PLoS Neglected Tropical Diseases
Public Library of Science
Toxocara species environmental contamination of public spaces in New York City
Volume: 14, Issue: 5
DOI 10.1371/journal.pntd.0008249
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### Notes

Abstract

Toxocara canis and Toxocara cati are helminth worms that infect dogs and cats, respectively. Infected dogs and cats will defecate thousands of Toxocara eggs into the environment. Humans are incidental hosts and are exposed when consuming contaminated soils via the fecal-oral route. After leaving the gastrointestinal tract, the Toxocara larvae will enter the vasculature and can migrate to any major organ system, including lungs, ocular, and central nervous system. Symptoms can range from mild muscle aches to severe asthma, blindness, and encephalitis. Humans are not definitive hosts of the parasite and cannot transmit Toxocara eggs to the environment or other humans. There is a need for research on the sanitary impact of Toxocara for both humans and animals, especially in large urban cities such as New York City. Poverty is also associated with higher rates of toxocariasis, with more contamination in poorer neighborhoods where animal control, deworming of pets, and less sanitary conditions exist. This study aims to understand further the disparity of lower socioeconomic areas having higher rates of contaminated parks and playgrounds, comparing the five boroughs of New York City.

Tyungu, McCormick, Lau, Chang, Murphy, Hotez, Mejia, Pollack, and Holland: Toxocara species environmental contamination of public spaces in New York City

## Introduction

Toxocara canis and Toxocara cati are ascarid nematodes that ubiquitously infect dogs and cats and can result in environmental contamination if the feces of infected animals contaminate community spaces. Eggs deposited in the soil can exhibit cryptobiosis when environmental conditions are not ideal and may survive for many years. [1] Human ingestion of embryonated eggs through contaminated soil, poor hygiene practices, or uncleaned vegetables, can result in paratenic zoonotic toxocariasis. [24] As Toxocara eggs develop in the soil, it is possible to detect the developmental progression of the helminth from germinal cells to the presence of viable infective larvae via microscopy. Eggs containing fully developed larvae are infectious to humans, whereas Toxocara at earlier stages of development are pre-infectious and cannot lead to toxocariasis. Following the ingestion of an embryonated egg, the third stage larva enters the bloodstream. It burrows through body tissues, where the worms can accumulate in the eye, brain, liver, or skin, leading to visceral or ocular larva migrans, blindness, subclinical cerebral infection, or covert infection which can diminish neurological cognition or result in developmental delays. [59]

Toxocariasis is an underreported and understudied disease in the United States. Both the parasite and human toxocariasis have been described as ‘enigmatic’ due to numerous deficiencies in the understanding of the organism, including the role of cerebral toxocariasis and the possible link to neurocognitive deficits and blindness in children. [2, 5] The US Centers for Disease Control and Prevention (CDC) identifies human toxocariasis as one of five neglected parasitic infections in the U.S., and it is possibly the most common helminthiasis in the United States after pinworm (Enterobius vermicularis ). [8, 10] Toxocariasis is a neglected disease of poverty due to its disproportionately high seroprevalence in areas of community poverty, especially among underrepresented minority populations living in poor areas. [1012]

Among the most enigmatic features of toxocariasis is its covert form, which generally is not associated with visceral larva migrans. Covert toxocariasis is subclinical, with only eosinophilia as a biomarker for suspicion of infection. [4] Covert toxocariasis has been linked to cognitive and developmental delays, lung dysfunction, and asthma. However, research is still in the nascent stages of understanding the full clinical spectrum of illness caused by T. canis and T. cati . [5] Overall, toxocariasis may be severely underdiagnosed due to the covert nature of the illness and gaps in medical knowledge; it is a disease that should not be overlooked as a cause of neurocognitive delay in children. [2, 4, 5, 7, 13, 14] A 1987 study in New York City (NYC) correlated T. canis seroprevalence to neurocognitive deficits in children. [5, 15, 16] Prior research has also shown that simple incubation of a Toxocara embryo allows it to become infective, and after infection, living larvae have been found in the brains of mice. [1] Mice with infected brains have significant impairment, including cellular damage, and may have different areas of brain involvement depending on the infecting Toxocara species. [17, 18] Covert toxocariasis may also represent an important environmental cause of asthma among disadvantaged American children. [19, 20]

Throughout the history of the National Health and Nutrition Examination Survey (NHANES), the prevalence of Toxocara within the United States population has varied, ranging from 5%-14% during analyses from 1988–1994 and 2011–2014. [11, 21] In both studies, Toxocara seropositivity was disproportionately higher in persons from lower socioeconomic status communities, particularly in Hispanic, non-Hispanic black communities, and those with less than a high-school education. [11, 21]

Few studies have been conducted in the United States, and most were published over two decades ago. [2228] In the few US studies, contamination rates ranged from 0.3%-27.5%, with the contamination rate defined as the number of eggs found per park or space evaluated. [2228]

Walsh et al. created a predicted probability model to estimate the seroprevalence of toxocariasis in NYC using estimates from the NHANES III and correlated findings with available sociodemographic information, which suggested higher levels of exposure to Toxocara species in low income, minority, and immigrant communities. [29] This study is the first geo-surveillance of Toxocara species in all five boroughs of NYC attempting to address the potential risk of infection within NYC communities by first examining soil from around the city.

