Enhanced passive surveillance dengue infection among febrile children: Prevalence, co-infections and associated factors in Cameroon.

Dengue virus (DENV) causes a spectrum of diseases ranging from asymptomatic, mild febrile to a life-threatening illness: dengue hemorrhagic fever. The main clinical symptom of dengue is fever, similar to that of malaria. The prevalence of dengue virus infection, alone or in association with other endemic infectious diseases in children in Cameroon is unknown. The aim of this study was to determine the prevalence of dengue, malaria and HIV in children presenting with fever and associated risk factors. Dengue overall prevalence was 20.2%, Malaria cases were 52.7% and HIV cases represented 12.6%. The prevalence of dengue-HIV co-infection was 6.0% and that of Malaria-dengue co-infection was 19.5%. Triple infection prevalence was 4.3%. Dengue virus infection is present in children and HIV-Dengue or Dengue- Malaria co-infections are common. Dengue peak prevalence was between August and October. Sex and age were not associated with dengue and dengue co-infections. However, malaria as well as HIV were significantly associated with dengue (P = 0.001 and 0.028 respectively). The diagnosis of dengue and Malaria should be carried out routinely for better management of fever.

Introduction

Dengue is currently regarded as the most prevalent mosquito-borne viral disease worldwide [1,2] and is described as a “neglected” tropical disease by the World Health Organization [3]. Over 50% of the world’s population live in dengue endemic countries [3-5]. Malaria is the most important mosquito-borne parasitic disease. Both infections share similar clinical symptoms [2]. The true impact of dengue in most African countries is unknown due to inadequate surveillance, misdiagnosis or lack of diagnosis, and low level of reporting or no reporting at all [6]. In Cameroon, dengue fever is not screened routinely, neither in children nor in adults. There is a licensed dengue vaccine available in many countries, but safety concerns and screening requirements limit its widespread use and impact. Only accurate diagnosis and appropriate clinical management can reduce the morbidity and mortality associated to dengue fever.

Few dengue studies have been conducted in Africa in general and Cameroon in particular. The first cases of dengue were found among 2 expatriates in Cameroon (German) in 2002, with serotype 1 (DENV-1) [7]. Another study showed that the seroprevalence of dengue in Cameroon was 35.9% in 2004 [8]. A study of prevalence and distribution of Flaviviridae, Togaviridae, and Bunyaviridae arboviral infections, done on adults in rural Cameroon, showed that 12.5% of cases were due to dengue type 2 virus (DENV-2) [9]. A cross sectional survey using a random cluster sampling method conducted in 2009 reported dengue cases in Yaoundé, Douala and Garoua, in adults, with a seroprevalence higher in Douala (60.8%) probably because of the climate (hot and humid) that promote the development of the transmitting vector, Aedes [10-11], compared to 23.3% in Garoua (hot and dry) and 9%, in Yaoundé (temperate). No case of dengue hemorrhagic fever (DHF) has been reported in Cameroon.

Cameroon is a tropical country where HIV, malaria and dengue co-exist as important infectious diseases. Dengue fever has been documented in Cameroon with two serotypes of viruses (DENV-1 and DENV-2). However, few data exist on dengue fever in children, on dengue-malaria and on HIV-dengue co-infection. The natural history and clinical manifestations of co-infection with HIV and dengue has not been established.

Therefore, the objective of this study was to determine the prevalence of dengue fever, dengue-malaria and dengue-HIV co-infections and associated risk factors in a pediatric population presenting with fever in Yaounde, Cameroon.

Material and methods

Ethics statement

The study received the approval of the National Ethical Committee, authorisation n° 2013/03/086/L/CNERSH/SP) of Cameroon. Infants were enrolled only when their parent or guardian gave written proxy-consent. Older children, aged above 5 gave their assent before enrollment.

Parents and Participants were ensured of confidentiality, by attribution of codified identification numbers.

We enrolled children and infants consulting at the Centre Mère Enfant–CME in Yaoundé for fever. Children aged 6 months to 15 years, presenting with fever (axillary temperature above 37.5°C) were enrolled in the study. They were screened clinically and only those presenting with fever without known reasons (abscess, urinary infections, pulmonary infections…) were retained in the study.

