Incidence of dengue illness in Mexican people aged 6 months to 50 years old: A prospective cohort study conducted in Jalisco.

BACKGROUND AND OBJECTIVES: The burden of dengue virus (DENV), a mosquito-borne pathogen, remains difficult to assess due to misdiagnosis and underreporting. Moreover, the large proportion of asymptomatic dengue cases impairs comprehensive assessment of its epidemiology even where effective surveillance systems are in place. We conducted a prospective community-based study to assess the incidence of symptomatic dengue cases in Zapopan and neighboring municipalities in the state of Jalisco, Mexico.
METHODS: Healthy subjects aged 6 months to 50 years living in households located in the Zapopan and neighboring municipalities were enrolled for a 24-month follow-up study (NCT02766088). Serostatus was determined at enrolment and weekly contacts were conducted via phone calls and home visits. Participants had to report any febrile episode lasting for at least two days. Suspected dengue cases were tested by reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR), detection of non-structural protein 1 (NS1), anti-DENV immunoglobulin G and M (IgG and IgM) assays.
RESULTS: A total of 350 individuals from 87 households were enrolled. The overall seroprevalence of anti-DENV IgG at enrolment was 19.4% (95% confidence interval [CI] 14.5-25.6) with the highest seroprevalence rate observed in the adult group. Over the 27-month study period from July 2016 to September 2018, a total of 18 suspected dengue cases were reported. Four cases were confirmed by RT-qPCR and serotyped as DENV-1. A fifth case was confirmed by the NS1 assay. The 13 remaining suspected cases were tested negative by these assays. Based on the 5 virologically confirmed cases, symptomatic dengue incidence proportion of 1.4% (95%CI 0.5-3.8) was estimated. No severe cases or hospitalizations occurred during the study.
CONCLUSION: Community-based active surveillance was shown as efficient to detect symptomatic dengue cases. CLINICAL TRIAL REGISTRATION: NCT02766088.

Introduction

Dengue is a viral disease caused by four types of dengue viruses (DENV-1, DENV-2, DENV-3, and DENV-4) [1] that are transmitted by mosquito vectors, being primarily Aedes aegypti and secondarily Aedes albopictus [2, 3]. Aedes aegypti is present in the tropical and subtropical regions and well adapted to urban habitats [4]. Aedes albopictus has spread in North America and some Southern European countries as it can accommodate to cooler temperate climates [4]. Although most dengue infections are asymptomatic, clinical manifestations can range from febrile illness to potentially fatal dengue shock syndrome [2, 5, 6]. The course of the disease develops in up to three phases, starting with an acute febrile episode of three to seven days [5]. Fever can be followed by the critical phase characterized by a systemic vascular leak syndrome lasting for one or two days [5]. Most patients recover from the hemorrhagic episode, though the disease can progress to potentially fatal dengue shock syndrome, severe bleeding or organ failure [6].

Worldwide, the global incidence of dengue increased from approximately 500,000 reported cases in 2000 to more than three million in 2015, and the highest number of cases ever reported occurred in 2019 [7]. It was estimated that between 100 and 400 million dengue infections annually occur worldwide [8], and that half of the global population, living within 128 countries, is now at risk [9]. Dengue is endemic in Asia and also in the Western Pacific, the Americas, Africa, and the Eastern Mediterranean region [7]. Moreover, arbovirus vectors are known to spread in the United States and Europe due to urbanization, increased mobility, and climate change [10]. The increase in dengue incidence is accompanied by explosive outbreaks that are seasonal and influenced by characteristics of the vector and the host [6, 7]. Following the re-infestation of Latin Americas by the Aedes aegypti mosquito during the 1960s, several outbreaks of dengue were reported in the region [11]. In 1977, DENV-1 caused an epidemic that began in Jamaica that expanded to Mexico by 1978 [11]. In 1981, DENV-4 was introduced in the Caribbean and caused epidemics in several countries including Mexico where some cases of dengue hemorrhagic fever were observed [11]. During the 1990s, several epidemics were caused by the DENV-3 type in Mexico. Between 2000 and 2010, increased dispersion of Aedes aegypti has amplified dengue virus circulation, leading to several outbreaks, including in Mexico in 2009, where around 250,000 cases were reported [11]. In the Americas, the number of cases annually reported has increased from around 400,000 in 2000 to more than three million in 2019, with more than 25,000 cases classified as severe [12]. In Mexico, reported numbers of probable and confirmed cases increased between 2018 and 2019 from 78,621 to 268,458 and 12,706 to 41,505, respectively [13]. The annual cost associated with dengue in the Mexican population was estimated to be around 170 million US dollars, accompanied by an annual average burden of 65 disability-adjusted life-years per million population [14]. In 2019, the overall rate for the country was 32.96 per 100,000 individuals, with the highest rate being in Jalisco (141.6/100,000) [13]. The increase in dengue incidence was particularly important in this state as estimates for 2016 and 2017 were 24.9 and 13.8 per 100,000 habitants, respectively [15, 16]. A large proportion of the increase was caused by DENV-2 in Mexico as the total number of DENV-2 cases progressed from 1,626 in 2017 to 12,637 in 2019 [13, 15].

Aside from the uncontrolled spreading of mosquito vectors, increased awareness by authorities and subsequent improvements in surveillance participate in the apparent increase in dengue infections. However, the lack of accurate incidence rates due to misdiagnosis and underreporting suggests that the burden of dengue might be even higher than the current estimates [7]. The underestimation of the dengue incidence arises from different factors: the high proportion of asymptomatic infections, the subjects with mild symptoms who do not seek treatment from a physician, the significant proportion of misdiagnoses due to the similarity of dengue symptoms and the underreporting of diagnosed cases [17, 18]. Compared to passive surveillance, active surveillance has the potential to detect mild and asymptomatic cases when coupled with serosurveillance and can help to understand the changes in epidemiology [17, 19]. Early detection of dengue would allow to improve the management of the disease and alleviate the overall burden of severe episodes and complications [20]. Moreover, estimates of unapparent, clinically apparent cases confirmed virologically would help assess the true disease burden and inform prevention strategies, including vaccine studies [21].

Currently, only one vaccine is available and licensed in about twenty countries [22]. Although this vaccine is efficacious and safe in seropositive individuals, a lower protection during the first two years, followed by an increased risk of severe dengue and hospitalization have been observed in seronegative vaccinated individuals [22]. Due to this risk of severe dengue in naïve individuals, vaccine use is limited to seropositive individuals. It is therefore important to achieve an understanding of disease dynamics in endemic areas to support further dengue vaccine development programs.

The present observational cohort study was conducted to primarily estimate the overall incidence of dengue infection confirmed by RT (reverse transcriptase)-PCR in subjects aged 6 months to 50 years living in a highly endemic area in Mexico.

Secondary objectives were to estimate a) the incidence of virologically (PCR or non-structural protein 1 [NS1]) confirmed and probable symptomatic dengue cases (by detection of IgG/IgM antibodies to dengue virus by ELISA or rapid immunochromatographic test) by age, gender, serotype (if applicable), and b) the prevalence of anti-DENV IgG antibodies at enrolment, overall and by age, as well as c) to describe the clinical presentations of dengue cases.

Methods

Study design and setting

This multi-center, prospective, household-based cohort surveillance study was conducted in geographically-defined communities in Latin America and Southeast Asia (NCT02766088). The initial protocol and study planning included seven countries. However, due to early study termination, only two sites in the Philippines and Mexico participated in the study. Here we report on the data collected during a 27-month period in Zapopan and neighboring municipalities in the state of Jalisco, Mexico, between July 14th, 2016 and September 14th, 2018.