## Methods

### Sample collection

Samples were collected from 3–5 cm below the surface after the top layer of soil was removed. Five samples were collected from each location on the same day, and each sample was from a different geographic area of the sample site, attempts were made to sample the entire site equally. These were combined before laboratory analysis into one larger sample weighing 100-150g. The samples were collected randomly during October 2015-July 2016. A sample size calculation was used to determine the level of precision using a metanalysis (Fakhri et al.) confidence interval of 16 to 27% soil contamination worldwide from 42,797 samples, 79 samples were needed for statistical significance, and a total of 91 samples were collected for this study.[30] A total of 91 sites were surveyed in New York City from October 2015-July 2016, and samples were collected from different sites at different times during this period. Of these, 27 (29.6%) were in Manhattan, 15 (16.5%) were in the Bronx, 13 (14.3) were in Brooklyn, 18 (19.8%) were in Queens, and 18 (19.8%) were in Staten Island. Sample sites were selected at random to maximize coverage of each borough, due to limited access, parts of Brooklyn and Staten Island were not tested.

#### Microscopic analysis

Samples were analyzed using a modified soil flotation technique derived from previously published floatation methods of helminth egg recovery from the soil and human stool. [31, 32] Sodium nitrate was chosen as the optimal flotation solution due to elevated specific gravity (1.25–1.35), best-published egg recovery results, and low laboratory cost. All specimens were sifted to remove large debris before being washed in 0.1% Tween 20 three times. Samples were subsequently mixed with approximately 10 to 14 ml sodium nitrate, specific gravity 1.30 (Vedco Feca Med, St Joseph, MO) enough to form a convex meniscus at the opening of the tube. With a coverslip in place, samples were centrifuged at 500 g for 5 minutes and allowed to stand for 5 minutes before viewing using a standard light microscope. Eggs, if present, were easily visualized and counted using this technique. Parasites were identified by structural characteristics, and embryonated forms were described if larvae were noted within the egg. Other incidentally discovered parasites were recorded and categorized using a subjective semiquantitative “+” system (Table 1). Coverslips were then washed into 2mL Eppendorf tubes with DNase-free water and saved at -20 degrees Celsius for later real-time PCR testing.

Table 1
Larvated, infectious Toxocara sp. were only found in the Bronx, the borough that had the highest prevalence of Toxocara contamination.
Prevalence of Toxocara sp. including the presence of larval forms in specimens collected from sites in each NYC borough.
BoroughPositives/Total% Toxocara positive (95% CI)Eggs (mean and range)Larvated forms
Bronx10/1566.7 (41.7–84.8)15.7 (1–102)+++
Staten Island7/1838.9 (20.3–61.4)3.4 (1–5)-
Queens6/1833.3 (16.3–56.3)1.8 (1–2)-
Brooklyn4/1330.8 (12.7–57.6)2.7 (1–7)-
Manhattan8/2729.6 (15.9–48.5)6.3 (4–12)-

#### Molecular methods

Parasite DNA was extracted from soil samples with MP FastDNA spin kits for soil (MP Biomedical, Santa Ana, CA) using supernatant from the coverslips from the previous centrifugation step. We processed this supernatant using bead beating for five minutes at 3,000 RPM, followed by a ten-minute heating step at 90°C to promote egg disruption. This same technique was utilized to extract DNA from T. canis and T. cati eggs for the standard controls. [33] The T. canis and T. cati DNA from these samples were quantified using a multi-parallel real-time quantitative polymerase chain reaction (qPCR) protocol. [33] All samples underwent qPCR testing using previously published probes with the following modifications [T. canis (5’-FAM-CCATTACCACACCAGCATAGCTCACCGA-3’-NFQ-MGB) and T. cati (5-FAM-TCTTTCGCAACGTGCATTCGGTGA-3’-NFQ-MGB)] and forward primers [T. canis (5’-GCGCCAATTTATGGAATGTGAT-3’) and T. cati (5’-ACGCGTACGTATGGAATGTGCT-3’)] and shared reverse primer 5’-GAGCAAACGACAGCSATTTCTT-3’) to both Toxocara species. [34] PCR was performed using an ABI ViiA 7 (Applied Biosystems, Foster City, CA) as previously described [33]. A sample was considered positive if there was detectable DNA at or before a cycle threshold of 38. The threshold of positivity was determined by T. canis/cati genomic DNA dilutions for the dynamic range. T. canis and T. cati eggs were isolated and washed with distilled water three times. Eggs were counted using a McMasters microscope slide (Advanced Equine Products, Issaquah, WA), and serial dilutions were used as standards. All samples were performed in duplicate and were repeated for discordant results. A standard curve was constructed using serial dilutions of 10,000 T. cani and T. cati eggs. Standards were used as positive controls with no-template for negative controls. A known concentration of plasmid internal control was added to all samples prior to bead beating to validate successful DNA extraction [35].

### Demographic data

Socioeconomic data were obtained from the websites of the US Census and the New York State Department of health. [36, 37]. Zipcodes were recorded from each sampling area, and New York City income data per zip code was obtained from a website of current census bureau income statistics for United States zip codes. [38]

### Statistical methods

Statistical analysis was carried out using GraphPad Prism version 7.0d (GraphPad, La Jolla, CA). The number of Toxocara eggs was compared by zip code, and Toxocara eggs per sample site were compared to individual boroughs using the Kruskal-Wallis test. Comparing the prevalence of Toxocara species by zip code or borough, a spearman rank was conducted comparing the prevalence of positive parks per borough and the median income per resident living in those zip codes.

