Modified Negative Staining of Heine for Fast and Inexpensive Screening of Cryptosporidium, Cyclospora, and Cystoisospora spp.

Khanna, Vinay; Tilak, Kriti; Ghosh, Archi; Mukhopadhyay, Chiranjay

doi:10.1155/2014/165424

Cited by 10 publications

(15 citation statements)

References 5 publications

(5 reference statements)

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“…Since the first human cryptosporidiosis cases, few studies on oocysts' behavior in fecal samples submitted to any temporary staining technique have been published. The major advances include using a direct wet mount (Garcia et al, 1982) or adding iodine (Ma & Soave, 1983), Schiff''s Periodic Acid (Horen, 1983), or Hein's staining protocol, which contains carbol fuchsin (Heine, 1982) and its modification with malachite green (Khanna et al, 2014).…”

Section: Discussionmentioning

confidence: 99%

Development of New Staining Procedures for DiagnosingCryptosporidiumspp. in Fecal Samples by Computerized Image Analysis

Loiola

Galvão

Santos

et al. 2021

Microscopy and Microanalysis

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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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Section: Discussionmentioning

confidence: 99%

Development of New Staining Procedures for DiagnosingCryptosporidiumspp. in Fecal Samples by Computerized Image Analysis

Loiola

Galvão

Santos

et al. 2021

Microscopy and Microanalysis

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“…Even with the aid of conventional staining methods for Cyclospora oocysts microscopic detection can be challenging (McHardy et al, 2014), but it remains the recommended diagnostic method. Of the various available stains, the modified Ziehl-Neelsen stain technique has been recommended for detecting Cyclospora oocysts in clinical samples (Brennan et al, 1996;Khanna et al, 2014).…”

Section: Staining Testsmentioning

confidence: 99%

“…To sum up, among the various available detection methods of Cyclospora oocyst based on morphology, the modified Ziehl-Neelsen staining and autofluorescence techniques have been recommended for the detection of oocysts in the clinical samples (Berlin et al, 1998;Khanna et al, 2014).…”

Section: Flow Cytometrymentioning

confidence: 99%

Advances in Cyclosporiasis Diagnosis and Therapeutic Intervention

Cui

et al. 2020

Front. Cell. Infect. Microbiol.

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Cyclosporiasis is caused by the coccidian parasite Cyclospora cayetanensis and is associated with large and complex food-borne outbreaks worldwide. Associated symptoms include severe watery diarrhea, particularly in infants, and immune dysfunction. With the globalization of human food supply, the occurrence of cyclosporiasis has been increasing in both food growing and importing countries. As well as being a burden on the health of individual humans, cyclosporiasis is a global public health concern. Currently, no vaccine is available but early detection and treatment could result in a favorable clinical outcome. Clinical diagnosis is based on cardinal clinical symptoms and conventional laboratory methods, which usually involve microscopic examination of wet smears, staining tests, fluorescence microscopy, serological testing, or DNA testing for oocysts in the stool. Detection in the vehicle of infection, which can be fresh produce, water, or soil is helpful for case-linkage and source-tracking during cyclosporiasis outbreaks. Treatment with trimethoprim-sulfamethoxazole (TMP-SMX) can evidently cure C. cayetanensis infection. However, TMP-SMX is not suitable for patients having sulfonamide intolerance. In such case ciprofloxacin, although less effective than TMP-SMX, is a good option. Another drug of choice is nitazoxanide that can be used in the cases of sulfonamide intolerance and ciprofloxacin resistance. More epidemiological research investigating cyclosporiasis in humans should be conducted worldwide, to achieve a better understanding of its characteristics in this regard. It is also necessary to establish in vitro and/or in vivo protocols for cultivating C. cayetanensis, to facilitate the development of rapid, convenient, precise, and economical detection methods for diagnosis, as well as more effective tracing methods. This review focuses on the advances in clinical features, diagnosis, and therapeutic intervention of cyclosporiasis.

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“…Published data from the French CNR-LE for cryptosporidiosis show that: i) even with around 250 notified cases each year, cryptosporidiosis is still largely underestimated in France, ii) cryptosporidiosis is predominant in immunocompetent individuals and especially in young children and young adults, and iii) cryptosporidiosis is over-represented in the summer [ 5 , 6 ]. The routine diagnosis of cryptosporidiosis still relies on light microscopy examination for many laboratories [ 7 , 8 , 9 , 10 ]. However, light microscopy examination lacks sensitivity, is time-consuming, and requires skilled technicians, making it an inefficient method for laboratories which are able to switch to PCR analysis [ 8 , 10 , 11 , 12 ].…”

Section: Introductionmentioning

confidence: 99%

Comparative Performance of Eight PCR Methods to Detect Cryptosporidium Species

et al. 2021

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Diagnostic approaches based on PCR methods are increasingly used in the field of parasitology, particularly to detect Cryptosporidium. Consequently, many different PCR methods are available, both “in-house” and commercial methods. The aim of this study was to compare the performance of eight PCR methods, four “in-house” and four commercial methods, to detect Cryptosporidium species. On the same DNA extracts, performance was evaluated regarding the limit of detection for both C. parvum and C. hominis specificity and the ability to detect rare species implicated in human infection. Results showed variations in terms of performance. The best performance was observed with the FTD® Stool parasites method, which detected C. parvum and C. hominis with a limit of detection of 1 and 10 oocysts/gram of stool respectively; all rare species tested were detected (C. cuniculus, C. meleagridis, C. felis, C. chipmunk, and C. ubiquitum), and no cross-reaction was observed. In addition, no cross-reactivity was observed with other enteric pathogens. However, commercial methods were unable to differentiate Cryptosporidium species, and generally, we recommend testing each DNA extract in at least triplicate to optimize the limit of detection.

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Modified Negative Staining of Heine for Fast and Inexpensive Screening of Cryptosporidium, Cyclospora, and Cystoisospora spp.

Cited by 10 publications

References 5 publications

Development of New Staining Procedures for DiagnosingCryptosporidiumspp. in Fecal Samples by Computerized Image Analysis

Development of New Staining Procedures for DiagnosingCryptosporidiumspp. in Fecal Samples by Computerized Image Analysis

Advances in Cyclosporiasis Diagnosis and Therapeutic Intervention

Comparative Performance of Eight PCR Methods to Detect Cryptosporidium Species

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