A structured questionnaire (see S1 Study Questionnaire) was used to collect some relevant clinical and socio-demographic data. Three milliliters of blood were collected in an EDTA-tube from each child presenting with fever complaints. This study was carried out during the months of April to October 2015; which are rainy months with maximum transmission rate of both dengue and malaria, best for the breeding of their vectors (Anopheles and Aedes). The following tests were done:

Malaria was diagnosed using gold standard method (microscopic examination of giemsa stained thick and thin blood smears). Dengue was diagnosed using the SD Bioline NS1 Ag+Ab combo rapid test according to the manufacturer’s instructions (Standard Diagnostics, Inc) and with ELISA CAPTURE DENGUE IgM/IgG. In order to confirm current dengue infection, in case of NS1 positive test, primer specific amplification was carried out as previously described [12-13]. HIV was diagnosed using rapid tests following the national algorithm for children aged more than 18 months (SD Bioline HIV-1/2 3.0 for the first test and Ora Quick Rapid HIV-1 Antibody Test for the second test on all samples). For younger children (less than 18 months) they were diagnosed using Roche amplicor DNA PCR version 1.5 according to the instructions from the manufacturer and as described by Nkenfou [14]. Hematological tests like CD4 count and full blood count were performed. Urine analysis was done using urine stick test as described by the manufacturer. Infants presented with positive urinary test were excluded from the study.

Definitions of cases

Current or recent dengue infection was defined on the basis of a positive result for NS1 Ag and / or IgM. Past infection was defined on a basis of a positive result of IgG alone.

Statistical analysis

Analyses of data obtained from questionnaires were carried out using Microsoft Excel 2010 and the Statistical Package for Social Sciences (SPSS) program (PASW Statistics 20). The Chi square test was used to determine the significance between different variables and the level of significance was 5%.

Results

1) Socio demographic description of the study population

A total number of 349 children fulfilled the selection criteria (fever with unknown clinical cause). They were 53.3% boys and 46.7% girls, aged 6 months to 180 months with a mean age of 3.2 years. These data are presented in Table 1. Most of the children (67.9%) slept under a protective bed net and 46.9% were living in an environment with stagnant water.

Table 1

Description of the study population.

	Total	%
Sex
Male	163	46.7%
Female	186	53.3%
Age (years)
‹2	168	48.1%
2–5	101	28.9%
›5	80	22.9%
Usage of Impregnated bed nets
Yes	237	67.9%
No	112	32.09%
Presence of stagnant water
Yes	122	31.2%
No	227	46.9%

2) Clinical characterization and prevalence of infections in the study population

All children enrolled presented an axillary temperature higher than 37.5°C. The highest temperature observed was 41°C, the lowest temperature observed was 37.8°C and the average temperature was 38.9°C. A total of 44 children (12.6%) were vomiting, 25 (7.2%) had diarrhea and 40 (11.5%) had fatigue. Out of 349 enrolled children,183 were infected by plasmodium (52.7% (183/349)), among which 54 were girls and 55 were boys (see Table 2). There were 183 cases of P. falciparum and 74 cases of P. vivax. All the P. vivax were in co-infection with P. falciparum.

Table 2

Overall prevalence or seroprevalence of different infections, co-infections and symptoms among the study participants.

Infection or symptoms	Frequency	Percent
Malaria (any infection with Plasmodium)	183	52.7
Plasmodium falciparum	183	52.7
Plasmodium vivax	74	21.3
HIV	44	12.6
Dengue (Overall cases)	101	29.1
IgG dengue	93	26.8
IgM dengue	70	20.2
Vomiting*	44	12.6
Diarrhea*	25	7.2
Fatigue*	40	11.5
Current dengue + malaria + HIV**	15	4.3
Current dengue + malaria**	68	19.5
Current dengue + HIV**	21	6.0

* These symptoms are present together with fever.

** Here we consider acute dengue not past exposure.

Forty-four out of the 349 enrolled children were HIV positive (12.6%) (see Table 2). Co-infection with malaria was found among 15 participants while HIV co-infection with Dengue was found among 21 children.

Children presenting dengue-positive test for NS1 Ag, IgM, IgG and IgM or IgG Abs Rapid Diagnostic Test (RDT) were 0.2% (1/349), 20.2% (70/349), 26.8% (93/349) and 29.1% (101/349) respectively (see Table 2).

Average CD4 count was 969 cells/mm3, normal for children aged 1–15 years. Children having the lower CD4 count (240–400 CD4 cells/mm3) were more likely to be positve for malaria (20% compared to 10.8%; X2 = 0.71, p = 0.4); for HIV (40% compared to 0%; X2 = 31.08; p<0.001) and for dengue(20% compared to 5.3%; X2 = 2.96, p = 0.085).

