Detection of intestinal parasites in human faecal samples using dissolved air flotation.

OBJECTIVE: Ova and parasite (O&P) examination is recommended for the laboratory diagnosis of agents causing parasitic infections; however, this exam requires scientific and technological improvements to enhance its diagnostic validity. Dissolved air flotation (DAF) is an efficient technical principle separating suspended solids in a liquid medium. We aimed to develop and validate a new procedure for intestinal parasite detection with DAF. METHODS: In this study, we collected samples from 500 volunteers, screened them by direct examination, and transferred the material to tubes using the Three Faecal Test (TF-Test) for triplicate DAF tests. We evaluated physical-chemical parameters and DAF prototype components through quantifying parasites recovered from floated and non-floated regions of the flotation column. The DAF operation protocol was validated with the gold standard results. RESULTS: The 10% saturated volume proportion and cationic surfactant showed regularity and high parasite recovery (80%). Modifications of the needle device did not influence parasite recovery (p > 0.05). Sensitivity, specificity, accuracy and kappa agreement obtained with the DAF protocol were 91%, 100%, 93% and substantial (k = 0.64), respectively. CONCLUSION: The DAF principle could be used to process faecal samples in routine laboratory exams, enabling intestinal parasite detection.

Use of the aqueous biphasic system as an alternative for concentration of Ascaris lumbricoides eggs, with non-toxic separation of faecal residues and fats.

OBJECTIVES: Human enteroparasites are considered a serious public health problem in underdeveloped countries located in world regions with tropical, subtropical and equatorial climates. These parasites are commonly diagnosed by the Parasitological Examination of Faeces (PEF), performed by conventional techniques and/or commercial kits that result in tests with low-to-moderate sensitivity, due to the use of destructive chemical solvents to parasite structures, and to present excess adipose substance and digestive residues in their microscopic slides. In order to improve the efficacy of these tests/examinations, this study aimed to investigate a new alternative for the PEF, with the use of Aqueous Biphasic System (ABS). METHODS: For this, four ABSs containing poly (ethylene glycol), PEG (PEG-4000 and PEG-6000), dipotassium phosphate and sodium citrate at different concentrations in the biphasic systems were evaluated with faecal samples containing eggs of Ascaris lumbricoides. RESULTS: The ABS consisting of PEG-4000 and dipotassium phosphate, at concentrations of 55% w/w and 20% w/w, respectively, achieved 100% satisfactory results compared to the conventional TF-Test technique in terms of preservation and concentration of A. lumbricoides eggs, with adequate separation of digestive residues, without using a centrifuge or chemical solvents that may cause harm to the parasites. CONCLUSIONS: This study presents ABS as a new low-cost technical principle for the detection of parasite eggs in PEF. The new technique is simple, fast, non-toxic, not harmful to the parasite and does not require a centrifuge.

UTILISATION DU SYSTÈME BIPHASIQUE AQUEUX COMME ALTERNATIVE À LA CONCENTRATION DES ŒUFS D'ASCARIS LUMBRICOIDES, AVEC SÉPARATION NON TOXIQUE DES RÉSIDUS FÉCAUX ET DES GRAISSES: OBJECTIFS: Les entéroparasites humains sont considérés comme un sérieux problème de santé publique dans les pays sous-développés situés dans des régions du monde aux climats tropicaux, subtropicaux et équatoriaux. L'examen parasitologique des matières fécales (EPMF) permet généralement de diagnostiquer ces parasites, à l'aide de techniques classiques et/ou de kits commerciaux, conduisant à des tests de sensibilité faible à modérée, dus à l'utilisation de solvants chimiques destructeurs pour les structures du parasite et résultant en excès de substances adipeuses et de résidus digestifs dans leurs lames microscopiques. Afin d'améliorer l'efficacité de ces tests/examens, cette étude visait à étudier une nouvelle alternative à l'EPMF, avec l'utilisation du Système Biphasique Aqueux (SBA). MÉTHODES: Pour cela, quatre SBA contenant du poly (éthylène glycol), du PEG (PEG-4000 et PEG-6000), du phosphate dipotassique et du citrate de sodium à différentes concentrations ont été évalués avec des échantillons de matières fécales contenant des œufs d'Ascaris lumbricoides. RÉSULTATS: Le SBA constitué de PEG-4000 et de phosphate dipotassique, à des concentrations respectives de 55% poids/poids et 20% poids/poids, a obtenu des résultats satisfaisants à 100% par rapport à la technique conventionnelle du TF-Test, en termes de préservation et de concentration des œufs de A. lumbricoides, avec une séparation adéquate des résidus digestifs, sans utiliser de centrifugeuse ni de solvants chimiques pouvant détruire les parasites. CONCLUSIONS: Cette étude présente le SBA comme un nouveau principe technique peu coûteux pour la détection des œufs de parasites dans l'EPMF. La nouvelle technique est simple, rapide, non toxique, n'endommage pas le parasite et ne nécessite pas de centrifugeuse.

Identification of Leishmania spp. promastigotes in the intestines, ovaries, and salivary glands of Rhipicephalus sanguineus actively infesting dogs.

