PLoS Negl Trop Dis plos plosntds PLOS Neglected Tropical Diseases 1935-2735 Public Library of Science San Francisco, CA USA 10.1371/journal.pntd.0010017 PNTD-D-21-00419 Research Article Medicine and health sciencesMedical conditionsInfectious diseasesBacterial diseasesTyphoid Medicine and health sciencesClinical medicineSigns and symptomsFevers Research and analysis methodsImmunologic techniquesImmunoassaysEnzyme-linked immunoassays Biology and life sciencesPhysiologyImmune physiologyAntibodies Biology and life sciencesImmunologyImmune system proteinsAntibodies Medicine and health sciencesImmunologyImmune system proteinsAntibodies Biology and life sciencesBiochemistryProteinsImmune system proteinsAntibodies People and placesPopulation groupingsEthnicitiesAsian peopleLao people Medicine and health sciencesPathology and laboratory medicineSerology Medicine and health sciencesMedical conditionsInfectious diseasesBacterial diseasesSalmonellaSalmonella typhi Biology and life sciencesMicrobiologyMedical microbiologyMicrobial pathogensBacterial pathogensSalmonellaSalmonella typhi Medicine and health sciencesPathology and laboratory medicinePathogensMicrobial pathogensBacterial pathogensSalmonellaSalmonella typhi Biology and life sciencesOrganismsBacteriaEnterobacteriaceaeSalmonellaSalmonella typhi Medicine and health sciencesEpidemiology An age-stratified serosurvey against purified Salmonella enterica serovar Typhi antigens in the Lao People´s Democratic Republic Typhoid fever serology in Lao PDR Hefele Lisa Conceptualization Formal analysis Funding acquisition Investigation Methodology Project administration Writing – original draft 1 2 Black Antony P. Conceptualization Funding acquisition Resources Supervision Writing – review & editing 2 Van Tan Trinh Methodology Resources 3 https://orcid.org/0000-0002-1819-2296 Tri Minh Nguyen Methodology Resources 4 5 https://orcid.org/0000-0003-2437-3568 Duc Hoang Nguyen Methodology Resources Supervision 4 5 https://orcid.org/0000-0001-5049-0544 Virachith Siriphone Methodology Resources 2 Muller Claude P. Investigation Methodology Supervision 1 https://orcid.org/0000-0002-5001-2128 Hübschen Judith M. Resources Supervision 1 https://orcid.org/0000-0003-2389-6631 Russell Paula Investigation Resources 6 7 https://orcid.org/0000-0003-3380-4446 Bartholdson Scott Josefin Methodology Resources 6 7 Ngoc Minh Chau Nguyen Methodology Resources 3 https://orcid.org/0000-0003-3672-9867 Thieu Nga Tran Vu Methodology Resources 3 https://orcid.org/0000-0003-1308-5755 Baker Stephen Conceptualization Funding acquisition Project administration Supervision Writing – original draft Writing – review & editing 6 7 * Department of Infection and Immunity, Luxembourg Institute of Health, Esch-sur-Alzette, Grand-Duchy of Luxembourg Lao-Lux Laboratory, Institut Pasteur du Laos, Vientiane, Lao People’s Democratic Republic The Hospital for Tropical Diseases, Wellcome Trust Major Overseas Programme, Oxford University Clinical Research Unit, Ho Chi Minh City, Vietnam University of Natural Sciences, Ho Chi Minh City Vietnam Vietnam National University, Ho Chi Minh City, Vietnam Cambridge Institute of Therapeutic Immunology and Infectious Disease, University of Cambridge School of Clinical Medicine, Cambridge Biomedical Campus, Cambridge, United Kingdom Department of Medicine, University of Cambridge School of Clinical Medicine, Cambridge Biomedical Campus, Cambridge, United Kingdom Bourret Travis J. Editor Creighton University, UNITED STATES

The authors have declared that no competing interests exist.

 * E-mail: sgb47@medschl.cam.ac.uk 13 12 2021 12 2021 15 12 e0010017 24 3 2021 22 11 2021 2021 Hefele et al This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

The epidemiology of typhoid fever in Lao People`s Democratic Republic is poorly defined. Estimating the burden of typhoid fever in endemic countries is complex due to the cost and limitations of population-based surveillance; serological approaches may be a more cost-effective alternative. ELISAs were performed on 937 serum samples (317 children and 620 adults) from across Lao PDR to measure IgG antibody titers against Vi polysaccharide and the experimental protein antigens, CdtB and HlyE. We measured the significance of the differences between antibody titers in adults and children and fitted models to assess the relationship between age and antibody titers. The median IgG titres of both anti-HylE and CdtB were significantly higher in children compared to adults (anti-HylE; 351.7 ELISA Units (EU) vs 198.1 EU, respectively; p<0.0001 and anti-CdtB; 52.6 vs 12.9 EU; p<0.0001). Conversely, the median anti-Vi IgG titer was significantly higher in adults than children (11.3 vs 3.0 U/ml; p<0.0001). A non-linear trend line fitted to the anti-CdtB and anti-HlyE IgG data identified a peak in antibody concentration in children <5 years of age. We identified elevated titers of anti-HlyE and anti-CdtB IgG in the serum of children residing in Lao PDR in comparison to adults. These antigens are associated with seroconversion after typhoid fever and may be a superior measure of disease burden than anti-Vi IgG. This approach is scalable and may be developed to assess the burden of typhoid fever in countries where the disease may be endemic, and evidence is required for the introduction of typhoid vaccines.

 Author summary

Typhoid fever is a serious bloodstream infection caused by the bacterium Salmonella Typhi. Estimating the burden of typhoid fever is complex due to the limitations, cost, and scalability of current diagnostic surveillance methods. The detection of specific antibody responses against the organism may be a more sustainable manner of measuring exposure and disease burden in endemic location. We measured antibody (IgG) in 937 serum samples (317 children and 620 adults) from across the Lao People`s Democratic Republic against a polysaccharide (Vi) and two experimental protein antigens, CdtB and HlyE, that may more appropriate markers of disease exposure. We measured the significance of the differences between antibody titers in adults and children and fitted models to assess the relationship between age and antibody titers. The median IgG titres against HylE and CdtB were significantly higher in children than adults. Conversely, the median IgG titres against Vi was significantly higher in adults than children. We identified a significant association between a peak in IgG titres against CdtB and HlyE in children aged under 5 years. These data are indicative of high level of typhoid fever exposure in children under 5 years of age in Lao PDR and we surmise that IgG titres against HylE and CdtB may be a superior measure of typhoid disease burden than IgG titres against Vi. Our approach is scalable and can be further validated to assess the burden of typhoid fever in countries where the disease may be endemic, and evidence is required for the introduction of typhoid vaccines.

