Development of New Staining Procedures for Diagnosing Cryptosporidium spp. in Fecal Samples by Computerized Image Analysis

Interpretation errors may still represent a limiting factor for diagnosing Cryptosporidium spp. oocysts with the conventional staining techniques. Humans and machines can interact to solve this problem. We developed a new temporary staining protocol associated with a computer program for the diagnosis of Cryptosporidium spp. oocysts in fecal samples. We established 62 different temporary staining conditions by studying 20 experimental protocols. Cryptosporidium spp. oocysts were concentrated using the Three Fecal Test (TF-Test®) technique and confirmed by the Kinyoun method. Next, we built a bank with 299 images containing oocysts. We used segmentation in superpixels to cluster the patches in the images; then, we filtered the objects based on their typical size. Finally, we applied a convolutional neural network as a classifier. The trichrome modified by Melvin and Brooke, at a concentration use of 25%, was the most efficient dye for use in the computerized diagnosis. The algorithms of this new program showed a positive predictive value of 81.3 and 94.1% sensitivity for the detection of Cryptosporidium spp. oocysts. With the combination of the chosen staining protocol and the precision of the computational algorithm, we improved the Ova and Parasite exam (O&P) by contributing in advance toward the automated diagnosis.

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Lactate delivery (not oxygen) limits hepatic gluconeogenesis when blood flow is reduced

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00319.2004 ◽

2006 ◽

Vol 290 (1) ◽

pp. E192-E198 ◽

Cited By ~ 3

Author(s):

Ken D. Sumida ◽

Jerry H. Urdiales ◽

Casey M. Donovan

Keyword(s):

Blood Flow ◽

Flow Rate ◽

Oxygen Delivery ◽

Liver Perfusion ◽

Lactate Concentration ◽

Oxygen Availability ◽

Limiting Factor ◽

Hepatic Gluconeogenesis ◽

Experimental Protocols ◽

The Impact

The purpose of this study was to determine, using the isolated liver perfusion technique, whether the limiting factor for hepatic gluconeogenesis (GNG) from lactate was precursor delivery or oxygen availability during reduced flow rates of 0.85 or 0.60 ml·min−1·g liver−1. After a 24-h fast, three different experimental protocols were employed. Protocol 1 examined the impact on GNG when reservoir lactate concentration was maintained but oxygen delivery was elevated via increases in hematocrit (Hct). Elevating the Hct from 22.5 ± 0.8% to 30.9 ± 0.4% at a blood flow of 0.89 ± 0.01 ml·min−1·g liver−1 increased the oxygen consumption (V̇o2) but did not augment GNG. Similarly, when the Hct was elevated from 22.5 ± 0.8% to 41.5 ± 0.7% at 0.59 ± 0.04 ml·min−1·g liver−1, V̇o2 was increased, but GNG was unaffected. Protocol 2 examined the impact on GNG when Hct was maintained but precursor delivery was elevated via increases in reservoir lactate concentration ([LA]). Specifically, elevating the [LA] from 2.31 ± 0.07 to 3.61 ± 0.33 mM at a flow rate of 0.82 ± 0.04 ml·min−1·g liver−1 significantly increased GNG. Similarly, elevating the [LA] from 2.31 ± 0.07 to 4.24 ± 0.37 mM at a flow rate of 0.58 ± 0.02 ml·min−1·g liver−1 increased GNG. Finally, we examined the impact of increasing both the oxygen and lactate delivery ( Protocol 3). Again, V̇o2 was elevated with increased oxygen delivery, but GNG was not augmented beyond that observed with elevations in lactate delivery alone, i.e., Protocol 2. The results indicate that, during decrements in blood flow, GNG is limited primarily by precursor delivery, not oxygen availability.

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Assessment of the first commercial multiplex PCR kit (ParaGENIE Crypto-Micro Real-Time PCR) for the detection of Cryptosporidium spp., Enterocytozoon bieneusi, and Encephalitozoon intestinalis from fecal samples

Diagnostic Microbiology and Infectious Disease ◽

10.1016/j.diagmicrobio.2019.04.004 ◽

2019 ◽

Vol 95 (1) ◽

pp. 34-37 ◽

Cited By ~ 6

Author(s):

Florent Morio ◽

Philippe Poirier ◽

Yohann Le Govic ◽

Adrien Laude ◽

Stéphane Valot ◽

...

