Staining blindly: an update on coloring techniques for fecal smears in parasitology: a scoping review

Abstract

Dye application for parasite highlighting in the Ova and Parasite exam is a common practice in parasitology diagnosis. Methods: A scoping review investigated how staining solutions interact with parasite structures. After screening 1334 papers, 35 met eligibility criteria. Results: Differentiating background from foreground in the fecal smear under light microscopy is the core of the research on this topic. Refractivity, unevenness of staining, size and temperature were explored to enhance staining protocols. Cryptosporidium spp. and Microsporidia were the main studied species. Conclusion: Studies on diagnostic efficacy outperform those that elucidate the physical–chemical interaction between dyes and parasites. An alternative approach involves technicians using computational tools to reduce subjectivity in fecal smear interpretation, deviating from conventional methods.

Plain language summary

What is this article about?

Coloring parasites during fecal exams has been widely used to find parasites in human feces. We searched for articles that could help us to answer the question: ‘How do dyes give color to parasites?’. Then, we filtered the information from a total of 1334 articles to 35.

What were the results?

Cryptosporidium spp. and Microsporidia are microbes that can be seen only through a microscope. Researchers were interested in these two species in the last 40 years. Differentiating parasites from dirt on a glass slide is the main problem researchers are trying to solve. The way the light goes through parasites under a microscope, variation of staining, size and temperature of dyes have been explored to identify what gives better results in coloring protocols.

What do the results of the study mean?

Little is known about the chemical interaction between dyes and parasites. On the other hand, there are many studies on how good coloring methods are and comparing protocols. An alternative to the conventional approaches in staining parasites is the use of computational tools to reduce doubt in the exam interpretation by technicians.

Plain language summary

Executive summary.

• Although the staining procedures for parasitology have been widely explored, there is scarce knowledge on the interaction between intestinal parasites and permanent or temporary dyes in the O&P exam.

• Size, shape, color uptake pattern, and refractivity are the main features highlighted throughout the articles for identifying parasites with light microscopy.

• The reviewed authors tend to value the technique's detection efficiency rather than the physical-chemical factors involved in the interaction between parasites and dyes.

• The coloring protocols intend to differentiate background from the foreground, where most of the parasites are placed in the fecal smear.

• Opportunistic pathogens such as Microsporidia and Cryptosporidium spp. raised the awareness of the researchers in the past 40 years.

• High-quality papers generally failed to answer the question: “Are the applied staining protocols original?”

• Moderate-quality articles mostly lack information about the proper application of statistical analysis of results.

• Low-quality articles failed to show information about preserving fecal samples and why the authors chose some specific dyes over others.

• Computational tools can be used to reduce subjectivity by identifying sample patterns that are difficult to technicians to evaluate precisely.

Regarding the Ova and Parasite examination (O&P), there are a variety of laboratory principles established in the literature for the identification of the main groups of protozoa (amoebas, flagellates, ciliates, coccidia); intracellular parasitic fungi (microsporidia) and, intestinal metazoans (nematodes, cestodes and trematodes). Different approaches for collection, preservation, transport and laboratory processing are recommended for each group of these parasites. At the same time, the professionals responsible for public and private laboratories demand decisions like the type of exam to be standardized and managing the infrastructure to carry out the laboratory routine (equipment, specific reagents and specialized staff). Although there have been advances in sample processing techniques and principles for performing the O&P exam [1], the interpretation of results is practically based on the same premises used in the past, especially concerning the preparation of fecal smears containing permanent and/or temporary staining for evaluation under conventional light microscopy. However, we have noticed the mechanism of binding dyes to parasites has not always been known or well explained in the literature. Thus, our review intends to track articles that seek to fill this knowledge gap and, therefore, serve as a basis for decision-making in diagnosing intestinal parasites by light microscopy.

We critically identified and analyzed the literature's state-of-the-art regarding the knowledge about the physical–chemical interactions between the parasite structures and the dyes usually applied to highlight eggs, cysts, oocysts and spores in the O&P exam. We were also interested in pointing out why some techniques are favored over others and understanding the trends in publication over the years.

Methods

Protocol & registration

The protocol was drafted using the Preferred Reporting Items for Systematic Reviews and Meta-analysis extension for Scoping Reviews Protocols (PRISMA-ScR), which is publicly available at the Open Science Framework since 17 October 2022.