The rate of current dengue-malaria co-infection was 19.5% (68/349) and current dengue-HIV co-infection was 6% (21/349).

Fifteen children presented positive tests for active dengue, malaria and HIV all together, representing 4.3% of the study population (see Table 2).

3) Risk factors associated with dengue and dengue co-infections in the study population

Infants sleeping under impregnated bed nets were less at risk of been infected by dengue virus (OR 2.1, 95%CI 0.12–0.39, P< 0.001) and Plasmodium (OR 2.55, 95%CI: 1.4–4, p<0.0001). Children living in stagnant water free environment were less at risk of being infected by the two pathogens (OR 8.85, 95%CI 4.6–16.7, p<0.001 and OR 2.7, 95%CI 1.7–44, P< 0.001 respectively for dengue and malaria). The peak transmission occurred during the months of August to October, corresponding to the shorter raining season in Yaoundé-Cameroon see Fig 1.

Fig 1

Seasonal distribution of dengue virus infection among children with fever attending hospitals in Yaounde.

Ig represents all cases of dengue infections (past and current infections). IgG represents cases of previous exposure to dengue. IgM represents current cases of dengue.

Sex and age were not associated with dengue or any dengue co-infections studied; see Tables 3 and 4 respectively.

Table 3

Sex distribution of the different infections (active and past) and symptoms in the study population.

	Sex		Total
	Female	Male	Total	X2, p value	OR; 95%CI
Ig dengue	54 (33.1%)	47 (25.5%)	101	2.410; 0.121	0.692; 0.435–1.102
IgG dengue	51 (31.3%)	42 (22.8%)	93	3.155; 0.076	0.650; 0.403–1.047
IgM dengue	33 (20.2%)	37 (20.1%)	70	0.001; 0.975	0.992; 0.586–1.677
P. falciparum	91 (56.5%)	92 (49.5%)	183	1.725; 0.189	0.753; 0.493–1.150
P. vivax	37 (23%)	37 (19.9%)	74	0.491; 0.484	0.832; 0.498–1.392
HIV	21 (12.9%)	23 (12.4%)	44	0.021; 0.884	0.954; 0.507–1.797
Vomiting	20 (12.3%)	24 (12.9%)	44	0.032; 0.859	1.059; 0.562–1.998
Diarrhea	10 (6.1%)	15 (8.1%)	25	0.486; 0.486	1.342; 0.586–3.076
Fatigue	15 (9.2%)	25 (13.4%)	40	1.538; 0.215	1.532; 0.778–3.018
Dengue + HIV	8 (4.9%)	13 (7%)	21 (6%)	0.665; 0.415	1.456; 0.588–3.606
Dengue + Malaria	38 (23.3%)	30 (16.1%)	68 (19.5%)	2.858; 0.091	0.633; 0.371–1.078
Dengue + Malaria + HIV	5 (3.1%)	10 (5.4%)	15 (4.3%)	1.126; 0.289	1.795; 0.601–5.366
Total	163	184	347

Note: For the ODD ratios, Male is considered as the reference. In the case of co-infections, only acute dengue was considered.

Table 4

Association between dengue (current and past), malaria and HIV.

	Dengue		Total
	No	Yes		X2; p	OR, 95% CI
	Ig dengue
Malaria-PF	115 (47.1%)	68 (67.3%)	183 (53%)	11.697; 0.001	2.311; 1.422–3.758
Malaria-PV	42 (17.2%)	32 (31.7%)	74 (21.4%)	8.877; 0.003	2.231; 1.306–3.808
HIV	25 (10.2%)	19 (18.8%)	44 (12.7%)	4.838; 0.028	2.048; 1.071–3.916
	IgG dengue
Malaria-PF	116 (46%)	67 (72%)	183 (53%)	18.453; 0.0001	3.021; 1.803–5.062
Malaria-PV	42 (16.7%)	32 (34.4%)	74 (21.4%)	12.691; 0.0001	2.623; 1.527–4.506
HIV	27 (10.6%)	17 (18.3%)	44 (12.7%)	3.598; 0.058	1.881; 0.972–3.639
	IgM dengue
Malaria-PF	132 (48%)	51 (72.9%)	183 (53%)	13.842; 0.0001	2.908; 1.632–5.180
Malaria-PV	47 (17.1%)	27 (38.6%)	74 (21.4%)	15.281; 0.0001	3.046; 1.715–5.411
HIV	29 (10.5%)	15 (21.4%)	44 (12.7%)	6.061; 0.014	2.332; 1.172–4.642
Total	244	101	345

Note: The absence of the other infection was considered as the reference.

