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?

Abstract

Background: Soil-transmitted helminths infect about one fifth 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 investigate 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. These samples 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, the other in a refrigerator for 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 part was put in a refrigerator for 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. 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; if that is not possible, Kato-Katz slides can be stored in a refrigerator for a maximum of 110 minutes.

Fig 1. Procedure of the Kato-Katz storing experiment.

Fig 2. Procedure of the whole sample storing experiment.

Fig 3. Procedure of the homogenizing experiment.

Fig 4. Kato-Katz storing experiment: Absolute median fecal egg counts (FEC) change over time for (A) hookworm (n = 67 for room temperature, n = 62 for refrigerator), (B) A. lumbricoides (n = 38) and (C) T. trichiura (n = 85).

Blue = refrigerator temperature, red = room temperature, A. lumbricoides = Ascaris lumbricoides, T. trichiura = Trichuris trichiura.

Fig 5

Whole sample storing experiment: Relative fecal egg counts (FEC) change over time for (A) hookworm (n = 259), (C) Ascaris lumbricoides (n = 163), (E) Trichuris trichiura (n = 313) and absolute FEC numbers and relative FEC change between day 0 and day 1 for hookworm (B), Ascaris lumbricoides (D) and Trichuris trichiura (F). For A, C and E: Dashed line = median. For B, D and F: Line = mean. Blue = refrigerator temperature, red = room temperature, A. lumbricoides = Ascaris lumbricoides, T. trichiura = Trichuris trichiura.

Fig 6

Homogenizing experiment: Relative fecal egg counts (FEC) differences from the overall mean per sample (over 7 observations) after different lengths of homogenization for (A) hookworm (n = 28), (B) A. lumbricoides (n = 19) and (C) T. trichiura (n = 52). Figures show boxplots and observed values (jittered). A. lumbricoides = Ascaris lumbricoides, T. trichiura = Trichuris trichiura.

Fig 7. Final recommendations on the procedure of the Kato-Katz technique.