI
116 88.7 119.9 94.2 121.3 101.6 123.8 115.9 119.5 136.8 119.5 138.1 73.3 80.7 82.8 108.5
174.1 119.6 188.2 114.8 179.3 151.3 179.5 149.3 205.6 182 192 183.2 146.1 131.3 160.5 138.9
225.5 190.2 225.5 152.8 254.9 209.7 218.7 185.9 255 218.4 246 216 212.3 195.5 208.4 195.5
28.9 18.9 29.6 13.2 33.1 22.7 28.5 19.2 37.4 23 43.2 19.8 41.5 25.1 39.1 23.3
74.2 32.9 88.4 62.1 98.9 96.5 122.2 112,7
Table 2 Parameters of the intensity of red colour for images of live and dead eggs of Ascaris suum using various staining techniques. Staining technique
Repetition Type of egg Intensity values for red colour Min Mean Max Std. deviation DASI (difference between Mindeath and Maxlive)
FluoCell Double Staining Kit (Calcein AM/PI) I
Dead
163 217
240 17.4
Live
33.1 58.9
129 20.5
Dead
161 223
250 24.9
Live
34.4 62.2
114 19.3
Dead
175 221
251 20.7
Live
55.5 84.9
134 20.6
Dead
175 225
255 25.4
Live
37.3 73.6
108 17
Dead
109 203
252 40.8
Live
24.4 52.9
80.8 13.4
II
Dead Live
117 188 20.8 48.9
I
Dead
II
Annexin V FITC Apoptosis Detection Kit
I
II
LIVE/DEAD BacLight Bacterial Viability Kit
Fluorescein diacetate þ propidium iodide
I
II
Youden's J Method parameters obtained (cut-point) (95% CL)
34
162.9
47
161.1
41
175.1
67
174.6
28.2
108.9
239 40.2 79.2 17.2
37.8
116.8
156 204
255 31.9
26
155.5
Live
53.7 86.8
130 19.4
Dead
164 228
254 26.8
2
175.6
Live
65.2 100
166 25.8
Bacterial Viability Kit (Molecular Probes Inc., USA) and a kit using fluorescein diacetate (Sigma-Aldrich, USA) and propidium iodine (MILLIPORE, USA). The majority of the dyes stained live eggs of A. suum green, whereas dead eggs were orange. A slightly different colouring of eggs was obtained with the kit LIVE/DEAD BacLight, consisting of two components, SYTO 9 and PI. According to the manufacturer, the SYTO 9 component penetrates into bacterial cells that are live and those with intact cellular structure, whereas the PI component stains dead cells exclusively. In our studies, live cells (not exposed
SE ¼ 1.00 (0.867e1.00) SP ¼ 1.00 (0.862e1.00) SE ¼ 1.00 (0.867e1.00) SP ¼ 1.00 (0.862e1.00) SE ¼ 1.00 (0.867e1.00) SP ¼ 1.00 (0.862e1.00) SE ¼ 1.00 (0.867e1.00) SP ¼ 1.00 (0.862e1.00) SE ¼ 1.00 (0.867e1.00) SP ¼ 1.00 (0.862e1.00) SE ¼ 1.00 (0.867e1.00) SP ¼ 1.00 (0.862e1.00) SE ¼ 1.00 (0.867e1.00) SP ¼ 1.00 (0.862e1.00) SE ¼ 0.96 (0.805e0.993) SP ¼ 1.00 (0.862e1.00)
to a high temperature) were stained yellow, consistent with the description, although a particularly clear colouring was obtained for egg sheaths. This is consistent with the results of previous studies conducted by Da˛ browska et al. (2014) and Karkashan et al. (2015). In the experiment performed by Karkashan, live eggs with intact sheath structure absorbed neither SYTO 9 nor PI. To facilitate the absorption of dyes, the researcher used sodium hypochlorite, which removed the outer protein layer of the egg sheath. However, this allowed only the staining of the inner lipoprotein layer by both dye A (SYTO 9), and dye B (PI). In our experiment, dye absorption
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M. Włodarczyk et al. / Experimental Parasitology 178 (2017) 7e13
occurred without removal of the outer protein sheath. The differences between own results and those obtained by Karkashan et al. (2015) may result from the use of eggs prepared differently for the experiment (e.g., obtaining mature eggs from the final section of the roundworm uterus or obtaining them from faeces, or the ‘whitening’ of eggs using sodium hypochlorite). It may also be a result of the observation method because Karkashan observed egg fluorescence using a confocal microscope, whereas in our studies, a traditional fluorescence microscope was used, in which individual layers emitting light mutually overlap. Nevertheless, this does not explain the relatively low intensity of red colour in live eggs in our experiment. For dead eggs, Karkashan et al. (2015) observed that both dyes penetrated into their interiors. Similarly, in our studies, this was observed for dead eggs stained an intermediate colour between green and red fluorescence, which indicates the presence of both dyes inside the eggs and, at the same time, hindered interpretation of the results. Gasol et al. (1999) encountered