Although no statistical significance was found between age and dengue Ab, the prevalence (IgM) and seroprevalence (IgG) increased steadily with age as shown in Fig 2. This may be reflective of cumulative exposure, underlining the fact that dengue transmission is high and endemic rather than epidemic. As well, as the child grows older, he become more independent in his movements and can pull away the bednet.

Fig 2

Diagram showing the variation of the prevalence of dengue in relation to age group.

Ig represents all cases of dengue infections (past and current infections). IgG represents cases of previous exposure to dengue. IgM represents current cases of dengue.

Table 4 shows a significant association between dengue, malaria and HIV (p <0.001). Current dengue infection was associated with Plasmodium falciparum as well as Plasmodium vivax both with p< 0.001 and with HIV with p = 0.014. Similarly, the association between co-infection of malaria as well as HIV with current dengue infections was statistically significant indicating an endemic co-existence and potential association between these infections. The possibility of a weakened immune system among pediatric patients could explain such association. Among the symptoms recorded during the consultation, diarrhea was associated with dengue, mainly the current infections (p = 0.036); see Table 5.

Table 5

Occurrence of specific symptoms among actively dengue infected compared to non-infected patients.

	IgM dengue
	No	Yes	Total	X2, p value	OR; 95% CI
Vomiting	32 (11.6%)	12 (17.1%)	44 (12.7%)	1.577; 0.209	1.584; 0.769–3.263
Diarrhea	24 (8.7%)	1 (1.4%)	25 (7.2%)	4.361; 0.036	0.153; 0.020–1.149
Fatigue	31 (11.2%)	9 (12.9%)	40 (11.5%)	0.152; 0.697	1.171; 0.530–2.588
Total	277	70	347

Discussion

The sero-prevalence and distribution of dengue has been poorly documented in Cameroon and more so in children. A total of six studies reported on dengue in Cameroon, with two including children. We found a dengue prevalence (NS1, anti-dengue IgM antibodies detection) of 20.2% and a seroprevalence of 26.8% for anti-dengue IgG antibodies. The result obtained in the present study is higher than the 9.8% previously reported by Demanou et al in Yaoundé among adults [11]. This was a cross sectional survey conducted in three main cities in Cameroon (Douala, Garoua and Yaounde), using a random cluster sampling design to document risk factors associated with anti-dengue virus Immunoglobulin G seropositivity in humans The higher rate of dengue in the present study may be explained by the higher susceptibility to infection in children and cases of mother to child transmission of dengue virus [15-16].

The primary clinical manifestation of dengue is fever, similar to that of malaria which is endemic in Cameroon [17]. Our findings showed a prevalence of 52.7% of malaria cases alone and 19.5% for malaria-dengue co-infections in our study population. The first documented cases of dengue-malaria co-infection among children in Cameroon was published in 2018 [18] with a prevalence of 12.5%. This percentage is lower than that obtained in our study. We had a proportion of 19.5% compared to 12.5% in the study of Monamele et al in 2018. The reason may be that the methodology used in the two studies for malaria diagnosis was different, we used microscopy, still considered as gold standard, while in the previous study the rapid diagnostic test was used. As well their population size of 33 malaria cases was smaller than our which was 183 cases.

Acute dengue-malaria co-infection could present more severe clinical symptoms than a single infection [19]. These findings show that there is need for more attention in case of fever to carry out a laboratory test for malaria or dengue before treatment.

Among the children enrolled, 12.6% were HIV infected. Cameroon is said to be in a generalized HIV epidemic, although with decreasing prevalence, from 5.5 to 3.4% [20-22]. Pediatric HIV is also a major public health problem although transmission is reduced due to the implementation of option B+ (Treat all HIV infected pregnant or breastfeeding mother despite CD4 count or viral load) for the prevention of mother to child transmission [23]. The percentage of HIV obtained in this pediatric study population is in agreement with the national prevalence at the time of the study. Our study identified 21 out of 349 cases of dengue-HIV co-infections, thus a prevalence of 6%. Viral coinfections are known to be more severe in infants compared to adults. Viral coinfections could even be worse especially for dengue and HIV as the two viruses share the common target cells such as CD4 positive cells [19]. Special care should then be taken while consulting HIV infected children.