Sand flies are recognized as the major vector of canine visceral leishmaniasis. However, in some areas of Brazil where sand flies do not occur, this disease is found in humans and dogs. There has been speculation that ticks might play a role in transmission of canine visceral leishmaniasis and the DNA of Leishmania spp. has been reported in whole ticks. We investigated the presence of Leishmania spp. promastigotes in the intestines, ovaries, and salivary glands of Rhipicephalus sanguineus ticks collected from tick-infested dogs in two cities of Brazil. We used 66 dogs that tested positive and 33 that tested negative for Leishmania spp. according to direct cytological examination assays. Ten ticks were collected from each dog and dissected to collect the intestines, ovaries, and salivary glands for immunohistochemistry (IHC) and diagnostic real-time polymerase chain reaction (RT-PCR). IHC results showed Leishmania spp. in 98, 14, and 8 % of the intestines, ovaries, and salivary glands, respectively. Real-time PCR showed that 89, 41, and 33 % of the tick intestine, ovary, and salivary glands, respectively, were positive for Leishmania spp. The verification of promastigotes of Leishmania spp. by two independent techniques in ticks collected from these urban region dogs showed that there is need for clarification of the role of ticks in the transmission of canine visceral leishmaniasis in Brazil.

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

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.

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.

Toward automating the diagnosis of gastrointestinal parasites in cats and dogs.

Diagnosing gastrointestinal parasites by microscopy slide examination often leads to human interpretation errors, which may occur due to fatigue, lack of training and infrastructure, presence of artifacts (e.g., various types of cells, algae, yeasts), and other reasons. We have investigated the stages in automating the process to cope with the interpretation errors. This work presents advances in two stages focused on gastrointestinal parasites of cats and dogs: a new parasitological processing technique, named TF-Test VetPet, and a microscopy image analysis pipeline based on deep learning methods. TF-Test VetPet improves image quality by reducing cluttering (i.e., eliminating artifacts), which favors automated image analysis. The proposed pipeline can identify three species of parasites in cats and five in dogs, distinguishing them from fecal impurities with an average accuracy of 98,6%. We also make available the two datasets with images of parasites of dogs and cats, which were obtained by processing fecal smears with temporary staining using TF-Test VetPet.

TF-Test techniques for the laboratory diagnosis of gastrointestinal parasites of humans and animals

Intestinal parasites inhabit the intestinal tract of humans and animals, causing damages whose severity depends on several factors related to the parasite and the host. Immunocompromised individuals are more likely to develop severe forms of parasitic infestation. The diagnosis of the gastrointestinal parasitosis is mainly performed by the examination of the feces, which consists of the direct visualization and identification of the parasites eliminated through the feces. These tests are generally low sensitive and the microscope slides contain a large number of impurities, which can impair the result of the diagnosis. In order to improve the diagnostic accuracy, a new parasitological technique called Three Fecal Test (TF­Test) was developed. To further improve its diagnostic accuracy, few modifications of the original protocols have been made with the years. In this study the performance of these new techniques to detect gastrointestinal parasites in human and animal fecal samples was described and discussed in relation to the performance of other conventional coprological tests. It could be concluded that the TF­Test conventional and modified can be used for the diagnosis of several human and animal parasites, with satisfactory results.

Automated Diagnostics: Advances in the Diagnosis of Intestinal Parasitic Infections in Humans and Animals.

The increasingly close proximity between people and animals is of great concern for public health, given the risk of exposure to infectious diseases transmitted through animals, which are carriers of more than 60 zoonotic agents. These diseases, which are included in the list of Neglected Tropical Diseases, cause losses in countries with tropical and subtropical climates, and in regions with temperate climates. Indeed, they affect more than a billion people around the world, a large proportion of which are infected by one or more parasitic helminths, causing annual losses of billions of dollars. Several studies are being conducted in search for differentiated, more sensitive diagnostics with fewer errors. These studies, which involve the automated examination of intestinal parasites, still face challenges that must be overcome in order to ensure the proper identification of parasites. This includes a protocol that allows for elimination of most of the debris in samples, satisfactory staining of parasite structures, and a robust image database. Our objective here is therefore to offer a critical description of the techniques currently in use for the automated diagnosis of intestinal parasites in fecal samples, as well as advances in these techniques.

Dissolved air flotation as potential new mechanism for intestinal parasite diagnosis in feces.

The parasitological examination of feces is recommended for the laboratory diagnosis of intestinal parasites due to its practicality, low-cost, and moderate diagnostic sensitivity. Dissolved Air Flotation (DAF) is an efficient technical principle used in other areas to separate dispersed solids. This study sought the preliminary evaluation of a proof-of-concept prototype as a tool for detecting species of parasites by adjusting DAF. Two DAF prototype units were developed to evaluate microbubbles' generation amidst fecal suspension and parasites' capture. For this evaluation, samples were screened and processed by the TF-Test technique (Control) and simultaneously by DAF device. The dimensional and attachment characteristics in the formation of the microbubbles were evaluated, and the percentage of parasitic recovery in floated and not-floated regions compared by Student's t-test. The second prototype unit proved to be more efficient in forming microbubbles with diameters between 34 and 170µm. The flotation tests showed a recovery of 73.27%, 58.12%, 37.85%, and 91.89% for Ascaris lumbricoides, Hymenolepis diminuta, Giardia duodenalis, and Strongyloides stercoralis, respectively. This study confirmed the selective interaction between microbubbles and parasite eggs and larvae during the flotation process using the DAF principle for the first time through imaging.