Ethical statement

This multi-center, prospective, household-based cohort surveillance study (NCT02766088) was conducted following the Internal Council on Harmonization good clinical practice guidelines and the Declaration of Helsinki. The study protocol, amendments, and other study-related documents were reviewed and approved by the study site’s independent ethics committee (research committee and research ethics committee of Hospital General de Occidente). Written and signed informed consent to participate were obtained from eligible individuals or their legally authorized representative. Additional assent from subjects below the legal age of consent was sought when applicable. The present manuscript was developed following the STrengthening the Reporting of OBservational studies in Epidemiology (STROBE) statement.

Participants

Study researchers used a community-based approach for the recruitment of subjects. Invitation to the families was made through seven promoters of the study (three social workers, two doctors, a nutritionist, and a nurse) who also lived in Zapopan and the neighboring municipalities. Each promoter contacted three to eight families. The percentage of participants recruited by each promoter varied between 8% and 20%. Flyers with the information about the study and contact center were distributed. Some families were recruited through other families already included in the study. To be eligible for inclusion in the study, individuals should a) be 6 months to 50 years of age, b) live in Zapopan or neighboring municipalities (state of Jalisco, Mexico), c) agree to go to the study site for visit(s) in case of acute febrile illness, observe the signs of dengue and understand how to measure and report body temperature, d) plan to remain at the same residence during the study follow-up period, and e) be reachable by phone. Several members of the same household could participate in the study. Written and signed informed consent to participate were obtained from eligible individuals or their legally authorized representative. Additional assent from subjects below the legal age of consent was sought when applicable.

Study procedures

A total of three visits were scheduled over a 24-months follow-up period (Fig 1). Socio-demographic information, medical and vaccination histories, as well as a blood sample, were collected at enrolment (Visit 1) through a questionnaire (S1 Appendix). The Month-12 and Month-24 visits occurred via telephone contact to collect serious adverse events (SAEs) and updated demographic, medical, and vaccination information. SAEs were defined as any death, life threatening event, hospitalization, or persistent disability that occurred during the study. SAEs related to blood drawn were considered as related to study procedure, other SAEs were also collected during the entire study period. Between the Month-12 and Month-24 visits, surveillance contacts for febrile illness were conducted once a week by telephone (95%) or in person (5%). Unscheduled visits at the study site were required in case of acute febrile illness to assess suspected dengue cases (SDC) (Fig 1). All SDCs were then followed in person.

Fig 1

Study design.

SAE, serious adverse event related to blood drawn. Virologically confirmed dengue case: confirmed by reverse-transcriptase quantitative polymerase chain reaction or detection of non-structural protein 1 antigen.

Determination of serostatus at study entry

Anti-DENV (serotypes 1, 2, 3, and 4) immunoglobulin G (IgG) were measured by indirect enzyme-linked immunosorbent assay (ELISA) from the blood sample taken at enrolment (Panbio Dengue IgG Indirect ELISA) [23].

Confirmation of dengue infection and serotype in suspected cases

Confirmation and type of DENV infection were determined from the blood samples taken during unscheduled visits for SDCs. Assays for the determination of DENV types were systematically performed using the reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) technique (Simplexa Dengue, Focus Diagnostics) [24]. Viral RNA extracted from serum samples was reverse-transcribed into cDNA, thereafter detected by real-time PCR. The assay amplifies four serotype-specific regions (dengue 1: NS5 gene; dengue 2: NS3 gene; dengue 3: NS5 gene; dengue 4: capsid gene) that allow discriminating the serotypes.

A rapid immunochromatographic test (SD Bioline Dengue Duo), consisting of a one-step assay designed to detect both dengue virus NS1 antigen and differential IgG/immunoglobulin M (IgM) antibodies to dengue virus, was primarily used to provide prompt laboratory results to the physician and the subject [25].

Due to the unavailability of the combined test, a one-step sandwich format microplate enzyme immunoassay (Platelia Dengue NS1 Ag) for the qualitative or semi-quantitative detection of dengue virus NS1 (NS1-antigen ELISA) [26] was performed in combination with IgG and IgM capture by ELISA (Panbio) [23].

Case definitions

A SDC was considered in the presence of body temperature ≥38.0°C within the past eight days lasting from 36 hours to seven days, potentially accompanied by other dengue symptoms (see S2 Appendix). The association of symptoms with a potential dengue episode was based on physician’s differential diagnosis.

A SDC presenting at the health care facility within five days following the symptom onset was defined as an early presenter. A SDC presenting at the health care facility six days or more after the onset of symptoms was defined as a late presenter.

Seven consecutive calendar days with body temperature <38.0°C, in the absence of antipyretic medication, were required to differentiate two episodes of suspected dengue [6].

A virologically confirmed symptomatic dengue case was an SDC confirmed by either RT-qPCR or NS1 antigen detection or both.

A probable symptomatic dengue case was defined as an SDC for which the RT-qPCR was either negative or not performed, had a negative or undetermined NS1 but (i) had a positive anti-DENV IgM text, or (ii) the anti-DENV IgG capture test was positive.

Dengue cases with warning signs had at least one aggravated symptom (see S2 Appendix).

Severe dengue had at least one severe symptom (see S2 Appendix).

Statistical methods

Sample size calculation was performed for the whole study that should have included four study sites. Calculation assumed an annual dropout rate of 5% and targeted the enrolment of about 1,750 subjects across the four centers. In Zapopan, approximately 300 to 500 subjects were to be recruited. The target study population aimed at including between 30% and 50% of adults.

Statistical analysis was performed on all eligible participants with available data. Demographic characteristics (age at Visit 1, gender and the number of participants enrolled per household) were summarized using descriptive statistics. The incidence proportions were calculated during the study period for the RT-qPCR confirmed, the virologically confirmed, and the probable symptomatic dengue cases. These proportions were also estimated by generalized estimating equations logistic regression model accounting for the clustering effect (the households). The 95% confidence interval (CI) accounting for the clustering effect was computed for all estimated incidence proportions. However, if the estimated design effect was less than 1, then the classical logistic regression model not accounting for the clustering effect was used to estimate the incidence proportion and 95% CI (see S2 Appendix for the definition of design effect). The clinical symptoms reported during SDCs were tabulated. The proportion of subjects with a DENV antibody IgG positive result at enrolment and accompanying 95% CI were estimated by age category using the same methodology as for the incidence proportions. All statistical analyses were performed using the statistical analysis systems (SAS) version 9.4.

Results

Demographics

A total of 350 individuals from 87 households were enrolled, located mainly in the municipalities of Zapopan (69.4%) and Guadalajara (21.3%); other less frequent municipalities were Tlaquepaque, Tonalá, Tlajomulco, and Chapala (Jalisco, Mexico). A total of 344 subjects completed the study; five (1.4%) were lost to follow-up and one participant died of causes unrelated to the study. The number of subjects enrolled per household ranged from one to seven (median: 4; interquartile range [IQR]: 4–4). Of the 350 enrolled subjects, 184 (52.6%) were women, 188 (53.7%) were under 18 years of age and the remaining 162 (46.3%) participants were adults up to 50 years of age (Table 1).

Table 1

Demographics and households’ characteristics.