Because malaria and dengue are both transmitted by the bite of insects (Anopheles and Aedes respectively), environmental conditions favoring their breeding have an impact on their transmission rate. Stagnant water provide breeding conditions for Anopheles and Aedes. Parents should make sure their children sleep under protective bed nets which are freely distributed to pregnant women by the Malaria Control Programme in Cameroon, especially children younger than 5 years and during high transmission period. Our findings showed that children sleeping under bednet were less at risk from been infected by dengue and malaria, as bednet acts as shield and protects them from insect bites. The reason being that they are less at risk of been bitten by mosquitoes, although bednets are generally thought to have less impact on dengue, due to the day biting of dengue transmitting mosquito.

Malaria and HIV may be risk factors for dengue infections or vice-versa. Malaria and dengue are transmitted by mosquitoes that multiply rapidly during rainy seasons and thus may co-occur temporally. So being exposed to malaria can be concurrent to exposure to dengue, this is why the two infections can co-exist in the same person, and one being the risk factor for another. On the other side, HIV may be a risk factor for dengue and the reciproque may apply, given that both viruses target the same cells in the body.

Conclusion

Dengue is common among Cameroonian children. Since Cameroon is endemic for malaria, fever may indicate either malaria or dengue. Clinicians should be aware of the presence of dengue in Cameroon and parents should properly use the bed net they are given to protect their children.

Management of patients with dengue fever and HIV infection should be studied, especially in infants where both viral diseases are sometimes more severe. Close monitoring may mitigate complications among HIV-infected individuals who contract dengue fever.

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27 Feb 2020

Dear Dr. Nkenfou,

Thank you very much for submitting your manuscript "DENGUE INFECTION AMONG FEBRILE CHILDREN: PREVALENCE, CO-INFECTIONS AND ASSOCIATED FACTORS" 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.

Two reviewers have provided reviews for this paper. Both reviewers highlighted that this is an interesting study on an important area that presents novel findings. However they also pointed out that the manuscript needs substantial revisions. The reviewers have provided very thorough comments to help with this revision. I echo their comments and ask the authors to thoroughly revise the manuscript inline with these comments, but also with attention throughout to consistency and clarity of meaning of terms used, ensuring the focus of the paper is clear, with results presented clearly, and that the discussion leads from the results.

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Two reviewers have provided reviews for this paper. Both reviewers highlighted that this is an interesting study on an important area that presents novel findings. However they also pointed out that the manuscript needs substantial revisions. The reviewers have provided very thorough comments to help with this revision. I echo their comments and ask the authors to thoroughly revise the manuscript inline with these comments, with attention throughout to consistency and clarity of meaning of terms used, ensuring the focus of the paper is clear, with results presented clearly, and that the discussion leads from the results.

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: This manuscript describes a cohort of acutely febrile children presenting to a single center in Yaounde, Cameroon, with the objective of describing incidence of dengue virus (DENV) infection, and associated co-infection with malaria and/or HIV among children with undifferentiated febrile illness. The authors describe the various tests used to detect DENV, malaria, and HIV. For DENV, the authors describe using the SD Bioline dengue Duo, an IgM/IgG ELISA, and PCR. However, it is not clear how they defined a case of DENV. For example, in line 92, the authors state that a positive DENV NS1 test result prompted confirmatory testing by PCR. Were samples that were NS1 positive but PCR negative not considered cases? What about discrepancies between NS1 and IgM results? This confusion is reflected later in the results.

The authors' use of the term prevalence throughout the manuscript is confusing. In the DENV literature, as well as that of most other infections, seroprevalence refers to presence of serum antibody, typically IgG, describing evidence of previous exposure/infection. The authors report anti-DENV IgG results as signifying previous infection, but the use of the term prevalence to describe both previous exposure and incident infection is confusing. Prevalence and incidence are not interchangeable.

Reviewer #2: See below

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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: The presentation of the results is confusing. A diagram of how many subjects were classified into each of the diagnostic groupings would help to clarify the information provided in Table 2. Specifically, Table 2 indicates that there were 101 cases of DENV overall, with 70 cases positive for IgM. This implies that the remaining cases were due to NS1 positivity or PCR positivity. Yet, the text (lines 139-140) states that only 1 child was positive for NS1. A footnote in Table 3 explains that the 101 cases included both previous exposure and current DENV infections. The logic behind the grouping of previous exposure, signified by positive DENV IgG, together with incident infection, signified by positive DENV IgM, into a single group (referred to as "Ig dengue" in tables 3-5 but "Ag dengue" in tables 6-7?) is neither intuitive nor explained. DENV IgG in combination with positive IgM might signify non-primary infection with a serotype that is heterologous to that of the previous infection. However this is not discussed and such heterologous non-primary DENV infections are not identified.