## Results

### Environmental survey results (Microscopy results)

Of the 27 Manhattan sample sites, Toxocara eggs were found in 29.6% of playgrounds with at least one egg being identified by microscopy. Of the Staten Island sites tested, 38.9% had Toxocara eggs found, of the 13 Brooklyn sites tested 30.8% had eggs found, Queens had 33.3% of sites contaminated, and the Bronx had 66.7% sites contaminated (Table 1). The overall prevalence of 38.5% (35/91) contaminated playgrounds in NYC.

Infective embryonated eggs (Fig 1A and 1B, Fig 2) were only found in the Bronx playgrounds, with over 70% of eggs recovered being in their larvated state. All other boroughs had eggs in the pre-infectious, unembryonated (unlarvated) form (Fig 1C, Fig 2).

Fig 1
Toxocara forms isolated in NYC specimens: a and b: Larvated ‘infective’ Toxocara eggs at 40x magnification, c: Unlarvated Toxocara egg at 40 x magnification, obtained in NYC study.
Fig 2
Toxocara was found in those sites represented by either blue triangles (unembryonated egg(s)) red stars (embryonated egg(s)). Toxocara was not found at sites marked by black circles. The highest density of Toxocara geo-contamination was found in the Bronx, which was the only borough where infected larvae were found. Sampling ArcMap of NYC, created using ArcGIS 10.6.1.Geospatial results of Toxocara sampling across NYC.

### Toxocara species results (qPCR results)

Of all the positive samples, six were found to have T. cati DNA present (Fig 3, Table 1). None of the parks were found to have T. canis DNA present. More than 4 of 6 parks that tested positive were from samples with the highest number of larvated eggs, accounting for the higher copy numbers of genomic DNA present in many of the samples (mean 19.5, 0 to 102 eggs). (Fig 3) The six positive samples had calculated egg counts from the standard dilutions using linear regression that correlated to 40 –Cycle threshold (Ct) (Spearman r = 0.999, p < 0.0001). One sample (Bk8) was negative by microscopy but positive by qPCR (13.958, 40-Ct). One egg of either T. canis or T. cati in 500 μl of water was consistently detected by qPCR with Ct values of 37 to 38.

Fig 3
Calculated egg count was derived from dilutions of a known amount of Toxocara cati eggs and correlated to 40 –Ct (Spearman r = 0.999, p < 0.0001).qPCR results, parks that were qPCR-positive were contaminated with T. cati.

### Association of geo-contamination and socioeconomic data

Contamination was more prevalent in lower socioeconomic neighborhoods. Manhattan, the borough with the highest median income ($97,255) of zip codes where sampling was performed, had the lowest rate of contamination (29.6%) compared to a contamination rate of 66.7% in the Bronx, which had the lowest median income ($26,131) (Fig 4). Queens, Brooklyn, and Staten Island had contamination rates from 30.8–38.9% and median incomes between $57,452-$69,998, Spearman r = -0.7, p = 0.233 (Fig 4). The Bronx sample sites had the highest mean egg burden (Bronx: 16.4, Queens: 1.8, Brooklyn: 1.9, Staten Island: 3.0, Manhattan: 5.9, p = 0.0365) and prevalence. (Fig 5) Overall, there was an inverse relationship between median income and the likelihood of Toxocara species contamination (Fig 5).

Fig 4
The line represents the median income of families living in the zip code where Toxocara testing was performed. Results aggregated by borough. (Spearman r = -0.7, p = 0.233).Association of positive Toxocara results with the median income of residents living in the ZIP codes of sampled sites.
Fig 5
Kruskal-Wallis P = 0.0365. Eggs were not equally distributed in the parks by borough. The Bronx* has the highest egg burden compared to the other boroughs.Toxocara eggs/park by borough.

## Discussion

In this first environmental study of Toxocara prevalence in NYC, approximately one-third of sites sampled throughout the five boroughs contained Toxocara eggs (Table 1). The distribution of contamination was not equal across the city. The Bronx had the highest Toxocara burden and prevalence, compared to the other boroughs (Figs 4 and 5). All boroughs, except the Bronx, had eggs in their unlarvated, pre-infectious form (Fig 1).

From the qPCR data, T. cati was the only species identified. This suggests that cats are the predominant reservoir causing contamination in New York City play areas, although dogs can transiently carry T. cati from eating cat feces but are not part of the parasites’ life cycle. [39] In fact, the finding of cat contamination is not entirely unprecedented, as NHANES data have shown high co-infection rates with toxocariasis and toxoplasmosis nationally, suggesting that cats may represent an important but often underappreciated source of Toxocara eggs. [40] There are several reasons for predominant feline contamination. Park fencing consists of vertical metal bars, which will effectively prevent dog entry into parks (unless pet owners allow access) but are unlikely to restrict stray cat access. Furthermore, people are more likely to remove feces from their dogs than cats. Cats can be infected with T. cati through all stages of life, whereas dogs are most severely infected as puppies. [41] Veterinary data in the New York City area shows that cats test positive for roundworm infections at least four times more frequently than dogs, regardless of the borough. [42] Furthermore, pet cats shed Toxocara more commonly than pet dogs. [43] Although the samples were not optimally processed for qPCR and the lack of T. cani DNA may be attributed to the small sample size.