Characteristics	Number of subjects, n (%)
Total number of enrolled subjects	350 (100)
Gender
Female	184 (52.6)
Male	166 (47.4)
Age groups
6–12 months	2 (0.6)
1–4 years	47 (13.4)
5–8 years	43 (12.3)
9–17 years	96 (27.4)
18–50 years	162 (46.3)
Age at enrolment
Mean (SD), years	20.1 (13.99)
Median (IQR), years	15.0 (8.0–32.0)
Min–Max	9 months–50 years
	Number of households, n’ (%)
Total number of enrolled households	87
Number of participants enrolled per households
1	1 (1.1)
2	5 (5.7)
3	12 (13.8)
4	49 (56.3)
5	15 (17.2)
6	3 (3.4)
7	2 (2.3)

n, number of subjects; n’, number of households; SD, standard deviation; IQR, interquartile range.

Serostatus at enrolment

Overall, 68 (19.4%) study participants were anti-DENV IgG positive at enrolment (Table 2). The highest anti-DENV IgG positivity rate (27.2%) was observed in the 18–50 years age group.

Table 2

Proportion of subjects with anti-DENV IgG positive result at enrolment (ATP cohort).

	Number of subjects, n (%)	Proportions estimated from GEEa, % (95%CIb)
Age groups
6–12 months (N = 2)	0 (0.0)	-
1–4 yearsc (N = 47)	3 (6.4)	6.4 (2.1–18.0)
5–8 years (N = 43)	5 (11.6)	11.8 (4.5–27.5)
9–17 years (N = 95)	16 (16.8)	16.6 (9.8–26.6)
18–50 years (N = 160)	44 (27.5)	27.2 (19.8–36.2)
Overall (N = 347)	68 (19.6)	19.4 (14.5–25.6)

DENV, dengue virus; IgG, immunoglobulin G; N, total number of subject tested; n, number of subjects with positive results; ATP, according-to-protocol.

a Proportions estimated from generalised estimating equations (GEE) logistic regression model taking the clustering effect (the households) into account.

b 95%CI = 95% confidence interval based on the robust variance estimate from the GEE model.

c % = (n/N) X 100 and 95%CI = Wald CI as the design effect is ≤1.

Virological endpoints

A total of 28 unscheduled visits were reported from July 2016 to September 2018, among which 18 were assessed as SDCs and further evaluated. Among the ten other unscheduled visits, six subjects had upper respiratory tract infections, two had gastrointestinal infections, one had urinary tract infection, and the last subject had influenza and urinary tract infection. None of the SDCs were hospitalized. Among the 18 SDCs, one subject was IgG positive at enrollment. Sixteen subjects were early presenters, and two subjects were late presenters. Five suspected cases were virologically confirmed, either by both RT-qPCR and dengue NS1 antigen assay (n = 4) or only by dengue NS1 antigen assay (n = 1). All four RT-qPCR-confirmed cases were serotyped as DENV-1. The remaining 13 SDCs were tested negative by RT-qPCR for all 4 DENV types, by NS1, by DENV IgG, and by DENV IgM. Three of the five virologically confirmed symptomatic dengue cases occurred during the dengue season (July to October), the other two were reported in December 2016 and November 2017, outside the dengue season. Two cases concomitantly occurred in the same household.

The overall incidence proportion of RT-qPCR- and virologically confirmed symptomatic dengue cases over the 27-month study period was 1.1% (95% CI 0.4–3.0) and 1.4% (95% CI 0.5–3.8), respectively. No probable SDCs were reported.

Clinical presentations

Among all five virologically confirmed cases, only one presented with at least one warning sign for dengue as defined by the World Health Organization [5]. Symptoms reported for the virologically confirmed symptomatic dengue cases and the other SDCs are presented in Table 3. All confirmed symptomatic dengue cases presented the following main signs of dengue: fever, headache, retroorbital pain, and myalgia. Three out of the five confirmed cases and ten out of the 13 non-confirmed SDCs had at least one digestive symptom. Two of the confirmed dengue cases and seven of the cases with no virological nor serological evidence of dengue infection had at least one respiratory symptom. Only one non-confirmed SDC had hemorrhagic manifestation (gingival bleeding and epistaxis). No severe cases were reported, and all patients recovered from their SDC with no complications.

Table 3

Summary of temperature at first visit and symptoms of suspected dengue cases.

		Virologically confirmed N = 5		Non-confirmed SDC N = 13		Total N = 18
Characteristics	Categories	n	%	n	%	n	%
Axillary temperature at first visit [°C]	<37.5	3	60.0	11	84.6	14	77.8
	37.5–38.0	0	0	0	0	0	0
	38.1–38.5	2	40.0	2	15.4	4	22.2
	38.6–39.0	0	0.0	0	0.0	0	0.0
	>39.0	0	0.0	0	0.0	0	0.0
At least one main sign	Yes	5	100	13	100	18	100
Fever	Yes	5	100	13	100	18	100
Headache	Yes	5	100	13	100	18	100
Retroorbital pain [eye pain]	Yes	5	100	9	69.2	14	77.8
Myalgia	Yes	5	100	11	84.6	16	88.9
Joint pain	Yes	3	60.0	8	61.5	11	61.1
Chills	Yes	2	40.0	2	15.4	4	22.2
Rash	Yes	1	20.0	4	30.8	5	27.8
Itching	Yes	1	20.0	1	7.7	2	11.1
At least one digestive symptom	Yes	3	60.0	10	76.9	13	72.2
Abdominal pain	Yes	3	60.0	8	61.5	11	61.1
Nausea or vomiting	Yes	3	60.0	9	69.2	12	66.7
Diarrhea	Yes	2	40.0	2	15.4	4	22.2
At least one respiratory symptom	Yes	2	40.0	7	53.8	9	50.0
Cough	Yes	2	40.0	5	38.5	7	38.9
Nasal Congestion	Yes	2	40.0	4	30.8	6	33.3
Sore throat	Yes	0	0.0	2	15.4	2	11.1
Dyspnea	Yes	0	0.0	1	7.7	1	5.6
At least one hemorrhagic manifestation	Yes	0	0.0	1	7.7	1	5.6
Petechiae	Yes	0	0.0	0	0.0	0	0.0
Purpura/ecchymosis	Yes	0	0.0	0	0.0	0	0.0
Hematemesis	Yes	0	0.0	0	0.0	0	0.0
Melena/hematochezia	Yes	0	0.0	0	0.0	0	0.0
Gingival bleeding	Yes	0	0.0	1	7.7	1	5.6
Epistaxis	Yes	0	0.0	1	7.7	1	5.6
Urinary tract bleeding	Yes	0	0.0	0	0.0	0	0.0
Unusual vaginal bleeding	Yes	0	0.0	0	0.0	0	0.0
At least one other sign	Yes	3	60.0	8	61.5	11	61.1
Pallor or cool skin	Yes	0	0.0	0	0.0	0	0.0
Conjunctivitis	Yes	1	20.0	1	7.7	2	11.1
Jaundice	Yes	0	0.0	0	0.0	0	0.0
Convulsion or coma	Yes	0	0.0	0	0.0	0	0.0
Lethargy or restlessness	Yes	0	0.0	2	15.4	2	11.1
Clinical fluid accumulation	Yes	1	20.0	0	0.0	1	5.6
Dizziness	Yes	1	20.0	2	15.4	3	16.7
Thoracic pain	Yes	0	0.0	0	0.0	0	0.0
Other	Yes	3	60.0	5	38.5	8	44.4

N, total number of SDCs; n, number of SDCs with the symptom; SDC, suspected dengue case.