As currently written, most of the results section simply restates information presented in the tables without providing additional interpretation. The tables can be presented in a more concise manner. There are seven tables, several of which (Tables 4 and 5 in particular) may be summarized in a sentence in the text or submitted as supplementary materials (see specific comments below).

Since this manuscript is focused on DENV and associated co-infections, consider restructuring the results section to report DENV incidence first, then the co-infections in the context of DENV infection.

Reviewer #2: See below

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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: The authors appropriately highlight the public health relevance, that DENV infection occurs in children in Cameroon, frequently concurrently with either malaria and/or HIV, and that additional investigation of the effect of concurrent infection on disease presentation is warranted. However, discussion of associations between DENV and co-infection, which perhaps are the most interesting findings of the study, is superficial. Finally, the conclusion ends on speculation about disease severity, which is not addressed in this manuscript.

Reviewer #2: See below

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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: Specific comments:

1. Lines 54-55 are redundant

2. Line 60: There is a licensed vaccine (Dengvaxia, Sanofi-Pasteur)

3. Lines 86-87: During which year(s) was the study performed? Also, the statement regarding transmission during the rainy months requires a reference.

4. Consider providing the questionnaire in supplementary materials.

5. Line 92: Please specify a reference for the ELISA if this was an in-house assay, or the manufacturer if this was a commercial assay.

6. Line 99: Please clarify what is a "positive" urinary test?

7. Line 115: Stating the upper age range as 180 months followed by the mean in years is awkward. Consider referring the upper limit as 15 years.

8. Line 117: Please clarify what "living in an environment with stagnant water" means.

9. Lines 128-129: 54 girls + 55 boys = 109 children. What about the other 74 children with malaria? Also Table 2 is referenced but does not include these data.

10. Table 2: Dengue (Overall cases) is reported as 101, while DENV co-infections were 15 + 68 + 21 = 104. All DENV cases were co-infections? Please clarify.

11. Table 2: Reporting of the CD4 count is poorly defined. Is this the mean or median? What does "Min:240-400" mean? Consider reporting this in the text instead of in the table, since the remainder of the table reports frequencies.

12. Lines 123-126 and table 2: Since this manuscript focuses on DENV, malaria, and HIV infection, consider reporting symptoms associated with these infections rather than the frequency of symptoms of the entire cohort.

13. Line 137: Is this the DENV case definition? What about the PCR testing referenced in the methods?

14. Table 4: Consider making this a supplementary table or excluding it altogether. The lack of association of any of the factors with sex can be concisely stated in the text. Additionally, the reference group should be identified when reporting odds ratios.

15. Table 5: Please clarify the reference group for odds ratios. Is it the odds of having a particular test result for the specified age group over the odds for all other ages? Or is there a reference group? Consider making this a supplementary table or excluding it altogether since the lack of association with age can be stated in the text.

16. Lines 206-207: Please clarify the statement regarding susceptibility to infection and mother-to-child transmission.

17. Line 224: The principal targets of DENV infection are monocytes/macrophages/dendritic cells, whereas the principal targets of HIV infection are T lymphocytes. These are not the same cells.

18. Line 231: Malaria and DENV are transmitted by mosquitoes, not flies.

19. Lines 232-234: These statements are unsupported. DENV and malaria are transmitted by different mosquito vectors with different feeding habits (daytime vs. night time). Thus exposure is inherently different.

Reviewer #2: See below

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Summary and General Comments

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

Reviewer #1: This manuscript reports the incidence of dengue virus (DENV) infection among children in Cameroon, along with associated co-infection with malaria and HIV. Given the paucity of data on DENV in Cameroon, the data offer an important contribution toward advancing knowledge of DENV in Africa. Unfortunately, this manuscript is disorganized and presents the data in a confusing fashion. The analyses are superficial and/or incomplete. These shortcomings detract from the potential impact of the study.

Reviewer #2: The authors describe results of an enhanced passive surveillance study wherein children aged 6 months to 15 years presenting with undifferentiated fever were enrolled and tested simultaneously (I believe) for malaria, dengue, and HIV. The authors note that co-infections may be missed using standard testing practices. The results are quite interesting and could represent an important contribution to the literature regarding the clinical overlap between dengue, malaria, and HIV. However, the manuscript would benefit from increased precision in presenting results and discerning current and prior dengue infection based upon serological results.