 Ministry of Foreign and European Affairs Luxembourg-Laos Partnership for Research and Capacity Building in Infectious Disease Surveillance II Black Antony P. Ministry of Foreign and European Affairs Luxembourg-Laos Partnership for Research and Capacity Building in Infectious Disease Surveillance II Hefele Lisa http://dx.doi.org/10.13039/100010269 Wellcome Trust 215515/Z/19/Z https://orcid.org/0000-0003-1308-5755 Baker Stephen This work was supported by the Ministry of Foreign and European Affairs, Luxembourg and the Luxembourg Institute of Health (project “Luxembourg-Laos Partnership for Research and Capacity Building in Infectious Disease Surveillance II”) to LH and APB and a Wellcome senior research fellowship to SB (215515/Z/19/Z). The funders had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication. PLOS Publication Stage vor-update-to-uncorrected-proof Publication Update 2021-12-23 Data Availability All relevant data are within the manuscript and its Supporting Information files. Introduction

Typhoid fever is a systemic disease caused by Salmonella enterica subspecies enterica serovar Typhi (S. Typhi), a bacterium transmitted via contaminated food or water. Globally, an estimated 128,000–161,000 people per year die as a consequence of this infection [1]. Typhoid fever is typically diagnosed clinically, with blood culture as confirmatory gold standard [2–5]. Additionally, despite its limited performance, the serological Widal test is still commonly used [3,6]. However, all current diagnostic tests for typhoid fever have limitations and new technologies are constantly being evaluated [7,8].

A lack of longitudinal incidence data in many countries where typhoid fever is suspected to be endemic is a major barrier for the introduction of typhoid conjugate vaccines (TCVs), as past or current typhoid fever disease burden represents the evidence base for vaccination policy[9]. Consequently, there is a need for new approaches that can assess the extent of typhoid fever infections without automated blood culture systems or expensive population-based studies. Serological markers to assess typhoid fever prevalence may be a suitable approach for generating disease estimates and accounting for subclinical infections. S. Typhi exposure in the general population is not routinely evaluated in cross-sectional studies, but serological assays have been used to measure seroprevalence [10–12].

Antibodies against hemolysin E (anti-HlyE) and cytolethal distending toxin subunit B homolog (anti-CdtB) have been identified as potential biomarkers for identifying typhoid fever cases/exposure [7,13–15]. HlyE and CdtB are expressed in S. Typhi and S. Paratyphi A, but uncommon in other Salmonella spp. [16]. Additionally, antibody responses against the capsular polysaccharide Vi antigen (anti-Vi), the major component of TCVs, have also been used to assess exposure [10,11,17]. The Vi antigen is only present in S. Typhi, S. Dublin and S. Paratyphi C, but is absent from S. Paratyphi A and most gastroenteritis-causing serovars[18].

Typhoid fever is a notifiable disease in the Lao People`s Democratic Republic (PDR), and several outbreaks have been reported between 2012 and 2017 [19–21]. A study estimated that the annual incidence of typhoid fever in Vientiane was 4.7 per 100,000 persons between 2015 and 2017 [22]. More recently, a study conducted in Vientiane over 18 years reported that the annual number of typhoid fever patients decreased from 2010 onwards; the estimated annual incidence of typhoid fever was 0.59 per 100,000 people in 2018 [20]. Healthcare access is inadequate in Lao PDR, with blood culture capability limited to only three laboratories nationally [21]. Consequently, typhoid fever surveillance and confirmation is inadequate, which impacts on an accurate assessment of the disease burden, and ultimately hindered the introduction of TCV. There is a need for better methods to estimate the burden of typhoid fever in endemic countries; specifically, to identify locations and age groups that have the highest exposure to S. Typhi.

To provide more data on the circulation of S. Typhi in Lao PDR, we conducted a serological, cross-sectional study using serum samples from different age groups and geographical areas of Lao PDR. This study is the first serology-based study for typhoid fever in Lao PDR and provides initial insights into age-associated exposure to S. Typhi and baseline antibody titers against Vi and HlyE and CdtB in the general population.

 Methods Ethical statement

The studies generating samples and data for this study were approved by the Lao National Ethics Committee (Cohort 1: 059/2013/NECHR, 022/2014/NECHR, 033/2017/NECHR, 032/2017/NECHR, 031/2017/NECHR, 056/2017/NECHR, and 038/2016/NECHR; Cohort 2: NECHR 059/2013 and 059/2014). Formal written informed consent was taken from all individuals enrolled in the previous studies, in the case of those aged under 16 years this was provided by a parent or guardian.

 Study population

The samples for this study originated from independent child and adult cohorts. The child cohort was comprised of 317 children and adolescents aged between 9 months and 15 years. These individuals were selected randomly (with respect to age and sex) from participants who were recruited within the framework of three other studies [23,24]. The studies generating these serum samples were conducted in central Lao PDR between 2017 and 2018. Two of these studies were cross-sectional seroprevalence studies focusing on vaccine-preventable diseases [23,24]. The third study was a hospital-based study focusing on transfusion-transmissible infections in blood transfusion recipients; only the control samples from this study were subjected to anti-Salmonella ELISAs. The adult cohort was comprised of 620 blood donors aged between 17 and 40 years who were recruited in the context of another research study between 2013 and 2015. The samples were randomly selected from a total of 5,018 and stratified by age, sex, and province. Typhoid fever vaccination is not part of the national immunization schedule in the Lao PDR and it is unlikely that participants of these studies received a typhoid fever vaccine.

 Serological testing

To determine the concentration of anti-Vi IgG antibodies, a commercial ELISA kit (Vacczyme, Binding site, UK) was employed according to the manufacturer’s instructions. Antibody concentrations were derived from the optical density (OD) data using a standardized curve-fitting 4-parameter logistic method.

Anti-HlyE IgG and anti-CdtB IgG in-house ELISAs were performed according to a previously described protocol, both antigens were also purified in house [7]. Briefly, 96 well flat-bottom ELISA plates (NunC 442404, Thermo Scientific) were coated overnight with 100 μl per well of the various antigens (final concentrations; 7 μg/ml of CdtB antigen and 1 μg/ml of HlyE antigen in 50 mM Carbonate Bicarbonate buffer). Coated plates were washed and blocked with 5% milk solution in Phosphate-buffered saline for two hours. After the blocking, plates were washed and incubated with 100μl sample (1:200 dilution) at room temperature. Plates were incubated with 100μl per well of alkaline phosphatase conjugated anti-human IgG (Sigma) for one hour at room temperature. Plates were developed using p-Nitrophenyl phosphate (SigmaFAST N1891, Sigma Aldrich, UK) substrate for 60 minutes at ambient temperature and the final absorbance was read at dual wavelengths (405 nm and 490 nm) using an automated microplate reader. Antibody concentrations in ELISA units (EU) were derived from the OD data using a standardized curve-fitting 4-parameter logistic method. If the measured antibody concentration was above or below the calculation range, the sample was tested again in a higher or lower dilution.

