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. 2021 Jun 21;15(6):e0009510. doi: 10.1371/journal.pntd.0009510

Prevalence and correlates of Helicobacter pylori infection among under-five children, adolescent and non-pregnant women in Nepal: Further analysis of Nepal national micronutrient status survey 2016

Suresh Mehata 1,*, Kedar Raj Parajuli 2, Narayan Dutt Pant 3, Binod Rayamajhee 4,5, Uday Narayan Yadav 6,7,8,9, Ranju Kumari Mehta 10, Priya Jha 11, Neha Mehta 12, Meghnath Dhimal 13, Dipendra Raman Singh 14
Editor: Jeffrey H Withey15
PMCID: PMC8263064  PMID: 34153049

Abstract

Most of the Helicobacter pylori infections occur in developing countries. The risk factors for H. pylori infections are poverty, overcrowding, and unhygienic conditions, which are common problems in under-privileged countries such as Nepal. Despite having a high risk of H. pylori infections, no national level study has been conducted to assess prevalence and correlates of H. pylori infection in Nepal. Therefore, we hypothesized that micronutrients such as iron, vitamin B12 deficiency, socio-economic status, and nutritional status correlate with the prevalence of H. pylori infection in Nepal.

We studied prevalence and correlates of H. pylori infection among under-five children, adolescents aged 10–19 years and married non-pregnant women aged 20–49 years using data from the Nepal National Micronutrient Status Survey 2016 (NNMSS-2016). H. pylori infection was examined in stool of 6–59 months old children and 20–49 years old non-pregnant women whereas the rapid diagnostic kit using blood sample was used among adolescent boys and girls.

Prevalence of H. pylori infection was 18.2% among 6–59 months old children, 14% among adolescent boys and 16% among adolescent girls aged 10–19 years; and 40% among 20–49 years non-pregnant women. Poor socioeconomic status, crowding, and unhygienic condition were found to be positively associated with higher incidence of H. pylori infections. No significant correlation was observed between nutritional and micronutrients status (iron or risk of folate deficiency) with H. pylori infection.

Findings from this study suggest that poverty-associated markers are primary contributors of H. pylori infections in Nepalese communities. To control acquisition and persistence of H. pylori infection in Nepal, we suggest improved management of safe drinking water and implementation of sanitation and hygiene programs, with a focus on those of lower socioeconomic status.

Author summary

Helicobacter pylori is associated with a wide spectrum of gastrointestinal diseases and is a common problem in tropical region where inter-human contact is the primary mode of disease transmission. Poor socio-economic status is a crucial fueling factor of H. pylori infection. In this study, the authors present data from Nepal national micronutrient status survey 2016 (NNMSS-2016) and investigated the risk factors associated with H. pylori infections among under-five years old children, 10–19 years old adolescent boys and girls, and 20–49 years non-pregnant women nationwide. Study findings corroborate that poverty-associated markers are the key driving factor of H. pylori infections in Nepal, which can have a manifold effect on nutrition and subsequent child growth retardation. Management of safe drinking water and implementation of sanitation and hygiene practices to decrease acquisition of H. pylori infection is a pressing need in rural parts of Nepal and among marginalized communities. The findings of this study highlight the varied prevalence of H. pylori by age group, gender, place of residence, ethnic group, and ecological regions of the country. Moreover, no significant correlation was observed between nutritional and micronutrients status with H. pylori infection.

Introduction

Helicobacter pylori, a gram-negative spiral-shaped bacterium previously known as Campylobacter pyloridis, was first isolated and identified by Warren and Marshall in 1982[1]. It colonizes the gastric milieu of more than half of the global population and is associated with gastric diseases such as peptic ulcer, chronic gastritis, gastric cancer, and mucosa associated lymphoid tissue (MALT) lymphoma[2, 3]. In addition, H. pylori has also been associated with some non-gastrointestinal diseases such as pre-eclampsia, autoimmune thyroid diseases, acute coronary diseases, myocardial infarction, hepatic encephalopathy, prostatitis, and psoriasis[4–7]. H. pylori is responsible for iron deficiency anemia, thrombocytopenia, fetal malformation, and fetal growth retardation in pregnant women. Epidemiological studies suggest that anemia due to iron deficiency is correlated with H. pylori infection. Gastric acidity caused by H. pylori can impair iron and vitamin B12 absorption and leads to anemia[8]. Further, among infants and children, the infection has been associated with chronic diarrhea and malnutrition[9, 10]. However, H. pylori infection associated as a protective factor for the diseases such as asthma, osteoporosis, inflammatory bowel disease, and esophageal cancer[4, 11, 12].

H. pylori is ubiquitous and the infection caused by the bacterium is a substantial global health problem, affecting 50% of the global population[2]. With infection rates of 50% of people in developed countries and 80% of people in developing countries, H. pylori infection is more concentrated in resource-limited countries[2]. In a meta-analysis, the overall global prevalence of H. pylori infection was 44.3%, 50.8% in developing countries and 34.7% in developed countries[13]. There was no significant difference between the H. pylori infection prevalence between males and females (46.3% versus 42.7%)[13]. Most studies have demonstrated an equal rate of H. pylori infection between males and females; however, one study reported male gender as a significant risk factor[14]. The prevalence varies by geographical area, race and ethnicity, while the prevalence among children ranges from 10% to >80%[11]. Reports have shown the prevalence of infection is generally higher in developing countries due to socio-economic status, poverty; and unhygienic and overcrowded conditions[1, 13, 15–18].

H. pylori infection is often acquired during childhood and later the risk of infection rapidly declines[19]. For example, in developing countries, the infection is acquired during age below five years in 70–90% cases and generally remains asymptomatic leading to long term clinical sequelae such as gastritis, peptic ulcer and stomach cancer, then the risk of infection declines rapidly in later age[2, 20, 21]. In developed countries, the prevalence of infection in children is lower, and the percentage of infected people increases with age, accounting for up to 60% of cases[20, 22, 23]. However, most of the infections in developed countries are also acquired during childhood[24]. Despite high socio-economic status and hygienic conditions in developed countries, certain racial and ethnic groups (Blacks and Hispanics in the USA) have a higher rate of H. pylori associated infections[24]. This may be attributed to genetic predispositions to infection, which are yet to be fully understood[24].

The prevalence of H. pylori related infections in Nepal varies from 16.3% to 70.5%[25] and the infection is highly associated with malnutrition, and diarrheal diseases, notably among children. In a hospital based cross-sectional study reported from Nepal, patients who live in crowded urban area with lesser frequency of foods, were found to be highly susceptible to acquire the H. pylori infection[26]. Therefore, we hypothesized that micronutrients such as iron, vitamin B12 deficiency, socio-economic status, and nutritional status correlates with the prevalence of H. pylori infection in Nepal.

The aim of this study was to assess prevalence and correlates (socio-demographic, nutritional status, and micronutrient status) of H. pylori infection in Nepal among under five children, 10–19 years old adolescents and 20–49 years old non-pregnant women, using nationally representative data.

Methods

Ethics statement

Ethical approval for this study was obtained from the the Ethical Review Board (ERB) of the Nepal Health Research Council (NHRC) (Reg. No.: 201/2015). Well informed written consent was ascertained from all study participants before they were included in the study. Parental consent was obtained for the participants of <18 years. Adolescents of below 18 years were asked to participate in the study at the time of survey in community, eligible and interested adolescents written parents’ consent was ascertained during the study.

Data sources

Data for this study was taken from the Nepal National Micronutrient Status Survey 2016 (NNMSS-2016), a national level survey on basic health and demographic indicators[27]. The detailed methodology used has been presented in the same report[27]. In brief, the survey used stratified multi-stage cluster sampling. The five development regions (Eastern, Central, Western, Mid-western, and Far western) and three ecological zones (Terai, Hill, and Mountain) were used as the basis of stratifications; a random sampling approach across the 15 strata was used in order to represent the nation. A total of 180 clusters or wards (30 clusters from the Mountain zone; 75 each from the Hill and Terai zones) were selected using probability proportionate to size, as the primary sampling units. Subsequently, systematic random sampling was used to select 24 households from each cluster. Total 4,309 households were selected for interview and the response was 99.7%. Further, the response rate for 1,709 children aged 6–59 months included in the study was 98.9%. Likewise, 1,025 and 1,865, 10–19 years old adolescent boys and girls were interviewed, respectively, with a response rate of 98% each. A total of 2,144 non-pregnant women aged 20–49 years were also interviewed with a response rate of 99%.