Eligibility criteria

The papers included in this review should have been written in English or Spanish, accessible for free, and contain the keywords within the title or abstract. In addition, the articles should cover light microscopy topics, and explore aspects of the membrane's ultrastructure of eggs, cysts and oocysts. We gave special attention to including papers that presented at least one of the several staining techniques as the primary technique to be investigated. Only papers focused on diagnosing intestinal parasites from fecal samples were considered. All the studies in which the main diagnostic approach was indirect (e.g., molecular and immunologic tests), or regarded epidemiological surveillance, and treatment of diseases, were discarded, even if dyeing solutions were superficially addressed. Studies that did not minimally discuss the relation between staining solutions and parasites and other reviews were excluded from this review.

Information sources

To identify relevant scientific evidence, we performed a comprehensive search in the following databases: PubMed, Scielo, LILACS, Web of Science, Cochrane Library, Brazilian Digital Library of Theses and Dissertations, Scopus and EMBASE. The searches were made from June 2021 to June 2022. The Endnote bibliography manager was used to organize, import, and export all the references. The library technician removed duplicates. We used the State-of-the-Art Through Systematic Review – StArt software as a computational tool to improve the quality of the application of the scoping review protocol. We have also used the Rayyan website to accelerate the collaborative work on the scoping review, especially helping the reviewers work blindly and independently to solve conflicts.

Choosing keywords

We performed a previous exploratory search of the most commonly used keywords in the bibliography databases. This search resulted in papers containing a range of keywords more likely to be used in our systematic search. The keywords should be listed on the MesH (MEDLINE) and DeCS (Virtual Health Library-VHL database) Medical Subject Headings. We defined the general search string as follows:

(“Staining and Labeling” OR “Coloring Agents”) AND (“Rosaniline Dyes” OR “Methylene Blue” OR “Methyl Green” OR “Eosine Yellowish-(YS)” OR “Rose Bengal” OR “Phosphotungstic Acid” OR Phenols OR “Dimethyl Sulfoxide” OR “Iodine Compounds” OR Hematoxylin) AND (Helminths OR Coccidia OR Amebiases OR Microsporidia OR Giardia)

The search string could be adjusted according to the specific requirements of different electronic databases. The final search strategies are summarized in the Supplementary Material (search strategy).

Selection of sources of evidence

We first selected the studies by reading the title and abstract. Two reviewers independently evaluated the resulting papers according to the pre-set eligibility criteria. We described and recorded all the conflicts between the reviewers. A third reviewer was requested to resolve any conflict in the reviewers' decisions. An article should be included if it agrees at least with two inclusion criteria. An article was excluded if it met at least two exclusion criteria. The number of resulting articles from this stage was recorded. In the second stage, we selected the papers by the full-article reading. Once all the articles had been read, a new selection step was carried out. Exclusions could be made since reading the articles completely provided a more discerning selection. However, it was not possible to include new studies at this point.

Data charting process

We abstracted data by developing a three-sheet extraction form to understand the trends in the evidence on the topic reviewed. The articles were randomly divided among six independent reviewers for mapping and recording the data of interest. Any inconsistencies in the forms were resolved by consensus among the reviewers. All the parameters we used to seek data within the articles can be seen in Table 1.

Table 1.

Parameters used to guide the data charting process performed by the reviewers, including the questions to assess the quality of the scientific evidence within the papers.

Extraction data form
Paper overview	Specific data	Quality evaluation of papers (YES = 1 point; NO = 0 point)
Authors	Chemical group of dyes	Are the parasitic concentration methods shown?
Year	Parasite structures the dyes are more likely to bind	Is the parasite staining described in detail?
Staining methods		After the collection, is there information about fecal preservation?
Species	Tools for measuring physical–chemical properties of solutions	Are the applied staining protocols unprecedented?
Concentration methods	Type of diagnostic method (qualitative, quantitative)	Was any diagnostic confirmation method used?
Main results (in brief)	Parasites morphometric measures	Did the authors show any reasons for choosing the dyes?
Comments	List parasite structures that interact with the stains	Are the statistical parameters appropriate to evaluate the staining protocols?