Safety outcomes

No SAEs related to study procedures were reported during the entire study period. One fatal outcome unrelated to the study procedures occurred during the study.

Discussion

This study was conducted to evaluate the incidence of symptomatic dengue infections among household members aged 6 months to 50 years in Jalisco, the Mexican state having reported the highest dengue incidence (141.6/100,000) in 2019 [13]. The active weekly surveillance conducted from July 2016 to September 2018 detected 18 suspected dengue cases among which five were virologically confirmed.

The number of virologically confirmed dengue cases was close to the number expected at study design, yielding an incidence proportion of 1.4%. Moreover, the overall seroprevalence in this community (19.4%; 95% CI 14.5–25.6) was close to estimates from a cross-sectional study conducted in children aged 6–17 years old from 22 endemic states in Mexico in which the seroprevalence in seven clustered states including Jalisco was 13.3% (95% CI 9.0–19.2) [27]. This suggests that an important proportion of the population could be naïve to dengue, even in the adult population where the highest seroprevalence was observed. The trend of increased seroprevalence with age is also consistent with the results of the cross-sectional study [27]. Moreover, the serotyping results from the four RT-qPCR-confirmed cases were in accordance with the serotypes observed by the Mexican surveillance system of dengue as DENV-1 was almost exclusively detected in cases reported in Jalisco in 2016 and 2017 [15, 16]. However, 2019 reports have shown a dramatic increase in DENV-2 as well as in the incidence of dengue in Jalisco [13].

The findings presented here contribute to the body of evidence about the burden of dengue in Jalisco, an area where recent changes in incidence and epidemiology have been reported [13]. As for the assessment of influenza A and B burden in Mexico [28], our surveillance approach was effective in detecting symptomatic dengue cases and showed that an important proportion of the studied population was naïve to dengue. Moreover, this setting would be appropriate for the development of prophylactic dengue vaccines as better characterizing the serostatus of the population and the yearly incidence of symptomatic dengue cases are important pre-requisite when planning vaccine efficacy trials and estimating the sample size of these trials.

Limitations

The recruitment process of participants by flyers provided by healthcare workers and the inclusion of multiple members from the same household constitute a selection bias. Moreover, the high proportion of weekly surveillance contacts made by telephone poses a risk of response bias. Another major limitation of our study is also related to the surveillance strategy, focusing on the acute febrile illness of at least two days of duration. The use of antipyretics or transient fever lasting less than two days may have hidden some potential dengue cases. Moreover, focusing on a clinical presentation that is common to many illnesses decreases the specificity of surveillance. As around three-quarters of dengue infections are asymptomatic, it is expected that overall dengue incidence was underestimated [5, 29]. An additional blood sample taken at study conclusion could have shed some light on the proportion of asymptomatic dengue infections that have occurred during the study conduct. Finally, atypical presentations of dengue have been described and may also have resulted in an underestimation of symptomatic cases [30, 31].

Conclusion

Active surveillance was effective in detecting symptomatic dengue cases. However, screening programs are needed to further identify the proportion of asymptomatic viremic infections and describe their contribution to the global epidemiology and transmission of dengue in Mexico. Improved surveillance may help understanding the changes in clinical presentations of dengue infection and assessing more accurately the burden of dengue. Nevertheless, current detection of incident cases may be sufficient for the development of efficient prevention strategies. Fig 2 provides a plain language summary of the findings of this study.

Fig 2

Plain language summary.

Socio-demographic questionnaire.

(PDF)

Click here for additional data file.

Supplementary methods.

Cases definitions and sample size calculation.

(DOCX)

Click here for additional data file.

18 Sep 2020

PONE-D-20-24564

Burden of dengue illness in Mexican people aged 6 months to 50 years old: results from a prospective, cohort study conducted in the Zapopan community

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If your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement.

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[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: I Don't Know

Reviewer #3: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: First i would like to thank the authors for this really magnificent work conducted as overall it is well done

1/ the title should be changed from zapopan community ---> Jalisco state since we included other municipality other than zapopan

2/ I would appreciate if the authors mention some statistics about dengue in those <6 months worldwide or in Mexico then justify why that population was not included here

3/ For method please report the exact date of beginning and finishing the study and also precise exact duration was it 2 years of 27 months (some parts written 2 years other 27 months)

4/ The participants recruitment seems some kind of biased (selection bias) as they are people who go frequently to hospitals (contact with doctor nurse..) and are mostly from same households ( families recruited each other )

--> Flyers process not elaborated , please precise the percentage of participants recruited by each promoter, by flyers, by other families , where no other procedure possible (radio, TV announcement maybe...)

---> the selection bias should be mentioned in limitations

5/ The Socio-demographic information, medical and vaccination histories , physical exam ---> how were they done , any standardized method or guideline like a questionnaire maybe ? if so please provide a copy of it as supplementary material

6/ Weekly surveillance contact by telephone or in person are two different methods with very different results as in person is more reliable else there is risk of response bias

--> give percentage of those followed by telephone and those by in person and how was that performed any standard questionnaire for that ?

---> mention response bias in limitations of the study

7/ The unscheduled visits apart from the 18 suspected cases SDC , what were the reasons for the other unscheduled cases ?(10 visits)

8/ The death case what was the age and the cause of death ?

9/ The confirmed cases or suspected : were all of them followed by in person visits or by telephone ?

10/ Early presenter and late presenter , this classification was not used in the results or the table for fever , so when was the fever reported for the different cases ? when did they report to the hospital ? ----> a timeline is appreciated

*else what are their clinical history (any chronic disease explaining their susceptibility to the disease) , their ages ?

11/ Were cases aware of the mosquito contact ? where did the contact happen ? the regions of the cases ?

12/ Serious adverse events SAE how were there defined ? any list of it ? how were they collected ? questionnaire ? physical exam ?

13/ Please include in the results that ''no severe cases' were reported'' and that ''all patients conditions did progress well with no complications''

14/ Please include in the method part a reference to STORBE checklist as justify the choice

Reviewer #2: I would like to give some suggestions for this paper.

-Throughout the paper: the paper should be consistent about the follow-up time (2 years or 27 months?). Also, there was no information on primary and secondary infections.

ABSTRACT - Conclusion: use a more specific word instead of “the surveillance approach.”

INTRODUCTION

-Paragraph 2: the paper should mention previous dengue outbreaks/epidemics.

METHODS

-The paper may mention the rationale of why the age range 6 months to 50 years was selected.

-The authors should describe how to identify suspected cases, such as mentioning the symptoms associated.

-Can the authors explain why the IgM test was not done?

RESULTS

-Clinical presentations: the authors should write more for this section. Many data from Table 1 should have been pointed out in this section, especially for the categories that had 0%.

DISCUSSION

-The first sentence of the Discussion should have the statistic for the incidence in 2019.

-Line 279 - 280: the paper should give the specific statistic (the incidence of DENV-1 compared to the others, how many percent increased of DENV-2 in 2019). Are there any reasons why the incidence dramatically increased in 2019?

-The paper should give a statistic about the population in Zapopan or neighboring municipalities (the living areas of participants) and discuss how their experimental results can represent the whole population.

-Line 286: I think the authors should explain more how their surveillance approach can help the development of vaccines.

