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Editor comments with author response

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Reviewer comments with author response

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Comments to the Author

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Reviewer #1: Partly

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: N/A

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

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Reviewer #1:

The manuscript is well presented and written; the authors have addressed the major and minor comments by other reviewers and the manuscript has gone significant improvement as suggested. The study is potentially important in providing method on sample collection and diagnostic of soil transmitted helminths.

Two major comments:

1. Although potassium dichromate is an appropriate stool preservative in tropical climates, I am not convinced that use of potassium dichromat is better than the use of other preservative such as alcohol/ethanol. Why is potassium dichromate for preservation was ideal for the trial location (Fijian islands)? Would that be also ideal to other endemic settings?

In the article van Papaiakovou et al. Plos NTD 2018, that is cited by the author, is mentioned that after considering various factors such as preservative cost, inhibitor resistance, toxicity, availability, associated labor, sample shipping requirements, the use of 95% ethanol as a preservative is recommended in most situations. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5773002/

A study by Kaisar et al (Parasitology et al 2017) https://pubmed.ncbi.nlm.nih.gov/28290266/ and ten Hove et al (Trans Roy Soc 2008) https://pubmed.ncbi.nlm.nih.gov/18177680/ also showed that ethanol preserved stool can give good result and can be transported from the rural setting to the central laboratory.

And I agree with the authors that combination of DNA extraction and bead-beating can optimize the result, especially for T. trichiura eggs as shown in the study of Kaisar: The percentage of positive result in Trichuris PCR is increased when using ethanol preservation (45·0%), bead-beating (51·7%) and a combination (55·0%) and all three methods showed significantly higher DNA loads.

The study by Ayana et al showed that DNA concentration in samples preserved in potassium dichromate where lower compared to that found in sample preserved in ethanol and RNAlater. However, the gain in detecting cases was more pronounced for T. trichiura and potassium dichromate. https://journals.plos.org/plosntds/article?id=10.1371/journal.pntd.0007778

At the time that this study was conceived (2016), potassium dichromate was established as a reliable preservative of stools in this setting [1]. Cost was a major factor when choosing potassium dichromate over ethanol as stated in our manuscript (lines 106-110):

“Ethanol has been validated for use as a preservative in the absence of cold chain [2-5], but transport in large volumes by air becomes restricted and costly due to its classification as a dangerous good. Potassium dichromate is commonly used in STH field trials to provide long-term preservation of stool at ambient temperatures and does not have the same freight restrictions [1, 4, 6].”

Later on, publications by Papaiakovou et al in 2018 [4] and Ayana et al in 2019 [2] support the use of ethanol as a preservative of stool for detection of T. trichiura. For future studies, we would consider alternative preservatives as mentioned in our Discussion (lines 425-427):

“In future, we could trial different DNA and RNA preservatives that are commercially available. These reagents are available in kits, are nontoxic, and can be easily deployed to a remote setting with tropical climates, but they are likely to be more expensive.”

2a. I wonder why there were no detection of Trichuris in the PCR? (S3 table) Would it be the negative effect of the preservation as mentioned by the author (e.g., multiple rinse process)?

Yes, this is a possibility that we mentioned in lines 428-430:

“The requirement for multiple rinses to remove the potassium dichromate may have also resulted in loss of free helminth DNA from degraded eggs, however most would be kept intact within eggs or larvae until DNA extraction was performed.”

For other possible reasons as to why we did not detect Trichuris by qPCR, see section 2c below.

2b. Was there explanation of the negative PCR results on positive KK for hookworm and Ascaris?

Yes, lines 408-413:

“Parasites detected by KK but not qPCR might be attributed to the over 4-fold increase in stool volume used for KK (41.7 mg) compared to qPCR (10 mg). We tried to limit this effect by maximising the volume of DNA tested (5 ul per reaction, in duplicate) in our multiplex qPCRs to achieve the highest sensitivity without compromising the reaction by introducing inhibitors.”

2c. From 6 Trichuris positive KK results, 3 have >160 EPG. It might be interesting to check in the ongoing study of the authors, whether use of other preservation such as ethanol, can prevent the negative findings.

Yes, we discuss this in lines 418-427:

“A possible limitation in this study is the use of potassium dichromate as a preservative. It is important to note that in the Myanmar study, which reported higher detection of T. trichiura by qPCR than KK, stool was placed directly at -20°C and not preserved prior to molecular analysis [7]. It is possible that the potassium dichromate in our study could have prevented detection of T. trichiura by qPCR, as the effects of this preservative was not accounted for in our T. suis experiment. When compared to potassium dichromate, RNA later and 96% ethanol have been reported to increase DNA yield for A. lumbricoides, T. trichiura and N. americanus [2]. In future, we could trial different DNA and RNA preservatives that are commercially available. These reagents are available in kits, are nontoxic, and can be easily deployed to a remote setting with tropical climates, but they are likely to be more expensive.”

2d. Is the prevalence of Trichuris low in the area (Fijian islands)? If the prevalence low, why use the stool samples from area to test the diagnostic tools and not looking for another region? Or test the prevalent helminths from the region. The author mentioned that work is currently underway to test a larger sample size. I would prefer to wait for the result to have is published here.

Data documenting STH infections in Fiji is limited. A study in 2005 of Fijian school children showed the prevalence of Trichuris by microscopy to be 17% [8].

Minor comment:

1. figure 4 needs better quality.

We have increased the resolution of Figure 4.

2. line 151: Samples were transported and stored at room temperature at The Murdoch Children’s Research Institute (MCRI) laboratories for testing within 2 years of collection.

I wonder whether the 40 samples were collected in the spread of 2 years, or the samples were hold in the field for 2 years? Or I misunderstood the statement?

Sorry for the confusion. The sentence has been amended for clarification. Lines 151-154:

“Samples were collected over a period of 16 months (July 2017 to November 2018) and then transported to The Murdoch Children’s Research Institute (MCRI) laboratories where testing was performed between May 2018 to February 2019.”

Reviewer #2:

Minor comment only:

Abstract: Line 33, bead beater was already mentioned in line 27, delete sentence

We have deleted the words “using the Mini-Beadbeater-24” (line 33).

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Reviewer #1: Yes: Aprilianto Eddy Wiria

Reviewer #2: No

References

1. Llewellyn S, Inpankaew T, Nery SV, Gray DJ, Verweij JJ, Clements AC, et al. Application of a Multiplex Quantitative PCR to Assess Prevalence and Intensity Of Intestinal Parasite Infections in a Controlled Clinical Trial. PLoS Negl Trop Dis. 2016;10(1):e0004380. Epub 2016/01/29. doi: 10.1371/journal.pntd.0004380. PubMed PMID: 26820626; PubMed Central PMCID: PMCPMC4731196.

2. Ayana M, Cools P, Mekonnen Z, Biruksew A, Dana D, Rashwan N, et al. Comparison of four DNA extraction and three preservation protocols for the molecular detection and quantification of soil-transmitted helminths in stool. PLoS Negl Trop Dis. 2019;13(10):e0007778. Epub 2019/10/29. doi: 10.1371/journal.pntd.0007778. PubMed PMID: 31658264; PubMed Central PMCID: PMCPMC6837582.

3. Kaisar MMM, Brienen EAT, Djuardi Y, Sartono E, Yazdanbakhsh M, Verweij JJ, et al. Improved diagnosis of Trichuris trichiura by using a bead-beating procedure on ethanol preserved stool samples prior to DNA isolation and the performance of multiplex real-time PCR for intestinal parasites. Parasitology. 2017;144(7):965-74. Epub 2017/03/16. doi: 10.1017/S0031182017000129. PubMed PMID: 28290266; PubMed Central PMCID: PMCPMC5471844.

4. Papaiakovou M, Pilotte N, Baumer B, Grant J, Asbjornsdottir K, Schaer F, et al. A comparative analysis of preservation techniques for the optimal molecular detection of hookworm DNA in a human fecal specimen. PLoS Negl Trop Dis. 2018;12(1):e0006130. Epub 2018/01/19. doi: 10.1371/journal.pntd.0006130. PubMed PMID: 29346412; PubMed Central PMCID: PMCPMC5773002.

5. Song SJ, Amir A, Metcalf JL, Amato KR, Xu ZZ, Humphrey G, et al. Preservation Methods Differ in Fecal Microbiome Stability, Affecting Suitability for Field Studies. mSystems. 2016;1(3). Epub 2016/11/09. doi: 10.1128/mSystems.00021-16. PubMed PMID: 27822526; PubMed Central PMCID: PMCPMC5069758.

6. Stracke K, Clarke N, Awburn CV, Vaz Nery S, Khieu V, Traub RJ, et al. Development and validation of a multiplexed-tandem qPCR tool for diagnostics of human soil-transmitted helminth infections. PLoS Negl Trop Dis. 2019;13(6):e0007363. Epub 2019/06/18. doi: 10.1371/journal.pntd.0007363. PubMed PMID: 31206520.

7. Dunn JC, Papaiakovou M, Han KT, Chooneea D, Bettis AA, Wyine NY, et al. The increased sensitivity of qPCR in comparison to Kato-Katz is required for the accurate assessment of the prevalence of soil-transmitted helminth infection in settings that have received multiple rounds of mass drug administration. Parasit Vectors. 2020;13(1):324. Epub 2020/06/26. doi: 10.1186/s13071-020-04197-w. PubMed PMID: 32580759; PubMed Central PMCID: PMCPMC7315547.

8. Thomas M, Woodfield G, Moses C, Amos G. Soil-transmitted helminth infection, skin infection, anaemia, and growth retardation in schoolchildren of Taveuni Island, Fiji. N Z Med J. 2005;118(1216):U1492. Epub 2005/06/07. PubMed PMID: 15937527.

9. Kim SH, Stothard JR, Rinamalo M, Rainima-Qaniuci M, Talemaitoga N, Kama M, et al. A first nation-wide assessment of soil-transmitted helminthiasis in Fijian primary schools, and factors associated with the infection, using a lymphatic filariasis transmission assessment survey as surveillance platform. PLoS Negl Trop Dis. 2020;14(9):e0008511. Epub 2020/09/26. doi: 10.1371/journal.pntd.0008511. PubMed PMID: 32976499; PubMed Central PMCID: PMCPMC7518615.

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