similar difficulties in the determination of bacterial cells with the Live/Dead BacLight kit using flow cytometry. To obtain a greater differentiation of colours, the researchers washed the filter with stained bacteria with isopropyl alcohol. Because both dyes components of the kit react with nucleic acids during competition for the binding site of one component, isopropanol removed the dye that was more weakly bound. An uneven absorption and binding of dyes to eggs of A. suum was also observed for the remaining dye kits, although to a lower degree. Some of the eggs showed yellow fluorescence caused by the penetration of both dyes into the inside of the eggs. Such eggs caused difficulties in interpretation of results and their classification as live or dead eggs. Difficulties with obtaining a uniform colour were also reported by de Victorica and Galvan (2003), and Jensen et al. (2009). These researchers found that some dyes may be toxic to live eggs and larvae and cause the leakage of components directly into the interior of dying eggs, thus making observation difficult. Therefore, according to the WHO (WHO, 2004), it is recommended to examine fluorescence dyes within several minutes after their application. An uneven intensity of colours may also be noted inside dead eggs, as seen on images in the report by Karkashan et al. (2015). This was likely due to various amounts of DNA, which both dyes bind (after penetration through intact structures of cells), emitting fluorescence. The eggs of A. suum used by these researchers, at the moment of inactivation with high temperature, were at various stages of development. Some contained developed larvae, which may have resulted in their more intense colour, in contrast to those containing an embryo at the initial stages of development. As a result of the these studies, the staining that would allow for the differentiation of eggs into live or dead has risks of a difficult and often faulty image for visual assessment. Therefore, for the determination of egg viability, additional instruments were used, which allowed the elimination of the risk of erroneous assessment. For this purpose, computer software was used to compare the mean intensity of colours, with red and green for live and dead eggs, respectively. The results obtained for the intensity of basic green colour were not satisfactory. A considerable common area described by the DAOI value between the results for live and dead eggs, particularly for fluorescein diacetate þ propidium iodide. This made it impossible to use this approach for the development of a reliable method for the identification of live and dead eggs. This partially results from properties of the digital description of colours, in which the yellow and orange colour indicated dead eggs in the microscopic image and is composed of the basic colours RGB, to which the basic green colour has a great contribution. The use of images within the basic red colour showed its usefulness for the effective
differentiation between live and dead eggs of A. suum. Additionally, selection of the staining method is of important for obtaining good parameters of the analytical method. The Annexin V FITC Apoptosis Detection Kit was the most beneficial. Good parameters of detection were obtained (DASI ¼ 41 and 67), with a small probability of obtaining false positive and false negative results, with a diagnostic sensitivity SE ¼ 1.00 (0.867e1.00) and specificity SP ¼ 1.00 (0.862e1.00). Analysis of parameters obtained while preserving the intra-laboratory repeatability conditions with a series of specimens in two repetitions, also confirmed the superiority of staining with the Annexin V FITC Apoptosis Detection Kit. The Youden J index for these staining series was nearly identical. However, for fluorescein diacetate þ propidium iodide, even the most favourable cut-off point for the differentiation between live and dead eggs of A. suum, may cause the occurrence of a certain percentage of wrongly classified eggs. The estimated parameter of diagnostic sensitivity of the method is SE ¼ 0.96 (0.805e0.993). It should be emphasized, however, that the presented study was conducted under experimental conditions. This is the first description of such a method for the estimation of colorimetric viability of stained eggs, which is the basis for further, more practical application. Therefore, there is a need of further investigations with the use of samples from naturally contaminated environmental samples. 