-Are there any surveillance approaches prior to this study? If no, the authors should say that this is the first surveillance approach in the country and explain the significance of their study. If yes, the authors should compare their approach with previous strategies and explain the novelty/effectiveness of their approach.

TYPOS/GRAMMARS

-Write the full name of the test (such as reverse-transcriptase quantitative polymerase chain reaction) only the first time the word appears in the paper.

-Pay attention to format the name of genes (italic, lower-case for genes in the virus?)

-Some sentences need to be revised. The authors may need people who use English fluently to help improve the manuscript.

-The word “Though” at the beginning of the sentences should be replaced by “However”

Reviewer #3: The study is well conducted, fits the journal scope, and has a clear scientific message. However, I’d like to suggest these comments to increase the clarity of some points in the study:

1- Title: the authors in their paper described the disease incidence and not burden, which is completely different. I recommend replacing the word “Burden” with “Incidence” in the title. For more information about "Disease burden" concept, authors can search on WHO or CDC websites in addition to reading some articles discussing this concept.

2- Abstract: the sentence “The 13 remaining suspected cases were not confirmed” may mislead the readers that the study conductors didn’t perform RT-qPCR or NS1 assays for these 13 participants. So, it better to state clearly that their results were negative by these assays.

3- Introduction: it covers a good background for the topic, is well written, and the aim was described clearly. However, the section about vaccination [line 107 to line 115 on page 6 of 23] is clearly out of the topic and I think it should be removed.

4- Methods: it’s organized and clear. But additional clarification for these points should be added:

- Is there a justification for choosing this age range? I mean why not people older than 50.

- This sentence “Seven consecutive calendar days with body temperature <38.0°C, in the absence of antipyretic medication, were required to differentiate two episodes of SDC” should be supported with a reference or guideline.

- The first paragraph of statistical methods [line 203 to line 207 on pages 9 and 10 of 23] is confusing and should be re-written.

5- Results: in table 2, the results of serostatus were presented for only 247 out of 250 enrolled participants (the results of three participants were missed). As the blood samples were taken at the enrollment visit from all participants, the cause of missing for these three samples should be described in the results section.

6- ِAbout data sharing policy: you clearly stated that the data will be available for research proposals approved by an independent review committee if they submit proposals to (www.clinicalstudydatarequest.com) website, however, you didn't provide a clear ID/URL for your data in this website's database which is obligatory according to the journal policy.

**********

6. 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: Yes: Nacir Dhouibi

Reviewer #2: Yes: Anh Phuc Nguyet Nguyen

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

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1 Dec 2020

Dear Editor,

We would like to thank you for the consideration of our manuscript entitled “Burden of dengue illness in Mexican people aged 6 months to 50 years old: results from a prospective, cohort study conducted in the Zapopan community” (PONE-D-20-24564). We also would like to extend our thanks to the reviewers for their careful review of the manuscript.

We have addressed all comments from the reviewers below and revised the manuscript using track-changes. The line numbers used in the responses refers to the line numbers in the track-changes version. We believe that these revisions have helped us to improve our manuscript.

Additionally, please find hereafter the amended disclosure sections:

Funding statement:

GlaxoSmithKline Biologicals SA funded this study (NCT02766088/GSK study identifier: 200318). GlaxoSmithKline Biologicals SA also provided support in the form of salaries for authors GH, SD, MDB and AG. GlaxoSmithKline Biologicals SA was involved in all stages of study conduct, including study design, data collection and analysis; GlaxoSmithKline Biologicals SA also covered all costs associated with the development and publication of this manuscript.

Competing Interests Statement:

GlaxoSmithKline Biologicals SA (GSK) funded this study and covered all costs associated with the development and publication of this manuscript. GH, SD, MDB and AG are employees of the GSK group of companies. SD and AG hold shares in the GSK group of companies. RD was an employee of the GSK group of companies at the time of the study. GAT received payments from the GSK group of companies, as part of the multi-center study. GH, SD, MDB, AG and GAT declare no other financial and non-financial relationships and activities. GIN declare no financial and non-financial relationships and activities and no conflicts of interest.

This does not alter our adherence to PLOS ONE policies on sharing data and materials, but please note that the anonymized data is only available upon request due to privacy reasons regarding patient’s privacy and sensitive data.

We look forward to future correspondence regarding our submission and are more than happy to provide further information on any questions or comments you may have.

With kind regards,

Melanie de Boer

GSK

Email melanie.x.de-boer@gsk.com

Journal's comment

"1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at:

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf"

Response: We have revised the main text, placement of tables and figure captions as well as the title page.

"2. We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For information on unacceptable data access restrictions, please see http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions.

In your revised cover letter, please address the following prompts:

a) If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially identifying or sensitive patient information) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent.

b) If there are no restrictions, please upload the minimal anonymized data set necessary to replicate your study findings as either Supporting Information files or to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. Please see http://www.bmj.com/content/340/bmj.c181.long for guidelines on how to de-identify and prepare clinical data for publication. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories.

We will update your Data Availability statement on your behalf to reflect the information you provide."

Response: As explained in the previously provided data sharing statement, the data set is only available upon request because of privacy reasons regarding study participants. Indeed, the data contain potentially identifying or sensitive patient information. The anonymized data is available to anyone upon request on https://www.clinicalstudydatarequest.com/ (instructions on how to request the data is available directly on the link). The study identifier is 200318.

3. We note that one or more of the authors have an affiliation to the commercial funders of this research study, GSK.

a. Please provide an amended Funding Statement declaring this commercial affiliation, as well as a statement regarding the Role of Funders in your study. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. You can update author roles in the Author Contributions section of the online submission form.

Please also include the following statement within your amended Funding Statement.

“The funder provided support in the form of salaries for authors [insert relevant initials], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.”

If your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement.

b. Please also provide an updated Competing Interests Statement declaring this commercial affiliation along with any other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc.

Within your Competing Interests Statement, please confirm that this commercial affiliation does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: ""This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests). If this adherence statement is not accurate and there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.

c. Please include both an updated Funding Statement and Competing Interests Statement in your cover letter. We will change the online submission form on your behalf.

Response:

a. Amended funding statement: “

GlaxoSmithKline Biologicals SA funded this study (NCT02766088/GSK study identifier: 200318). GlaxoSmithKline Biologicals SA also provided support in the form of salaries for authors GH, SD, MDB and AG. GlaxoSmithKline Biologicals SA was involved in all stages of study conduct, including study design, data collection and analysis; GlaxoSmithKline Biologicals SA also covered all costs associated with the development and publication of this manuscript.”

The role of the authors as mentioned in the ‘author contributions’ section is correct and accurately depicts the role of each author in the study.

“RD, GIN and AG participated to the conception and design of the study. GAT, RD, GIN and MDB contributed to the acquisition of data. GAT, RD, MDB and SD participated to the analysis and interpretation of data. All authors revised the article critically for important intellectual content and provided final approval of the submitted version.”

b. The competing Interests Statement already discloses the financial relationships of the authors with the funder. We have amended it to include the funder’s role and the adherence statement with justification on data access restriction.

“GlaxoSmithKline Biologicals SA (GSK) funded this study and covered all costs associated with the development and publication of this manuscript. GH, SD, MDB and AG are employees of the GSK group of companies. SD and AG hold shares in the GSK group of companies. RD was an employee of the GSK group of companies at the time of the study. GAT received payments from the GSK group of companies, as part of the multi-center study. GH, SD, MDB, AG and GAT declare no other financial and non-financial relationships and activities. GIN declare no financial and non-financial relationships and activities and no conflicts of interest.