Similar results have been recently reported in several cities in Europe [44, 45] and have important implications in determining strategies to reduce contamination and risk for human infection. [39] In a recent metanalysis by Fakhri et al., the average soil contamination with Toxocara in the USA was 4 to 23% playgrounds in NYC likely are have concentrations of animal exposure and results in higher egg counts.[30] Several considerations exist regarding why Toxocara qPCR was discordant for samples that were positive or negative by microscopy. One may be due to the way the specimens were saved and prepared for qPCR. The qPCR is not a direct comparison to microscopy as samples were first processed for microscopic analysis, and qPCR was performed on the supernatant obtained from the coverslips of the slides. The specific transfer method after microscopy could account for a loss of eggs. Quantitative PCR was performed on the supernatants removed from the slide subsequent to the microscopy examination and not directly from the soil specimen. Given the low number of eggs (1–2) in many specimens, any loss could result in a negative qPCR. Specimens with embryonated eggs have an abundance of Toxocara DNA, often with very high calculated genomic equivalents, while non-larvated cells have only a single copy of DNA. Thus, PCR may better be able to identify samples with larvated cells present. Sample Bx15 was outside the dynamic range of the standards, and this is likely because several eggs were in the larvated stage and would have many copies of the target sequence, giving an exaggerated calculated egg count (Fig 3). Sample Bk8 was negative by microscopy, but positive by qPCR with significant 40-Ct value (13.958), showing that microscopy is subjective and can miss visualizing eggs. Prior literature studies [46, 47] suggest that these eggs can adhere to plastic and may have been lost during the rinsing of the coverslip and slide when the eggs were transferred into the Eppendorf tube in preparation for freezing. Other limitations are samples were collected in different seasons, with changes in environmental temperatures influencing embryonated eggs. These temperature changes could have influenced the larvated egg results found throughout the boroughs. Geography can also influence the prevalence, burden, and stages of embryonated eggs in the environment. Staten Island and Manhattan are not contiguous with the mainland and can have decreased migrating dogs or cats, therefore impacting the results.

The distribution of Toxocara contaminated parks was not homogenous across the city but was more prevalent in areas of lower socioeconomic status. Of the five boroughs, the Bronx has the lowest median income but the highest level of soil contamination with the highest number of infectious, larvated parasites. Based on embryonated/larvated eggs, the risk of Toxocariasis from soil ingestion was highest in the Bronx. It is unknown whether these areas have more pets or stray cats, but given the association with the lower-income, it is likely that the higher contamination rate and the higher infectious potential may be related to the ability to pay for regular veterinary check-ups and deworming of pets. Animals that frequent contaminated areas are likely to become re-infected even after deworming if neighboring animals have not been dewormed. Brooklyn, Queens, and Staten Island have higher median incomes with 30–38% geo-contamination. Manhattan has the highest median income but the lower percentage of contamination at 29.6%, but still indicating that over one-quarter of Manhattan sites tested have Toxocara present. Because there was variability in the soil weights between playgrounds (100 gm to 150 gm), there is a potential for sampling bias, since increased grams of soil can mean more eggs available to be seen by microscopy or detected by qPCR. This discrepancy may have increased the number of eggs in the Bronx (Fig 5). The overall high level of parasites throughout all boroughs may be a result of the ‘pet boom’; the number of household U.S. cats and dogs has more than doubled in the past four decades, contributing to an increasing problem of stray cats in poorer neighborhoods. [48]

A disparity of higher Toxocara environmental contamination distribution in poorer neighborhoods is present and likely translates into an actual health disparity, but this remains to be evaluated as there are other ways that Toxocara can be transmitted to humans. Recent definitive data on the seroprevalence of Toxocara infection in children and adults living in those areas are lacking. However, results from a 1987 study did find higher seroprevalence in children and adolescents from poorer neighborhoods in NYC. [15] Furthermore, both Toxocara NHANES studies suggest minority individuals and those with lower socioeconomic status are at greater risk of Toxocariasis. [11, 21] Therefore, the prudent approach, given the potentially serious health consequences of Toxocara infection, especially for young children and adolescents, is to assume that transmission to humans will occur and to make every effort to reduce environmental contamination by Toxocara as much as possible and as soon as possible.

Beyond its importance as a human health disparity, Toxocara soil contamination is a One Health issue; it impacts the cleanliness of the environment and may impact the health of domestic pets and wildlife. Infective eggs represent an environmental risk and potential health hazard to children with pica. Other paratenic hosts of this parasite include rodents, birds, or rabbits, which, when infected, continue to contribute to the lifecycle of the parasite. Finally, other larvated parasites were seen by microscopy but unable to be definitively identified in these samples, posing a potential infectious risk to visiting animals or children.

## Conclusions

Toxocara species are common pet parasites that are found in the sand and soil of almost one-third of sample sites in NYC, especially in poorer neighborhoods. The predominant species in NYC appears to be T. cati. Preventive measures should be taken. These include improved fencing of play areas to prevent feral cat entry, deworming of domestic pets according to national veterinarian guidelines, better control of stray cats and dogs, picking up feces of pets, avoiding consumption of food that may have become contaminated, restriction of children with pica from play areas, and frequent handwashing after visiting play areas before ingestion of food or snacks.