5. Conclusion These studies confirm the usefulness of fluorescent dyes for the viability assessment of the eggs of intestinal parasites of A. suum. The staining methods may be an alternative to time-consuming traditional methods based on subjective evaluation by a researcher. The additional use of computer software for the analysis of images within the range of red colour allows the avoidance of erroneous assessment in the case of doubtful staining. The advantage of this method is also obtaining more repeatable and reliable results for the classification of live and dead eggs. Acknowledgments Funding: This work was supported by the Polish Ministry of Science and Higher Education [grant number NN 305 6013 39]. References Bazeli, M., 2007. Biological Analysis of Sewage Sludge. BIOM Biological Laboratory (In Polish). Pracownia Biologiczna BIOM. Wyd. PPR STUDIO K2. Piła. Błaszczyk, K., Krzysko-Łupicka, T., 2014. Overview of the research methods for _ sewage sludge used in Poland. Inzynieria i Ochr. Srodowiska 17 (1), 117e133. Da˛ browska, J., Zdybel, J., Karamon, J., Kochanowski, M., Stojecki, K., Cencek, T., 2014. Assessment of viability of the nematode eggs (Ascaris, Toxocara, Trichuris) in sewage sludge with the use of LIVE/DEAD Bacterial Viability Kit. Ann. Agric. Environ. Med. 21 (1), 35e41. De˛ bowska, R., Bazela, K., Eris, I., 2007. Apoptoza i ochronne działanie kwasu foliowego. Dermatol. Estet. 9 (2), 83e90. Directive 86/278/EEC, 1986. Council directive on the protection of the environment, and in particular of the soil, when sewage sludge is used in agriculture. Off. J. Eur. Communities L 181, 6e12. de Victorica, J., Galvan, M., 2003. Preliminary testing of a rapid coupled methodology for quantitation/viability determination of helminth eggs in raw and treated wastewater. Water Res. 37, 1278e1287. Final Report by Milieu Ltd, 2008. WRc and RPA for the European Commission DG Environment under Study Contract DG ENV.G.4/ETU/2008/0076 R. Environmental, economic and social impacts of the use of sewage sludge on land. Milieu Ltd, (Belgium), Brussels. Gantzer, C., Gaspard, P., Galvez, L., Huyard, A., Dumouthier, N., Schwartzbrod, J., 2001. Monitoring of bacterial and parasitological contamination during various treatment of sludge. Water Res. 35 (16), 3763e3770. Gasol, J.M., Zweifel, U.L., Peters, F., Furhman, J.A., Hagstrom, A., 1999. Significance of size and nucleic acid content heterogeneity as measured by flow cytometry in natural planktonic bacteria. Appl. Environ. Microbiol. 65, 4475e4483. Greiner, M., Pfeiffer, D., Smith, R.D., 2000. Principles and practical application of the
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Polish Standard PN-Z-19000-4, 2001. Soil Quality e Assessment of the Soil Sanitary Conditions e Detection of Eggs of the Intestinal Parasites Ascaris lumbricoides and Trichuris trichiura (In Polish). Regulation of the Minister of the Environment of 6 February 2015 on municipal sewage sludge, 2015. J. Laws volume 257. Sadowska, J., Grajek, W., 2009. Analysis of physiological state of single bacterial cell using fluorescent staining methods. Biotechnologia 4 (87), 102e114. Sergeant, E., 2015. Epitools Epidemiological calculators., AusVet Animal Health Services and Australian Biosecurity Cooperative Research Centre for Emerging Infectious Disease. Shapiro, H.M., 2000. Microbial analysis at the single-cell level: tasks and techniques. J. Microbiol. Methods 42, 3e16. US. Environmental Protection Agency, 1999. Environmental Regulations and Technology: Control of Pathogens and Vector Attraction in Sewage Sludge. EPA/625/ R-92/013. Revised edition. U.S. EPA, Washington, D.C. WHO, 2004. Integrated Guide to Sanitary Parasitology. World Health Organization, Geneva, Switzerland. WHO-EM/CEH/121/E. WHO, 2006. Guidelines for the safe use of wastewater, excreta and greywater. In: Policy and Regulatory Aspect, 1. World Helth Organization, Geneva.