This does not alter our adherence to PLOS ONE policies on sharing data and materials, but please note that the anonymized data is only available upon request due to privacy reasons regarding patient’s privacy and sensitive data.”

4. Please amend either the abstract on the online submission form (via Edit Submission) or the abstract in the manuscript so that they are identical.

Response: Both abstracts should now be identical.

5. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information

Response: We have added captions for the Supplementary Appendix 1 and 2 (lines 555-557) and revised the supplementary files accordingly.

6. Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please delete it from any other section.

Response: We have moved the ethics statement in the method section (lines 152-165).

Reviewer #1

1/ the title should be changed from zapopan community ---> Jalisco state since we included other municipality other than zapopan

Response: We have revised the title to “Incidence of dengue illness in Mexican people aged 6 months to 50 years old: results from a prospective, cohort study conducted in the Jalisco state”

2/ I would appreciate if the authors mention some statistics about dengue in those <6 months worldwide or in Mexico then justify why that population was not included here

Response: This epidemiological study aimed at enrolling a population in the age range that would be considered subsequently for an efficacy trial of a candidate vaccine. It is unlikely that infants younger than 6-month old would be enrolled in such trials for the reasons explained below.

In dengue endemic countries, most infants have dengue maternal antibodies at birth. The titers progressively decline in the first months of life. Maternally acquired DENV-specific antibodies play a dual role in infants during the first year of life: they confer protection at birth, and then they decline to a lower level capable of increasing the risk of severe DENV infection through antibody-dependent enhancement.

In addition, maternal antibodies may interfere with the response to primary infant vaccination (as observed for instance for measles or pertussis). There seems to be little opportunity for an immunogenic vaccine response among infants before 6 months of age.

We did not amend the manuscript with this rationale as we were asked to keep the text about vaccination to a minimum.

3/ For method please report the exact date of beginning and finishing the study and also precise exact duration was it 2 years of 27 months (some parts written 2 years other 27 months)

Response: We have added clarifications about the 27-month study period and harmonize throughout the text. Please note that the study lasted 27 months but that the follow-up period for each enrolled subject was planned for 24 months. Exact date of study start and conclusion were added in the methods (lines 144-146).

4/ The participants recruitment seems some kind of biased (selection bias) as they are people who go frequently to hospitals (contact with doctor nurse..) and are mostly from same households ( families recruited each other )

--> Flyers process not elaborated , please precise the percentage of participants recruited by each promoter, by flyers, by other families , where no other procedure possible (radio, TV announcement maybe...)

---> the selection bias should be mentioned in limitations"

Response: We have added the number families contacted by the promoters and the proportion of patients recruited by each promoter (lines 170-171). We have added the selection bias in the limitation section (lines 364-366).

5/The Socio-demographic information, medical and vaccination histories, physical exam ---> how were they done, any standardized method or guideline like a questionnaire maybe? if so please provide a copy of it as supplementary material.

Response: Socio-demographic information were collected through a questionnaire in Spanish. We have added the information in the methods (lines 186-187) and provided the questionnaire as supplementary appendix 1.

6/ Weekly surveillance contact by telephone or in person are two different methods with very different results as in person is more reliable else there is risk of response bias

--> give percentage of those followed by telephone and those by in person and how was that performed any standard questionnaire for that?

---> mention response bias in limitations of the study "

Response: We have included the percentage of telephone contact (95%) and in person visits (5%) in bracket in the methods (line 194) and added a sentence about the risk of response bias (lines 366-367).

7/ The unscheduled visits apart from the 18 suspected cases SDC, what were the reasons for the other unscheduled cases? (10 visits)

Response: We have added as sentence in the "Virological endpoints" section (lines 299-301)

8/ The death case what was the age and the cause of death?

Response: The subject who died belonged to the 9-17 years group. As the death was not related to the study, this information and the cause of death are not provided to the reader.

9/ The confirmed cases or suspected: were all of them followed by in person visits or by telephone ?

Response: They were all followed in person. We have added lines 199-197.

10/ Early presenter and late presenter, this classification was not used in the results or the table for fever , so when was the fever reported for the different cases ? when did they report to the hospital ? ----> a timeline is appreciated

*else what are their clinical history (any chronic disease explaining their susceptibility to the disease) , their ages ? "

Response: We have added the numbers of early and late presenters (lines 303-304). However, we did not collect clinical history or chronic conditions for SDCs.

11/ Were cases aware of the mosquito contact? where did the contact happen? the regions of the cases ?

Response: Questions about mosquito contact were not asked to the subjects attending an ad-hoc visit. Regions of the cases is related to the study site location and enrollement criteria, i.e. Zapopan or neighboring municipalities (Jalisco state, Mexico), as indicated in the methods.

12/ Serious adverse events SAE how were there defined? any list of it? how were they collected? questionnaire? physical exam?

Response: Only SAEs following blood drawn were considered as related to the study procedure. Definition of all SAEs was added to the methods (lines 189-192).

13/ Please include in the results that ''no severe cases' were reported'' and that ''all patients conditions did progress well with no complications''

Response: We have added the recommended text to the "Clinical presentation" section (lines 325-326).

14/ Please include in the method part a reference to STORBE checklist as justify the choice

Response: We have added the reference to the STROBE checklist in the ethical statement that now lies in the Methods (lines 163-165).

Reviewer #2

Throughout the paper: the paper should be consistent about the follow-up time (2 years or 27 months?). Also, there was no information on primary and secondary infections.

Response: The 27-month period is for the study duration. Follow-up of each subject was 24 months. We have clarified this in the methods (lines 144, 184). We have also added the information about IgG status of the SDCs in the "Virological endpoints" section (lines 302-303).

ABSTRACT - Conclusion: use a more specific word instead of “the surveillance approach .”

Response: Changed to "Community-based active surveillance"

"INTRODUCTION

-Paragraph 2: the paper should mention previous dengue outbreaks/epidemics. "

Response: We have added a brief history of dengue epidemics in the Americas, focusing on Mexico (lines 80-88).

"METHODS

-The paper may mention the rationale of why the age range 6 months to 50 years was selected . "

Response: This epidemiological study aimed at enrolling a population in the age range that could be subsequently considered for an efficacy trial of a candidate vaccine. In dengue endemic countries, most infants have dengue maternal antibodies at birth, that protect them from early dengue infection. However, maternal antibody levels wane over few months. Children younger than 6-month old are thus unlikely to be considered for vaccination. In addition, maternal antibodies may interfere with the response to primary infant vaccination (as observed for instance for measles or pertussis).

The upper age limit is also related to the potential vaccine trial, limiting our assessment to age groups in which most individuals are healthy and immunocompetent. We broadly targeted adolescents and young adults as the incidence of dengue was shown to peak in the age range of 10 to 20 years in Mexico.

We did not amend the manuscript with this rationale as we were asked to keep the text about vaccination to a minimum.

The authors should describe how to identify suspected cases, such as mentioning the symptoms associated.

Response: This is already presented in the first paragraph of the case definitions (lines 224-228) and complemented by Supplementary Appendix 2.

Can the authors explain why the IgM test was not done?

Response: IgG/IgM test was done and used to assess suspected dengue cases. We have amended lines 132-135 to clarify this.

"RESULTS

-Clinical presentations: the authors should write more for this section. Many data from Table 1 should have been pointed out in this section, especially for the categories that had 0%. "

Response: We have added few sentences about the main observations from table 3 in the "clinical presentation" section. However, we believe that describing the null categories has less interest for the reader.