 Data analysis

Anti-Vi IgG data containing left-censored values were analyzed using methods described in the NADA package [25]. The left-censored data were stored using an indicator variable: The first variable contained the measured titer data and values below the calculation limit were stored as the lowest limit (7.4 U/ml). The second variable indicated if the value was a true measurement or censored. Summary statistics of the anti-Vi IgG data were calculated using robust regression on order statistics to account for left-censoring of the data. Antibody titers measured by ELISA were log transformed. After analysis of normality of independent variables in the different groups (using the Shapiro-Wilk test) and homogeneity of the variances between the groups (using Levene’s test), non-parametric statistical tests were employed. Wilcoxon test or Kruskal-Wallis test followed by Dunn’s multiple comparison tests with Bonferroni correction were used to test the significance of the differences between the antibody titer measured in groups. In case of the left-censored anti-Vi IgG data, a generalized Wilcoxon test was used (“cendiff”, NADA package [25]). The Spearman correlation coefficient or Kendall´s tau (in case of censored data) were calculated to measure the association between antibody levels determined by ELISA.

In order to assess the relationship between age and anti-HlyE IgG and anti-CdtB IgG antibody levels, both a linear regression model and a generalized additive model were fitted to the data. Generalized additive models are regression-based models that estimate non-linear trends for the predictor variable without making assumptions about the shape of the function. The anti-Vi IgG data was fitted as a function of age using Akritas–Theil–Sen non-parametric regression to account for the left-censored data. A p value <0.05 was considered statistically significant. Data analyses were conducted using R [26] with tidyverse [27], ggbeeswarm [28], ggpubr [29], mgcv [30], rstatix [31], fitdistrplus [32], plotrix [33] and NADA [25].

 Results Population characteristics

In total, sera from 937 participants originating from a range of provinces across Lao PDR were included in the study (Table 1 and S1 Fig). The majority of children in the child cohort were from Vientiane (249/317; 78.6%) and most (171/317; 53.9%) were female. The age of the children ranged from 0 to 15 years, with a median age of 8 years. The majority (373/620; 60.2%) of the adult participants were male (Table 1) and over a third (232/620; 37.4%) were students. The age of the adult participants ranged from 17 to 40 years (median 26 years).

10.1371/journal.pntd.0010017.t001 Characteristics of study participants in adult and child cohorts.
Children (n = 317) Adults (n = 620)
Study year 2017–2018 2013–2015
Age (years) min—max 0–15 17–40
mean 7.56 26.34
median 8 26
Sex (%) male 46.06 60.16
female 53.94 39.84
Province (%) Attapeu 0.00 13.23
Bolikhamxay 21.45 0.00
Huaphan 0.00 10.00
Khammouane 0.00 17.42
Luang Namtha 0.00 11.61
Luang Prabang 0.00 16.13
Vientiane Province & Capital 78.55 15.65
Phongsaly 0.00 4.35
Xayabouli 0.00 11.61
Profession (%) office worker - 28.06
soldier - 25.48
student - 37.42
other - 9.03
The seroprevalence of anti–S. Typhi IgG antibodies

We measured IgG antibodies targeting HlyE, CdtB, and Vi antigen in serum from the 937 participants. Overall, the anti-Vi antibody titers ranged from 7.4 U/ml to 600 U/ml, the anti-HlyE IgG antibody titers ranged from 12.6 EU to 5163.2 EU, and the anti-CdtB IgG antibody titers ranged from 2.8 to 1466.1 EU (Table 2). Notably, 469/937 (50.1%) of the samples generated anti-Vi IgG titers that were below the calculation limit of 7.4 U/ml. The mean anti-HlyE, anti-CdtB, and anti-Vi IgG titers among all participants were 453.8 EU, 16.8 EU and 7.5 U/ml, respectively (Table 2).

10.1371/journal.pntd.0010017.t002 Anti–<italic>S</italic>. Typhi serum IgG antibody titers in the adult and child cohorts.
N N cens Median Mean sd Max Min
anti-HlyE IgG (EU) All data 937 0 234.32 453.83 672.37 5163.20 12.64
Cohort 1: Children 317 0 351.74 734.59 980.75 5163.20 28.34
Cohort 2: Adults 620 0 198.14 310.29 362.72 4520.40 12.64
anti-CdtB IgG (EU) 1 All data 935 0 16.80 77.15 176.20 1466.10 2.75
Cohort 1: Children 317 0 52.59 178.68 270.01 1466.10 3.73
Cohort 2: Adults 618 0 12.87 25.07 40.57 470.18 2.75
Anti-Vi IgG (U/ml)
All observations All data 937 469 7.53 27.84 59.01 600.00 7.40
Cohort 1: Children 317 218 3.02 10.34 23.15 204.60 7.42
Cohort 2: Adults 620 251 11.32 36.71 69.00 600.00 7.40
Uncensored observations2 All data 468 0 24.49 52.78 75.71 600.00 7.40
Cohort 1: Children 99 0 15.12 28.47 35.20 204.57 7.42
Cohort 2: Adults 369 0 29.92 59.29 82.11 600.00 7.40

N = total number per group; N cens = number of observations below the calculation limit (censored values); sd = standard deviation

1Two participants whose samples were repeatedly below the calculation limit in the anti-CdtB IgG assay were excluded from the analysis

2Robust regression on order statistics were used to calculate summary statistics, due to the high number of observations below the limit of calculation

The distribution of antibody responses to the various antigens in adults and children is shown in Fig 1. These data demonstrated a clear delineation between the distribution of antibody titers between children and adults. For example, there was a significant difference in median anti-HlyE IgG titer between children (351.7 EU) and adults (198.1 EU; p<0.0001, Wilcoxon test) (Fig 1A). Similarly, the median anti-CdtB IgG was significantly higher in children than adults (52.6 vs 12.9 EU; p<0.0001, Wilcoxon test) (Fig 1B). We also observed a difference between the anti-Vi IgG titers in children and adults; however, contrary to the protein antigens, the median anti-Vi IgG titer was significantly higher in adults than children (11.3 vs 3.0 U/ml; p<0.0001, Wilcoxon test) (Fig 1C, Table 2). Data from both children and adults was available from Vientiane. We observed the same difference between the distributions of antibody titers between children and adults in this subset of data (S2 Fig).

10.1371/journal.pntd.0010017.g001 The distribution of anti–<italic>S</italic>. Typhi serum IgG titers in children and adults in Lao PDR.

Each dot shows the antibody titer of an individual sample for (A) anti-HlyE IgG, (B) anti-CdtB IgG, and (C) anti-Vi IgG with an underlying boxplot. The dashed line in panel C represents the censoring limit, all data points below were treated as left-censored data. Differences between groups were assessed using Wilcoxon rank sum test followed by Dunn’s post-hoc test with Bonferroni correction: ****p<0.0001.