Critical appraisal of individual sources of evidence

We designed the data charting parameters to identify the results that directly or indirectly answer the central question of this review. We intended to relate the data to each other to locate and highlight the need for studies on this topic. To assess the quality of scientific evidence, we developed a score (0 to 7) based on guiding questions (Table 1). For each ‘yes’ answer, we assigned 1 point, and for each ‘no’, 0 points. We considered low-quality papers with a score under 3, moderate quality for those between 4 and 5, and high-quality articles for those with scores of 6 and 7.

Results

After screening 1334 titles and abstracts, and 110 full-text articles, 35 papers published from 1974 to 2021 fulfilled our eligibility criteria (Figure 1). Most of the 1224 papers at the title and abstract reading stage were excluded because the authors intended to cover treatment, sero-epidemiological surveys, and studies on sensitivity and specificity, performed mainly by molecular and immunological techniques. A total of 75 reports were excluded at the final screening stage. The main reason was that they did not address the studies for fecal samples (32.87%) but for tissue sections, body fluids, water samples, or in vitro cultures. Even if the fecal samples were correctly addressed, 14 documents (19.17%) needed to properly show the discussion on how coloring solutions tend to bind to intestinal parasites.

Figure 1.

PRISMA flow diagram for scoping reviews which included searches of databases and registers only.

We intended to critically handle the data in two perspectives: synthesis of information about the trend of scientific divulgation (e.g., amount of publications over the years), and, synthesis of information that fill the gaps of knowledge on the topic (e.g., the range of the explored dyes, their chemical classes and the reasons why they were chosen). A summary table containing the studied species, the concentration techniques used in each study, the staining methods, and the parasites' morphological features displayed in the O&P exam for both permanent and temporary procedures is available in Supplementary Table S1.

The main targeted species present within the reviewed articles from 1974 to 2021 are shown in Figure 2. We divided the results on the applying of permanent and non-permanent protocols by species in Figure 3 and, as for the chemical group of dyes used in both type of protocols are detailed in Figure 4.

Figure 2.

The main targeted species in the reviewed articles from 1974 to 2021.

Studies on different Microsporidia species overlap over the years.

Figure 3.

Permanent and non-permanent protocols applied to different species of intestinal parasites in the reviewed articles.

Figure 4.

Number of publications by chemical group of dyes used in both permanent and temporary protocols.

The mean score obtained from evaluating the scientific quality of papers was 4.97/7.0, with a standard deviation of 0.527. The mean, mode, median and standard deviation values by classification of groups are shown in Table 2, raw data is available in Supplementary Table S2.

Table 2.

Statistical mean, mode, median and standard deviation of the scored articles regarding the quality of scientific evidence (by classification group).

Classification	Mean	Mode	Median	Standard deviation	N
Low	3	3	3	0	3
Moderate	4.59	5	5	0.492	22
High	6.40	6	6	0.516	10
Total	4.97	5	5	0.527	35

Discussion

General findings

The research in parasitology staining shows a massive empiric profile over the years [2], even though some attempts to reduce the subjectivity or better control of experiments have been developed, such as quantification of structures in the smear [3], or building scores based on measuring the intensity of stain uptake in the nucleus, flagella, axonemes, median bodies, the cytoplasm of parasites and contrast with the background [4,5].

The motivation for choosing new staining protocols for the O&P exam is the attempt to solve the inconvenience of conventional methods, such as labor intensity, time-consuming procedures [2,6–10], costs [2,8,9,11], lack of studies on certain parasite species [12]; avoiding invasive procedures for diagnosis [13,14]; and reducing the eyestrain of microscopists in the laboratory routine while reading the slides [8,15]. The health risks of the prolonged handling of toxic substances and contaminated material [10,16], the production of bio-waste [7], and the effort to develop a simultaneous coloring method for different species [17] also figure as reasons to improve parasitological staining techniques.

The differentiation between the background and foreground, where most parasitic structures can be found in the fecal smear under light microscopy, seems to be the core of the research in staining procedures for parasitological purposes [5,11–13,18–22]. The identification tends to be more challenging if there is no distinction between parasites and structures such as yeasts, bacteria, spores, lipid droplets and other elements [3,11,12,22]. In addition, over clearing [10] or overstaining [5,12] parasites are equally undesirable for diagnosis.

