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Background: Techniques for diagnosing intestinal parasites need technological advancements in the preanalytical (collection/processing) and analytical (detection) stages. The dissolved air flotation (DAF) technique effectively recovers parasites from processed feces for routine diagnosis. Artificial intelligence (AI) is a practical and affordable alternative to modernize the analysis stage of microscopy images and generates high efficiency in the parasitological examination of feces., Methods: The objective of this study was to standardize a laboratory protocol for stool processing using the DAF technique in conjunction with an automated diagnosis of intestinal parasites (DAPI) system. A total of 400 samples were obtained to perform the tests with the use of DAF to verify the recovery of the parasites as a function of the chemical reagent (polymer and surfactant), the volume of the flotation tube, and standardization of smear assembly on a microscopy slide, with automated analysis by DAPI. The DAF protocol that obtained the most satisfactory results in terms of parasite recovery (P < 0.05) and slide positivity was compared with the Three Fecal Test (TF-Test) protocol with manual (microscopists) and automated (DAPI) evaluation. We compared the sensitivity with the modified TF-Test technical protocol and the diagnostic agreement with the gold standard (Kappa) result., Results: There was no significant difference in the parasite recovery between the 10 ml and 50 ml tubes (P > 0.05). The surfactants showed a range of parasite recoveries between 41.9% and 91.2% in the float supernatant. We obtained a maximum positivity of 73% of the assembled slides when we applied DAF processing with 7% CTAB surfactant and 57% positivity with the modified TF-Test technique. Regarding diagnostic performance, the TF-Test-modified and DAF techniques used in fecal processing for subsequent computerized analysis by AI presented sensitivities of 86% and 94%, with kappa agreements of 0.62 and 0.80 (substantial), respectively., Conclusions: The DAF protocol defined in this study and the DAPI system are innovative processes for parasite recovery and fecal debris elimination that are favorable for effectively detecting pathogenic structures in laboratory diagnosis., (© 2024. The Author(s).) more...

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.) more...

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., (© 2022 John Wiley & Sons Ltd.) more...

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., (Copyright © 2021. Published by Elsevier B.V.) more...

Intestinal parasites are responsible for several diseases in human beings. In order to eliminate the error-prone visual analysis of optical microscopy slides, we have investigated automated, fast, and low-cost systems for the diagnosis of human intestinal parasites. In this work, we present a hybrid approach that combines the opinion of two decision-making systems with complementary properties: (DS 1 ) a simpler system based on very fast handcrafted image feature extraction and support vector machine classification and (DS 2 ) a more complex system based on a deep neural network, Vgg-16, for image feature extraction and classification. DS 1 is much faster than DS 2 , but it is less accurate than DS 2 . Fortunately, the errors of DS 1 are not the same of DS 2 . During training, we use a validation set to learn the probabilities of misclassification by DS 1 on each class based on its confidence values. When DS 1 quickly classifies all images from a microscopy slide, the method selects a number of images with higher chances of misclassification for characterization and reclassification by DS 2 . Our hybrid system can improve the overall effectiveness without compromising efficiency, being suitable for the clinical routine - a strategy that might be suitable for other real applications. As demonstrated on large datasets, the proposed system can achieve, on average, 94.9%, 87.8%, and 92.5% of Cohen's Kappa on helminth eggs, helminth larvae, and protozoa cysts, respectively., (Copyright © 2020 Elsevier Ltd. All rights reserved.) more...

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. more...

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., (© 2019 John Wiley & Sons Ltd.) more...

Objectives: Laboratory diagnosis of Schistosoma mansoni eggs is routinely performed by conventional quantitative techniques through the parasitological examination of human faeces. However, the diagnostic sensitivity of this type of exam varies from low to moderate. We aimed to develop a new parasitological technique called TF-Test Quantified (TFT-Quant), for the quantitative detection of S. mansoni eggs in human faeces., Methods: Four study stages were performed using 43 mice infected by S. mansoni cercariae. These experiments allowed the definition of an operational protocol for TFT-Quant and a comparison of this new technique to the conventional Helm-Teste technique., Results: The results show a good diagnostic efficacy of TFT-Quant, which reached 100% in sensitivity and specificity, indicating an 'Almost Perfect' Kappa (k) agreement., Conclusions: This new technique provided a quantitative gain in the detection of S. mansoni eggs, largely free of faecal debris. The next stage of this study is the field validation of the TFT-Quant technique with the use of human faecal samples from an endemic region for mansonic schistosomiasis in Brazil (Microregion Jequitinhonha, State of Minas Gerais). In parallel to this validation, computational algorithms will be developed to allow the automated quantitative diagnosis of S. mansoni eggs., (© 2019 John Wiley & Sons Ltd.) more...

Due to its important zoonotic potential, cryptosporidiosis arouses strong interest in the scientific community, because, it was initially considered a rare and opportunistic disease. The parasitological diagnosis of the causative agent of this disease, the protozoan Cryptosporidium spp., requires the use of specific techniques of concentration and permanent staining, which are laborious and costly, and are difficult to use in routine laboratory tests. In view of the above, we conducted the feasibility, development, evaluation and intralaboratory validation of a new parasitological technique for analysis in optical microscopy of Cryptosporidium spp. oocysts, called TF-Test Coccidia, using fecal samples from calves from the city of Araçatuba, São Paulo. To confirm the aforementioned parasite and prove the diagnostic efficiency of the new technique, we used two established methodologies in the scientific literature: parasite concentration by centrifugal sedimentation and negative staining with malachite green (CSN-Malachite) and Nested-PCR. We observed good effectiveness of the TF-Test Coccidia technique, being statistically equivalent to CSN-Malachite. Thus, we verified the effectiveness of the TF-Test Coccidia parasitological technique for the detection of Cryptosporidium spp. oocysts and observed good concentration and morphology of the parasite, with a low amount of debris in the fecal smear., (Copyright © 2016 Elsevier B.V. All rights reserved.) more...