The anti-HlyE IgG and anti-CdtB IgG titers demonstrated a significant positive correlation with each other (Spearman´s rho = 0.5; p<0.00001) (Fig 2A). Notably, the correlation coefficient of anti-HlyE IgG and anti-CdtB IgG was substantially higher among children (Spearman´s rho = 0.72; p<0.00001) (Fig 2B) than among adults (Spearman´s rho = 0.35; p<0.00001) (Fig 2C). Conversely, the anti-Vi IgG titers did not exhibit a strong correlation with the anti-CdtB IgG titers (all data: Kendall´s tau = -0.03, p = 0.14; children: Kendall´s tau = -0.11, p<0.0001; adults: Kendall´s tau = 0.03, p = 0.21) or with anti-HlyE IgG titers (all data: Kendall´s tau = -0.03, p = 0.14; children: Kendall´s tau = -0.09, p = 0.003; adults: Kendall´s tau = 0.12, p = 0.004) (Fig 2).

10.1371/journal.pntd.0010017.g002 Correlation of anti–<italic>S</italic>. Typhi serum IgG titers against three antigens in children and adults in Lao PDR.

A, B and C show the correlation between anti-HlyE IgG and anti-CdtB IgG for all data, cohort 1 and cohort 2 respectively. D, E and F show the correlation between anti-Vi IgG and anti-HlyE IgG for all data, cohort 1 and cohort 2 respectively. G, H and I show the correlation between anti-Vi IgG and anti-CdtB IgG for all data, cohort 1 and cohort 2 respectively. Black lines indicate predicted values from linear regression analysis in A, B and C and from Akritas-Thiel-Sen regression lines in D, E, F, G, H and I. Censored observations in D, E, F, G, H and I were plotted as vertical dashed lines.

 The relationship between age and anti–S. Typhi serum IgG antibodies

We fitted a linear model to investigate the relationship between anti-HlyE IgG titers and age. We found a significant negative relationship between anti-HlyE IgG titers and age (p<0.0001, adjusted R2 = 3.4%). To assess the possibility of a non-linear relationship between age and anti-HlyE IgG titers, we fitted a generalized additive model (GAM) (Fig 3A). The fitted, non-linear trend in the GAM differed significantly (p<0.0001) from the linear trend fitted in the linear regression model (GAM: p<0.0001, adjusted R2 = 15.5%, deviance explained = 16.2%). When comparing the overall model fit, the GAM demonstrated a better fit with the data than the linear model (GAM: AIC = 2649.85, BIC = 2698.65; linear model: AIC = 2767.8, BIC = 2782.3). Similarly, the GAM describing the relationship between anti-CdtB IgG titers and age (Fig 3B) was superior in terms of fit in comparison to the linear model (GAM: p<0.0001, adjusted R2 = 30.4%, deviance explained = 30.9%, AIC = 2788.01, BIC = 2835.6; linear model: p< 0.0001, adjusted R2 = 17.4%, AIC = 2939.80, BIC = 2954.32) (Fig 3). The GAM for the anti-HlyE IgG and anti-CdtB IgG titers suggested the highest prevalence of antibody was in children aged <5 years (Figs 3A, 3B and S3). The fitted GAM trends identified a prominent decrease in antibody titers until the age of 20 years. S4 and S5 Figs show the GAM for anti-HlyE IgG and anti-CdtB IgG antibody titers as a function of age, by province in adults. We observed an upward trend of anti-HlyE IgG antibody titers with age in most provinces. For the anti-CdtB IgG data however, this trend was not consistent and differed by province. Lastly, the anti-Vi IgG data was fitted as a function of age, which showed a positive relationship using Akritas–Theil–Sen non-parametric regression to account for the censored data (likelihood r = 0.33, p<0.0001, Kendall`s tau = 0.21; p<0.0001) (Fig 3C); suggesting that anti-Vi IgG increases with age. In addition, we assessed the relationship between antibody titers and age in the Vientiane subset for which data from children and adults were available. A comparable pattern was observed: Anti-HlyE IgG and anti-CdtB IgG titers were highest in children and then decreased with age, while anti-Vi IgG titers were highest in adults (S6 Fig).

10.1371/journal.pntd.0010017.g003 Results of generalized additive and linear models assessing anti–<italic>S</italic>. Typhi IgG antibody prevalence in children and adults in Lao PDR as a function of age.

Non-linear smooths were fitted for age in the model for anti-HlyE IgG (A) and anti-CdtB IgG (B) data. The tick marks on the x-axis are observed data points. In panel C, the Akritas-Thiel-Sen regression line relating to the anti-Vi IgG titer data as function of age was plotted in order to account for the censored values (censored observations were plotted as vertical dashed lines). %dev. = the percent of the total model deviance explained.

 Trends in anti–S. Typhi serum IgG antibodies regarding sex, occupation, and location

There was no significant difference for any of the anti-S. Typhi antibodies between male and female children or adults. We next investigated differences in anti-S. Typhi serum IgG antibodies according to occupation and study site in adults. A Kruskal Wallis test revealed a significant difference of anti-HlyE IgG among occupation groups (p<0.0001) (S7 Fig). A post-hoc Dunn´s test with Bonferroni correction determined significant differences between the general population with unspecified occupation (“other”) (median anti-HlyE IgG = 312.4 EU) and students (median anti-HlyE IgG = 157.6 EU; p<0.001), between office workers (median anti-HlyE IgG = 259.2 EU) and students (p<0.0001), and between office workers and soldiers (median anti-HlyE IgG = 187.5 EU; p<0.05). Likewise, there was a significant difference between anti-CdtB IgG titers in students (median anti-CdtB IgG = 10.1 EU; p<0.05) and soldiers (median anti-CdtB IgG = 13.2 EU), the general population with unspecified occupation (median anti-CdtB IgG = 17.1 EU) and students (p<0.01) and between students and office workers (median anti-CdtB IgG = 15.2 EU; p<0.001). There was no significant difference in anti-Vi IgG titers between the occupation groups.

Further Kruskal Wallis tests revealed significant differences in anti-HlyE IgG titers, anti-CdtB IgG titers, and anti-Vi IgG titers between the different provinces (p<0.0001, p<0.01 and p<0.05 respectively) (Fig 4). A post-hoc Dunn´s test with Bonferroni correction identified a significant difference between several provinces. The distribution of anti-HlyE IgG titers differed between Khammuane (median anti-HlyE IgG = 255.9 EU) and Phongsaly (median anti-HlyE IgG = 144.0 EU; p<0.05), Attapeu (median anti-HlyE IgG = 142.3 EU; p<0.01) and Xayabouli (median anti-HlyE IgG = 157.8 EU; p<0.01), and between Vientiane (median anti-HlyE IgG titer = 272.0 EU; p<0.01) and Attapeu and Xayabouli (p<0.05) (Fig 4A). The median anti-CdtB IgG titres differed significantly between Khammouane (median anti-CdtB IgG = 15.3 EU) and Xayabouli (median anti-CdtB IgG = 9 EU; p<0.05) and between Vientiane (median anti-CdtB IgG = 16.1 EU) and Xayabouli (p<0.01) (Fig 4B). The only significant difference in median anti-Vi IgG titers was identified between Khammouane (median anti-Vi IgG = 20.3 U/ml) and Luang Prabang (median anti-Vi IgG = 7.8 U/ml; p<0.05) (Fig 4C).