Automated Diagnosis of Canine Gastrointestinal Parasites Using Image Analysis.

Because canine intestinal parasites are considered cosmopolitan, they carry significant zoonotic potential to public health. These etiological agents are routinely diagnosed using microscopic examination commonly used because of its low cost, simple execution, and direct evidence. However, there are reports in the literature on the poor performance of this test due to low to moderate sensitivity resulting from frequent errors, procedures and interpretation. Therefore, to improve the diagnostic efficiency of microscopic examination in veterinary medicine, we developed and evaluated a unique new protocol. This system was tested in a study involving four genera of highly prevalent canine intestinal parasites in an endemic region in São Paulo state, Brazil. Fecal samples from 104 animals were collected for this research. The new protocol had a significantly higher (p < 0.0001) number of positive cases on image data, including parasites and impurities, and was elaborate to test them with the TF-GII/Dog technique, with a moderate agreement and Kappa index of 0.7636. We concluded that the new Prototic Coproparasitological Test for Dogs (PC-Test Dog) allowed a better visualization of the parasitic structures and showed a favorable result for the diagnosis of intestinal parasites in dogs.

A historical review of the techniques of recovery of parasites for their detection in human stools.

Since the early 20th century, the detection of intestinal parasites has improved with the development of several techniques for parasitic structures recovery and identification, which differ in sensitivity, specificity, practicality, cost, and infrastructure demand. This study aims to review, in chronological order, the stool examination techniques and discuss their advantages, limitations, and perspectives, and to provide professionals and specialists in this field with data that lays a foundation for critical analysis on the use of such procedures. The concentration procedures that constitute the main techniques applied in routine research and in parasitological kits are a) spontaneous sedimentation; b) centrifugation-sedimentation with formalin-ethyl acetate; and c) flotation with zinc sulfate solution. While selecting a technique, one should consider the purpose of its application and the technical-operational, biological, and physicochemical factors inherent in the procedures used in stool processing, which may restrict its use. These intrinsic limitations may have undergone procedural changes driven by scientific and technological development and by development of alternative methods, which now contribute to the improvement of diagnostic accuracy.

A historical review of the techniques of recovery of parasites for their detection in human stools

Abstract Since the early 20th century, the detection of intestinal parasites has improved with the development of several techniques for parasitic structures recovery and identification, which differ in sensitivity, specificity, practicality, cost, and infrastructure demand. This study aims to review, in chronological order, the stool examination techniques and discuss their advantages, limitations, and perspectives, and to provide professionals and specialists in this field with data that lays a foundation for critical analysis on the use of such procedures. The concentration procedures that constitute the main techniques applied in routine research and in parasitological kits are a) spontaneous sedimentation; b) centrifugation-sedimentation with formalin-ethyl acetate; and c) flotation with zinc sulfate solution. While selecting a technique, one should consider the purpose of its application and the technical-operational, biological, and physicochemical factors inherent in the procedures used in stool processing, which may restrict its use. These intrinsic limitations may have undergone procedural changes driven by scientific and technological development and by development of alternative methods, which now contribute to the improvement of diagnostic accuracy.

Identification of Leishmania spp. promastigotes in the intestines, ovaries, and salivary glands of Rhipicephalus sanguineus actively infesting dogs

Sand flies are recognized as the major vector of canine visceral leishmaniasis. However, in some areas of Brazil where sand flies do not occur, this disease is found in humans and dogs. There has been speculation that ticks might play a role in transmission of canine visceral leishmaniasis and the DNA of Leishmania spp. has been reported in whole ticks. We investigated the presence of Leishmania spp. promastigotes in the intestines, ovaries, and salivary glands of Rhipicephalus sanguineus ticks collected from tick-infested dogs in two cities of Brazil. We used 66 dogs that tested positive and 33 that tested negative for Leishmania spp. according to direct cytological examination assays. Ten ticks were collected from each dog and dissected to collect the intestines, ovaries, and salivary glands for immunohistochemistry (IHC) and diagnostic real-time polymerase chain reaction (RT-PCR). IHC results showed Leishmania spp. in 98, 14, and 8 % of the intestines, ovaries, and salivary glands, respectively. Real-time PCR showed that 89, 41, and 33 % of the tick intestine, ovary, and salivary glands, respectively, were positive for Leishmania spp. The verification of promastigotes of Leishmania spp. by two independent techniques in ticks collected from these urban region dogs showed that there is need for clarification of the role of ticks in the transmission of canine visceral leishmaniasis in Brazil.