"DISCUSSION

-The first sentence of the Discussion should have the statistic for the incidence in 2019 ."

Response: We have added the values presented in the introduction.

Line 279 - 280: the paper should give the specific statistic (the incidence of DENV-1 compared to the others, how many percent increased of DENV-2 in 2019). Are there any reasons why the incidence dramatically increased in 2019?

Response: As our study ended in September 2018, we would prefer to not discuss 2019 data in the results. The increased incidence is mentioned in the introduction as background information about the study location. We can however mention that the incidence of dengue globally is thought to increase in the future, most likely as a collateral effect of global warming. We have nevertheless added a sentence about the impressive increase of DENV-2 cases between 2017 and 2019 (lines 98-100) in Mexico.

The paper should give a statistic about the population in Zapopan or neighboring municipalities (the living areas of participants) and discuss how their experimental results can represent the whole population.

Response: Statistics about dengue in Jalisco and Mexico are provided in the discussion. As we did not calculate incidence, our observations could not be discussed in the light of the available data for dengue incidence in the state of Jalisco. We nevertheless believe that our observations are representative of the population living in Jalisco.

Line 286: I think the authors should explain more how their surveillance approach can help the development of vaccines.

Response: We have completed the sentence to clarify the statement (lines 360-363).

Are there any surveillance approaches prior to this study? If no, the authors should say that this is the first surveillance approach in the country and explain the significance of their study. If yes, the authors should compare their approach with previous strategies and explain the novelty/effectiveness of their approach.

Response: Such surveillance approach was already used for the assessment of influenza A and B co-circulation and burden in Mexico. We have added this information and the reference (28) in lines 356-357.

"TYPOS/GRAMMARS

-Write the full name of the test (such as reverse-transcriptase quantitative polymerase chain reaction) only the first time the word appears in the paper.

-Pay attention to format the name of genes (italic, lower-case for genes in the virus?)

-Some sentences need to be revised. The authors may need people who use English fluently to help improve the manuscript.

-The word “Though” at the beginning of the sentences should be replaced by “However”

Response: We have revised the text and corrected it where needed.

Reviewer #3

1- Title: the authors in their paper described the disease incidence and not burden, which is completely different. I recommend replacing the word “Burden” with “Incidence” in the title. For more information about "Disease burden" concept, authors can search on WHO or CDC websites in addition to reading some articles discussing this concept.

Response: We have revised the title to “Incidence of dengue illness in Mexican people aged 6 months to 50 years old: results from a prospective, cohort study conducted in the Jalisco state”

2- Abstract: the sentence “The 13 remaining suspected cases were not confirmed” may mislead the readers that the study conductors didn’t perform RT-qPCR or NS1 assays for these 13 participants. So, it better to state clearly that their results were negative by these assays.

Response: We have corrected the sentence as suggested by the reviewer.

3- Introduction: it covers a good background for the topic, is well written, and the aim was described clearly. However, the section about vaccination [line 107 to line 115 on page 6 of 23] is clearly out of the topic and I think it should be removed.

Response: We have reduced the paragraph but kept some information about the vaccine as the present study aims at supporting the design of future efficacy study for novel vaccines.

"4- Methods: it’s organized and clear. But additional clarification for these points should be added:

- Is there a justification for choosing this age range? I mean why not people older than 50. "

Response: This epidemiological study aimed at enrolling a population in the age range that could be subsequently considered for an efficacy trial of a candidate vaccine. In dengue endemic countries, most infants have dengue maternal antibodies at birth, that protect them from early dengue infection. However, maternal antibody levels wane over few months. Children younger than 6-month old are thus unlikely to be considered for vaccination. In addition, maternal antibodies may interfere with the response to primary infant vaccination (as observed for instance for measles or pertussis).

The upper age limit is also related to the potential vaccine trial, limiting our assessment to age groups in which most individuals are healthy and immunocompetent. We broadly targeted adolescents and young adults as the incidence of dengue was shown to peak in the age range of 10 to 20 years in Mexico.

We did not amend the manuscript with this rationale as we were asked to keep the text about vaccination to a minimum.

This sentence “Seven consecutive calendar days with body temperature <38.0°C, in the absence of antipyretic medication, were required to differentiate two episodes of SDC” should be supported with a reference or guideline.

Response: Study protocol was developed following the WHO guidelines for diagnosis of dengue. We have repeated the ref 6 in line 234.

The first paragraph of statistical methods [line 203 to line 207 on pages 9 and 10 of 23] is confusing and should be re-written.

Response: We have revised the sentence for clarity.

5- Results: in table 2, the results of serostatus were presented for only 247 out of 250 enrolled participants (the results of three participants were missed). As the blood samples were taken at the enrollment visit from all participants, the cause of missing for these three samples should be described in the results section.

Response: There were 350 subjects enrolled in Mexico, among whom 68 were positive, 279 were negative, and 3 had equivocal results. Equivocal results mean that the level of measured dengue IgG antibodies were not high enough to conclude of a dengue infection or not. The 3 subjects with equivocal lab results were excluded from the stat analysis in the ATP cohort.

6- ِAbout data sharing policy: you clearly stated that the data will be available for research proposals approved by an independent review committee if they submit proposals to (www.clinicalstudydatarequest.com) website, however, you didn't provide a clear ID/URL for your data in this website's database which is obligatory according to the journal policy.

Response:

We have added details and explanation on data request in the response letter as requested by the journal. The URL provided leads directly to the instructions on how to request a data set and the study ID is 200318.

Submitted filename: DeAntonio et al - response letter.docx

Click here for additional data file.

3 Mar 2021

PONE-D-20-24564R1

Incidence of dengue illness in Mexican people aged 6 months to 50 years old: results from a prospective, cohort study conducted in the Jalisco state

PLOS ONE

Dear Dr. de Boer,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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PPlease modify the manuscript with the grammar suggestions provided by the reviewers.

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Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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Reviewer #4: All comments have been addressed

**********

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**********

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Reviewer #1: I Don't Know

Reviewer #2: I Don't Know

Reviewer #3: Yes

Reviewer #4: Yes

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Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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Reviewer #1: (No Response)

Reviewer #2: Below are some suggestions about grammars to help improve this manuscript

-Throughout the paper, the author should write either “the state of Jalisco” or simply “Jalisco” instead of “Jalisco state”

-Suggested title: “Incidence of dengue illness in Mexican people aged 6 months to 50 years old: a prospective cohort study conducted in Jalisco”

-Line 41: no space between the number 19.4 and %

-Line 46: “cases were tested negative by these assay”

-Line 49: delete the word “conduct”

-Line 70: “Worldwide, the global incidence of dengue”

-Line 85-88: these three sentences are all simple sentences and should be reworded and/or combined together

-Line 112-113: the phrase “using serotype-specific polymerase chain reaction (PCR) and anti-dengue immunoglobulin G (DENV IgG) assays” is not relevant to the previous clause in the sentence. The author may consider deleting that phrase.

-Line 124: replace the word “vaccinees” by “vaccinated people” or “vaccinated individuals”

-Line 146: 14th, 2016 and September 14th, 2018.