10.1371/journal.pntd.0010017.g004 The distribution of anti–<italic>S</italic>. Typhi serum IgG titers in adults in Lao PDR by province.

Each dot shows the measurement of an individual sample for (A) anti-HlyE IgG, (B) anti-CdtB IgG and (C) anti-Vi IgG with an underlying boxplot. Differences between groups were assessed using Kruskal-Wallis test followed by Dunn’s post-hoc test with Bonferroni correction: *p<0.05, **p<0.01. ATP = Attapeu, HPN = Huaphan, KHM = Khammouane, LNT = Luang Namtha, LPB = Luang Prabang, VTN = Vientiane, PSL = Phongsaly, XAY = Xayabouli. The dashed line in panel C represents the censoring limit, all data points below were treated as left-censored data.

 Discussion

The only reliable method for assessing the disease burden of typhoid fever is establishing population-based surveillance studies and introducing a standardized blood culture system. This approach recently uncovered a large, previously unobserved, burden of typhoid fever in sub-Saharan Africa [34]. These studies are complicated, expensive, and not sustainable outside of research funding. Additionally, clinical criteria, blood culture sensitivity, and site-specific nuances need to be taken into account throughout the study and can impact heavily on the corrected and uncorrected incidence figures. Serology may be a more scalable approach for typhoid fever surveillance. Serology is a fraction of the cost of population-based blood culture studies and could be used to highlight disease “hotspots” and identify which component of the population should be targeted for TCV introduction.

The antibody dynamics of active typhoid fever and carriage are yet to be fully characterized. However, both HlyE and CdtB have been shown to be informative S. Typhi antigens for serological investigations [15,16,35]. Anti-HlyE IgM, IgA and IgG responses are known to be elevated in confirmed typhoid fever cases in comparison to healthy controls [14]. Likewise, anti-CdtB IgM responses were higher in typhoid fever cases compared to controls using recombinant CdtB in an indirect ELISA [13]. As the principal component of typhoid fever vaccines, the Vi polysaccharide is the S. Typhi antigen most commonly used in serological studies [10,11,17].

We found that half of the participants had anti-Vi IgG antibody concentrations <7.4 U/ml, with an estimated median titer of 7.5 U/ml. Notably, the median baseline concentrations were higher in adults than children and we observed a positive association between anti-Vi IgG titer and age. These findings are largely consistent with previous reports [10,36]. The median anti-Vi titer after removing those below the calculation limit was 24.5 U/ml overall and 29.9 U/ml in adults, which was higher than previously reported median titers in healthy adults from the non-typhoid fever endemic countries Spain and Germany (8.6 U/ml and 21 U/ml, respectively) [37,38]. Conversely, the median anti-HlyE IgG and anti-CdtB IgG titers were substantially higher in children than in adults. The non-linear trend fitted to the HlyE IgG data identified a peak in antibody concentration in children below the age of 5 years; this peak was followed by a decrease and a secondary increase after the age of 20 years. The anti-CdtB IgG data followed largely the same trajectory; however, the increasing trend after the age of 20 years was not observed. The anti-HlyE IgG and anti-CdtB IgG titres correlated reasonably well with each other in the adult and children cohort. Notably, the anti-Vi IgG data, however, did not correlate well with either of the other two antibody profiles. A low correlation between the anti-Vi IgG titers and anti-HlyE/anti-CdtB IgG titers was surprising and could be due to different durability profiles. In contrast to the low correlation (tau -0.09; all data) between anti-CdtB IgG and anti-Vi IgG in our study, the IgM measurements against both of these antigens have demonstrated a moderate correlation (rho = 0.77) [7]. Data regarding the relationship between anti-S. Typhi antibody responses is currently lacking, indicating the need for further investigations.

Our data, measuring the presence of antibodies developed against the two protein antigens, anti-HlyE IgG and anti-CdtB IgG, potentially indicates greater S. Typhi exposure in children than adults. This contrasts a hospital based surveillance study in Vientiane, which reported no typhoid fever in children over the course of two years [22]. In a retrospective study in a central hospital in Vientiane, from 2000 to 2018, the median age of patients with confirmed typhoid fever was 21 years [20]. However, these hospital-based surveillance studies were performed in general hospitals, which have a low number of pediatric patients as they are admitted to more specialist facilities or have febrile illness managed in the community. Age-related Typhoid fever incidence patterns vary between high and low incidence settings: the incidence is typically highest in children in high incidence settings but more equally distributed in low incidence settings. In the global context, children are disproportionally affected by typhoid and paratyphoid fever with the highest incidence occurring in children aged between 5 and 9 years [39]. Serological testing is not a marker of active disease, but our data suggests children in the sampled locations in Lao PDR have been exposed to S. Typhi. A follow-up study specifically focusing on febrile disease and comparing blood culture data with serological testing in children in multiple provinces would allow a more accurate confirmation of the typhoid fever burden in children in Lao PDR.

Our study has limitations; the samples selected for this study were collected over different years and under the sampling framework of other studies. The data associated with the studies did not incorporate any health-related information including whether the participants had ever been diagnosed with typhoid fever. Serum samples from children were only available from two provinces in the Lao PDR. In Pongsaly province, fewer serum samples were available compared to the other locations, limiting our data analyses for this province. Additionally, the correlation between typhoid fever diseases and the antibody titer data was not yet established, complicating the interpretation of the titer data considerably. We currently have no cut-off for seroconversion with these antigens, so we used precise titers as a direct comparison between age groups and locations. Lastly, we cannot exclude the possibility of cross-reactive antibody responses, as HlyE and CdtB may be found in other bacteria [40,41].

In conclusion we identified a high prevalence of anti-HlyE and anti-CdtB IgG in the serum of children residing in Lao PDR. As these antigens are conserved within invasive Salmonella and known to stimulate an antibody response during infection our data suggest these may be tractable markers of disease exposure in typhoid fever endemic locations. Additional validation is required, but our approach is cost effective and scalable and may be developed to assess the burden of typhoid fever in countries.

 Supporting information Map of study sites generating serum samples for anti-S. Typhi IgG serology in the Lao PDR.

PSL = Phongsaly, LNT = Luang Namtha, HPN = Huaphan, LPB = Luang Prabang, XAY = Xayabouli, VTN = Vientiane, BLX = Bolikhamxay, KHM = Khammouane, ATP = Attapeu. The map was created with QGIS (QGIS Development Team, 2018). The data regarding the administrative boundaries of Lao PDR were obtained from the Humanitarian Data Exchange website https://data.humdata.org/dataset/lao-admin-boundaries, dataset provided by the National Geographic Department of Lao PDR, 2019) and recreated under a CC BY-IGO license. Projection used: EPSG 4326 –WGS 84.

