Diagnosis of soil-transmitted helminths using the Kato-Katz technique: What is the influence of stirring, storage time and storage temperature on stool sample egg counts?

Background Soil-transmitted helminths infect about one ninth of the world's population and have a negative impact on health. The Kato-Katz technique is the recommended method to detect soil-transmitted helminth eggs in stool samples particularly in programmatic settings however, some questions in its procedure remain. Our study aimed to study the effect of storage time, storage temperature and stirring of stool samples on fecal egg counts (FECs). Methodology/Principal findings In the framework of a clinical trial on Pemba Island, United Republic of Tanzania, 488 stool samples were collected from schoolchildren, which were evaluated in three experiments. In the first experiment (n = 92), two Kato-Katz slides were prepared from the same stool sample, one was stored at room temperature and the other in a refrigerator for up to 50 hours, and each slide was analyzed at nine time points (20, 50, 80, 110, 140 minutes, 18, 26, 42 and 50 hours). In the second experiment (n = 340), whole stool samples were split into two, one part was stored at room temperature and the other in a refrigerator for up to 48 hours. From each part one Kato-Katz slide was prepared and analyzed at three time points over two days (0, 24 and 48 hours). In the third experiment (n = 56), whole stool samples where stirred for 15 seconds six times and at each time point a Kato-Katz slide was prepared and analyzed. Mean hookworm FECs of Kato-Katz slides stored at room temperature steadily decreased following slide preparation. After two hours, mean hookworm FECs decreased from 22 to 16, whereas no reduction was observed if Kato-Katz slides were stored in the refrigerator (19 vs 21). The time x storage interaction effect was statistically significant (coefficient 0.26, 95% CI: 0.17 to 0.35, p < 0.0001). After 24 hours mean hookworm FECs dropped close to zero, irrespective of the storage condition. Whole stool samples stored at room temperature for one day resulted in a mean hookworm FEC decrease of 23% (p < 0.0001), compared to a 13% reduction (p < 0.0001) if samples were stored in the refrigerator. Fecal egg counts of A. lumbricoides and T. trichiura remained stable over time regardless of storage temperature of whole stool samples. Finally, we found a significant reduction of the variation of hookworm and T. trichiura eggs with increasing rounds of stirring the sample, but not for A. lumbricoides. However, for hookworm we observed a simultaneous decrease in mean FECs, making it difficult to draw recommendations on stirring samples. Conclusions/Significance Our findings suggest that stool samples (i) should be analyzed on the day of collection and (ii) should be analyzed between 20-30 minutes after slide preparation; when that is not possible, Kato-Katz slides can be stored in a refrigerator up to about 110 minutes. Author summary Intestinal worms, such as hookworm, Ascaris lumbricoides and Trichuris trichiura, infect one in nine people worldwide and, in children, they can lead to cognitive and physical impairment. An accurate detection of these infections is important to treat patients and control the spread of intestinal worms. Currently, the Kato-Katz technique is the most commonly used diagnostic method, in particular, in programmatic settings but there are still several open questions about it. In this study, we aimed at exploring the effect of storage time, storage temperature and stirring of stool samples on the number of worm eggs detectable by the Kato-Katz technique. We included 488 stool samples collected from schoolchildren on Pemba Island, United Republic of Tanzania. We found that, at room temperature, hookworm eggs rapidly disappear from Kato-Katz slides, but storing them in a refrigerator preserves eggs up to 110 minutes. For A. lumbricoides and T. trichiura no relevant trend in fecal egg counts in Kato-Katz slides over time and storing temperature was detected. However, storing whole stool samples in the refrigerator overnight did not prevent hookworm eggs from disappearing and, thus, we recommend analyzing all samples on the day of collection. For A. lumbricoides and T. trichiura fecal egg counts remained stable in whole stool samples over time, regardless of storage temperature. Finally, we found that stirring samples reduced the variation of fecal egg counts for hookworm and T. trichiura, but not for A. lumbricoides and, in the case of hookworm, fecal egg counts decreased with increasing rounds of stirring the sample.

Automated summary

This study aimed to investigate the impact of storage time, temperature, and stirring on fecal egg counts detectable by the Kato-Katz technique. Results showed that hookworm eggs rapidly decreased at room temperature, but could be preserved for about 110 minutes in a refrigerator. There was no significant trend observed for Ascaris lumbricoides and Trichuris trichiura fecal egg counts over time and storage temperature.