Sample collection

Blood collected at the time of interview, and stool samples collected later that day or the following morning were used for H. pylori detection. From, 20–49 years old non-pregnant women and 6–59 months old children 11ml of venous blood was collected by the trained phlebotomists, while 6 ml was collected from 10–19 years old adolescent boys and girls. For children butterfly needles were used to collect venous blood. Blood samples were collected in three vacutainers (two 3ml purple top with EDTA and one 5ml blue top) following the standard precautions[27]. Some parameters such as, anemia (using a HemoCue Hb 301 analyzer) and RBC folate deficiency was assessed in the field by a phlebotomist using red blood cell folate microbiological assay[28].

Anthropometric measurements

Anthropometric measurements were collected to study the nutritional status of the children, adolescents, and adult women. A standard height/length-measuring board (Short-Board) was used to measure recumbent length of <2 years old children and standing height of >2 years old children, adolescent boys and girls, and adult women. For weight measurement, an electronic SECA digital scale (UNICEF Electronic Scale/Uni scale) was used.

Height-for-age (HAZ), weight-for-age (WAZ), and weight-for-length/height (WHZ) z scores were calculated using the WHO standards. The z-score <-1.96SD for stunting, underweight, and wasting were classified as HAZ, WAZ, and WHZ, respectively.

Variables

H. pylori infection

Among children and non-pregnant women H. pylori infections were detected by testing antigen in stool samples[29], while among adolescents 10–19 years, the QuickVue Rapid Test Kit (RTK) (Quidel Corporation, San Diego, CA 92121, USA) for examination of H. pylori specific antibody (IgG) in whole blood of adolescent boys and girls was used. The QuickVue H. pylori RTK detects IgG antibodies specific to H. pylori produced by individuals colonized or infected with the organism. Approximately, 50μL (2 hanging drops) of whole blood was added to the test cassette from the Purple Top Vacutainer. Results were recorded as positive, negative or invalid after 5 minutes of samples load and assays were performed following the manufacturer’s instructions. The reported sensitivity and specificity of QuickVue kit with biopsy was 90% and 78% respectively[30]. QuickVue has not been previously neither used nor validated for Nepalese population thus, we did not establish any cut off value for the sensitivity and specificity of QuickVue in this study. The validation of QuickVue in the local population in future studies is anticipated. Fresh stool samples collected were stored in a cold box of <10°C and transferred to the pathologist. Then the pathologist segregated 1 gram of stool to a second sterile cryovial which was later transferred to Siddhi Polyclinic Laboratory (one of the most advanced molecular laboratory of Nepal) in Kathmandu to test for H. pylori antigen using cold chain box. All the frozen stool samples were stored in Walter Reed/AFRIMS Research Unit Nepal (WARUN) until laboratory tests were performed for the investigation of H. pylori antigens. H. pylori antigen detection was performed using an enzyme-linked immunoassay (ELISA) test kit on a Mago clinical analyzer (Erba Lachema, Czech Republic). Positive and negative controls were used in each analytical test; and the absorbance for positive control was at least 0.8 OD units while that for negative control was less than 0.09 OD units. The reported sensitivity and specificity were 96% and 83%, respectively[31].

External quality control of the laboratory analysis

All laboratories that analyzed this study samples participated in CDC external quality assurance (EQA) programs. The EQA program also includes QA for ferritin, RBC folate, etc. The precision for the measurement of ferritin was >90–95% with <0.5% bias. The precision and bias for folate measurement was >90% and <4.0% respectively.

Ferritin

Ferritin, a blood protein, is a WHO recommended iron status indicator in human body, and low serum ferritin level indicates iron deficiency (WHO, 2001). To assess iron status, serum ferritin level was measured in venous blood samples collected from study participants and the geometric mean ferritin was calculated.

Data analysis

All analyses were performed using the software Stata 15 (StataCorp LLC, Texas, USA). Adjusted prevalence ratio (APR) was calculated using multiple Poisson regression, with all covariates (age, gender, education, ecological zone, place of residence, wealth status, caste/ethnicity, nutritional status, serum ferritin, risk of folate deficiency and BMI) included simultaneously in the model considering the cluster sampling design. The wealth status was calculated based on household assets recommended by Demographic and Health Survey such as: type of toilets, cooking fuels, source of water and energy, type of housing, number of living rooms, ownership of household assets, etc. Principal component analysis was used to calculate the wealth index and further wealth quintile was categorized using ranked technique to translate it to the ordinal scale. P<0.05 was considered to be statistically significant.

Results

Prevalence and correlates of H. pylori infection among children aged 6–59 months

Overall, 18.2% of children aged 6–59 months were infected with H. pylori. Study findings revealed a higher prevalence of H. pylori infection among participants aged 4–5 years (26%), a lower prevalence observed among participants aged below one year (9%), and that the prevalence of H. pylori increases increased with age. By ethnicity, the highest prevalence was observed among Muslims (31%) followed by Dalits (25%), whereas the lowest prevalence was observed among Brahmins/Chhetris (15%). Similarly, the highest prevalence was observed among those who resides in urban areas compared to those in rural areas (25% versus 19%, respectively). Moreover, the highest prevalence of H. pylori was observed among poor households (23%) compared to non-poor (17%) and adolescents who were at risk of folate deficiency (29.2%) (Table 1).

Table 1. Prevalence and correlates of H. pylori among preschool children (6 to 59 months) by background characteristics, nutritional, and micronutrient status.

Distribution (%) H. pylori result of 6–59 months children Correlates of H. pylori Total (N)
Positive (%) Negative (%) aPR 95%CI P
Age group (years)
<1 9.0 8.9 91.1 1 144
1–2 21.3 11.0 89.0 1.16 0.55–2.41 0.698 309
2–3 21.9 22.2 77.8 2.42 1.18–4.95 0.016 348
3–4 24.0 22.7 77.3 2.52 1.30–4.89 0.006 362
4–5 23.8 26.2 73.8 2.97 1.48–5.93 0.002 360
Gender
Male 54.7 20.0 80.0 1 778
Female 45.3 19.4 80.6 0.96 0.74–1.23 0.725 745
Caste/ethnicity
Brahmin/Chhetri 31.0 14.6 85.4 1 542
Terai-Madhesi Other Castes 14.1 19.5 80.5 1.37 0.72–2.58 0.338 116
Dalits 18.3 25.1 74.9 1.63 1.08–2.47 0.019 316
Janajatis 32.6 20.2 79.8 1.18 0.80–1.73 0.401 489
Muslims 3.9 31.0 68.0 2.18 1.24–3.84 0.007 48
Ecological Region
Hill 42.9 18.1 81.9 1 644
Mountain 8.0 23.3 76.7 1.04 0.68–1.59 0.855 250
Terai 49.1 20.5 79.5 1.44 0.99–2.11 0.058 629
Place of residence
Urban 12.4 24.7 75.3 1 202
Rural 87.6 19.0 81 0.67 0.44–1.03 0.065 1321
Province
Province 1 14.6 25.3 74.7 1 231
Province 2 23.2 16.9 83.1 0.47 0.21–1.06 0.067 174
Bagmati Province 20.4 21.4 78.6 1.05 0.62–1.76 0.860 206
Gandaki Province 8.9 12.5 87.5 0.61 0.28–1.33 0.218 135
Lumbini Province 16.3 19.9 80.1 0.72 0.42–1.22 0.221 287
Karnali Province 6.0 24.7 75.3 1.05 0.59–1.88 0.858 137
Sudoorpachim Province 10.6 17.9 82.1 0.70 0.42–1.18 0.182 353
Wealth status
Poorest 21.0 22.5 77.5 1 422
Poorest 20.0 24.3 75.7 1.06 0.76–1.47 0.732 321
Middle 19.4 18.1 81.9 0.81 0.55–1.18 0.272 266
Richer 20.2 14.4 85.6 0.55 0.35–0.87 0.011 279
Richest 19.4 19.0 81.0 0.71 0.41–1.23 0.221 235
Stunting (HAZ)
No 63.7 18.8 81.2 1 943
Yes 36.3 20.8 79.2 1.06 0.76–1.49 0.732 578
Underweight (WAZ)
No 69.9 20.1 79.9 1 1062
Yes 30.1 18.2 81.8 0.73 0.49–1.10 0.138 459
Wasting (WHZ)
No 88.0 19.8 80.2 1 1353
Yes 12.0 17.0 83.0 1.02 0.59–1.76 0.931 164
Iron deficiencya
No 77.1 20.4 79.6 1 1166
Yes 22.9 17.8 82.3 1.06 0.77–1.46 0.739 317
Risk of folate deficiencyb
    No 94.2 19.3 80.7 1 1401
    Yes 5.8 29.2 70.8 1.32 0.96–1.81 0.091 75
Total 100 19.6 80.4 1523
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Abbreviations: HAZ: Height-for-Age Z scores; WAZ Weight-for-Age Z scores; WHZ: Weight-for-Height Z scores

aBiomarker was regression-adjusted to a pooled country reference to adjust for inflammation, using CRP and AGP (ferritin) or AGP only. Iron deficiency defined as inflammation-adjusted serum ferritin <12 μg/L[32].

bFolate cutoff based on the risk of megaloblastic anemia defined as RBC folate <305.0 nmol/L[32].