(TIF)

 The distribution of anti–<italic>S</italic>. Typhi serum IgG titers in children and adults in Vientiane, Lao PDR.

Each dot shows the antibody titer of an individual sample for (A) anti-HlyE IgG, (B) anti-CdtB IgG, and (C) anti-Vi IgG with an underlying boxplot. The dashed line in panel C represents the censoring limit, all data points below were treated as left-censored data. Differences between groups were assessed using Wilcoxon rank sum test followed by Dunn’s post-hoc test with Bonferroni correction: ***p<0.001, ****p<0.0001.

(TIF)

 Results of generalized additive models assessing anti–<italic>S</italic>. Typhi IgG antibody prevalence in children and adults in Lao PDR as a function of birth year.

Non-linear smooths were fitted for birth year in the model for anti-HlyE IgG (A) and anti-CdtB IgG (B) data. Shaded bands represent the pointwise 95%-confidence interval.

(TIF)

 Results of the generalized additive model assessing anti-HlyE IgG titer in adults in Lao PDR as a function of age by province.

Shaded bands represent the pointwise 95%-confidence interval. ATP = Attapeu, HPN = Huaphan, KHM = Khammouane, LNT = Luang Namtha, LPB = Luang Prabang, VTN = Vientiane, PSL = Phongsaly, XAY = Xayabouli.

(TIF)

 Results of the generalized additive model assessing anti-CdtB IgG titer in adults in Lao PDR a function of age by province.

Shaded bands represent the pointwise 95%-confidence interval. ATP = Attapeu, HPN = Huaphan, KHM = Khammouane, LNT = Luang Namtha, LPB = Luang Prabang, VTN = Vientiane, PSL = Phongsaly, XAY = Xayabouli.

(TIF)

 Results of generalized additive and linear models assessing anti–S. Typhi IgG antibody prevalence in children and adults in Vientiane, Lao PDR as a function of age.

Non-linear smooths were fitted for age in the model for anti-HlyE IgG (A) and anti-CdtB IgG (B) data. The tick marks on the x-axis are observed data points. In panel C, the Akritas-Thiel-Sen regression line relating to the anti-Vi IgG titer data as function of age was plotted in order to account for the censored values (censored observations were plotted as vertical dashed lines). %dev. = the percent of the total model deviance explained.

(TIF)

 The distribution of anti–<italic>S</italic>. Typhi serum IgG titers in adults in Lao PDR by occupation.

Each dot shows the measurement of an individual sample for (A) anti-HlyE IgG, (B) anti-CdtB IgG and (C) anti-Vi IgG with an underlying boxplot. Differences between groups were assessed using Kruskal-Wallis test followed by Dunn’s post-hoc test with Bonferroni correction: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. If not specified otherwise, differences in titer data were non-significant. Participants whose occupation is not specified are grouped into “other”. The dashed line in panel C represents the censoring limit, all data points below were treated as left-censored data.

(TIF)

We wish to acknowledge investigators, especially Vilaysone Khounvisith and Phonethipsavanh Nouanthong, from the named studies for providing access to the samples and data of the study participants. We are thankful to our collaborators at Luxembourg Development Cooperation, the Lao Red Cross and Provincial and district hospitals and health centres. Latdavone Khenka and Bounta Vongphachanh helped with the experiments. We wish to acknowledge Dr. Paul Brey and staff at the Institut Pasteur du Laos for their support.

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Lancet Infect Dis. 2019;19: 369–381. doi: 10.1016/S1473-3099(18)30685-6 30792131 Gargi A, Reno M, Blanke SR. Bacterial toxin modulation of the eukaryotic cell cycle: are all cytolethal distending toxins created equally? Front Cell Infect Microbiol. 2012;2: 124. doi: 10.3389/fcimb.2012.00124 23061054 Hunt S, Green J, Artymiuk PJ. Hemolysin E (HlyE, ClyA, SheA) and related toxins. Adv Exp Med Biol. 2010;677: 116–126. doi: 10.1007/978-1-4419-6327-7_10 20687485 10.1371/journal.pntd.0010017.r001 Decision Letter 0 Bourret Travis J Associate Editor Chattopadhyay Sujay Deputy Editor 2021 Bourret, Chattopadhyay This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Submission Version 0

16 Jun 2021

Dear Dr. Baker,

Thank you very much for submitting your manuscript "An age-stratified serosurvey against purified Salmonella enterica serovar Typhi antigens in the Lao People´s Democratic Republic" 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. 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.

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:

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Associate Editor

PLOS Neglected Tropical Diseases

Sujay Chattopadhyay

Deputy Editor

PLOS Neglected Tropical Diseases

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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 objectives as set out are noble and would answer key research questions in this area.

The study design should have had an element of longitudinal collection of serological data on the study population to understand how long the markers of typhoid exposure persist in the individuals and how this relates to patient presentation or carriage status

Sample size and analytical framework are well designed, and no concerns on ethics

Reviewer #2: The methodology of the study is clearly presented and articulated.

However, the specificity of the 3 antibodies for S. Typhi (vs other non-typhoidal serovars for instance), and their detectability in the serum are not compared or discussed. This absence limits the conclusion of the study but could be included as an additional experiment testing the presence of these antibodies in the blood of patients after typhoid fever was detected (for this purpose, the sample collection could be unrestrained to Lao if necessary). Alternatively, references to previously published work providing this comparison (if existing) should be included and discussed.

Minor point:

- In the “Study population” subsection of the Methods, the author should mention that the populations included in the study have not received the Vi polysaccharide-based vaccine.

- The authors may comment if both cohorts (adult and children) had similar living conditions (access to running water, etc).

Reviewer #3: 1. I realize that these blood samples were from other studies, but is there any way to determine if any samples were from people vaccinated for typhoid fever? If not, could this be stated in the manuscript.

2. The adult blood donor age range from 17 – 40 years seems a little bit narrow. How were the individuals randomly selected? What was the overall range of the 5,018 samples that the 640 adult samples were randomly selected from for the study?

3. Could more details be provided about the commercial anti-Vi IgG ELISA kit, such as were the manufacturer’s instructions followed exactly without any modifications. If modifications, what were those modifications? Wavelength? As this is a critical method of the manuscript providing the reader with enough detail to potentially replicate it is vital.

4. Why was HlyE IgG ELISA included in the study? The reference cited for the in-house developed ELISA does not appear to include hlyE gene, therefore what is the specificity and sensitivity of this ELISA?

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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: Analysis as presented is well thought out and clearly done including Tables and Figures.

Major concerns:

Page 8, Line 207: We observed an upward trend of antibody titers with age in some provinces; however, this trend t was not consistent and differed by province. Lastly, the anti-Vi IgG data was fitted as a function of age, which showed a positive relationship using Akritas–Theil–Sen non-parametric regression to account for the censored data (likelihood r = 0.33, p<0.0001, Kendall`s tau = 0.21; p<0.0001) (Figure 3); suggesting that anti-Vi IgG increases with age.