-Line 177: replace the word “take” by “measure”

-Line 219: the author may consider changing the phrase “As an alternative to the combined test was not available” to “Due to the unavailability of the combined test”

-Line 270: no comma between “(69.4%)” and “and Guadalajara”

-Line 271: should have a comma between “Tlajomulco” and “and Chapala”

-Line 272: should not begin a sentence by a number

-Line 355: “dengue in Jalisco, in an area where”

-Line 364: the author can consider adding a subheader called “Limitations”

-Line 380: change the word “successful” to “effective”

Reviewer #3: (No Response)

Reviewer #4: There is an issue that the authors need to clarify between lines 135 abd 147. Other than that, my comments are minor.

**********

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Reviewer #1: Yes: Nacir Dhouibi

Reviewer #2: Yes: Anh Phuc Nguyet Nguyen

Reviewer #3: No

Reviewer #4: No

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Submitted filename: PONE-D-20-24564R1_Comments due 15 Jan 2021.docx

Click here for additional data file.

30 Mar 2021

March 30, 2021

Dear Editor,

We would like to thank you for the consideration of our revised manuscript entitled “Burden of dengue illness in Mexican people aged 6 months to 50 years old: results from a prospective, cohort study conducted in the Zapopan community” (PONE-D-20-24564R1).

We have addressed all additional comments from the reviewers below and revised the manuscript using track-changes. We hope that the revised manuscript will fully meet PLOS ONE’s publication criteria.

We look forward to future correspondence regarding our submission and are more than happy to provide further information on any questions or comments you may have.

With kind regards,

Melanie de Boer

GSK

Email melanie.x.de-boer@gsk.com

Comments:

Reviewer #2

-Throughout the paper, the author should write either “the state of Jalisco” or simply “Jalisco” instead of “Jalisco state”

Reply: We made the correction throughout the text

-Suggested title: “Incidence of dengue illness in Mexican people aged 6 months to 50 years old: a prospective cohort study conducted in Jalisco”

Reply: We have modified the title as suggested.

-Line 41: no space between the number 19.4 and %

Reply: The space between numerals and the % has been removed (line 38 of the revised version with tracked changes).

-Line 46: “cases were tested negative by these assay”

Reply: We have revised the sentence as suggested (line 43 of the revised version with tracked changes).

-Line 49: delete the word “conduct”

Reply: Deleted (line 46 of the revised version with tracked changes)

-Line 70: “Worldwide, the global incidence of dengue”

Reply: We have inserted “global” in the sentence (line 67 of the revised version with tracked changes).

-Line 85-88: these three sentences are all simple sentences and should be reworded and/or combined together

Reply: We have revised the sentences to improve the flow (lines 81-87 of the revised version with tracked changes).

-Line 112-113: the phrase “using serotype-specific polymerase chain reaction (PCR) and anti-dengue immunoglobulin G (DENV IgG) assays” is not relevant to the previous clause in the sentence. The author may consider deleting that phrase.

Reply: We have deleted that part of the sentence (lines 111-112 of the revised version with tracked changes).

-Line 124: replace the word “vaccinees” by “vaccinated people” or “vaccinated individuals”

Reply: “Vaccinees” has been changed for “vaccinated individuals” (line 120 of the revised version with tracked changes).

-Line 146: 14th, 2016 and September 14th, 2018.

Reply: Corrected (lines 141-142 of the revised version with tracked changes).

-Line 177: replace the word “take” by “measure”

Reply: Replaced as suggested (line 168 of the revised version with tracked changes).

-Line 219: the author may consider changing the phrase “As an alternative to the combined test was not available” to “Due to the unavailability of the combined test”

Reply: Revised as suggested (line 208 of the revised version with tracked changes).

-Line 270: no comma between “(69.4%)” and “and Guadalajara”

Reply: Corrected (line 257 of the revised version with tracked changes).

-Line 271: should have a comma between “Tlajomulco” and “and Chapala”

Reply: Corrected (line 38 of the revised version with tracked changes).

-Line 272: should not begin a sentence by a number

Reply: Corrected (line 259 of the revised version with tracked changes).

-Line 355: “dengue in Jalisco, in an area where”

Reply: We have removed “in” before “an area where” (line 342 of the revised version with tracked changes).

-Line 364: the author can consider adding a subheader called “Limitations”

Reply: We have added the subheading for limitations (line 350 of the revised version with tracked changes).

-Line 380: change the word “successful” to “effective”

Reply: Revised as suggested (line 366 of the revised version with tracked changes).

Reviewer #4

Methods

LINES 135-136

However, due to early study termination, only two sites in the Philippines and Mexico participated in the study.

LINES 146-47

However, due to early study termination, only two sites in the Philippines and Mexico participated in the study.

Mention this sentence just once in the paper.

Reply: We have deleted the sentence from lines 146-47 (lines 149-151 of the revised version with tracked changes)

If the Philippines participated, where are the data and the analyses? Please clarify

Expound briefly on “early study termination”

Reply: As the global, pre-defined analysis could not be performed, data from the Philippines and Mexico were analyzed separately. Data and results for the Philippines are described in another paper currently under consideration for publication in Asian Biomedicine.

In December 2017, GSK notified its longstanding collaboration partners, Institution Oswaldo Cruz (Fiocruz) and the Walter Reed Army Institute of Research and the US Army Medical Materiel Development Agency (WRAIR/USAMMDA), of the intention of GSK’s Vaccines Investment Board (VIB) to deprioritize the development of the dengue purified inactivated vaccine (DPIV) candidate. This decision was made due to the scientific challenges and development risks associated with the candidate vaccine. This decision entailed the early termination of the associated observational studies sponsored by GSK.

Page 16 Top row

Conjunctivis (spelling)

Reply: Corrected to “conjunctivitis”, thank you!

Page 12 Table 1

Age groups: The authors listed 5 levels, 1-4 and 5-8 years could be combined as they did not differ much in values and that the range would be closer to 9-17 and 18-50 years

Reply: We acknowledge that 1-4 and 5-8 age groups do not differ much in values and could have been combined. However, the stratification was made as per protocol and merging both group would require an additional analysis that would take time while having little impact on our observations and the overall message of the paper.

LINE 256 household ranged from one to seven (median: 4).

The authors reported IQR in Table 1 but not in LINE 256

Reply: IQR is provided is Table 1 for the median age at enrolment. We have nevertheless added the IQR in text for the number of participants by household (lines 261-262 of the revised version with tracked changes).

LINES 247-248 incidence proportions. All statistical analyses were performed using the statistical analysis systems version 9.4. Cite the source of the system used

Reply: We have added the software, SAS, in brackets in the sentence (line 253 of the revised version with tracked changes).

LINE 233 Statistical analysis was performed on all evaluable eligible participants

What is evaluable?

Reply: We have removed evaluable and inserted “with complete data” instead. We hope that this revision clarifies the meaning of the sentence (lines 238-239 of the revised version with tracked changes).

Submitted filename: DeAntonio et al - response letter.docx

Click here for additional data file.

5 Apr 2021

Incidence of dengue illness in Mexican people aged 6 months to 50 years old: a prospective cohort study conducted in Jalisco

PONE-D-20-24564R2

Dear Dr. de Boer,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Humberto Lanz-Mendoza

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

22 Apr 2021

PONE-D-20-24564R2

Incidence of dengue illness in Mexican people aged 6 months to 50 years old: a prospective cohort study conducted in Jalisco

Dear Dr. de Boer:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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Thank you for submitting your work to PLOS ONE and supporting open access.

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on behalf of

Dr. Humberto Lanz-Mendoza

Academic Editor

PLOS ONE