Multivariate Poisson regression analysis was used to ascertain the prevalence ratio of H. pylori infections among children aged 6–59 months. In the adjusted model, the higher prevalence of H. pylori infection was observed in Muslims (aPR2.15;95%CI:1.20–3.84) and Dalits (aPR 1.63;95% CI:1.13–2.36) as compared to Brahmins/Chhettris. Compared to poorest quintile, the lower prevalence of H. pylori infection was observed among the richer quintile participants (aPR 0.55; 95%CI:0.35–0.87).

Younger children had a lower likelihood of H. pylori infection; the prevalence ratio of H. pylori infection was higher among preschool children aged over four years (aPR 2.97; 95%CI: 1.48–5.93), 3–4 years (aPR: 2.52; 95%CI: 1.30–4.89) and for those between 2–3 years (aPR: 2.42; 95%CI:1.18–4.89), compared to those aged less than one year (Table 1).

No significant difference in prevalence ratio was observed by nutritional status (stunting, wasting and underweight), iron and folic acid deficiency status of children with H. pylori infection (Table 1).

Prevalence and correlates of H. pylori infection among adolescent boys aged 10–19 years

Overall, 14% of adolescent boys and 16% of adolescent girls were infected with H. pylori in Nepal. The prevalence was highest among adolescents who had a higher level of education (boys 28% and girls 30%) and those aged 15–19 years (boys 18% and girls 20%). Also, the highest prevalence was observed among adolescent boys who resides in Sudoorpachim Province (20%) and in urban areas (19%), followed by those in Terai-Madhesi ethnic groups and those who reside in the mountains (17% each). Additionally, among adolescent girls, the highest prevalence was observed among Muslims (25%) followed by those who reside in Sudoorpachim Province (21%) and in the Terai (17%) (Tables 2 and 3).

Table 2. Prevalence and correlates of H. pylori among adolescent boys by background characteristics, nutritional status, and micronutrient status.

Distribution
(%)		 H. pylori results Correlates of H. pylori Total (N)
Positive (%) Negative (%) aPR 95%CI P
Age group (years)
10–14 58.3 10.5 89.5 1 599
15–19 41.7 17.8 82.2 1.30 0.88–1.93 0.192 424
Education
Never attended school/ Primary 32.9 9.1 90.9 1 327
Secondary 61.9 14.8 85.2 1.65 0.97–2.80 0.064 647
Higher 5.2 27.8 72.2 3.13 1.22–8.00 0.017 49
Caste/ethnicity
Brahmin/Chhetri 35.7 15.2 84.5 1 434
Terai-Madhesi Other Castes 13.1 17.4 82.6 1.15 0.56–2.38 0.702 70
Dalits 14.4 10.4 89.6 0.62 0.36–1.07 0.087 159
Janajatis 33.9 11.9 88.1 0.94 0.61–1.45 0.787 338
Muslims 2.9 12.2 87.8 0.83 0.27–2.59 0.750 22
Ecological Region
Hill 41.8 11.4 88.6 1 434
Mountain 6.9 17.3 82.7 1.23 0.67–2.27 0.503 157
Terai 51.3 14.8 85.2 1.65 0.97–2.81 0.064 432
Place of residence
Urban 13.8 18.6 81.4 1 142
Rural 86.2 12.8 87.2 0.59 0.36–0.96 0.034 881
Province
Province 1 16.4 11.7 88.3 1 179
Province 2 22.7 13.3 86.7 0.85 0.43–1.65 0.625 106
Bagmati Province 17.0 13.0 87.0 1.10 0.50–2.43 0.813 132
Gandaki Province 9.7 6.1 93.9 0.64 0.26–1.55 0.320 102
Lumbini Province 18.8 15.9 84.1 1.17 0.65–2.08 0.603 205
Karnali Province 4.4 19.8 80.2 1.55 0.74–3.24 0.248 87
Sudoorpachim Province 11.0 18.0 82.0 1.31 0.70–2.49 0.400 212
Wealth status
Poorest 18.6 16.4 83.6 1 251
Poorer 20.3 10.9 89.1 0.62 0.34–1.13 0.117 211
Middle 22.2 12.4 87.6 0.51 0.28–0.92 0.025 209
Richer 17.5 14.5 85.5 0.60 0.32–1.14 0.121 165
Richest 21.5 14.1 85.9 0.53 0.26–1.08 0.079 187
Stunting (HAZ)
No 66.6 12.2 87.8 1 636
Yes 33.4 15.5 84.5 1.37 0.96–1.95 0.086 343
BMI (Wt/ht2)
<18.5 66.4 12.3 87.7 1 684
18.5–24.9 31.3 15.1 84.9 0.96 0.62–1.50 0.869 323
≥25.0 2.3 28.4 71.6 1.89 0.68–5.23 0.220 16
Iron deficiency a
No 95.2 13.8 86.2 1 975
Yes 4.8 11.0 89.0 1.02 0.31–3.31 0.976 37
Total 100 14.0 86.0 1023
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Abbreviations: HAZ: Height-for-Age Z scores; BMI: Body Mass Index

aBiomarker was regression-adjusted to a pooled country reference to adjust for inflammation, using CRP and AGP (ferritin) or AGP only. Iron deficiency defined as inflammation-adjusted serum ferritin <15 μg/L[32].

bFolate cutoff based on the risk of megaloblastic anaemia defined as RBC folate <305.0 nmol/L[32].

Table 3. Prevalence and correlates of H. pylori among adolescent girls by background characteristics, nutritional status, and micronutrient status.

Distribution (%) H. pylori results Correlates of H. pylori Total (N)
Positive (%) Negative (%) aPR 95%CI P
Age group (years)
10–14 55.3 12.5 87.5 1 994
15–19 44.7 20.1 79.9 1.49 1.08–2.07 0.016 817
Education
Never attended school/ Primary 34.5 14.9 85.1 1 587
Secondary 61.6 15.6 84.4 0.99 0.73–1.36 0.973 1149
Higher 3.9 29.6 70.4 1.53 0.78–3.03 0.220 74
Caste/ethnicity
Brahmin/Chhetri 33.2 13.8 86.2 1 694
Terai-Madhesi Other Castes 11.4 14.1 85.9 1.06 0.56–2.01 0.863 119
Dalits 16.4 22.8 77.2 1.45 0.92–2.27 0.107 314
Janajatis 36.7 14.8 85.2 1.09 0.77–1.56 0.623 648
Muslims 2.3 25.1 74.9 1.75 1.03–2.97 0.039 36
Ecological Region
Hill 44.1 15.3 84.7 1 767
Mountain 7.5 12.8 87.2 0.73 0.49–1.08 0.118 284
Terai 48.4 17.0 83.0 1.33 0.89–1.99 0.168 760
Place of residence
Urban 9.8 15.4 84.6 1 207
Rural 90.2 16.0 84.0 0.88 0.53–1.47 0.635 1604
Province
Province 1 16.4 15.4 84.6 1 291
Province 2 21.1 15.2 84.8 0.85 0.45–1.59 0.602 196
Bagmati Province 17.1 13.8 86.2 1.02 0.60–1.73 0.937 205
Gandaki Province 10.4 9.0 91.0 0.71 0.43–1.17 0.181 184
Lumbini Province 18.2 18.4 81.6 1.01 0.62–1.67 0.954 365
Karnali Province 5.4 21.8 78.2 1.36 0.80–2.33 0.257 164
Sudoorpachim Province 11.5 20.6 79.4 1.39 0.85–2.27 0.190 406
Wealth status
Poorest 23.0 19.2 80.8 1 486
Poorer 22.2 14.4 85.6 0.83 0.58–1.18 0.309 417
Middle 20.1 15.5 84.5 0.82 0.53–1.26 0.359 332
Richer 18.5 16.1 83.9 0.81 0.47–1.39 0.445 310
Richest 16.2 13.6 86.4 0.62 0.36–1.04 0.071 266
Stunting (HAZ)
No 65.5 14.3 85.7 1 1097
Yes 34.5 18.2 81.8 1.20 0.93–1.54 0.163 594
BMI (Wt/ht2)
<18.5 58.5 14.0 86.0 1 1038
18.5–24.9 39.2 18.5 81.5 1.12 0.82–1.53 0.485 730
≥25.0 2.3 21.3 78.7 1.54 0.73–3.25 0.255 40
Iron deficiency a
No 82.2 15.3 84.7 1 1487
Yes 17.8 18.9 81.1 1.20 0.89–1.53 0.227 322
Risk of folate deficiencyb
    Yes 16.2 14.3 85.7 1 239
    No 83.8 16.3 83.7 0.74 0.53–1.02 0.066 1481
Total 100 15.9 84.1 1811
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Abbreviations: HAZ: Height-for-Age; BMIZ: BMI-Body Mass Index