Keyword(s):

Multiplex Pcr ◽

Real Time ◽

Real Time Pcr ◽

Enterocytozoon Bieneusi ◽

Fecal Samples ◽

Cryptosporidium Spp ◽

Encephalitozoon Intestinalis

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Protozoan pathogens Blastocystis and Giardia spp. in roof-harvested rainwater: the need to investigate the role of the common brushtail possum (Trichosurus vulpecula) and other potential sources of zoonotic transmission

Journal of Water Sanitation and Hygiene for Development ◽

10.2166/washdev.2019.064 ◽

2019 ◽

Vol 9 (4) ◽

pp. 780-785

Author(s):

Edward Waters ◽

Warish Ahmed ◽

Kerry Ann Hamilton ◽

Deniss Plaksins ◽

Damian Stark

Keyword(s):

Entamoeba Histolytica ◽

Disease Outbreaks ◽

Brushtail Possum ◽

Limited Information ◽

Fecal Samples ◽

Dientamoeba Fragilis ◽

Cryptosporidium Spp ◽

Blastocystis Spp ◽

Polymerase Chain

Abstract Globally, protozoan pathogens are an increasingly important cause of reported disease outbreaks, with the majority of documented outbreaks between 2004 and 2010 reported in Australia. While the microbiological contamination of roof-harvested rainwater (RHRW) has been well studied, limited information is available regarding contamination with protozoan pathogens. In this study, rainwater (n = 134) and possum fecal samples (n = 20) were screened for the presence of several protozoan pathogens, including Blastocystis spp., Cryptosporidium spp., Giardia spp., Dientamoeba fragilis, and Entamoeba histolytica using the multiplex real-time polymerase chain reaction. While Cryptosporidium spp. was only detected in two possum fecal samples (10%) and Giardia spp. was only detected in three RHRW samples (2.23%, n = 134), Blastocystis spp. was detected in both possum feces (25%) and RHRW (5.22%) samples. Dientamoeba fragilis and Entamoeba histolytica were not detected in any samples. These findings highlight protozoan pathogens as a potentially important area of focus for rainwater quality assessment. Furthermore, while possums are suggested as a potential source of Blastocystis spp. in RHRW, sources of this pathogen in RHRW warrant further investigation.

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Evaluation of fecal smear methods for research on Cryptosporidium spp. oocysts in the feces of dairy calves

Semina Ciências Agrárias ◽

10.5433/1679-0359.2022v43n2p585 ◽

2022 ◽

Vol 43 (2) ◽

pp. 585-598

Author(s):

Ana Paula Molinari Candeias ◽

◽

Gabrieli Maria Huff ◽

Adriana Fiorini Rosado ◽

André Luis Vriesman Beninca ◽

...

Keyword(s):

Molecular Analysis ◽

False Negative ◽

Microscopic Analysis ◽

Dairy Calves ◽

Fecal Samples ◽

Genetic Sequencing ◽

Cryptosporidium Spp ◽

Negative Results ◽

False Negative Results ◽

Fecal Smear

The objective of this study is to compare the direct fecal smear (DFS) and centrifugal sedimentation (CS) methods in the detection of Cryptosporidium spp. oocysts in fecal samples of dairy calves. One hundred and fourteen fecal samples were collected from calves aged up to six months from 10 dairy farms located in Palotina and Francisco Alves, Paraná, Brazil. The microscopic analysis revealed the presence of Cryptosporidium spp. oocysts in 51.75% (59/114) of the samples in both methods. In CS, 48.25% (55/114) of the samples were positive, while in DFS slides, only 6.14% (7/114) were positive. Only 4 samples were positive exclusively in DFS. To ensure that there were no false-negative results in the microscopic analysis, the 55 samples that were negative in both DFS and CS were selected for molecular analysis using the nested PCR (nPCR). Of these 55 samples, 24% (13/55) were positive and forwarded for sequencing part of the genome, which made it possible to identify C. parvum, C. bovis and C. ryanae. Besides the characterization of the Cryptosporidium species, it was possible to identify bacteria of the genus Acinetobacter interfering directly in the analyzed samples. The microscopic analysis also revealed higher sensitivity when CS was used to make the fecal smears. However, some samples that were negative in this technique had positive PCR results. Thus, molecular analysis is indicated to confirm cases of Cryptosporidium spp. Further studies are necessary to prove the specificities of the used primers since the results obtained in nPCR were positive for the protozoan but, when genetic sequencing was performed, Acinetobacter spp. was identified.