## Acknowledgements

The authors thank all current and past collaborators for their contributions to Toxocara research and acknowledge the inability to site all peer-reviewed publications. DLT is grateful to those who have helped with advice and education in regard to this research, especially Susan Little DVM Ph.D. DACVM, Yonghua Li MD, William Borkowski MD, Mona Rigaud MD, Celia Holland BSc Ph.D., and Gloria Heresi MD. Special thanks to Dwight Bowman Ph.D. for providing Toxocara cani eggs for qPCR validation, and Anne Zajac DVM MS Ph.D. for providing feline samples infected with Toxocara cati eggs for qPCR validation.

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R Mejia, Y Vicuna, N Broncano, C Sandoval, M Vaca, M Chico, et al. A novel, multi-parallel, real-time polymerase chain reaction approach for eight gastrointestinal parasites provides improved diagnostic capabilities to resource-limited at-risk populations. The American journal of tropical medicine and hygiene. 2013;88(6):, pp.1041–7. , doi: 10.4269/ajtmh.12-0726

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JF Durant, LM Irenge, R Fogt-Wyrwas, C Dumont, JP Doucet, B Mignon, et al. Duplex quantitative real-time PCR assay for the detection and discrimination of the eggs of Toxocara canis and Toxocara cati (Nematoda, Ascaridoidea) in soil and fecal samples. Parasites & vectors. 2012;5:, pp.288, doi: 10.1186/1756-3305-5-288

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DM Deer, KA Lampel, N Gonzalez-Escalona. . A versatile internal control for use as DNA in real-time PCR and as RNA in real-time reverse transcription PCR assays. Lett Appl Microbiol. 2010;50(4):, pp.366–72. , doi: 10.1111/j.1472-765X.2010.02804.x .

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Hygiene NYCDoHaM. Epiquery: NYC Interactive Health Data System—[NYC Population Data 2010] 2010. Available from: https://a816-healthpsi.nyc.gov/epiquery/.

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Carney K, Morales, A,. Income by Zip Code, 2016 [cited 2016 Aug 10]. Available from: https://www.incomebyzipcode.com.

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PA Overgaauw, F van Knapen. . Veterinary and public health aspects of Toxocara spp. Veterinary parasitology. 2013;193(4):, pp.398–403. Epub 2013/01/12. , doi: 10.1016/j.vetpar.2012.12.035 .

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JL Jones, D Kruszon-Moran, K Won, M Wilson, PM Schantz. . Toxoplasma gondii and Toxocara spp. co-infection. The American journal of tropical medicine and hygiene. 2008;78(1):, pp.35–9. .

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Little SE. Toxocara cati (Proceedings). In: Care UA, editor. CVC in Washington, DC Proceedings; April 1, 2010; Washington DC: DVM360.com; 2010. p. Toxocara cati (Proceedings) CVC in Washington, D.C. Proceedings.

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A Lucio-Forster, JS Mizhquiri Barbecho, HO Mohammed, BG Kornreich, DD Bowman. . Comparison of the prevalence of Toxocara egg shedding by pet cats and dogs in the U.S.A., 2011–2014. Veterinary Parasitology: Regional Studies and Reports. 2016;5:, pp.1–13. , doi: 10.1016/j.vprsr.2016.08.002.

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M Vanhee, A-C Dalemans, J Viaene, L Depuydt, E Claerebout. . Toxocara in sandpits of public playgrounds and kindergartens in Flanders (Belgium). Veterinary Parasitology: Regional Studies and Reports. 2015;1–2:, pp.51–4. , doi: 10.1016/j.vprsr.2016.03.002.

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D Otero, AM Alho, R Nijsse, J Roelfsema, P Overgaauw, L Madeira de Carvalho. . Environmental contamination with Toxocara spp. eggs in public parks and playground sandpits of Greater Lisbon, Portugal. Journal of infection and public health. 2018;11(1):, pp.94–8. , doi: 10.1016/j.jiph.2017.05.002 .

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A Kleine, A Springer, C Strube. . Seasonal variation in the prevalence of Toxocara eggs on children's playgrounds in the city of Hanover, Germany. Parasites & vectors. 2017;10(1):, pp.248, doi: 10.1186/s13071-017-2193-6

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A Kleine, E Janecek, P Waindok, C Strube. . Flotation and adherence characteristics of Toxocara canis and T. cati and a reliable method for recovering Toxocara eggs from soil. Veterinary parasitology. 2016;227:, pp.35–41. , doi: 10.1016/j.vetpar.2016.07.023 .

48

Andrew N. Rowan Ph.D. THSotUS. Animal Sheltering Trends in the U.S. A historical lesson from—and for—U.S. animal shelters. 2017 Jan 21, 2009.

12 Nov 2019

Dear Dr Mejia,

Thank you very much for submitting your manuscript "Toxocara species environmental contamination in public spaces in New York City" (#PNTD-D-19-01601) for review by PLOS Neglected Tropical Diseases. Your manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important problem, but raised some substantial concerns about the manuscript as it currently stands. These issues must be addressed before we would be willing to consider a revised version of your study. We cannot, of course, promise publication at that time.

We therefore ask you to modify the manuscript according to the review recommendations before we can consider your manuscript for acceptance. Your revisions should address the specific points made by each reviewer.

(1) A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript.

(2) Two versions of the manuscript: one with either highlights or tracked changes denoting where the text has been changed (uploaded as a "Revised Article with Changes Highlighted" file); the other a clean version (uploaded as the article file).

