PARATEST STABILITY STUDY, SYSTEM USED FOR DIAGNOSIS OF INTESTINAL PARASITES Rev.00 January 10, 2011 1. Introduction Intestinal parasitic diseases represent a serious public health issue in underdeveloped and developing countries, affecting adults and children. The latter may suffer losses in physical and intellectual development resulting from the action of the parasites on the host, such as nutritional and blood deprivation, inflammation, tissue injuries, gastrointestinal and digestive disorders among others. Although mortality is not too high in human illnesses by intestinal parasites, there are still reports of death by hyperparasitism in the country, demonstrating the neglect that these health disorders have been submitted to. Accurate diagnosis of parasitic diseases, with subsequent referral to the appropriate therapy, represents an important action in individual and collective health that leads to improved quality of life. With this objective, the system PARATEST is evaluated in this study as an alternative approach to parasitological diagnosis that offers less biohazard and more practicality to carry out the examinations, while maintaining the quality standard of results. 2. Objectives This study aims to evaluate the stability of the system for diagnosis PARATEST in relation to: a) Kit storage temperature in the original conditions of production, i.e. before addition and dilution of sample (feces) in the diluent / preservative liquid.
b) Kit storage temperature, after addition and dilution of feces in the diluent / preservative liquid. c) Kit storage time, especially at room temperature (22 ºC - 25 ºC) in the production original condition, i.e., before addition and dilution of biological sample (feces) in the diluent / preservative liquid. d) Kit storage time, especially at room temperature (22 ºC - 25 ºC), after dilution of feces in the diluent / preservative liquid. 3. Method 3.1. System (kit) PARATEST The System (Kit) PARATEST produced by the company Diagnostek consists of a plastic bottle whose cap is equipped with a filter membrane with pores of about 266 µm which retain much of the debris and residues from fecal bolus and allow parasitic forms that can be identified by microscope to pass through. The bottle from the Kit PARATEST contains a volume of 7 ml diluent / preservative liquid of 5% buffered formalin with sodium phosphate buffer at ph 7.0. For stool examinations about two grams of feces newly issued by the patient are diluted in this solution, and from the suspension thus obtained, a small fraction is dropped on a microscope slide and examined microscopically by a specialist in parasitology. The diagnosis is made by morphological identification of the parasitic forms observed on microscopic examination. All bottles in the Kit have lot identification, with information on the week, month and year of manufacture, which contributes to the accuracy of stability analyses. 3.1.1. Selection of PARATEST Kit batches for stability studies For stability analysis in relation to temperature, the variable related to the lot was fixed, using bottles from the same batch at different temperatures.
For stability analysis in relation to storage time, before the addition of fecal samples, bottles from different lots produced on different dates were used. For the stability analysis in relation to time after the addition of fecal sample, bottles from the same batch, with same date of manufacture were used. 3.1.2. Concept of stability for PARATEST Kit and the analysis criterion With regard to time or temperature, with and / or without diluted biological sample, the stability of the lots of PARATEST Kit is defined as the capacity of preservation (fixation) and sustenance of the integrity of parasitic forms in the different conditions tested. This stability was measured based on parasite morphology analyzed after being kept at different temperatures and different times. Therefore, the criterion for samples analyses to verify the stability of the kit was based on the morphology of the parasitic forms as described by microscopists according to the following convention: a) Intact and well parasitic forms: when the analyst noted parasitic forms with identical or very close morphology to the original morphology described in the literature and observed in fresh stool specimens and not (control sample), only diluted in the preservative liquid of PARATEST (morphological gold standard) at the time of analysis. b) Changed and not parasitic forms: when the analyst noted parasitic forms with different and distorted morphology in relation to the original morphology described in the literature and compared to that observed in fresh stool specimens not (control sample), only diluted in the preservative liquid of PARATEST (morphological gold standard) at the time of analysis. 3.1.3. Control samples
The controls of the tests at different times and different temperatures were made by comparison to intact / well parasitic forms (the morphological gold standard) examined simultaneously in real time to test samples. This control was obtained from fresh fecal samples known to be positive which were diluted in the preservative solution of PARATEST at the exact moment (time zero) of microscopic analysis, at room temperature, and monitored microscopically over time. 3.2. Description of tests 3.2.1. Evaluation of the diluent / preservative liquid stability from PARATEST Kit in its original condition of production, in relation to storage temperature For this test, three bottles from the PARATEST Kit were used, and from the same production batch, with production date less than a week, which were kept at different temperatures before the addition of stool sample (Bottle X - room temperature, between 22ºC and 25ºC; Bottle Y - refrigerated at 4 º C and Bottle Z greenhouse at 37 º C) for 7 days before the beginning of the study. The purpose was to determine whether the change in temperature would affect the stability of the diluent / preservative liquid. During testing, two grams of the same fresh fecal sample, poly-infected, and known positive for protozoan cysts and helminth eggs and larvae, were added to these three bottles. Immediately after the feces homogenization, an aliquot of the suspension from each bottle was dripped on a microscope slide and examined under an optical microscope by a specialist in parasitology, aiming at the description of morphology. Morphological analysis was performed in comparison with the gold standard of the pre-set control in this study and the morphology was described as agreement morphology, which is also defined in this study, according to item 3.1.2. After the first examination, immediately after dilution of the stool (time zero), the bottles were incubated at the initial temperatures and their contents were re-examined after 24
hours (time 1) and 48 hours (time 2). The results of these analyses are described in Section 4.1, Table 1. The option to use bottles belonging to lots manufactured less than a week was only a safety margin to eliminate possible variable related to storage time. 3.2.2. Evaluation of the diluent/preservative liquid stability in the PARATEST Kit, in relation to temperature variation after addition of stool For this test, three bottles from the Kit PARATEST were used, from the same production batch, with production date with less than a week, which were kept at room temperature until the time of the test (bottle M, bottle N and bottle O). Two grams of the same fresh fecal sample, poly-infected, and known positive for protozoan cysts and helminth eggs and larvae were added to each bottle. Immediately after homogenization in the diluent / preservative liquid from the PARATEST Kit, an aliquot of the suspension from each of the three bottles was dripped on a slide and examined under an optical microscope, settling upon these initial analyses (zero time at room temperature) the gold standard of morphological forms of the parasite forms observed. The qualitative results of these immediate analyses represented controls for the subsequent analysis of the contents of these bottles subjected to different temperatures. Next, one of these bottles were incubated at room temperature (bottle M), the second was incubated under refrigeration at 4 ºC (bottle N), and the third was incubated at 37ºC (bottle O). Over seven days, the tests were performed after 24 hours, 72 hours, 120 hours and 168 hours, when a new aliquot from each of the three bottles was dripped on a microscope slide for analysis. These subsequent microscopic examinations were performed by the same analyst, aiming at the description of morphology. This was compared to the gold standard, pre-defined in this study and in line with the convention of the morphological pattern description, also previously defined in this study (as per item 3.1.2). The results of these tests are described in section 4.2, Table 2.
The option to use bottles belonging to lots manufactured less than a week was only a safety margin to eliminate possible variable related to storage time. 3.2.3. Evaluation of the diluent/preservative liquid stability in the PARATEST Kit, compared to the storage time at room temperature (22 ºC - 25 ºC) before adding the biological sample fecal For this test, seven bottles of PARATEST Kit have been used, from different batches, and produced in different dates over a period of three years, namely: Bottle k, one week of production; Bottle P, one month of production; Bottle Q, three months of production; Bottle R, six months of production; Bottle S, one year of production; Bottle T, two years of production, and Bottle U, 3 years of production. room temperature until the time of the test (bottle M, bottle N and bottle O). Two grams of the same fresh fecal sample, poly-infected, and known positive for protozoan cysts and helminth eggs and larvae were added to each bottle. Immediately after homogenization in the diluent / preservative liquid of PARATEST Kit (time zero), an aliquot of the suspension from each of the three bottles was dripped on a slide and examined under an optical microscope. Morphological analysis of the parasite forms was confronted with the gold standard of the control sample, prepared in real time, examined at time zero and after one week. The test was conducted at room temperature. As a control, two tests were made for each bottle: time zero and after one week (time 1). The results of these tests are presented in section 4.3, Table 3. 3.2.4. Evaluation of the diluent / preservative liquid stability in PARATEST Kit, compared to the time of storage at room temperature (22ºC - 25ºC), after addition of the biological sample fecal For this test three bottles (bottle G, bottle H, and bottle I) PARATEST Kit have been used, belonging to the same lot, manufactured with less than a week before the test. This time of manufacture of the lot was random, aiming to eliminate only the variable time of the
test. Room temperature was adopted to perform the test during the whole period of analysis, standardized to a month. Two grams of the same fresh fecal sample, poly-infected, and known positive for protozoan cysts and helminth eggs and larvae were added to each bottle. Immediately after homogenization in the diluent/ preservative liquid of PARATEST Kit, an aliquot of the suspension from each of the three bottles was dripped on a slide and examined under an optical microscope (time zero). Morphological analysis of the parasitic forms, at time zero, represented the morphological gold standard, which was confronted with the parasitic forms in the bottles throughout the month. The results of these tests are presented in section 4.4, Table 4. 4. Results 4.1. Stability of diluent / preservative liquid of PARATEST Kit in its original condition of production, in relation to temperature variation As shown in the description of parasitic forms in Table 1, it was found that stability was not affected at the three tested PARATEST Kit storage temperatures, ensuring the sample conservation and morphology preservation of the parasitic forms, in optimized conditions, compared to the gold standard of control. After the analysis at time zero, the bottles were kept for over 48 hours at the original storage temperatures, without the morphology of the parasitic forms being affected. It was noted, however, that the evaporation of preservative / diluent liquid of PARATEST Kit is accelerated at the temperature of 37ºC.
Table 1: Influence of PARATEST Kit storage temperature on the morphology of the parasitic forms. Initial temperature of the diluent / preservative liquid Description of parasitic forms over time Time Zero (Immediate examination) Time 1 (After 24 hours) Time 2 (After 48 hours) Note Room T (22 o C-25 o C) (Bottle X) Similar to the gold standard 4 o C T (Bottle Y) Similar to the gold standard 37 o C T (Bottle Z) Similar to the gold standard Control* Represents the gold standard * Gold Standard 4.2. Result of the diluent / preservative liquid of PARATEST Kit stability evaluation in relation to temperature variation after the addition of stool As shown in Table 2 below, the variation in incubation temperature of PARATEST after the addition of fecal samples did not exert any deleterious effect on the conservation of parasitic forms for up to seven days (168 hours) after the date of dilution. However, it was observed an increased evaporation of the preservative liquid at the temperature of 37ºC.
Table 2 - Influence of temperature variation followed by addition of the biological sample on the parasitic forms. Description of parasitic forms over time Bottle / (temperature) Time Zero (Immediate examination) Time 1 24 hours Time 2 72 hours Time 3 120 hours Time 4 168 hours Bottle M Room T (22 o C-25 o C) Bottle N ( 4 o C T) Bottle O (37 o C T) Control* (gold standard) 4.3. Result of the diluent / preservative liquid in PARATEST Kit stability evaluation, compared to the time of storage at room temperature (22 ºC - 25 ºC) before adding the biological fecal sample As shown in Table 3 below, the test performed as described in Section 3.2.3. showed that the kit is stable before addition of the biological sample for a period of up to three years when stored at room temperature. It also revealed that after dilution of stool, parasitic forms are kept intact and for a period of up to four weeks.
Table 3. Stability evaluation of the Kit in relation to storage time prior to sample dilution. Description of parasitic forms over time Bottle / (production time) Time Zero (Immediate examination) Time 1 (1 week) Time 2 (2 weeks) Time 3 (3 weeks) Time 4 (4 weeks) Bottle K (1 week) Bottle P (1 month) Bottle Q (3 months) Bottle R (6 months) Bottle S (1 year) Bottle T (2 years) Bottle U Ongoing Ongoing Ongoing (3 years) study study study Control* * Gold Standard
4.4. Result of stability evaluation of the diluent / preservative liquid of PARATEST Kit, compared to the storage time at room temperature (22 ºC - 25 ºC), after adding the biological sample fecal As shown in Table 4 below, the parasitic forms are kept intact and for up to four weeks after dilution in the diluent / preservative liquid of PARATEST Kit. These results confirm those presented in section 4.3. Table 4. Influence of fecal sample permanence time in the diluent/preservative liquid on the morphology of the parasitic forms. Description of parasitic forms over time Bottle Room T (22 o C-25 o C) Time Zero (Immediate examination) Time 1 (1 week) Time 2 (2 weeks) Time 3 (3 weeks) Time 4 (4 weeks) Bottle G * Bottle H Bottle I Control* * Gold Standard 5. Conclusion
Based on stability studies described above it was concluded that: Regarding the variable Temperature : - The system PARATEST without the addition of biological samples (feces) maintains stability when stored at room temperature or under refrigeration or at 37 ºC for a period of three years ensuring the integrity and preservation of forms of the parasite when added to diluent /preservative liquid. Diagnostek recommends storage at room temperature (23 o C 25 o C). - PARATEST system after the addition of the biological sample (feces) maintains intact and well the parasitic forms at room temperature or refrigerated, as at the temperature up to 37ºC, for a period of one week. Diagnostek recommends storage, after biological sample dilution, at room temperature and stresses that temperatures around 37ºC could accelerate the evaporation of the diluent/ preservative liquid. Regarding the variable Time - The system PARATEST without the addition of biological samples (feces) is stable for a period of three years at room temperature, keeping parasitic forms intact and well, when the fecal sample is added to the liquid diluent/ preservative. - The system PARATEST, after the addition of biological samples (feces) maintains the parasitic forms intact and well at room temperature for at least thirty days (four weeks). Diagnostek recommends that examination be made within 10 days after the addition of stool in the diluent / preservative liquid.