MAJOR COMMENTS

- Please place the study type in the abstract and title (presumably enhanced passive surveillance?). Provide further details about the methods and very basic descriptive statistics (number enrolled, age, etc,) in abstract.

- I don’t know that it is reasonable to conclude that DENV IgG-positive alone represents ‘dengue-infected’ – they may have had historical infection. I do agree that DENV IgM is more likely to be DENV (but may also represent ZIKV or some other cross-reactive flavivirus). I also agree that NS1-positive for DENV = acute infection. Similarly, for ‘co-infections’ I would not report that malaria smear + / DENV IgG + represents a co-infection.

- Please be more precise in your presentation of findings – for example, in discussion line 209 – our findings showed a prevalence of 52.7% of malaria cases. You found that 52.7% of children presenting for care with acute undifferentiated fever had evidence of malaria sporozoites (correct?).

- Please comment on how this testing algorithm differs from common practice in Cameroon (I’m guessing that malaria testing would be the first step, stop if positive?) and how your findings may indicate that an expanded approach may be needed.

- Relatedly, please comment on ‘next steps’ needed to better understand the clinical presentation and epidemiology of dengue and malaria / HIV coinfections in Cameroon. Your findings are very interesting and suggest that there may be opportunities to better understand how clinical severity and complications of dengue infection may be changed in the setting of co-infection.

MINOR COMMENTS

- Please add ‘Cameroon’ and the general study type to the full title

- Introduction.

o Line 60. There is in fact a licensed vaccine for dengue available in many countries (though not, to my knowledge, in Cameroon), however there are restrictions on the use given safety concerns.

- Has there been an entomological survery done in Cameroon to document the geographic range of Aedes aegypti and Aedes albopictus? If so, would be good to mention. If not, would also be good to mention this knowledge gap.

- Would refer to DENV ‘serotypes ’ 1 and 2

- Please clarify that HIV, malaria, and dengue testing was performed for all individuals?

- Was there a time limit for the duration of fever? (I.e., less than 10 days?)

- Did you ask if the children slept under a bednet during the day? This is the biting period for Aedes mosquitoes

- For table 1, the percents for stagnant water don’t add up to 2100%

- Table 2: CD4 counts – is this the median?

- Results, would change the ORs so that they reflect your description of results (e.g., line 155, ‘were less at risk of being infected’ – change OR to be (1/8.85)

- Tables 6 and 7 seem to be mixed up in the text

- Tables 6-7 – does ‘Ag dengue’ refer to ‘any immunoglobulin, IgM or IgG, to DENV’?

- Table 6 – Does VIH refer to HIV?

- For all tables, footnotes defining abbreviations and the relevant diagnostic tests would be valuable

- It is unclear that is meant in line 206 about ‘findings are supported by reports showing more susceptibility to infection and cases of mother to child transmission of dengue virus’

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

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Reviewer #2: No

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

16 Jul 2020

Submitted filename: Reply to reviewers.docx

Click here for additional data file.

9 Sep 2020

Dear Dr. Nkenfou,

Thank you very much for submitting your manuscript "ENHANCED PASSIVE SURVEILLANCE OF DENGUE INFECTION AMONG FEBRILE CHILDREN: PREVALENCE, CO-INFECTIONS AND ASSOCIATED FACTORS IN CAMEROON" 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:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

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

Important additional instructions are given below your reviewer comments.

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

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

Sincerely,

Hannah E Clapham

Associate Editor

PLOS Neglected Tropical Diseases

Emily Gurley

Deputy Editor

PLOS Neglected Tropical Diseases

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

In addition to Reviewer 2 comments below, the AE has the following additional comments to help with the editing as suggested by Reviewer 2. This paper details an important study, but the analysis and the description of the results requires substantial editing.

Abstract:

Severe disease is generally refered to as severe dengue at the moment: https://www.who.int/news-room/fact-sheets/detail/dengue-and-severe-dengue.

“Dengue overall seroprevalence was 29.1%. Malaria cases were 52.7% and HIV cases represent 12.6%.” this line is unclear suggest rephrasing, seroprevalence suggests past infection, where as malaria and HIV are refered to as cases- please clarify what was being assessed.

“But malaria either due to P vivax or P

36 falciparum was associated to dengue infection, P=0.001 and 0.003 respectively. Likewise, HIV was

37 significantly associated to dengue p=0.028.” It is unclear what is being assessed here- suggest rephrasing.