aBiomarker was regression-adjusted to a pooled country reference to adjust for inflammation, using CRP and AGP (ferritin) or AGP only. Iron deficiency defined as inflammation-adjusted serum ferritin <15 μg/L[32].

bFolate cutoff based on the risk of megaloblastic anaemia defined as RBC folate <305.0 nmol/L[32].

Tables 2 and 3 present adjusted prevalence ratios of H.pylori infection among adolescent boys and girls in Nepal by background characteristics. In the adjusted model, among adolescent boys, compared to those who never attended school or had some primary level education, a higher prevalence was observed among those who had higher-level education (aPR: 3.13; 95%CI: 1.22–8.00). Compared to the urban dwellers, a lower prevalence was observed among those who reside in rural areas (aPR: 0.59; 95% CI: 0.36–0.96). No statistically significant differences in prevalence were observed by age group, caste/ethnicity, ecological regions, provinces, height for age, BMI, or iron deficiency status.

Among adolescent girls, a higher prevalence of H. pylori infection was observed among adolescents aged 15–19 years (aPR: 1.49; 95%CI: 1.08–2.07) compared to those aged 10–14 years. By caste/ethnicity, a higher prevalence was observed among Muslims (aPR: 1.75; 95% CI:1.03–2.97) compared to Brahmins/Chhetris. No statistically significant differences in prevalence were observed by ecological region, province, wealth status, height for age, BMI, iron deficiency status, or the risk of folate deficiency (Table 3).

Prevalence and correlates of H. pylori infection among non-pregnant women aged 20–49 years

Overall, 40% of women of reproductive age had H. pylori infection. The study also revealed that the highest prevalence was observed among Muslims (61%), those who reside in Karnali Province (48%) and those who had never attended school or had some primary education (44%).

Table 4 shows the adjusted prevalence ratio of H. pylori among non-pregnant women in Nepal by socio-demographic status. In the adjusted model, a higher prevalence of H. pylori infection was observed among women belonging to ethnic groups: Indigenous/Janajatis (aPR: 1.25; 95%CI: 1.05–1.48) and Muslims (aPR: 1.56; 95%CI: 1.06–2.29) compared to Brahmins/Chhetris. No statistically significant differences in prevalence were observed by age groups, education, ecological regions, place of residence, wealth status, height for age, BMI, the iron deficiency status, and the risk of folate deficiencies.

Table 4. Prevalence and correlates of H. pylori among Non-Pregnant Women by background characteristics, nutritional status, and micronutrient status.

Distribution (%) H. pylori Correlates of H. pylori Total (N)
Positive (%) Negative (%) aPR 95%CI P
Age group (Years)
20–24 23.3 38.6 61.4 1 395
25–34 40.0 40.8 59.2 1.04 0.85–1.27 0.732 702
≥35 36.7 41.1 58.9 1.03 0.83–1.27 0.804 640
Education
Never attended school/primary 50.8 44.1 55.9 1 932
Secondary 33.8 37.7 62.3 0.86 0.70–1.06 0.162 544
Higher 15.4 34.3 65.7 0.80 0.61–1.05 0.103 243
Caste/ethnicity
Brahmin/Chhetri 38.2 35.3 64.7 1 708
Terai Madhesi Other Castes 8.5 37.3 62.7 1.16 0.84–1.64 0.366 90
Dalits 14.5 43.7 56.3 1.21 0.94–1.55 0.149 270
Janajatis 36.9 44.1 55.9 1.25 1.05–1.48 0.013 637
Muslims 1.8 61.1 38.9 1.56 1.06–2.29 0.023 30
Ecological Regions
Hill 42.7 38.1 61.9 1 746
Mountain 6.5 41.6 58.4 1.00 0.79–1.25 0.986 295
Terai 50.8 42.4 57.6 1.18 0.93–1.48 0.167 696
Place of residence
Urban 13.8 42.1 57.9 1 248
Rural 86.2 40.1 59.9 0.90 0.74–1.09 0.300 1,489
Province
Province 1 17.2 47.7 52.3 1 294
Province 2 19.5 34.3 65.7 0.63 0.42–0.97 0.036 171
Bagmati Province 22.4 37.7 62.3 0.85 0.64–1.14 0.273 247
Gandaki Province 10.8 31.1 68.9 0.73 0.47–1.13 0.160 211
Lumbini Province 16.2 42.2 57.8 0.84 0.60–1.13 0.240 321
Karnali Province 5.0 48.1 51.9 1.18 0.86–1.62 0.300 157
Sudoorpachim Province 9.0 47.9 52.1 1.02 0.78–1.32 0.911 336
Wealth status
Poorest 14.9 44.8 55.2 1 376
Poorer 18.8 39.7 60.3 0.97 0.79–1.18 0.741 371
Middle 19.6 38.2 61.8 0.93 0.72–1.21 0.604 331
Richer 20.1 39.0 61.0 1.00 0.75–1.34 0.965 321
Richest 26.6 39.5 60.5 1.07 0.78–1.48 0.674 338
Height
≥145 cm 89.8 40.4 59.6 1 1,560
<145 cm 10.2 41.1 58.9 0.98 0.78–1.23 0.861 174
BMI (Wt/ht2)
<18.5 12.8 40.8 59.2 1 232
18.5–24.9 61.4 41.2 58.8 1.00 0.82–1.22 0.986 1,093
≥25.0 25.8 38.5 61.5 0.93 0.73–1.17 0.516 409
Iron deficiencya
No 81.7 41.4 58.6 1439
Yes 18.3 33.5 66.5 0.90 0.74–1.09 0.280 291
Risk of folate deficiencyb
    No 90.2 40.1 59.9 1 1,453
    Yes 9.8 42.5 57.5 1.04 0.82–1.30 0.756 191
Total 100 40.40 59.60 1,737
Open in a new tab

Abbreviations: BMI: Body Mass Index

aBiomarker was regression-adjusted to a pooled country reference to adjust for inflammation, using CRP and AGP (ferritin) or AGP only. Iron deficiency defined as inflammation-adjusted serum ferritin <15 μg/L[32].

bFolate cutoff based on the risk of megaloblastic anemia defined as RBC folate <305.0 nmol/L[32].

Discussion

To our knowledge, this is the first study to include samples from all over Nepal including various age groups to gauge the prevalence of H. pylori colonization or infection among different age population of Nepal. In this study, we included children of <5 years, adolescents aged 10–19 years, and non-pregnant women aged 20–49 years. There are earlier studies on the prevalence of H. pylori infection in Nepal, though mostly in hospital settings with small sample size and not generalizable to the national population level[25, 26, 33].

Overall, 18% of pre-school children aged 6–59 months were infected with H. pylori with a higher prevalence of H. pylori infection among participants aged 4–5 years (26%). In a study from Vietnam, 39% of children up to 6 years were infected with H. pylori, but as in our study, they also reported a higher prevalence of infection with increased age[34]. A higher rate of H. pylori infection among children than that observed in our study was also reported in Brazil (70%)[35]. Moreover, the prevalence of H. pylori infection is lower in developed countries compared to developing countries[36, 37]. In this study, the prevalence of H. pylori infection was significantly correlated with socio-demographic factors such as ethnicity, place of residence, and wealth status of the study population. A higher rate of infection was found among children belonging to poor socioeconomic status those living in crowded spaces with poor hygienic conditions. In developing countries, the correlation between H. pylori infection and socioeconomic status is inconsistent. Some authors[38–40] have reported inverse correlations, a few have noted no relationship[41, 42] and one[43] has reported a significant correlation. The most common mode of transmission of H. pylori infection are the faeco-oral route and through contaminated water[14]. Additionally, cases of gastro-oral H. pylori transmission have been reported[44]. These modes of infection will have a higher likelihood of occurrence among children of poor socioeconomic status, living in a crowded area and in unhygienic conditions. We postulate that women and children of lower socioeconomic status may have higher exposure to polluted water, such as that of ponds, hand-pumps, in the Terai region, which could explain the high prevalence of H. pylori infection among women and children of this area compared to other ecological regions in Nepal. A study by Bainganawt et al. (2014) supports the idea that the use of water from sources such as rivers and lakes increase the odds of H. pylori infection in pregnant women. Therefore, it is worth mentioning that improving water, sanitation, and hygiene practices may decrease the prevalence of H. pylori among children and women in Nepal.

Gender is considered as a potential risk factor of H. pylori infection[45]; thus, adolescent participants were segregated as boys and girls to compare the H. pylori incidence based on the gender. Among the adolescent population, 14% of boys and 16% of girls were positive for the anti-H. pylori IgG in rapid antibody tests performed by QuickVue kit. The prevalence was highest among adolescents who had a higher level of education (boys, 28%, and girls, 30%) and those aged 15–19 years (boys, 18%, and girls, 20%); highly educated people are more likely to be aware of hygiene practices and are therefore expected to have a lower infection rate of H. pylori. In the Nepalese context, adolescents aged 15–19 years who need to stay away from home in rented accommodation for their high school education may be exposed to external sources of H. pylori, including contaminated food from fast food retailers or street vendors with poor sanitation practices. Another reason could be the low socio-economic status of adolescents and their families which may force them to live in crowded and unhealthy environments where they have a higher likelihood of contact with H. pylori. Therefore, it is pertinent to consider socio-economic status and living/residential environments, lifestyle, and health behavior of an individual while designing interventions to control the H. pylori infections among adolescents.

Overall, 40% of non-pregnant women aged 20-49 years had an H. pylori infection, similar to results reported in Uganda (45.2%)[42]. The high rate of H. pylori infection was found among women belonging to marginalized communities, particularly among Dalits, Indigenous groups, and Muslims indicating that H. pylori poses a significant health concern among these groups in Nepal. This suggests that marginalized communities in the study population had poor access to clean food and clean water, unhygienic living conditions, and were of lower socioeconomic status. In the present study, we did not find an association between low birth weight and H. pylori infection, as demonstrated by a previous study reported in Uganda[46]. In this study, the prevalence of the infection varied according to the geographical location from 18% to 61%[42]. However, higher rates (56% to 74%) were reported from a U.S.–Mexico border population, and the prevalence again varied according to locality[47]. As in our study, Baingana et al. (2014) found an association between urban residence and education level and the rate of H. pylori infection among pregnant women[42]. The rate of H. pylori infection largely depends upon geographic area, age, race, and socioeconomic status[1].

In our study, we did not find any significant correlation between iron deficiency status and risk of folate deficiency with H. pylori infection across the age groups, which was in accordance with the findings of other studies[48, 49]. However, some studies have reported association of H. pylori infection with iron deficiency, such that reduced serum ferritin levels were measured in H. pylori infected cases[50–52]. In addition, there are reports of reversal of chronic iron deficiency anemia through eradication of H. pylori infection, without any additional anemia targeted treatments[53]. Iron being essential factor for H. pylori growth, this bacterium may compete with host for iron uptake creating iron deficiency in hosts[54]. However, from analysis of different available literature, it can be concluded that there is no universal agreement on correlation of H. pylori infection with iron deficiency. Nonetheless, the discrepancy seen in the results from different studies may also be due to different sample size or different age group of patients used in different studies, as the correlation may be age specific.

Strengths and limitations

This study is the largest nationwide epidemiological study to provide a comprehensive understanding of H. Pylori prevalence and correlated potential risk factors in Nepal. The study used systematic random sampling to estimate the prevalence, which enabled the recruitment of participants from diverse demographic backgrounds. This study has a few limitations. Because this study is cross-sectional, causality cannot be inferred from the findings. We were unable to account for several confounding factors, such as household sanitation status, family history and dietary habits, and thus residual confounding could not be eliminated. Future research should account for these factors. Furthermore, serology may create bias in differentiating past and present infection as antibody titers may remain high even after treatment. H. pylori positive rate was 26.2% in children at the age of 4–5 years, and 38.6% in non-pregnant women at the age of 20–24 years. However, the positive rates in adolescent boys and girls were very low at 10.5%-20.1%. While the H. pylori infection was evaluated by H. pylori antigen in the stool in children under five and non-pregnant women, it was evaluated only by rapid test kit in adolescent boys and girls. The differences in positive rate is probably due to the inadequate sensitivity and specificity of the both the test kits, the reported positive and negative predictive value of rapid test kit were 81% and 88%, respectively[31]. The positive and negative predictive values of ELISA test kit were 96% and 91%, respectively[32]. Hence, it is difficult to compare the data of adolescent boys and girls with the data obtained from subjects of other ages. Moreover, this study did not evaluate the health status of participants, such as chronic inflammatory diseases, can lead to decreased serum ferritin independent of H. pylori infection, and may have impacted the results. Also, this study did not account for prior treatment with antibiotics or proton pump inhibitors. This study also did not assess the correlation of infection rate with symptoms and disease process. This study did not include adult men and no persons older than 49 years.

Conclusion

In conclusion, the results of this study affirmed a sizeable prevalence of H. pylori infection in Nepal and was varied as per gender, age group, ethnic group, place of residence, and ecological region. We also reinforce the need for water, sanitation, and hygiene programs focused on marginalized communities and people of lower socio-economic status, aiming to improve hygiene practices, living conditions, and lifestyle behaviors that could decrease acquisition of H. pylori infection at both the household and community levels. This study do not find any correlation between nutritional and micronutrients status (iron and risk of RBC folate deficiencies) with H. pylori infection. Findings of this study suggest poverty-associated markers are the major drivers of H. pylori related infections in Nepalese communities. This study demonstrates the need for a population-based national study in order to understand the dynamics of H. pylori infection and transmission in Nepal.

Acknowledgments

The authors would like to thank the team members from Ministry of Health and Population, New ERA, UNICEF, EU, USAID, and the CDC for conducting this research. We are very grateful to the Government of Nepal for providing the permission to use the data for analysis and publication of this work. Our sincere thank goes to Mr Todd Lewis for proof reading and editing the manuscript.

Data Availability

All relevant data are within the paper.

Funding Statement

The authors received no funding for this work.

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PLoS Negl Trop Dis. doi: 10.1371/journal.pntd.0009510.r001

Decision Letter 0

Jeffrey H Withey, Robert C Reiner

3 Nov 2020

Dear Dr Mehata,

Thank you very much for submitting your manuscript "Prevalence and determinants of Helicobacter pylori infection among under-five children, adolescent and non-pregnant women in Nepal: A further analysis of Nepal national micronutrients status survey 2016" for consideration at PLOS Neglected Tropical Diseases. I sincerely apologize for the long delay in returning a decision on this manuscript. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments. The reviewers were in agreement that many important details are missing from the current manuscript, as well as appropriate statistical analyses.

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When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed, point by point list of your responses to the review comments and a description of the changes you have made in the manuscript to address each concern. 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).

Important additional instructions are given below your reviewer comments.

Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. 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,

Jeffrey H Withey

Associate Editor

PLOS Neglected Tropical Diseases

Robert Reiner

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: The lack of specific hypotheses makes the study way to exploratory and unfocused, despite the substantial data available regarding the burden of H pylori and the role of age, sex and poverty-related determinants

What rapid test kit was used, brand, model, date, etc. What was its Se and Sp, and impacts on results? Same for the stool antigen. Given that this is the main outcome of the study, it needs to be described in detail in the methods. And the impact of the differences between the two tests on the conclusion needs to be discussed also. How many samples were tested per plate and with repetitions or tittering levels?

What explains higher prevalence in adults when virtually no increasing trend is seen in younger individuals?

Data sources, describe age of adolescents and pregnant women, instead of reporting them in the sample collection section.

Stata’s name is not “Software for Statistics and Data Science”, please provide the proper reference in standard format

Is age considered an effect modifier? What about the differences in tests? Prevalence in adolescents is substantially lower most likely due to the tests

Numerical covariates cannot just be adjusted as having a continuous linear relationship with the logodss of the H pylori prevalence without first verifying that this relationship is indeed linear.

Reviewer #2: - health status of the participants should be evaluated and clarified.

- H. pylori infection was evaluated only by rapid test kit in adolescent boys and girls.

- the definition of the wealth status needs to be clarified in the text.

- the definition of "risk of folate deficiency" needs to be clarified in the text.

Reviewer #3: The tests for H. pylori (stool antigen test (monoclonal?) and serum/whole blood antibody test) are not named. This is needed information as it can influence the reliability of the results. It is not stated at all here what was tested in blood (likely antibodies, something like “to assess H. pylori infection antibodies against the pathogen were detected in whole blood using XX Test from YY company etc”). It is also important to know how many freeze and thaw cycles have been done to the stool samples. This might have a strong impact on the antigen test. It should be also clear whether the producer of the antigen test allows the testing of frozen stool samples. The temperatures of storage should also be stated.

Line 145-150: the response rate seems extremely high. Was this addressed in the cited reference? How is it explained?

Line 189: it should read probably IgG/IgM? It would sound better IgG and IgM

Line 188-190: it comes the question why those aged 10-19 were tested by blood test and not stool antigen test and vice versa.

Line 198-190: “Chi-square test was used to measure the association between the exploratory and outcome variables” This sentence is not clear. Chi-square can detect differences between groups but not measure the association, the formulation of the sentence is for a regression analysis. What were outcome variables? If I understand right there was only 1 (being infected or not).

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

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: Why does table 1 lacks p-values or some test to compare groups? Also, given the sampling approach, bivariate results are probably not reasonable estimates. Bivariate tables can be integrated with regression tables, and avoid double description of the same results. There is too much redundancy in the results, the way the tables are presented

Too much emphasis on comparing boys with girls despite of not being a leading study hypothesis and no remarkable sex-related differences.

Cannot just say prevalence in differences parts of the paper due to using two very different tests

Reviewer #2: Results need to be reconsidered (see below).

Reviewer #3: Generally a lot of overly detailed results with minimal relevant information. Probably most tables could be provided as supplementary material. All results of an age group should be provided as one block. As there are no confidence intervals in the descriptive data tables it is very hard to see which differences are at all relevant.

Line 215-216: probably “highest” and “lowest” is meant here.

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

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?

-Is public health relevance addressed?

Reviewer #1: Findings confirm that poverty is the main factor associated with the disease, as know from the literature. What is the novelty?

In the discussion, comparison of results need to take into account what tests were used for each study in each country. Even within this study, the changes in prevalence by age suggests to be test-dependent

Reviewer #2: Conclusion is clear.

Reviewer #3: Some very important points are not addressed at all in the discussion. There were no adult men and no persons older than 49 years in study, important points as they limit the statements that can be made about the prevalence of H. pylori infection in Nepal.

Line 389: the sentence is formulated confusingly. In the other study children above 10 were highly infected? How high? This point should be addressed as the data of the study is also from Nepal and puts into question whether methodological problems may be the cause (e.g., how was H. pylori infection detected in the other study?, If it was stool antigen testing maybe it was from fresh samples?)

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

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: The English writing requires some minor adjustments and typographical mistakes

Somewhat lengthy introduction with an excessive emphasis on developed countries in the introduction, which are not relevant for comparison to the settings of Nepal. I suggest focusing on more comparable settings to the study population

Reviewer #2: Editorial modification is required (see below).

Reviewer #3: Line 91-92: what is meant here by marginally? Is it then not relevant?

Line 92-93: “H pylori is cause of gastrointestinal blood loss, reduction in iron absorption and increase in iron absorption by bacteria.” This sentence does not have a reference. It is also too general with stating H. pylori is a cause of GI blood loss. Is it a relevant cause?

Line 95-97: these are more associations between H. pylori and the diseases mentioned then causing a clear decrease.

Line 101-104: Probably here the study from Hooi et al (Global Prevalence of Helicobacter pylori Infection: Systematic Review and Meta-Analysis) is mentioned. It would be better to cite the original study (the meta-analysis) then papers citing it. It also looks strange to cite 2 papers (14 and 15) for the same data.

Line 110-112: the sentence is not clear? Bloating and vomiting are risk factors for H. pylori infection? In what time period, compared to whom? Most person have bloating or vomiting sometime in their lives. Also family history of gastric cancer is probably not a risk factor for H. pylori infection but rather an association with (most likely the risk factor for cancer is H. pylori infection.

Line 113-117: In the first sentence it is not clear what is meant by longer period. The second sentence is repeating the first (acquired at an early age is during childhood). In developed countries time of infection for most persons is childhood. The whole sentence is rather confusing and too long, it should read probably something like “…acquired during childhood after which the risk of infection rapidly declines…”.

Some sentences in the manuscript are very long and not formulated clearly. For example in line 81-85 it reads as if the lymphoma was colonizing the stomach: “Helicobacter pylori (H. pylori), a gram-negative and spiral-shaped bacterium previously known as Campylobacter pyloridis then C. pylori, was first isolated and identified by Warren and Marshall in 1982 (1), has been associated with gastric diseases such as peptic ulcer, chronic gastritis, gastric cancer, mucosa associated lymphoid tissue (MALT) lymphoma (2, 3) which colonizes in the gastric milieu of more than half of the global population.”

Italicization and abbreviations are not consistent in the text.

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

Summary and General Comments

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: This is an interesting effort, but unfocused and without clear hypotheses relevant to the global community in a topic where substantial evidence already exists. The manuscript needs a clear direction and a central topic

Reviewer #2: In this manuscript, Mehata et al investigated the prevalence of H. pylori infection among the Nepalese population. They found that poverty is associated with high prevalence of H. pylori infection.

1) In the Abstract section (l.39), mucosa associated lymphoid tissue lymphomas are indolent in nature and are rarely a cause of death, so this sentence needs to be corrected.

2) In the Background section, the authors wrote that gastric acidity caused by H. pylori leads to reduced vitamin B12 (l.91-92), but this is not true. Hypochlorhydria which occurs as a result of atrophic gastritis caused by H. pylori infection leads to reduced vitamin B12. This misleading sentence needs to be corrected.

3) In the Background section, the reviewer could not understand the sentence "reduction in iron absorption and increase in iron absorption by bacteria" (l.93). This sentence is confusing and needs to be edited.

4) The paragraph in the Background section from line 98 to123 has conflicting sentences within itself and is not so important for the present study. The authors should consider deleting or carefully re-editing this paragraph.

5) Although the authors hypothesized that supply of micronutrients determines the prevalence of H. pylori infection in Nepal (l.127-128), there is no sentence in the Results or Discussion verifying this hypothesis. Furthermore, the only micronutrient assessed in this study, other than iron, is folate (described as "risk of folate deficiency"), which was not associated with H. pylori infection. The authors should investigate other additional micronutrients, such as vitamin B12, selenium, etc., if they wish to prove their hypothesis. In addition, the definition of "risk of folate deficiency" needs to be clarified in the text.

6) Were all the participants healthy? The authors are using serum ferritin as the parameter of iron status. Some diseases, such as chronic inflammatory diseases, can lead to decreased serum ferritin independent of H. pylori infection, so the health status of the participants should be evaluated and clarified.

7) H. pylori positive rate was 26.2% in children at the age of 4-5, and 38.57% in non-pregnant women at the age of 20-24. However, the positive rates in adolescent boys and girls were very low at 10.5%-20.1%. While the H. pylori infection was evaluated by H. pylori antigen in the stool in children under five and non-pregnant women, it was evaluated only by rapid test kit in adolescent boys and girls. This decreased positive rate is probably due to the inadequate sensitivity of the rapid test kit. Hence, it is difficult to compare the data of adolescent boys and girls with the data obtained from subjects of other ages. The authors need to explain this limitation in the text.

8) How did the authors judge the wealth status of the participants? This needs to be clarified in the text.

9) There are a number of errors in the Discussion section (e.g. l.370 the authors describe that H. pylori infection was significantly associated with residence area in children under the age of 5, but none of the provinces or place of residence were significantly associated with H. pylori infection./ l.405 Dalits is not significantly associated with H. pylori infection./ etc.). The authors need to carefully check the manuscript and correct the errors.

10) The reference needs to be edited to match the style of this journal.

11) There are many grammatical errors in the text. These need to be corrected.

Reviewer #3: The study tries to estimate the prevalence of H. pylori infected individuals in Nepal using data and samples already available from an earlier population survey.

Line 124-130: if the prevalence of infection in Nepal is already known then it should be stated more clearly what this study adds to already available data (the study aim states “study was to assess prevalence and determinants of H. pylori”). Different study population, micronutrient data?

On the same line the sentence "Hence, we hypothesized that supply of micronutrients determines the prevalence of H. pylori

infection in Nepal." is unclear as then it is not stated how this hypothesis was then tested.

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

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.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

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.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

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 https://journals.plos.org/plosntds/s/submission-guidelines#loc-methods

PLoS Negl Trop Dis. doi: 10.1371/journal.pntd.0009510.r003

Decision Letter 1

Jeffrey H Withey, Robert C Reiner

5 Feb 2021

Dear Dr Mehata,

Thank you very much for submitting your manuscript "Prevalence and correlates of Helicobacter pylori infection among under-five children, adolescent and non-pregnant women in Nepal: Further analysis of Nepal national micronutrient status survey 2016" for consideration at PLOS Neglected Tropical Diseases.

There was no response to reviewers' comments included with this revision- only a response to editorial comments. This paper cannot be evaluated further until you respond to each of the reviewers comments directly and submit a revised manuscript with this point by point response. These reviewer comments were included in the original decision letter. I have copied them at the bottom of this letter as well.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

When you are ready to resubmit, please upload the following:

[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. 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).

Important additional instructions are given below your reviewer comments.

Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. 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,

Jeffrey H Withey

Associate Editor

PLOS Neglected Tropical Diseases

Robert Reiner

Deputy Editor

PLOS Neglected Tropical Diseases

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

There is no response to reviewers comments included with this revision- only a response to editorial comments. This paper cannot be evaluated further until you respond to each of the reviewers comments directly and submit a revised manuscript with this point by point response.

Reviewer #1: The lack of specific hypotheses makes the study way to exploratory and unfocused, despite the substantial data available regarding the burden of H pylori and the role of age, sex and poverty-related determinants

What rapid test kit was used, brand, model, date, etc. What was its Se and Sp, and impacts on results? Same for the stool antigen. Given that this is the main outcome of the study, it needs to be described in detail in the methods. And the impact of the differences between the two tests on the conclusion needs to be discussed also. How many samples were tested per plate and with repetitions or tittering levels?

What explains higher prevalence in adults when virtually no increasing trend is seen in younger individuals?

Data sources, describe age of adolescents and pregnant women, instead of reporting them in the sample collection section.

Stata’s name is not “Software for Statistics and Data Science”, please provide the proper reference in standard format

Is age considered an effect modifier? What about the differences in tests? Prevalence in adolescents is substantially lower most likely due to the tests

Numerical covariates cannot just be adjusted as having a continuous linear relationship with the logodss of the H pylori prevalence without first verifying that this relationship is indeed linear.

Reviewer #2: - health status of the participants should be evaluated and clarified.

- H. pylori infection was evaluated only by rapid test kit in adolescent boys and girls.

- the definition of the wealth status needs to be clarified in the text.

- the definition of "risk of folate deficiency" needs to be clarified in the text.

Reviewer #3: The tests for H. pylori (stool antigen test (monoclonal?) and serum/whole blood antibody test) are not named. This is needed information as it can influence the reliability of the results. It is not stated at all here what was tested in blood (likely antibodies, something like “to assess H. pylori infection antibodies against the pathogen were detected in whole blood using XX Test from YY company etc”). It is also important to know how many freeze and thaw cycles have been done to the stool samples. This might have a strong impact on the antigen test. It should be also clear whether the producer of the antigen test allows the testing of frozen stool samples. The temperatures of storage should also be stated.

Line 145-150: the response rate seems extremely high. Was this addressed in the cited reference? How is it explained?

Line 189: it should read probably IgG/IgM? It would sound better IgG and IgM

Line 188-190: it comes the question why those aged 10-19 were tested by blood test and not stool antigen test and vice versa.

Line 198-190: “Chi-square test was used to measure the association between the exploratory and outcome variables” This sentence is not clear. Chi-square can detect differences between groups but not measure the association, the formulation of the sentence is for a regression analysis. What were outcome variables? If I understand right there was only 1 (being infected or not).

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

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: Why does table 1 lacks p-values or some test to compare groups? Also, given the sampling approach, bivariate results are probably not reasonable estimates. Bivariate tables can be integrated with regression tables, and avoid double description of the same results. There is too much redundancy in the results, the way the tables are presented

Too much emphasis on comparing boys with girls despite of not being a leading study hypothesis and no remarkable sex-related differences.

Cannot just say prevalence in differences parts of the paper due to using two very different tests

Reviewer #2: Results need to be reconsidered (see below).

Reviewer #3: Generally a lot of overly detailed results with minimal relevant information. Probably most tables could be provided as supplementary material. All results of an age group should be provided as one block. As there are no confidence intervals in the descriptive data tables it is very hard to see which differences are at all relevant.

Line 215-216: probably “highest” and “lowest” is meant here.

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

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?

-Is public health relevance addressed?

Reviewer #1: Findings confirm that poverty is the main factor associated with the disease, as know from the literature. What is the novelty?

In the discussion, comparison of results need to take into account what tests were used for each study in each country. Even within this study, the changes in prevalence by age suggests to be test-dependent

Reviewer #2: Conclusion is clear.

Reviewer #3: Some very important points are not addressed at all in the discussion. There were no adult men and no persons older than 49 years in study, important points as they limit the statements that can be made about the prevalence of H. pylori infection in Nepal.

Line 389: the sentence is formulated confusingly. In the other study children above 10 were highly infected? How high? This point should be addressed as the data of the study is also from Nepal and puts into question whether methodological problems may be the cause (e.g., how was H. pylori infection detected in the other study?, If it was stool antigen testing maybe it was from fresh samples?)

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

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: The English writing requires some minor adjustments and typographical mistakes

Somewhat lengthy introduction with an excessive emphasis on developed countries in the introduction, which are not relevant for comparison to the settings of Nepal. I suggest focusing on more comparable settings to the study population

Reviewer #2: Editorial modification is required (see below).

Reviewer #3: Line 91-92: what is meant here by marginally? Is it then not relevant?

Line 92-93: “H pylori is cause of gastrointestinal blood loss, reduction in iron absorption and increase in iron absorption by bacteria.” This sentence does not have a reference. It is also too general with stating H. pylori is a cause of GI blood loss. Is it a relevant cause?

Line 95-97: these are more associations between H. pylori and the diseases mentioned then causing a clear decrease.

Line 101-104: Probably here the study from Hooi et al (Global Prevalence of Helicobacter pylori Infection: Systematic Review and Meta-Analysis) is mentioned. It would be better to cite the original study (the meta-analysis) then papers citing it. It also looks strange to cite 2 papers (14 and 15) for the same data.

Line 110-112: the sentence is not clear? Bloating and vomiting are risk factors for H. pylori infection? In what time period, compared to whom? Most person have bloating or vomiting sometime in their lives. Also family history of gastric cancer is probably not a risk factor for H. pylori infection but rather an association with (most likely the risk factor for cancer is H. pylori infection.

Line 113-117: In the first sentence it is not clear what is meant by longer period. The second sentence is repeating the first (acquired at an early age is during childhood). In developed countries time of infection for most persons is childhood. The whole sentence is rather confusing and too long, it should read probably something like “…acquired during childhood after which the risk of infection rapidly declines…”.

Some sentences in the manuscript are very long and not formulated clearly. For example in line 81-85 it reads as if the lymphoma was colonizing the stomach: “Helicobacter pylori (H. pylori), a gram-negative and spiral-shaped bacterium previously known as Campylobacter pyloridis then C. pylori, was first isolated and identified by Warren and Marshall in 1982 (1), has been associated with gastric diseases such as peptic ulcer, chronic gastritis, gastric cancer, mucosa associated lymphoid tissue (MALT) lymphoma (2, 3) which colonizes in the gastric milieu of more than half of the global population.”

Italicization and abbreviations are not consistent in the text.

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

Summary and General Comments

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: This is an interesting effort, but unfocused and without clear hypotheses relevant to the global community in a topic where substantial evidence already exists. The manuscript needs a clear direction and a central topic

Reviewer #2: In this manuscript, Mehata et al investigated the prevalence of H. pylori infection among the Nepalese population. They found that poverty is associated with high prevalence of H. pylori infection.

1) In the Abstract section (l.39), mucosa associated lymphoid tissue lymphomas are indolent in nature and are rarely a cause of death, so this sentence needs to be corrected.

2) In the Background section, the authors wrote that gastric acidity caused by H. pylori leads to reduced vitamin B12 (l.91-92), but this is not true. Hypochlorhydria which occurs as a result of atrophic gastritis caused by H. pylori infection leads to reduced vitamin B12. This misleading sentence needs to be corrected.

3) In the Background section, the reviewer could not understand the sentence "reduction in iron absorption and increase in iron absorption by bacteria" (l.93). This sentence is confusing and needs to be edited.

4) The paragraph in the Background section from line 98 to123 has conflicting sentences within itself and is not so important for the present study. The authors should consider deleting or carefully re-editing this paragraph.

5) Although the authors hypothesized that supply of micronutrients determines the prevalence of H. pylori infection in Nepal (l.127-128), there is no sentence in the Results or Discussion verifying this hypothesis. Furthermore, the only micronutrient assessed in this study, other than iron, is folate (described as "risk of folate deficiency"), which was not associated with H. pylori infection. The authors should investigate other additional micronutrients, such as vitamin B12, selenium, etc., if they wish to prove their hypothesis. In addition, the definition of "risk of folate deficiency" needs to be clarified in the text.

6) Were all the participants healthy? The authors are using serum ferritin as the parameter of iron status. Some diseases, such as chronic inflammatory diseases, can lead to decreased serum ferritin independent of H. pylori infection, so the health status of the participants should be evaluated and clarified.

7) H. pylori positive rate was 26.2% in children at the age of 4-5, and 38.57% in non-pregnant women at the age of 20-24. However, the positive rates in adolescent boys and girls were very low at 10.5%-20.1%. While the H. pylori infection was evaluated by H. pylori antigen in the stool in children under five and non-pregnant women, it was evaluated only by rapid test kit in adolescent boys and girls. This decreased positive rate is probably due to the inadequate sensitivity of the rapid test kit. Hence, it is difficult to compare the data of adolescent boys and girls with the data obtained from subjects of other ages. The authors need to explain this limitation in the text.

8) How did the authors judge the wealth status of the participants? This needs to be clarified in the text.

9) There are a number of errors in the Discussion section (e.g. l.370 the authors describe that H. pylori infection was significantly associated with residence area in children under the age of 5, but none of the provinces or place of residence were significantly associated with H. pylori infection./ l.405 Dalits is not significantly associated with H. pylori infection./ etc.). The authors need to carefully check the manuscript and correct the errors.

10) The reference needs to be edited to match the style of this journal.

11) There are many grammatical errors in the text. These need to be corrected.

Reviewer #3: The study tries to estimate the prevalence of H. pylori infected individuals in Nepal using data and samples already available from an earlier population survey.

Line 124-130: if the prevalence of infection in Nepal is already known then it should be stated more clearly what this study adds to already available data (the study aim states “study was to assess prevalence and determinants of H. pylori”). Different study population, micronutrient data?

On the same line the sentence "Hence, we hypothesized that supply of micronutrients determines the prevalence of H. pylori

infection in Nepal." is unclear as then it is not stated how this hypothesis was then tested.

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.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

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 https://journals.plos.org/plosntds/s/submission-guidelines#loc-methods

PLoS Negl Trop Dis. doi: 10.1371/journal.pntd.0009510.r005

Decision Letter 2

Jeffrey H Withey, Robert C Reiner

30 Mar 2021

Dear Dr Mehata,

Thank you very much for submitting your manuscript "Prevalence and correlates of Helicobacter pylori infection among under-five children, adolescent and non-pregnant women in Nepal: Further analysis of Nepal national micronutrient status survey 2016" 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.

One reviewer still has concerns with the first revisions of this manuscript and you must submit a detailed response to these concerns along with the second revision of the manuscript.

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.

When you are ready to resubmit, please upload the following:

[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).

Important additional instructions are given below your reviewer comments.

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,

Jeffrey H Withey

Associate Editor

PLOS Neglected Tropical Diseases

Robert Reiner

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: The hypothesis posed in the introduction are related to nutrition-related covariates, but also include socioeconomic status, preventing a focused analysis and manuscript. This reflects in the introduction, results and section, that try to address any potential factor associated to the outcome, without focus or set direction. Similarly, the micronutrient-related hypotheses end up receiving little attention and are lost in the manuscript, particularly in the central messages and conclusions in the abstract and discussion

Was serum ferritin deficiency analyzed as a potential factor associated to h. pylori, similar to folate deficiency, or only the association of the actual values of serum ferritin were associated to h pylori? A similar approach should be used for folate and serum ferritin, and if not, it should be justified.

The sensitivity and specificity of the QuickVue should be reported in the paper, and it’s low specificity addressed. Similarly, Se/Sp for ELISA assays used should be reported in the methods, to be able to understand how these characteristics impact the results in the different age groups.

Where underage adolescents asked for consent to participate?

Adjusting by province may result in over adjusting and take away the effect of many variables such as differences in nutritional deficits between groups.

Using PCA to determine a wealth index is an not sufficient description to address what that index implies and how can it be interpreted, as it does not provide an standardize well known measure but instead it results in a data-specific index. Additionally, dichotomization of a data-rich summary probably reduces excessively the diversity and variance of the wealth index. Multiple, well-defined categories will probably would be a better fit for this data, particularly because it is trying to capture the diversity within a whole country.

Analysis of numerical covariates assuming linear relationships can lead to model misspecification and the shape of the actual association should be assessed before deciding on a linear or other type of fit, making sure that the model specification responds to the data

No rationale is presented for analyzing boys separate from girls

P values are used to define significance, but the actual values are not presented and without them is hard to assess the significance level

Reviewer #2: (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: (No Response)

Reviewer #2: (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?

-Is public health relevance addressed?

Reviewer #1: The discussion continues to be unfocused, particularly without attention to the nutritional factors that were supposed to be the main hypothesis

No attention is given to the broader age differences and trends between the three study groups, the role of tests used and the difference that age implies to the epidemiology of h pylori, as age trends seem to be present in children and adolescents, but not in adults.

The limitations of the study are only listed and not really discussed or addressed as they may have important challenges to the conclusions and validity of the results

Reviewer #2: (No Response)

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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: English writing, only in the abstract: under five children, adolescent and … / We studied prevalence, / data from Nepal Micronutrient Survey / while blood sample was used /

No hypothesis listed in abstract.

Review comments are submitted with the understanding that the authors will respond to each comment by making changes in the actual manuscript to address each comment. Responses in the letter are useful, but actual changes in the manuscript are necessary, as readers will probably have similar concerns as those of reviewers.

Reviewer #2: (No Response)

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Summary and General Comments

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 changes made in this revised version of the manuscript do not feel that are properly addressing the concerns of this reviewer, as changes feel mainly superficial. A proper assessment of the role of nutritional deficiencies in H pylori prevalence would require more in-depth attention and focus

Reviewer #2: (No Response)

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PLoS Negl Trop Dis. doi: 10.1371/journal.pntd.0009510.r007

Decision Letter 3

Jeffrey H Withey, Robert C Reiner

27 May 2021

Dear Dr Mehata,

We are pleased to inform you that your manuscript 'Prevalence and correlates of Helicobacter pylori infection among under-five children, adolescent and non-pregnant women in Nepal: Further analysis of Nepal national micronutrient status survey 2016' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

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Robert Reiner

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

PLoS Negl Trop Dis. doi: 10.1371/journal.pntd.0009510.r008

Acceptance letter

Jeffrey H Withey, Robert C Reiner

16 Jun 2021

Dear Dr Mehata,

We are delighted to inform you that your manuscript, "Prevalence and correlates of Helicobacter pylori infection among under-five children, adolescent and non-pregnant women in Nepal: Further analysis of Nepal national micronutrient status survey 2016," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

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PLOS Neglected Tropical Diseases

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

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