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Please provide a short caption, including credits, uploaded as a separate "Other" file. If your image is from someone other than yourself, please ensure that the artist has read and agreed to the terms and conditions of the Creative Commons Attribution License at http://journals.plos.org/plosntds/s/content-license (NOTE: we cannot publish copyrighted images).

(4) If applicable, we encourage you to add a list of accession numbers/ID numbers for genes and proteins mentioned in the text (these should be listed as a paragraph at the end of the manuscript). You can supply accession numbers for any database, so long as the database is publicly accessible and stable. Examples include LocusLink and SwissProt.

(5) To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see http://journals.plos.org/plosntds/s/submission-guidelines#loc-methods

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While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

We hope to receive your revised manuscript by Jan 11 2020 11:59PM. If you anticipate any delay in its return, we ask that you let us know the expected resubmission date by replying to this email.

To submit a revision, go to https://www.editorialmanager.com/pntd/ and log in as an Author. You will see a menu item call Submission Needing Revision. You will find your submission record there.

Sincerely,

Celia Holland

Guest Editor

PLOS Neglected Tropical Diseases

Christine Budke

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: See general comment to the Authors

Reviewer #2: Details must be provided of sampling methodology (e.g. when during the year, etc) and sampling strategy (type of sample [random, convenience, etc]; sampling frame, etc). As far as I can see the sampling was not representative so may be subject to bias – this should be discussed. The authors say it is a ‘geo-surveillance’ study but provide no information on whether the sample is representative or geographic risk. Some degree of precision needs to be provided for prevalence estimates (i.e. 95% CI) and information should be provided for sample size and justification for sample size. Saying the Bronx has a 67% prevalence of positive samples is difficult to interpret without some measure of precision.

Viability testing can be done by egg incubation which is probably a better approach than microscope alone. This requires some discussion as to how useful is microscopy alone.

Reviewer #3: (No Response)

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: See general comment to the Authors

Reviewer #2: A useful addition to this manuscript would be to use the NHANES Toxocara seropostivity data (if available for these NYC areas which the authors refer to in the text) to map geographic risk and associate this with the findings of this environmental survey. This would thus link environmental contamination risk with actual human exposure. Surely risk of positive samples by area could be modelled statistically? Such modelling could control for factors such as socioeconomic level, etc.

Table 1 should provide more information showing number of samples and number of locations, proportions of samples positive from each location (parks vs. playgrounds, by microscopy, PCR, or either), whether any sample was positive at each location, etc.

Figure 3 indicates that qPCR correlates perfectly with egg count. Is this true?

Figure 5 should show medians rather than mean. One outlier for the Bronx biases the mean.

Reviewer #3: (No Response)

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

Reviewer #1: See general comment to the Authors

Reviewer #2: Limitations need to be discussed more extensively (see comments above)

Reviewer #3: (No Response)

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: See general comment to the Authors

Reviewer #2: The introduction could be cut significantly without affecting the message of the manuscript.

Reviewer #3: (No Response)

--------------------

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: The ms PNTD-D-19-01601 describes a study aiming at evaluating the environmental contamination of New York City with the eggs of the zoonotic nematodes Toxocara spp. The study has been carried out properly and it is well described, although I suggest some refinements to the Authors before considering it fully suitable for publication.

The concept that “infective Toxocara eggs are more predominant in poor socio-economical settings” is redundant throughout the ms and explained and discussed too much. I suggest the authors to give such an explanation of the higher presence in the Bronx rather than in the other boroughs once, in a detailed way, without reiterating it various times. The Introduction should also be reworded accordingly, as the ingestion of infective Toxocara eggs by a child poses relevant sanitary issues regardless the family income or the socioeconomic setting of the borough. A child may put in his/her mouth contaminated fingers in Central Park as also in the Bronx. Than, if some areas are more contaminated than others, this increases the risk but the sanitary importance and consequences are the same. Thus, the Study has the primary aim to evaluate the presence of contamination by Toxocara eggs in NYC and then (secondary objective) to compare its level in the five boroughs (see end of the Author summary).

Examples of repetitions: In the last three lines of the abstract this is reported twice, half of Page 3, first line of Page 4

I suggest the Authors to avoid wording like “we” “our”, etc, and rather use an impersonal style.

The full name of the parasites (Toxocara canis and Toxocara cati) should be reported only the first time the nematodes are cited, or at the begin of a sentence. All the other times , they should read T. canis or T. cati (abbreviating the genus). Please amend the text accordingly.

Author summary

First line: add “respectively” after “cats”

Sentence from “Since” to “York City”: I would say that the need of such research relies on the sanitary impact of Toxocara for both humans and animals, rather than because humans are not definitive hosts of the parasite

Introduction

Half of page 3: ….questions still exist surrounding these enigmatic parasites (and not this enigmatic parasite): Toxocara canis and Toxocara cati are two distinct species.

Last three lines: Toxocara should be in italics (as also at the end of Page 9, and in other parts of the text, please check).

Methods and Results

The Methods section should contain details of the areas that have been sampled and the samples that have been examined. How the areas within the boroughs have been selected and the samples have been collected. How many samples per each single area. These details should not be present in the Results section, that instead should include only details on the positive samples.

Delete “(The dog form of Toxocara)” at Page 8

First two lines of Page 9: cats are source of contamination, not vectors (this term has another meaning in veterinary parasitology). Also, dogs may carry T. cati egg after ingestion of cat faeces, thus the eggs are transported passively in dogs’ intestine and eliminated via the faeces. It is a passive transportation, not an infection as it seems by the sentence. Please reword for clarity.

