EVALUATION OF DIFFERENTIAL EFFICIENCY OF STAINING METHODS FOR CRYPTOSPORIDIUM OOCYSTS

Iakovenko D. V., Pokhil S. I., Tymchenko O. M., Chigirinskaya N. A., Kostyria I. A.

METHODS AND METHODOLOGIES

Scentific article

Cryptosporidiosis (ICD-10-CM А07.2) is an ubiquitous parasitic disease («watery diarrhea») that is caused by the protozoans of the genus Cryptosporidium (type Apicomplexa) that are capable of invasion, reproduction and parasitism inside the enterocytes of the gut tract mucosal filli. Cryptosporidiosis as a diagnosis is established based on the epidemiological and clinical data, as well as results of laboratory tests. Among the large quantity methods of cryptosporidiosis laboratory diagnostics the light and luminescent microscopy remain the most popular in the regular laboratory practice and are based on the determination of the parasitic oocysts in the smears of the biomaterials (mainly fecal). The aim of the study is to evaluate the differential efficiency of six the staining methods for Cryptosporidium oocysts that are most commonly used in the medical practice: negative stain – NSM; modified (cold) Ziehl-Neelsen stain – mZN; modified (cold) Kinyoun’s acid-fast stain – mKAF; modified Köster’s stain – mКSA; auramine-phenol stain – APM; auramine-rhodamine stain – ARM. Object and methods. The object of this study were the suspensions of C. parvum oocysts (n = 6) that were obtained from samples fecal of children and calves by the centrifugal sedimentation in the formalin-ethyl-acetate mix. Smears (n=36) were prepared from 5.0 μl of homogenized suspensions of oocysts. They were dried, fixed and stained using NSM, mZN, mKAF, mКSA, APM, ARM as described in previously published papers. Slides of the smears stained with NSM, mZN, mKAF, mКSA were analyzed by light microscopy and those that were stained with APM, ARM by luminescent microscopy (total increase of objects ×1000-1500, oil-immersion). Microscopically examination to evaluate the differential efficiency of staining methods for Cryptosporidium oocysts included the definition: the total number of oocysts (TNO); a regularly rounded form of oocysts (RFO) and their thick wall (TWO); mean dimensions of the oocysts (i.e. their diameter – DIO); the internal structure inherent in oocysts (ISO). Results and discussion. Obtened results show that luminescent microscopy can detect 2-5 times more Cryptosporidium oocysts (TNO) in the same smears of their suspensions than light microscopy. All proven staining methods (NSM, mZN, mKAF, mKSA, APM, ARM) allow microscopy to visualize such differential signs of Cryptosporidium oocysts as RFO, TWO, DIO. C. parvum oocysts had mean diameter 4,9-5,2 μm and the certain look, depending on the method used to stain them: NSM – look like as transparent round spots («ghosts») on a dark green background; mZN, mKAF – red discs on a pale green background; mKSA – pink and pink-purple on a pale green background; APM, ARM – rings or discs whith characteristically bright green fluorescence against a dark background. Only the ARM has allowed the identification criterion ISO to be found at a satisfactory level (oocysts were light green, and internal sporozoite – yellow-green color). Conclusions. Among the six tried out of staining methods for Cryptosporidium oocysts, which are most often used in medical practice, relatively higher differential efficiency is characterized by ARM. However, this method has such disadvantages as a multi-stage and long-term staining protocol and the need to use phenol, which is a highly dangerous substance. Further research should be aimed at creating easier-to-reproduce, safe and effective staining method for Cryptosporidium oocysts.

Cryptosporidium oocysts, staining methods, differential efficiency

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«Bulletin of problems biology and medicine» Issue 4 part 1 (146), 2018 year, 198-203 pages, index UDK 619:616.993.1

10.29254/2077-4214-2018-4-1-146-198-203