The method for parasitic concentration in the sample also plays an essential role in the staining procedures outcomes, especially because parasites can hide under debris [18,23], and the use or not of various fixatives interferes with the uptake of dyes [4,12,18,24]. The interpretation of the results regarding the interaction between preservatives and dyes can be controversial. Some authors suggest that the uptake of color is enhanced when trichrome staining is applied to PVA (polyvinyl alcohol) fixed samples [25]. In contrast, others concluded that the same combination is worse than performing the technique without preservatives [18]. Yet, some authors suggest that when trichrome staining is used in formalin-fixed smears, the results are inferior compared with other preservatives [4,14]. Ziehl-Neelsen technique, in turn, tends to perform equally in fresh and formalin-fixed stools for detecting Taenia spp. eggs [24].

Using safranin to stain Cyclospora cayetanensis resulted in uniform coloration of oocysts [14]. Except for that, none of the dyes provide homogeneous staining of oocysts, cysts, eggs and larvae. Variability of staining patterns, and the level of uptake of those stains have been pointed out with some frequency among the reviewed authors [6,11,14,23–29].

Parija [15] and Khubnani [19] for example, diverge while describing Hymenolepis nana and Taenia spp. structures in microscopy slides, both using unconcentrated samples. Still, Moodley [11] and Cozon [30], report different patterns of staining for Cryptosporidium spp. when colored with Ziehl-Neelsen method after Sheater's and Potassium Dichromate concentration, whereas, Ponce [20] and Parija [31] did not describe the parasitic structures for the same staining methods without any fecal concentration.

Cryptosporidium spp. under Safranine-Methylene Blue appeared as vivid orange pink after Zinc Sulfate or Formalin-Ethyl acetate fecal concentration for Baxby [6], but uneven reddish orange for Moodley [11].

In addition, generic terms regarding staining such as ‘stained well’ [25], ‘stained poorly’ [7], ‘fair staining’ [18], ‘faintly visible’ [28], ‘visible’ [25], ‘often partly stained’ [17], might be too imprecise due to its subjectivity, thus, compromising data analysis and replicability.

Observations on unstained parasites in the fecal smear usually appear more in those studies from amoebae and coccidia's groups of parasites rather than those that target helminths.

For Cryptosporidium spp. and C. cayetanensis [6,14,26], ‘ghost’ oocysts are often reported at random or as a result of failing to perform the staining procedures (e.g., steps of fixation and dehydration) [6,14]. The inherent fragility of those structures due to the staining protocol [21,26], or the exposure to the toxic high environmental oxygen [28] are also reasons for the variation in the staining pattern.

Modifications in the staining procedure have been made to overcome the protocol limitations. Among them stands out the fecal smear heating to better penetration of dyes through the membranes of parasites [2,6,17,32]. In addition, variation in time of exposure to dyes [17,20], and the use of chemical reagents such as DMSO (Dimethyl Sulfoxide) for increasing the penetration of stains has been suggested [16].

On the other hand, the uneven uptake of staining can be explored to identify the vital status of parasites [28,32], the life cycle stage and the morphological differences between species that are easily mistaken [24–27]. A certain level of association can be made between the maturity stage of parasites and how colors are displayed in the fecal smear. Atypical forms of Cryptosporidium spp. oocysts stained by Ziehl-Neelsen, Kinyoun, or Safranin-Methylene Blue techniques are structurally distinct from the others [26]. This condition may be related to immature stages of thick and thin-walled oocysts or even can be derived from different strains of Cryptosporidium spp. Entamoeba coli mature cysts stain poorly compared with immature ones [25]. The same is seen in Taenia spp. eggs that are covered by a thick embryophore composed of prismatic keratin blocks and a colloid substance that shows acid-fast properties that changes the color along egg maturation [24]. The analysis of parasite staining features is also useful when the differentiation of Dientamoeba fragilis from Blastocystis hominis is needed by the visualization of the peripheral rim of cytoplasm containing the nuclei [23]. Also, the viability of Entamoeba histolytica oocysts is verifiable through the application of vital stains. Erythrophagocytic trophozoites tend to be more resistant to deterioration and remain unstained in the fecal smear. Although it is not possible yet to distinct structures between E. histolytica and E. dispar by light microscopy, it is known that erythrophagocytic trophozoites can be pathogenic [28]. Regarding Ancylostoma caninum, viable eggs also remain unstained while non-viable are colored with varying accuracy depending on the incubation time [32].