Comment: what is the context of these titres in relation to active disease and carriage in endemic settings? Granted that they provide guidance on exposure, they are a good indicator and guide for deployment of vaccines to reduce carriage and transmission in the community.

Can they be used for rapid detection of disease?

Page 9, Line 232: The distribution of anti-HlyE IgG titers differed between Khammuane (median anti-HlyE IgG = 255.9 EU) and Phongsaly (median anti-HlyE IgG = 144.0 EU),Attapeu (median anti-HlyE IgG = 142.3 EU) and Xayabouli (median anti-HlyE IgG = 157.8 EU), and between Vientiane (median anti-HlyE IgG titer = 272.0 EU) and Attapeu and Xayabouli (Figure 4A).The median anti-CdtB IgG titres differed significantly between Khammouane (median anti-CdtB IgG= 15.3 EU) and Xayabouli (median anti-CdtB IgG = 9 EU) and between Vientiane (median anti-CdtB IgG = 16.1 EU) and Xayabouli (Figure 4B). The only significant difference in median anti-Vi IgG titers was identified between Khammouane (median anti-Vi IgG = 20.3 U/ml) and Luang Prabang (median anti-Vi IgG = 7.8 U/ml) (Figure 4C).

Comment: what is the reason for the variation according to geographic distribution?

Any specific risk attributes unique to the specific study sites?

Reviewer #2: In general, the results are presented in a very clear and exhaustive manner.

Minor point:

- Line 207-208 (referring to Figures S3-S4): the authors wrote “We observed an upward trend of antibody titers with age in some provinces; however, this trend t was not consistent and differed by province.“ This statement should be detailed/refined. For instance, the trend seems consistent between provinces for anti-HyE IgG but not for anti-CdtB IgG. Was the population size of some provinces limiting the analysis? If yes, this should be mentioned in the text.

Reviewer #3: 5. Lines 213-225, it would be good to put in the p-Values for those significant differences.

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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: Page 10, Line 252: We found that half of the participants had anti-Vi IgG antibody concentrations <7.4 U/ml, with an estimated median titer of 7.5 U/ml.

Comment: How do these titres correlate with actual typhoid disease?

Page 11, Line 276: A follow up study specifically focusing on febrile disease and comparing blood culture data with serological testing in children in multiple provinces would allow a more accurate confirmation of the typhoid burden in children in Lao PDR.

Comment: This correlation of active disease and serology is crucial to provide a diagnostic basis for use of serological testing suggested for diagnostic purposes.

At the moment, the investigators should cite lack of this correlation as a major limitation in interpretation of their - one cannot tell if the raised levels of antigens is marker for low-grade exposure, active disease or passive carriage

Reviewer #2: While the data obtained are exhaustively analyzed and described, the interpretation of the results remains superficial. The study – in particular, the discussion section - would highly beneficiate from a more in-depth biological contextualization and a consideration of the results in a broader context.

Here are several elements that may be further discussed:

(1) Differences observed between antibodies:

- The 3 different IgG should be further introduced. E.g., CdtB stands for Cytolethal Distending Toxin B, and HlyE stands for Haemolysin E. Both are bacteria toxins produced by several bacteria species. The specificity of these antigens to S. Typhi should be introduced (see also “methods” comments).

- A key result of the study is the absence of correlation between the anti-Vi polysaccharide IgG titers and the anti-toxin (CdtB /HlyE) IgG titers, and their different distribution in the age-stratified population. This result was unexpected and the authors should provide some hypotheses in the discussion. Could this be explained by different duration of the seroconversions? By the specificity or detectability of the antibodies? (see also “methods” comments).

- As one purpose of the study is to orientate future survey strategies and ease the introduction of a vaccination program, the authors may provide more direct guidance on which antibody detection should be favored for future serological surveys, and possibly comment on the availability of the different antibodies and their production cost.

(2) Difference observed between age, location, occupation:

- How could the difference of serology depending on the location and occupation be interpreted? Could it be linked with different access to running water, food origin, etc? Could the occupation be commented on in terms of Salmonella exposure? Could the province be commented on in terms of previous outbreak locations?

- As the authors checked both the serology as a function of the age and as a function of the year of birth, it would be interesting to discuss if the antibody distribution in the age-stratified population may result from a different exposure depending on the year (considering the date of S. Typhi outbreaks) or from the timing of biological processes.

- Can these results be put in context with the situation in neighboring countries with a high prevalence of S. Typhi?

Reviewer #3: 6. The authors do not discuss why there is no correlation between anti-Vi titers and anti-CdtB or anti-HlyE titers, it seems if this is a critical issue that should be discussed by the authors. The authors discuss the differences with age, but if all of these are signs of previous typhoid fever infection, would you not expect a correlation between the major polysaccharide antigen and potential toxins. Additionally, why do you think there is an age difference in the antigens that are inducing an immune response? Many enteric pathogens produce cytolethal distending toxin (CDT) is it possible that this is due to a cross-reactivity or response to other enteric pathogens more common in children such as Campylobacter. All of these should be discussed for the paper.

7. Is it possible that the children’s samples only coming from two provinces, which were significantly higher for anti-CdtB or anti-HlyE titers could be biasing some of the results for the different provinces? This should be addressed by the authors and how this bias was avoided.

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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: (No Response)

Reviewer #2: Minor points on the text:

- In the introduction, lines 61-62, the authors wrote: “A lack of longitudinal incidence data in many countries where typhoid is suspected to be endemic is a major barrier for the introduction of typhoid conjugate vaccines (TCVs)” I suggest that the authors add a sentence explicitly explaining why this constitutes a major barrier for TCV introduction.

- Direct references to given subfigure panels (for instance “Figure 1A”) are often missing in the text. It would help the readability of the results to specify systematically which part of the figure the text is referring to.

- Regarding the availability of data and materials, the authors state “All data generated or analyzed during this study are included in this published article and its supplementary information files.” Yet, it is unclear how to get access to the full raw anonymized dataset of the epidemiological study. As such data could be directly used for other investigations, it would be critical to provide this information.

Minor points on the Figures:

- Statistical information should be added in Figure 3.

- “ns” for “non-significant” could be added in Figure 5C.

Reviewer #3: 1. The authors switch between using “typhoid fever” and “typhoid” randomly throughout the manuscript. One term should be used consistently throughout the manuscript to avoid reader confusion.

2. The reference section should have genus and species names italicized for proper formatting.

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

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 a manuscript that provides data on serological surveillance for typhoid specific antigen markers that maybe useful for assessment of exposure to typhoid in disease endemic settings. What is not clear is how long these markers persist in the individuals in the community and are they specific in magnitude/persistence in acute disease compared to carriage? It would have made more sense to have some longitudinal data to assess and answer this question.