Discussion

Last paragraph: Rodents, birds, rabbit are paratenic hosts, not accidental. It would also be interesting to add at least a brief mention on what other “larvated parasites” were found in the samples

Reviewer #2: A study potentially of interest to readers of PLoS NTDs providing up-to-date data on environmental contamination with Toxocara spp in NYC. There are significant weaknesses with respect to the information provided on sampling strategy and the data presented.

Reviewer #3: The aim of the study by Tyungu and co-workers was to give an overview about the contamination rate with eggs of the zoonotic agent Toxocara spp. in soil of playgrounds in New York City, wherefore soil samples from 91 different playgrounds allocated in all five boroughs of NYC were collected. The prevalence varied between different boroughs. The authors draw the conclusion that the income and therefore socioeconomic conditions might be a valuable explanation for the discrepancies between the boroughs. Furthermore, the authors discriminated between Toxocara canis and T. cati by qPCR, leading to the assumption, that playgrounds are predominately contaminated with eggs of T. cati.

Overall, the manuscript deals with an interesting topic and provides a comprehensive insight into the contamination rate in New York City with Toxocara eggs. However, I have general concerns about the manuscript in its present form (e.g., data reliability if 100 g vs. 150 g soil was used – see comment below). Methods and results are partially superficial and further information on the study design, e.g. seasonal distribution of sampling, on the statistical analyses and molecular analyses is necessary before it may be recommended for publication. Please consider my comments and recommendations below to improve the manuscript.

Introduction:

The Introduction comprises important aspects for the study, but sometimes a clear structure is missing. Rearranging the chapter with a clear structure will definitively make it easier to follow and will help to enhance the understanding why the study is important.

“Stool” is the term in human medicine, for animals the term “feces” is used in veterinary medicine.

It is now generally accepted that the third-stage larva (L3), not the second stage, is the infective stage (see Brunaska et al. 1995: Toxocara canis: ultrastructural aspects of larval moulting in the maturing eggs. Int J Parasitol. 25:683–90.)

Regarding reference 15: There are further, more recent, large-scale studies on the association of Toxocara seropositivity and neurocognitive deficits, which could be mentioned here like reference 5, or: Erickson, L. D., Gale, S. D., Berrett, A., Brown, B. L., Hedges, D. W., 2015. Association between toxocariasis and cognitive function in young to middle-aged adults. Folia Parasitol. (Praha) 62:048.

The general migration route of Toxocara larvae in paratenic hosts should be mentioned as the accumulation in different organs leads to different clinical aspects of the disease.

I am not sure if the last sentence of the Introduction really reflects the study objectives. Contamination rates of public places give only an overview about a potential risk of infection and cannot automatically be extrapolated to the health disparities between different communities.

Methods:

How was the sampling frequency at the sampling sites? Just once or at different time points during the study period?

Analysed samples weighed 100-150 g. This is a huge variation (plus 50% in case of 150 g vs. 100g) and may have influenced the chance of egg recovery and egg recovery rates, possibly leading to biased results. This needs at least to be mentioned in the Discussion. (e.g., were the Bronx samples those with the highest sample weights or were the 6 qPCR positive samples those with the highest weights?)

What was the concentration of Tween20 used for washing? What were the ratios of soil sample and flotation/washing solution?

Please give “g” instead of rpm throughout - rpm is not a useful unit because force varies with the radius of the centrifuge. Using g allows other researchers to replicate experiments.

Please use degree Celsius instead of Fahrenheit to be consistent with the rest of the manuscript.

Please give the sequence of the reverse primer(s).

How was the DNA for the standard curve isolated from Toxocara eggs? With the same procedure used for the samples? And what egg numbers were used to construct the standard curve?

Statistical analyses: It is unclear what the difference between the two tests (Toxocara eggs by zip code and Toxocara eggs by individual boroughs) is, and why both a Kruskal-Wallis test and an ANOVA was conducted. The ANOVA does not compare prevalence, as egg count data was used (same as in the Kruskal-Wallis test?), please rephrase (prevalence would be presence/absence data). Were post-hoc tests conducted to clarify which boroughs differed significantly?

Please note that if sample sites were sampled more than once, this repeated sampling would need to be accounted for in the statistical analyses.

An alternative statistical approach could be Generalized Linear (Mixed) Modeling, including the predictors borough, sampling month (for reasoning see below), and sample site as a random factor (in case of repeated sampling).

Results:

The Results section needs more detail, as several aspects remain unclear.

Did the 91 sampling sites result in 91 samples or were these sites tested multiples times? And how was the overall prevalence for NYC?

It would be easier to follow if additionally the total numbers for each sampling area are given, e.g. 8/27 (29.6%) for Manhattan.

Can the authors provide further data on the contamination intensity of the soil, like shown in Figure 5? Results of statistical analyses should also be mentioned, including which test produced which P-value. Which boroughs differed significantly – Bronx compared to all other ones?

Which other parasite stages were detected besides Toxocara eggs? – there is a brief hint in the Discussion, but it is of interest which parasites were detected as they might have zoonotic potential as well.

How was the success rate of the qPCR? Where the qPCR positives the samples with the highest egg counts in microscopy (please give counted egg numbers for the qPCR positive samples).