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Occurrence of Cryptosporidium spp. and Giardia spp. in pigs at weaning

Semina Ciências Agrárias ◽

10.5433/1679-0359.2016v37n6p4157 ◽

2016 ◽

Vol 37 (6) ◽

pp. 4157 ◽

Cited By ~ 1

Author(s):

Denise Junqueira Matos ◽

Marcelo Vasconcelos Meireles ◽

Willian Marinho Dourado Coelho ◽

Katia Denise Saraiva Bresciani

Keyword(s):

Management Practices ◽

Sao Paulo ◽

São Paulo ◽

Fecal Samples ◽

Paulo State ◽

Cryptosporidium Spp ◽

Good Management ◽

Cryptosporidium Oocysts ◽

São Paulo State

Cryptosporidium spp. and Giardia spp. are parasites and have been reported in many species of mammals, including humans. The goal of this research was to investigate the occurrence of Cryptosporidium spp. and Giardia spp. in 45-days-old pigs. Fecal samples of 107 pigs were collected at three alternate days in piggeries in Araçatuba, São Paulo State, Brazil. Cryptosporidium oocysts were observed in 4.7% (5/107) of animals by Kinyoun acid-fast stain method and cysts of Giardia spp. were observed in 1.9% (2/107) of the animals by the method of Faust. Of all animals, 85 presented feces with normal consistency and 22 showed diarrhea in at least one collection. Two diarrheic samples showed positivity for both Cryptosporidium spp. and Giardia spp. From these results it is possible to infer that the occurrence of Cryptosporidium and Giardia was low because of the good management practices and both protozoa were not associated the presence of symptoms.

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Occurrence of gastrointestinal protozoans in cats from Londrina, Paraná, Brazil

Semina Ciências Agrárias ◽

10.5433/1679-0359.2020v41n1p213 ◽

2020 ◽

Vol 41 (1) ◽

pp. 213

Author(s):

Keila Jimenez Torrico ◽

Nelson Jessé Rodrigues dos Santos ◽

Hugo Luca Abate ◽

Felippe Danyel Cardoso Martins ◽

Luiz Daniel de Barros ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Giardia Duodenalis ◽

Positive Sample ◽

Pcr Analysis ◽

Fecal Samples ◽

Cryptosporidium Spp ◽

Specific Pcr ◽

Identity Match ◽

Pcr Products ◽

The City

Protozoans are widely distributed, and several species may parasitize the digestive tracts of cats (Felis catus), and can be transmitted to humans. The present study aimed to evaluate the species and occurrence of gastrointestinal protozoans in cats in the city of Londrina, Paraná, Brazil. A total of 206 cat fecal samples were tested, of which 141 were from shelter animals, and 65 were from pets owned by local people. Samples were processed by parasitological techniques. Coproparasitological techniques (Willis, Faust and Ziehl-Neelsen) were performed for detection of protozoan parasites. Subsequently, all samples were processed by PCR protocols specific to Toxoplasma gondii, Giardia spp., and Cryptosporidium spp. PCR products from positive samples were selected for sequencing. No samples were found to be positive for Cryptosporidium spp. using the Ziehl-Neelsen technique. Using specific PCR protocols, 1/206 (0.48%) samples tested positive for Cryptosporidium spp. After purification, this one positive sample was sequenced, and it demonstrated a 100% identity match to Cryptosporidium muris. Using specific PCR protocols, 13/206 (9.22%) cat fecal samples tested, including 2/65 (3.08%) pet cat fecal samples, were positive for T. gondii. PCR analysis revealed that 37/206 (17.96%) of cat fecal samples were positive for Giardia spp., including 27/141 (19.15%) of shelter cat fecal samples, and 10/65 (15.38%) pet cat fecal samples (p = 0.5124). When sequenced, these positive samples showed a 100% identity match with Giardia duodenalis. This study demonstrated that infections with Cryptosporidium spp., Toxoplasma gondii, and Giardia duodenalis are present in the population of both pet cats and shelter cats in the city of Londrina. This poses a risk to public health, because these parasites have a high zoonotic potential.