Reviewer #2: The study systematically compares the presence of 3 anti–S. Typhi IgG antibodies in the blood of Laotian of different age, sex, location, and occupation. The outcome of the study could be a cornerstone for the development of a vaccination strategy.

Strong points: The investigation is performed properly, the manuscript is presented clearly and the conclusions well justified. Due to the low health surveillance in Lao, the collection of this data is a tour de force and offers very high public health relevance.

Weak points: The manuscript currently lacks biological contextualization of the investigation and discussion of the results in a broader context (see more detailed comments in “conclusions”).

Reviewer #3: (No Response)

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

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Figure Files:

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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. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

 10.1371/journal.pntd.0010017.r002 Author response to Decision Letter 0 Submission Version 1

21 Jul 2021

Submitted filename: Cover letter_210720.doc

 10.1371/journal.pntd.0010017.r003 Decision Letter 1 Bourret Travis J Associate Editor Chattopadhyay Sujay Deputy Editor 2021 Bourret, Chattopadhyay This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Submission Version 1

15 Sep 2021

Dear Dr. Baker,

Thank you very much for submitting your manuscript "An age-stratified serosurvey against purified Salmonella enterica serovar Typhi antigens in the Lao People´s Democratic Republic" 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.

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,

Travis J Bourret

Associate Editor

PLOS Neglected Tropical Diseases

Sujay Chattopadhyay, PhD

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 #2: The authors have provided satisfying answers and text edits to my methods-related comments.

Reviewer #3: Methods are fine and clearly stated.

Reviewer #4: The objectives of the study are clearly spelled out. The study design is good; the population is clearly described and a large enough size of study participants are recruited to make the results statistically meaningful. There are no questions regarding ethical or regulatory requirements.

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

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 #2: The authors have provided satisfying answers and text edits to my results-related comments.

Reviewer #3: There are no issues with the results.

Reviewer #4: The results are clearly presented and the data support the conclusions. Figures and tables are clear.

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

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 #2: The authors have replied to all the points previously raised in their point-by-point answer. However, in most cases, they could not directly improve the results and conclusions within the manuscript, but rather detailed in their rebuttal the current limitations that restricted the study (lack of current knowledge on the antibody responses, lack of epidemiological information in Lao, etc.). Given these circumstances, the authors pushed the analysis and interpretation to their best extend and avoided drawing conclusions or interpretations that could not be directly supported by the data.

Reviewer #3: Conclusions from the study are supported by the data from the study, but a little more in depth thought as to the meaning of the results would help to strengthen the manuscript.

Reviewer #4: Conclusions are borne out by the data. Limitations of the study are also addressed by the authors.

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

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 #2: The authors have considered and implemented my editorial suggestions.

Reviewer #3: (No Response)

Reviewer #4: (None)

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

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 #2: While the limited conclusions of the study may impair its visibility, it also highlights the necessity to support and develop similar studies until reaching a broader understanding of typhoid epidemiology. However, I regret that the authors were unable to consequently revised the text to contextualize their study as suggested. This would have made the manuscript more attractive for a broader audience (only minor edits were implemented in this regard despite the editor's recommendation to significantly revise the manuscript). In its current state, as the manuscript mainly displays epidemiological observations but no or limited interpretations of the results, it may fit best a journal dedicated to epidemiological reports.

Reviewer #3: Overall, I think the manuscript has been greatly improved, but there are still a couple of minor issues that should be resolved prior to publication:

As the central finding of the study is that children have higher titers for HlyE and CdtB compared to adults, however this includes adults from numerous provinces and children from only two provinces. It would be interesting to see if this continues to be true by narrowing the analysis down to just the two provinces that all the children samples came from for the study (e.g. Vientiane & Bolikhamxay).

I would like to see them expand the thoughts on the low correlation between anti-Vi titers and anti-HlyE/anti-CdtB titers, as this seems critical to the study, but the authors do not offer a very detailed explanation other than different durability profiles. Is there anything in the literature to support this thought? Anything in the literature to offer alternative ideas?

For the difference in children versus adults that is opposite from the hospital based study in Vientiane, what do other typhoid fever studies in other countries suggest about the results? Have other studies found differences between serum based versus hospital based studies in other countries?

Overall, I think a little bit more thought on the meaning of the results is needed for the discussion prior to publication. Otherwise it is a solid paper.

Reviewer #4: (No Response)

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

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Reviewer #2: No

Reviewer #3: No

Reviewer #4: 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, 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. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

References

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article's retracted status in the References list and also include a citation and full reference for the retraction notice.

Submitted filename: Hefele_L.SBaker.review.docx

 10.1371/journal.pntd.0010017.r004 Author response to Decision Letter 1 Submission Version 2

6 Oct 2021

Submitted filename: Cover letter_041021.doc

 10.1371/journal.pntd.0010017.r005 Decision Letter 2 Bourret Travis J Associate Editor Chattopadhyay Sujay Deputy Editor 2021 Bourret, Chattopadhyay This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Submission Version 2

22 Nov 2021

Dear Dr. Baker,

We are pleased to inform you that your manuscript 'An age-stratified serosurvey against purified Salmonella enterica serovar Typhi antigens in the Lao People´s Democratic Republic' 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.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

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

Best regards,

Travis J Bourret

Associate Editor

PLOS Neglected Tropical Diseases

Sujay Chattopadhyay

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 #2: (No Response)

Reviewer #4: The methodology is adequately described and backed by literature references. The plots neatly describe the data. This together with the large study size allows the authors to draw convincing and useful conclusions.

**********

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 #2: (No Response)

Reviewer #4: The results clearly show, as reported, that antibody responses against typhoid fever antigens HlyE and CdtB are notably higher in children than in adults. However, when using the bacterial polysaccharide antigen Vi, antibody responses are higher in adults than in children.

**********

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 #2: (No Response)

Reviewer #4: The conclusions are supported by the data.

The limitations are addressed and described. The authors discuss how their data will further our understanding.

The study is of great public health relevance and is discussed.

**********

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 #2: (No Response)

Reviewer #4: Accept

**********

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 #2: The authors have thoughtfully considered all my comments and edited the manuscript accordingly. I don't have further requests. Therefore, I support the manuscript for its publication in PNTD.

Reviewer #4: There are no remaining issues.

**********

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 #2: Yes: Virginie Stévenin

Reviewer #4: No

Submitted filename: Hefele_L.SBaker.review_2.docx

 10.1371/journal.pntd.0010017.r006 Acceptance letter Bourret Travis J Associate Editor Chattopadhyay Sujay Deputy Editor 2021 Bourret, Chattopadhyay This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

7 Dec 2021

Dear Professor Baker,

We are delighted to inform you that your manuscript, "An age-stratified serosurvey against purified Salmonella enterica serovar Typhi antigens in the Lao People´s Democratic Republic," 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.

Soon after your final files are uploaded, the early version of your manuscript will be published online unless you opted out of this process. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.

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

Best regards,

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases