Gates Open Res Gates Open Research 2572-4754 F1000 Research Limited London, UK 10.12688/gatesopenres.13124.1 Research Note Articles Molecular detection of intestinal helminths and protozoa among young children in Dosso Region, Niger

[version 1; peer review: 2 approved with reservations]

 Aiemjoy Kristen Conceptualization Data Curation Formal Analysis Investigation Methodology Project Administration Software Supervision Visualization Writing – Original Draft Preparation Writing – Review & Editing https://orcid.org/0000-0003-1886-2699 a 1 Arzika Ahmed M. Methodology Project Administration Supervision 2 Cook Catherine Data Curation Investigation Methodology Project Administration Supervision Writing – Review & Editing 3 Lebas Elodie Data Curation Investigation Methodology Project Administration Supervision Writing – Review & Editing 3 Pilotte Nils Investigation Writing – Review & Editing https://orcid.org/0000-0002-8447-7425 4 5 Grant Jessica R. Investigation Writing – Review & Editing 4 Williams Steven A. Conceptualization Investigation Supervision Writing – Review & Editing https://orcid.org/0000-0002-4881-7496 4 5 Lietman Thomas M. Funding Acquisition Supervision Writing – Review & Editing https://orcid.org/0000-0001-8216-0240 3 Keenan Jeremy D. Funding Acquisition Supervision Writing – Review & Editing 3 Department of Medicine, Division of Infectious Diseases and Geographic Medicine, Stanford University School of Medicine, Stanford, California, 94305, USA The Carter Center, Niamey, Niger Francis I. Proctor Foundation for Research in Ophthalmology, University of California San Francisco, San Francisco, California, 94143, USA Department of Biological Sciences, Smith College, Northampton, Massachusetts, USA Department of Molecular and Cellular Biology, University of Massachusetts, Amherst, Massachusetts, USA kaiemjoy@stanford.edu

No competing interests were disclosed.

 23 4 2020 2020 4 38 9 4 2020 Copyright: © 2020 Aiemjoy K et al. 2020 This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Eukaryotic parasites are significant contributors to childhood illness in Niger. While helminthiases have received national attention through mass deworming efforts, the epidemiology of intestinal protozoa in Niger remains underexamined. This study employed real-time PCR diagnostics to describe the prevalence of two schistosomes, four soil-transmitted helminths, and one protozoan parasite in Boboye Department, Dosso Region. Prevalence was assessed using bulk stool specimens collected from a population-based sample of 86 children residing in 9 communities. Anthropometric measurements were used to calculate child growth z-scores and stool consistency was graded. Helminths were absent from the study population, with the exception of a single Schistosoma haematobium infection (1/86; 1.2%). Giardia duodenalis was the only protozoa present, detected in 65% (56/86) of children. Prevalence of G. duodenalis peaked in 2-year-olds with 88% (15/17) positivity. The population was generally undernourished, though growth indices did not differ significantly between children with and without G. duodenalis infection.

 Schistosoma soil-transmitted helminths protozoa molecular diagnostics Gates Foundation OPP1032340 University of California, San Francisco This study was supported by the Gates Foundation (OPP1032340) and the University of California San Francisco (UCSF) Catalyst Program. The Salesforce Foundation provided user licenses to Salesforce.com and cloud storage. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Introduction

Eukaryotic parasites are significant contributors to childhood illness in Niger. In 2012, the World Health Organization estimated that 10–49% of Nigeriens lived with intestinal or urogenital schistosomiasis, while more than two-thirds of children required preventative chemotherapy for soil-transmitted helminths (STH) 1 . In 2015, over 75% of pre-school aged children across Niger were targeted for preventative anthelmintic treatment 2 . While mass deworming programs have drawn attention to the public health significance of helminthiases, the epidemiology of intestinal protozoa in Niger is not well described 3 . However, limited data suggest that protozoa may cause an appreciable fraction of clinically evaluated enteric infections 4, 5 .

Real-time polymerase chain reaction (qPCR) assays targeting high-copy-number genetic elements have been validated for many globally burdensome parasites and have been shown to provide greater sensitivity and specificity than traditional copromicroscopy 6– 8 . This study makes use of these significant strides in diagnostic accuracy to evaluate the age-dependent prevalence of seven eukaryotic pathogens in nine rural communities of the Niger River Valley.

 Methods Study background

We conducted a cross-sectional study to evaluate the prevalence of helminthiasis, schistosomiasis and intestinal protozoa infection in Boboye Department, Dosso Region, Niger. The study was nested within the Macrolides Oraux pour Réduire les Décès avec un Oeil sur la Résistance (MORDOR) trial, a cluster-randomized trial investigating the effects of community mass administration of azithromycin on child health and mortality (Clinicaltrials.gov ID NCT02048007). Details of the MORDOR study design are available elsewhere 9 .

This nested sub-study occurred in 9 of the 30 MORDOR study communities (see Figure 1) in Niger during a regularly scheduled study visit between May and June of 2017. All children residing in the study communities who were sampled for participation in the parent trial were eligible to participate. In the 9 communities, 447 children age 0–4 years were eligible to participate and 354 participated in the MORDOR study visit, of whom 86 provided a bulk stool sample.

<italic toggle="yes">Giardia duodenalis</italic> prevalence by study site in Dosso Region, Niger.

Study communities are indicated by red dots on the map. The prevalence is listed as the number of samples that tested positive for Giardia duodenalis over the total number of samples tested followed by the percentage.

 Sample collection and analysis

On the day of the study visit, participating children gathered in a central location in the community. Trained field examiners performed three height and weight measurements per child in accordance with standard World Health Organization (WHO) protocols 10 . Stool was collected at the time of the study visit, by instructing caregivers to have their children defecate in a potty chair lined with a plastic bag. After defecation, the caregiver returned the stool sample to the field collection team. The field examiner then collected a 0.5-mL specimen and placed it in an empty sterile 2-mL tube. No media was added to the samples. The stool samples were immediately placed on ice and transported to a -20°C freezer by the end of the day.

Isolation of total DNA from bulk stool and rectal swabs followed a procedure optimized for the qPCR detection of intestinal helminths. Multi-parallel qPCR targeted the following species: the STH Ancylostoma duodenale 6 , Ascaris lumbricoides 11 , Necator americanus 6 , and Trichuris trichiura 6 ; the trematode flukes Schistosoma haematobium 12 and Schistosoma mansoni 12 ; and the protozoan parasite Giardia duodenalis 13 . All qPCR assays targeted highly repetitive non-coding elements with the exception of the G. duodenalis assay, which targets the small subunit ribosomal RNA gene. Procedures for sample collection, DNA isolation, qPCR, and quality control followed previously described protocols and standards 14 .

The median height and weight measurements were used to calculate height-for-age (HAZ), weight-for-age (WAZ), and weight-for-height (WHZ) z-scores according to WHO child growth standards 15 . We used generalized estimating equations to evaluate differences in anthropometry z-scores according to G. duodenalis positivity adjusting for age and accounting for clustering by community. All analyses were run in R v3.5.3.

 Ethical approval

Ethical committees from the Niger Ministry of Health and the University of California (San Francisco, CA, USA) granted approval for this study. Verbal informed consent was obtained in French from all caregivers. Verbal consent was obtained rather than written consent because of mixed literacy levels in the study population.

 Results

Stool samples were collected from 86 children residing in 9 communities. The median age was 2 years old (IQR 1-4). Overall, 59.3% (51/86) of participants were female. The mean child growth z-scores were less than zero for all indicators (HAZ = -1.53 [SD 1.39], WAZ = -1.55 [SD 1.22], WHZ = -0.97 [SD 1.1]).

G. duodenalis infection was detected in 65% (56/86) children living in 7 of the 9 surveyed communities. The 2 communities for which no infections were observed, Goberi Peulh and Tombo, only contained 1 and 3 total participants, respectively. For the 7 communities in which G. duodenalis was detected, prevalence ranged from 30% (3/10) to 89% (16/18) ( Figure 1). The prevalence of G. duodenalis increased with age, with 88.2% (15/17) testing positive. Only 1 helminth infection was detected ( S. haematobium) ( Table 1).

Prevalence of intestinal parasites among young children in Boboye Department, Dosso Region, Niger.
Variable Age of child
< 1 year 1 year 2 years 3 years 4 years Total
N children 11 20 17 13 25 86
Helminths  
  Ancylostoma duodenale 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%)
  Ascaris lumbricoides 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%)
  Necator americanus 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%)
  Schistosoma haematobium 0 (0%) 0 (0%) 0 (0%) 0 (0%) 1 (4%) 1 (1.2%)
  Schistosoma mansoni 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%)
  Trichuris trichiura 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%)
Protozoa  
  Giardia duodenalis 6 (54.5%) 9 (50.0%) 15 (88.2%) 10 (76.9%) 16 (76.0%) 56 (65.1 %)

Children who tested positive for G. duodenalis had a -0.18 (95% CI: -0.77–0.40) lower height-for-age Z-score, a 0.22 (95% CI: -0.3–0.75) higher weight-for-age z-score and a 0.52 (-0.07–1.1) higher weight-for-height Z-score. However, none of these differences were statistically significant at the 0.05 level. Individual-level de-identified anthropometric, demographic and infection data are available (see Underlying data) 16 .

 Discussion

In conclusion, we found that young children residing in the MORDOR study area in rural Niger had a high prevalence of G. duodenalis and a low prevalence of helminthic infections as measured by PCR in stool specimens. The population was generally undernourished, with all three anthropometric indices below average. Lower HAZ scores were observed among children positive for G. duodenalis, similar to prior observations in rural Amhara Region, Ethiopia 17 . However, there was no significant difference in growth indices between children with and without G. duodenalis infection, though this could be related to sample size.

The administrative Region of Dosso is situated in the Niger River Valley, a more densely populated region with higher rates of infectious disease than the majority of the nation 18 . In 2016, children 4 years of age and younger represented 81.1% of diarrhea cases, 43.7% of dysentery cases, and 37.7% of intestinal parasitisms recorded in Dosso Region, yet the etiological agents of these complaints remain unreported 4 . Given the high rates of G. duodenalis observed in the present study – albeit within a limited sample – it is possible that the contributions of intestinal protozoa to public health in the Niger River Valley warrant further investigation.

The absence of STH and S. mansoni in this cohort may relate to the success of national mass drug administration programs. In 2015, over 800,000 citizens of Dosso Region received praziquantel through mass drug administration, including over 200,000 living in Boboye Department; over half a million received albendazole, with over 100,000 residing in Boboye Department 4 . However, without data on baseline prevalence and community-level anthelmintic distribution in this population, conclusions cannot be drawn.

The low prevalence of S. haematobium may be due to testing stool rather than urine, the standard specimen type for this species. Though S. haematobium predominantly evacuates via urine, this species was detected in the feces of a single child. Ectopic elimination in the brain and intestine has been observed in S. haematobium, sometimes attributed to high parasite loads 19 . Whether a greater number of children would have tested positive for S. haematobium by urine analysis cannot be known but could be an area of further research.

These findings indicate that G. duodenalis infections may be a significant contributor to child morbidity in the Dosso Region of Niger.

 Data availability Underlying data

Open Science Framework: Molecular detection of intestinal helminths and protozoa among young children in Dosso Region, Niger. https://doi.org/10.17605/OSF.IO/FMTYH 16 .

This project contains the following underlying data:

NigerParasitePCR.csv. (Demographic, anthropometric variables and infection status for each participant.)

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

 Acknowledgements

We would like to thank the children and guardians who participated in this study for their time and invaluable contributions. We would also like to acknowledge Jacqueline R. M. A. Maasch (University of Pennsylvania) for contributing the figures in this paper and for collaborating on data analysis.

 World Health Organization: Investing to Overcome the Global Impact of Neglected Tropical Diseases: Third WHO Report on Neglected Tropical Diseases 2015. World Health Organization;2015;3. Reference Source World Health Organization, Department of Control of Neglected Tropical Diseases: Integrating Neglected Tropical Diseases into Global Health and Development: Fourth WHO Report on Neglected Tropical Diseases.2017. Reference Source Squire SA Ryan U : Cryptosporidium and Giardia in Africa: current and future challenges. Parasit Vectors. 2017;10(1):195. 28427454 10.1186/s13071-017-2111-y 5397716 Secretariat Général du Ministère de la Santé Publique du Niger [Secretary General of the Niger Ministry of Public Health]: Annuaire Des Statistiques Sanitaires Du Niger: Année 2016.2017. Reference Source Soumana A Kamaye M Saidou D : Intestinal parasitosis in children less than five years of age in Niamey, Niger. Mali Med. 2016;31(4):19–28. 30079652 Pilotte N Papaiakovou M Grant JR : Improved PCR-Based Detection of Soil Transmitted Helminth Infections Using a Next-Generation Sequencing Approach to Assay Design. PLoS Negl Trop Dis. 2016;10(3):e0004578. 27027771 10.1371/journal.pntd.0004578 4814118 Easton AV Oliveira RG O’Connell EM : Multi-parallel qPCR provides increased sensitivity and diagnostic breadth for gastrointestinal parasites of humans: field-based inferences on the impact of mass deworming. Parasit Vectors. 2016;9(1):38. 26813411 10.1186/s13071-016-1314-y 4729172 Mejia R Vicuña Y Broncano N : A novel, multi-parallel, real-time polymerase chain reaction approach for eight gastrointestinal parasites provides improved diagnostic capabilities to resource-limited at-risk populations. Am J Trop Med Hyg. 2013;88(6):1041–1047. 23509117 10.4269/ajtmh.12-0726 3752800 Keenan JD Bailey RL West SK : Azithromycin to reduce childhood mortality in sub-Saharan Africa. N Engl J Med. 2018;378(17):1583–1592. 29694816 10.1056/NEJMoa1715474 5849140 Physical status: The use and interpretation of anthropometry. Report of a WHO Expert Committee. World Health Organ Tech Rep Ser. 1995;854:1–452. 8594834 Pilotte N Maasch JRMA Easton AV : Targeting a highly repeated germline DNA sequence for improved real-time PCR-based detection of Ascaris infection in human stool. PLoS Negl Trop Dis. 2019;13(7):e0007593. 31329586 10.1371/journal.pntd.0007593 6675119 Seignon K Grant J Pilotte N : Development of a sensitive quantitative PCR assay for the detection of Schistosoma mansoni and Schistosoma haematobium in patients. In preparation. Verweij JJ Schinkel J Laeijendecker D : Real-time PCR for the detection of Giardia lamblia. Mol Cell Probes. 2003;17(5):223–225. 14580396 10.1016/s0890-8508(03)00057-4 Maasch J Arzika AM Cook C : Rectal swabs as an alternative sample collection method to bulk stool for the real-time PCR detection of Giardia duodenalis. Amerigan Journal of Tropical Medicine and Hygiene. Sumbitted 2019. World Health Organization: WHO child growth standards: length/height-for-age, weight-for-age, weight-for-length, weight-for-height and body mass index-for-age: methods and development.2006. Reference Source Aiemjoy K : Molecular detection of intestinal helminths and protozoa among young children in Dosso Region, Niger.2020. http://www.doi.org/10.17605/OSF.IO/FMTYH Aiemjoy K Gebresillasie S Stoller NE : Epidemiology of Soil-Transmitted Helminth and Intestinal Protozoan Infections in Preschool-Aged Children in the Amhara Region of Ethiopia. Am J Trop Med Hyg. 2017;96(4):866–872. 28167597 10.4269/ajtmh.16-0800 5392634 Leslie J Garba A Oliva EB : Schistosomiasis and soil-transmitted helminth control in Niger: cost effectiveness of school based and community distributed mass drug administration [corrected]. PLoS Negl Trop Dis. 2011;5(10):e1326. 22022622 10.1371/journal.pntd.0001326 3191121 Loridant S Mouahid G Cornu M : Case Report: Hemianopia: From Suspected Glioblastoma to the Diagnosis of Ectopic Schistosomiasis Haematobium Infection in a Traveler Returning from the Republic of the Congo. Am J Trop Med Hyg. 2018;99(1):94–96. 29714164 10.4269/ajtmh.18-0028 6085791 10.21956/gatesopenres.14306.r29090 Reviewer response for version 1 Gaye Oumar 1 Referee Sow Doudou 2 Co-referee University Cheikh Anta Diop de Dakar, Dakar, Senegal University Gaston Berger of Saint-Louis, Saint-Louis, Senegal

Competing interests: No competing interests were disclosed.

 29 7 2020 Copyright: © 2020 Gaye O and Sow D 2020 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

Regarding the question 2 on methodology and results interpretation, authors should take into account comments below:

Methods section

How did authors select the 9 study communities among the 30 MORDOR communities? To be clarified.

Author must explain how did they choose the 86 children enrolled in this study among the 354 MORDOR participants, or it was an arbitrary choice? To be clarified.

Why did authors targeted S. haematobium in stool samples? S haematobium is responsible of urogenital schistosomiasis and can be found in some cases in stool samples. Did they have previous data showing the importance of this parasite in stool samples?

In the reference 14, the authors mention positive controls but it is not clear what the positive controls were.

It is not considered appropriate to consider Ct values above 38 as negative, high Ct values can be an indicator of an (upcoming) problem of PCR contamination or non-specificity of the PCR. 

 Results section

Authors did not show any results on the intensity of infection. Authors should precise the median and/or the range of ct results to give an estimate of the intensity of infection.

Did authors compare real time PCR to microscopy results? If yes, results should be shown to support future epidemiological survey based on microscopy as PCR is expensive particularly in large surveys.

Is the work clearly and accurately presented and does it cite the current literature?

Yes

If applicable, is the statistical analysis and its interpretation appropriate?

Yes

Are all the source data underlying the results available to ensure full reproducibility?

Yes

Is the study design appropriate and is the work technically sound?

Partly

Are the conclusions drawn adequately supported by the results?

Yes

Are sufficient details of methods and analysis provided to allow replication by others?

Yes

Reviewer Expertise:

parasitology, molecular biology, epidemiology

We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however we have significant reservations, as outlined above.

 10.21956/gatesopenres.14306.r29071 Reviewer response for version 1 Ojurongbe Olusola 1 Referee https://orcid.org/0000-0001-6967-9187 Department of Medical Microbiology and Parasitology, Ladoke Akintola University of Technology, Osogbo, Nigeria

Competing interests: No competing interests were disclosed.

 24 7 2020 Copyright: © 2020 Ojurongbe O 2020 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

This manuscript reported a study carried out in Dosso Region, Niger on Molecular detection of intestinal helminths and protozoa among young children.

The authors reported high prevalence of Giardia duodenalis and generally undernourished  population. The authors concluded that G. duodenalis infections may be a significant contributor to child morbidity in the Dosso Region of Niger. However, extremely low sample size of this manuscript makes it difficult to accept this conclusion. These, as well as other major concerns, must be addressed before this manuscript can be considered acceptable for indexing.

The presentation of the DNA extraction methodology is not clear. The author stated that “the Isolation of total DNA from bulk stool and rectal swabs followed a procedure optimized for the qPCR detection of intestinal helminths”. This procedure was not referenced and and there was no mentioning of kit used. There are many procedure optimized for DNA extraction in stool. The author should properly explain what was done

The author also should briefly explain what he meant by “multi-parallel” in the result section.

The authors stated that “Goberi Peulh and Tombo, only contained 1 and 3 total participants” and no infection was observed in theses 2 communities. I will suggest that the names of these communities should not be listed as being free from parasitic infection as the sample size of 1 and 3 is too little to be considered.

“The prevalence of G. duodenalis increased with age, with 88.2% (15/17) testing positive” the author should correct this statement by including age “2 years old” in the sentence.

I will suggest that the author should not start discussion with “in conclusion”.

The author reported high prevalence of G. duodenalis in rural Niger. While the author attributed low STH and S. mansoni to the success of national mass drug administration programs, nothing was discussed as to why G. duodenalis infection was high. This should be discussed.

If according to the author there is a mass administration of PZQ that lead to the absence of STH and S. mansoni, it is not clear why the author is assuming that there will be a higher prevalence of SH in the same community if urine are to be tested. Both SM and SH are susceptible to PZQ. So the explanation of high load or ectopic egg elimination may not be tenable. 

Is the work clearly and accurately presented and does it cite the current literature?

Yes

If applicable, is the statistical analysis and its interpretation appropriate?

Yes

Are all the source data underlying the results available to ensure full reproducibility?

Yes

Is the study design appropriate and is the work technically sound?

Partly

Are the conclusions drawn adequately supported by the results?

No

Are sufficient details of methods and analysis provided to allow replication by others?

No

Reviewer Expertise:

Molecular Parasitology and genetics.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.