When parasites are analyzed under light microscopy, their refringence is frequently noticed through the microscope eyepiece [9,11,14,15,20,21,26,30,33]. Indeed, this feature can be used to indicate the maturity of oocysts [26]; to differentiate oocysts from non-refractile yeasts lower down in the smear [11]. Non-excysted oocysts of Cryptosporidium spp. are more refringent than excysted oocysts [30]. Moreover, verifying the refringence helps to identify Giardia lamblia cysts by its morphology, which means a ‘halo’ between the cyst wall and the cytoplasm [15], probably due to the shrinkage after the dehydrating process [21]. Refractility is absent in Cystoisospora belli [20] and C. cayetanensis [14] oocysts at 100X magnification. However, the latter appears slightly green and refractile at 400X.

Histochemical studies on Acanthogyrus sp. (formerly Acanthosentis) suggests a strong association between the maturation stage of the egg and changes in the refractivity and affinity to acid or basic dye. It can be explained by the thickness of the membranes, and the most prominent organic species or substances in each phase of the egg development (e.g., tyrosyl, tryptophanyl, chitin, mucopolysaccharides); this phenomenon is known as metachromasia [34].

Additionally, variations in shape and size of structures can suggest different species of microsporidia and Hymenolepis spp. in the O&P exam [9,24,27,35].

Considerations on the period of publication & targeted species

The studies on coccidia parasites, mainly Cryptosporidium spp., raised constant awareness of researchers over the years while staining helminth eggs or larvae did not represent a concern in the time frame of publications. The acid-fast property of organisms first explored by Robert Koch for Mycobacterium tuberculosis [36] was the main guiding principle for searching for intestinal parasites in the last 40 years. For that, stools must be non-concentrated, and the fixed fecal smears undergo different fixation, coloration, and discoloration steps to be analyzed under a light microscope.

From 1983 to 2003, studies on Cryptosporidium spp. followed by Microsporidia species, increased on an average of once a year (Figure 2). Those infections did not pose a threat until the report of the first cases of HIV infection in humans [37]. Since cryptosporidiosis, microsporidiosis (caused by Enterocytozoon bieneusi and Septata intestinalis infection) and cyclosporiasis can present themselves as opportunistic infections [13,14,38], scientists probably increased their efforts to improve the diagnostic methods in a costless and less invasive way [39–41]. The plateaus observed from 2003 to 2021 for staining-related studies on those species indicate that the advances in light microscopy approaches were slowed down gradually. We suggest that it started to happen due to the ever-increasing preference of researchers for improving other diagnostic tools, such as serological and molecular techniques.

Considerations on staining methods & chemical group of dyes

As Cryptosporidium spp. oocysts and Microsporidia spores behave like acid-fast microorganisms, studies that evaluated permanent staining techniques were more common than those that used temporary staining methods for those agents (Figure 3). However, the authors use temporary staining methods (e.g., iodine) for screening purposes.

The parasites' morphology, mainly regarding size, determines the type of staining technique chosen by the authors. Applying temporary stains for helminths overlaps initiatives with permanent stains for the same parasite group. Articles that explore temporary staining are usually directed to larger parasites. Authors that studied the species Ancylostoma caninum, Eimeria spp., Schistosoma mansoni, Strongyloides papillosus and Toxocara spp. did not explore the use of permanent staining, probably because these parasites require less effort to be located and quantified on the microscope slide.

The ‘misleading’ factor in the O&P exam is associated with the difficulty of distinguishing parasites from other-like structures present in the slide [11,12,23]. The inner structures such as the nucleus, sporoblasts, sporocysts, and membranes, are more challenging to visualize in smaller species [2,14]. Even though there were some attempts to apply differential staining methods, exploring morphometric measurement and quantification is still rare.

Triarylmethanes, represented by Lactophenol Cotton-Blue, Carbol-fuchsin, and Malachite Green stains, are the most frequently used chemical group of dyes and follow the ascending pattern of the permanent staining techniques over the years (Figure 4). Usually, triarylmethanes are combined with azo compounds such as Chromotrope 2R to highlight coccidia and microsporidia species in fecal smears. Trichrome-based stains and Ziehl Neelsen, Kinyoun, and their modifications are usually prepared by combining triarylmethanes and azo compounds to differentiate the background from the desired parasitic structures. Iodine for both temporary and permanent preparations and phenothiazines (e.g., Methylene Blue) are among the most used dyes. Diarylmethanes (e.g., Auramine-phenol) figure as an alternative method to light microscopy since a fluorescence microscope is needed.

Considerations on parasite structures & binding to the dyes

Triarylmethanes successfully color the background of non-fixed smears due to the presence of the acidic sulfonic groups [42]. The uptake of color from azo compounds such as carmoisin by Ascaris lumbricoides eggs is given by the electric field interaction between positively charged egg wall proteins and negatively charged molecules of dye [5].

By its turn, Iodine is one of the oldest reagents used in the microtechnique [43]. Iodine molecules diffuse rapidly through the cell lipidic bilayer and are weakly bound by proteins, which color brown [44]. Iodine exhibits affinity to glycogen [43], which can be the reason for the successful staining of helminth eggs, even though this process is not well described throughout the articles [15,19,35]. Giardia cysts are deeply stained probably due to the interaction between dye and the set of karyosomes, axoneme and median bodies [18]. Instead, E. coli/E. histolytica trophozoite forms are severely damaged by the iodine solutions [21,25] and its cysts are faintly stained [15,28]. C. cayetanensis cysts are completely unstained when submitted to iodine staining [14]. Iodine does not interfere with the affinity of the structures to the other dyes, but it prevents the stain from being washed in the decoloration steps in the permanent staining protocols. Because of this property, it is known as a ‘trapping agent’ [44].

Colorless dyes (e.g., phosphotungstic acid), instead, act as competitors for one or more dyes in the solution; then, the uptake of color is reduced through selective coloring [12,43]. The reduction of phosphotungstic acid in the formulation was applied to enhance background staining and avoid Fast Green stain being fastly replaced by aniline blue in the counterstaining steps [12]. The application of colorless dyes has been widely used in the papers of this review, especially for permanent preparations [2,9,12–14,17,18,20,25,27,28,35,45].

The dye's molecules' size is considered relevant in penetrating the cysts, oocysts, eggs, and larvae structures [43,46]. This statement tends to be true; however, it is probably not statistically accurate, at least when A. caninum eggs are submitted to viability assessment with Methylene Blue, Eosin Y and Methyl Red [32]. The contradiction may be explained because the ‘size’ of a particular dye is not strictly related to its molecular weight but to the properties of anion aggregation within the solution [44].

Heating fecal smears to facilitate the coloring process has already been studied in this field of research [2,6,17,32]. The particles' diffusion ratio increases as the temperatures rise during the coloration steps. At the same time, covalent bonds in the protein chain are weakened, rendering structures more permeable and particles less prone to aggregate. However, this process does not enlarge the amount of dye that is attached to the structures [44].

The most described structures from all the reviewed papers were: i) for helminths: egg shells, embryos, blastomeres, oncosphere, hooklets and larvae; and ii) for protozoa: nucleus, cytoplasm, operculum, oocysts, sporozoites, sporocysts, sporoblasts, cyst wall, space between membranes, coiled filament arrangement also described as belt-like structure or stripes in microsporidia, bacteria, yeasts, and general debris as well as mucus plugs, and plant cells. The authors do not necessarily give details on how those structures interact with the stains from a physical-chemical perspective; however, most describe the results of these interactions presented by displaying different colors under the light microscope.

Considerations on the quality of papers

High-quality papers generally failed to answer the question: “Are the applied staining protocols unprecedented?” In addition to the latter, moderate-quality articles mostly lack information about the proper application of statistical analysis of results. Indeed, inside the moderate group, descriptive statistics [7,12,22,30,31,35,47] is more often used than inferential statistical analysis [4,5,24,29,32].

On the other hand, the most answered question within the same group was “Any diagnostic confirmation method used to attest the species of parasites?” The diagnostic confirmation was performed using well-established parasitology techniques, generally represented by permanent staining procedures such as Trichrome and Ziehl-Neelsen. The low-quality articles failed to show information about preserving fecal samples and why the authors chose some specific dyes over others.

Around 60% of the reviewed papers did not present any unprecedented staining method for parasite detection in the O&P exam. Instead, most authors dealt with modifications to the conventional approaches. The scores for all the reviewed articles can be seen in the Supplementary Material (main table).

Limitations & possible improvements

Morphometric measurement is essential for species determination [9,23,24,27,35], and needs expertise and care [20]. However, measuring and quantifying parasites could be described in more detail better throughout the articles. The research in the O&P exam seems to be more empirical than chemically and statistically accurate, which means that the authors could be, in certain ways, hostages of subjectivity. Usually, the main outcome of staining-related articles is their efficiency in parasite detection. Identification based solely on the patterns of color uptake [24–28,32] prevents the authors from acquiring knowledge about the specific bounds that allow dyes to highlight some structures over others. In addition, the supportive literature is based on studies with staining fixed tissue sections, which can interfere with the conclusions on the dyes' behavior regarding fresh or fixed fecal smears.

Methods that can stain different species simultaneously [7,10,15,17,19,20,22,25,31,35,45,48] bring advantages to the diagnosis in parasitology either because they can detect coinfection cases or because they optimize the workflow in a highly demanding laboratory service.

The alternatives for comparing or confirming the light microscopy diagnosis rely on the use of a fluorescence microscope by applying nonspecific stains such as auramine-phenol [14,26,47], and exploring autofluorescence [3], performing immunofluorescence techniques [30], electronic [13], phase-contrast microscopy [33] and molecular assay [27]. In addition, the development of coloring procedures addressed to computer-assisted approaches has been proposed to improve the detection of Cryptosporidium spp. oocysts [49,50] and helminth ova [51] in the fecal smear.

Computers have been abundantly used to aid the diagnostics of intestinal parasites [51,52]. Starting from a digital image captured from a processed microscope slide, image analysis algorithms can detect (define the rough location of objects of interest), segment (delineate the object of interest), and classify (determine if an object is a parasite and, if so, its species) parasites. To do so, the algorithm has to model specific pattern of characteristics that can discriminate between objects of interest and background, and furthermore, distinguish between the different classes of objects. The data in which the algorithm works on (features) derived from the image pixel values, which initially is a representation of the pixel region's color, but can be expanded by feature extraction steps to describe other characteristics such as shape and texture. The more standardized the data is, the easier it becomes to model and identify the distinguishing patterns (either through hand-crafted equations or machine learnt models).

For instance, if the object of interest is the only object in the image to be of a specific color, distinguishing it from the others is a trivial task. However, if more intricate characteristics must be taken into account (e.g., fine-grained texture, size, and shape), the harder it is to create a feature representation where the object is clearly distinct. This challenge may be overcome if the interactions between dying solutions intended for computer-aided diagnosis and, parasitic structures could be deeply explored. Thus, mitigating the undefined color's uptake patterns in images acquired from several modalities of microscopy.

The use of computer diagnostic tools could reduce subjectivity in interpreting shape, size, texture, the intensity of color uptake, and even other computer-encoded patterns that technicians may not be able to evaluate precisely in the O&P exam. In addition, it allows the researchers to feed image databases that can be revised at any time, attributing qualities such as traceability and the possibility of remote cooperation.

Conclusion

The complex interplay among parasites and staining solutions is influenced by a nuanced combination of factors, including reagent concentration, the type of fecal sampling and preservation method, techniques for fecal concentration, the life cycle stage of intestinal parasites, and the convenience of observation for technicians. Studies on staining methods for Cryptosporidium spp. and Microsporidia detection guided the trends in the publication of parasitology staining over the years. The procedures required fresh unconcentrated smears and permanent staining techniques that exploit the oocysts or spores' acid-fast characteristics. However, most studies have focused on replicating conventional protocols for detecting parasite structures. Authors tend to value the technique's detection efficiency rather than the physical-chemical factors involved in the interaction between parasites and dyes. Size and color uptake patterns are the most described characteristics throughout the reviewed papers, however statistical approaches to evaluate those features are still rare.

Funding Statement

This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Finance Code 001.

Financial disclosure

This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Finance Code 001. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Competing interests disclosure

The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Writing disclosure

No writing assistance was utilized in the production of this manuscript.