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Prevalence of Cryptosporidiosis in Ostriches from Central and South Parts of Iraq

The Iraqi Journal of Veterinary Medicine ◽

10.30539/ijvm.v44i1.937 ◽

2020 ◽

Vol 44 (1) ◽

pp. 63-67

Author(s):

Al- Zubaidei H. Hamza

Keyword(s):

Infection Rate ◽

Morphological Characterization ◽

Wide Spread ◽

Traditional Methods ◽

Fecal Samples ◽

Cryptosporidium Spp ◽

Significant Difference ◽

June July ◽

First Time

The prevalence of Ostriches cryptosporidiosis was determined for the first time in central and south parts of Iraq to study the effects of age, sex and months on the infection rate and to record the morphological characterization of Cryptosporidium spp in Ostriches. A total of 200 Ostriches fecal samples were examined by traditional methods for detection of the parasite. The total infection rate was 11% (22/200), and the highest infection rate was 12.26% (13/106) in chicks ( 9 months), while the lowest infection rate was 9.57% (9/94) recorded in the adult (> 9 Months). The study included seven provinces in the central and south parts of Iraq: Wasit, Baghdad, Babylon, Diyala, Karbala, Al-Najaf and Al-Qadisiyah. The highest infection rate was reported in Al-Najaf (23.53%) compared with the lowest rate (0%) in Wasit province. March reported an infection rate of 50%, while the lowest rate (0%) was registered during the summer months: June, July and August with a significant difference (P0.05) among months of the study. Morphologically, the study indicated the wide spread of Cryptosporidium parasites in Ostriches in both central and south area of Iraq

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Assessment of Infections with Microsporidia and Cryptosporidium spp. in Fecal Samples from Wild Primate Populations from Cameroon and Democratic Republic of Congo

International Journal of Primatology ◽

10.1007/s10764-015-9820-x ◽

2015 ◽

Vol 36 (2) ◽

pp. 227-243 ◽

Cited By ~ 3

Author(s):

Christelle Butel ◽

Steve Ahuka Mundeke ◽

Pascal Drakulovski ◽

Donika Krasteva ◽

Eitel Mpoudi Ngole ◽

...

Keyword(s):

Democratic Republic ◽

Democratic Republic Of Congo ◽

Republic Of Congo ◽

Fecal Samples ◽

Cryptosporidium Spp

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Golden hamster (Mesocricetus auratus) as an experimental model forLeishmania(Viannia)braziliensisinfection

Parasitology ◽

10.1017/s0031182012002156 ◽

2013 ◽

Vol 140 (6) ◽

pp. 771-779 ◽

Cited By ~ 34

Author(s):

ADRIANO GOMES-SILVA ◽

JOANNA GARDEL VALVERDE ◽

RAQUEL PERALVA RIBEIRO-ROMÃO ◽

ROSA MARIA PLÁCIDO-PEREIRA ◽

ALDA MARIA DA-CRUZ

Keyword(s):

Golden Hamster ◽

Mesocricetus Auratus ◽

Spleen Weight ◽

Limiting Factor ◽

Experimental Conditions ◽

Adequate Model ◽

Skin Ulcers ◽

American Tegumentary Leishmaniasis ◽

History Of ◽

Experimental Protocols

SUMMARYThe lack of an adequate model forLeishmania(Viannia)braziliensisinfection is a limiting factor for studying American tegumentary leishmaniasis (ATL). The golden hamster (Mesocricetus auratus) is a promising model because besides being highly susceptible to dermotropicLeishmaniainfection, the lesions are very similar to cutaneous leishmaniasis (CL) in humans. However, differentLeishmaniaisolates or species and/or protocols have resulted in different outcomes, whereas no study has evaluated the reproducibility ofL. braziliensisinfection in this model. The natural history ofL. braziliensisinfection in 34 hamsters was evaluated by using a single parasite isolate in 8 independent experiments under similar experimental conditions. Clinical, histological and immunological analyses were performed. The hamsters presented skin ulcers similar to those observed in ATL. The intra-experiment lesion increment tended to show an intermediary variance. Histological analysis of infected skins showed granulomatous reaction, scarce amastigotes, and Schaumann's bodies. Blood lymphocytes proliferated in response to leishmanial antigens. The severity of the infection was positively correlated to spleen weight, and the titres of anti-LeishmaniaIgG antibodies. Our findings indicate that the hamster is an appropriate model for immunopathogenesis studies of CL caused byL. braziliensis, supporting its use in clinical, vaccine and chemotherapy experimental protocols.

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