What is the meaning of the correlation to the 40-Cycle threshold? In the methods it is only mentioned that a sample was considered positive if there was detectable DNA at or before a cycle threshold of 38. Did additional samples become positive at Ct 40? What is the lower limit of quantification by the conducted qPCR extrapolated from the standard curve (this is an important information to draw conclusions on the possible co-existence of T. canis eggs in the qPCR positive samples or might explain failure to identify most of the samples and to detect T. canis at all)? Did the calculated egg count correspond to the egg count determined by microscopy?

It is widely described that there are seasonal variations in the occurrence of Toxocara eggs in soil. Therefore, a breakdown how the egg burden may have varied between months would be an interesting additional information (if not possible for each borough, than for the whole city). This may provide an explanation why larvated eggs could only be detected in the Bronx. As known, embryonation accelerates with ascending temperatures and humidity. So, maybe the samples in the Bronx were collected after warmer periods? This is briefly mentioned in the Discussion, but without details on when each borough was sampled it remains unclear how much this has impacted the results. Multivariate analyses (see above) would allow to tease these effects apart.

Furthermore, the correlation between median income and percent positive places could also be tested statistically.

Discussion:

The limitations and the bias of the conducted qPCR, which was successful in six samples only, are well and plausible discussed. In all 6 samples T. cati DNA was detected (can co-contamination with T. canis be excluded – again, what was the sensitivity of the qPCR?). Even though T. cati is described to be the predominant species contaminating playgrounds in other cities, a samples size of 6 is too low to draw a conclusion for the whole city (consider also e.g. sensitivity of the qPCR; furthermore, you cannot exclude that all other positive samples contained T. canis eggs – be more cautious with your conclusions).

How can the contamination rates be classified compared to other metropoles or cities with similar climatic conditions? There are several publications available e.g. Fakhri et al., 2018: Toxocara eggs in public places worldwide - A systematic review and meta-analysis. Environmental pollution, 242:1467-1475.

The human infection risk has been associated with a certain number of eggs per grams soil (e.g. Woodruff et al. 1981: Toxocara ova in soil in the Mosul District, Iraq, and their relevance to public health measures in the Middle East. Ann Trop Med Parasitol. 75:555-7). This is a relevant point when stating that infection risk was highest in the Bronx. Authors should give numbers of detected eggs in their samples in the manuscript, e.g. by including a table.

Cleaning of play areas is mentioned - please include cleaning frequencies in public places in NYC in the MS.

Figures/Table:

Figure 1: The provided pictures of Toxocara eggs are not well focused, please provide better pictures if possible.

Figure 2: As PLOS NTD is an international journal for scientists all over the world, please use the metric system.

Figure 4: The income per zip code is not a series of consecutive measurements, therefore the points should not be connected via a line.

Figure 5: It would be nice to illustrate which boroughs differed significantly – Bronx compared to all other ones?

Table 1: Please write 30.8 instead of 30.77 to be consistent with the other numbers. Are the +++ a semiquantitative measurement or just an estimation by the authors? This should be mentioned.

Write species names such as Toxocara in italics throughout (check the text, figures and references).

The manuscript should be formatted according to the journals guidelines and checked for typos.

--------------------

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

5 Mar 2020

23 Mar 2020

Dear Dr Mejia,

Thank you very much for submitting your manuscript "Neglected infection of poverty: Toxocara species environmental contamination in public spaces in New York City" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Celia Holland

Guest Editor

PLOS Neglected Tropical Diseases

Christine Budke

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #3: (No Response)

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #3: (No Response)

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

Reviewer #3: (No Response)

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #3: (No Response)

--------------------

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #3: I am comfortable with the changes made in response to my previous comments.

However, I do not agree with the title change in the revised manuscript. Leaving aside that none of the reviewers has asked for this change, human Toxocara infections are not only related to poverty, but more stringent to risk groups. For example, farmers, or veterinarians show significantly higher seropositivy than other professional groups. High prevalences in tropical countries are not only related to poverty, but also the climate, allowing eggs to become infective very fast and, additionally, enable survival in moist climates. Even though I agree that poverty (i.e. poor hygiene, but not necessarily low income in well developed countries) is one of various risk factors for humans to become infected, the study conducted by the authors is not related to the infection of poverty per se, it just shows higher egg contamination rates of sandpits in a borough with comparatively low income. If people in the Bronx are more frequently infected than in other boroughs cannot be answered or extrapolated from contaminated sandpits only.

--------------------

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Reviewer #3: No

Figure Files:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

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Reproducibility:

To enhance the reproducibility of your results, PLOS recommends that you deposit laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see http://journals.plos.org/plosntds/s/submission-guidelines#loc-materials-and-methods

23 Mar 2020

24 Mar 2020

Dear Dr Mejia,

We are pleased to inform you that your manuscript 'Toxocara species environmental contamination in public spaces in New York City' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

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Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Celia Holland

Guest Editor

PLOS Neglected Tropical Diseases

Christine Budke

Deputy Editor

PLOS Neglected Tropical Diseases

***********************************************************

1 May 2020

Dear Dr Mejia,

We are delighted to inform you that your manuscript, "Toxocara species environmental contamination in public spaces in New York City," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

We have now passed your article onto the PLOS Production Department who will complete the rest of the publication process. All authors will receive a confirmation email upon publication.

The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles (Editorial, Viewpoint, Symposium, Review, etc...) are generated on a different schedule and may not be made available as quickly.

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Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Serap Aksoy

Editor-in-Chief

PLOS Neglected Tropical Diseases