Author summary:

“Dengue

45 fever, transmitted by a mosquitoe as well as malaria is common among Cameroonian children.” Suggest rephrasing.

Introductions:

“Both infections share similar clinical symptoms” requires reference.

There is now a vaccine for dengue- https://www.who.int/immunization/diseases/dengue/revised_SAGE_recommendations_dengue_vaccines_apr2018/en/

Results:

P9 Line 137: Rephrase to a complete sentence.

Similar points throughout results, please ensure all sentences are complete sentences.

What are the (%) in Table 3? This is currently very unclear, as is the interpretation from this table for when the peak transmission occurred. Much clarification needed.

What are the (%) in Tables 5 and 6? This requires clarification/editing.

LIne 182: I think the reference to table 6 and 7 are reversed. In addition, please provide further description of the co-infection relationship. At the moment in the text it is hard to understand what is meant.

I agree with reviewer 2, there needs to be further clarity on the past dengue infections and current dengue infections definition. In addition, how was a co-infection with dengue and the other pathogens defined? I think this should be current dengue infection, but it is not clear if this was how it was defined. This needs additional care and clarity for dengue, due to the acute nature of the infection and that the test used measure IgG and IgM (and clarity is needed on the interpretation of IgG positivity in this assay). Table 2 and the discussion of the results in the text needs to be clearer.

Similarly, in the discussion what is meant by “prevalence” is this the % of febrile cases that are current dengue, or past dengue? I don’t think either is the “prevalence”. Please rephrase.

“Our findings are supported by reports showing more susceptibility to

207 infection and cases of mother to child transmission of dengue virus 16-17.” Please be clear which of your results are supported by this.

The dengue- HIV co-infection discussion requires clarification.

“the common target cells”. Please state the target cells, and provide references.

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: See below

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

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: See below

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

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: See below

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

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: See below

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

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 manuscript has been improved from the prior submission; the authors’ thoughtful revisions are much appreciated. The discussion, in particular, is more solid. The classification of dengue illnesses as past and current remains a confusing aspect of the manuscript, and multiple small typological errors / areas for further clarification remain.

General comments:

- Not necessary to separate out IgM+ / IgG- and IgM+ / IgG+, this makes the presentation of results confusing. The timeline of specimen collection relative to onset of symptoms may influence whether IgM alone, IgM/IgG or IgG alone were detected but it is safe to say that detectable IgM+ is suggestive of acute / recent DENV infection. IgG+ alone suggests prior exposure. This simplification of presentation alone would clarify the abstract, results, table 2, and discussion.

- Relatedly, please comment on the timing of specimen collection relative to the onset of symptoms (median and IQR). Perhaps add to table 1.

- The manuscript would benefit from a detailed review to correct typological errors and general flow of language. This reviewer has pointed out a few specific areas to improve the flow of language below, but multiple other small typos remain. (e.g., abstract, line 40 ‘pic’ should be ‘peak’; Line 137, ‘with minimum been 1 day’ should be ‘with a minimum of 1 day’)

Minor comments:

- It is standard convention to define dengue virus as DENV, then use it throughout to refer to serotypes. ‘dengue’ is fine to use when describing dengue illnesses.

- The use of the term ‘prevalence’ is applied to very distinct concepts, i.e., % of febrile illnesses identified as dengue, seroprevalence. For percent of illness cases identified as dengue, perhaps move away from prevalence and describe as ‘the proportion of febrile illnesses identified as dengue’, for example.

- Line 70. Dengvaxia may prevent some infections but the major contribution appears to be a reduction in hospitalized illness, albeit with an increase in the incidence of severe disease among young, dengue -naïve children.

- Line 136 and Table 1. Water flasks or stagnant water? Does water flasks mean ‘containers’?

- Lines 142-146. For each of these infections, are symptoms reported for single infections (i.e., HIV alone, no dengue or malaria) or for a given infection regardless of coinfections? Consider a supplementary table showing symptoms for each virus alone, coinfections, comparing severity and clinical presentation.

- Lines 166-167 is very confusing. Hopefully can be simplified as in first general comment above.

- Table 2. Please be explicit in table about the fact that you are considering acute dengue (not prior dengue) in coinfection estimates (as you have added to the text).

- Table 3 – perhaps turn into a figure showing percents by month?

- Supplemental table 4. Please indicate what is the referent group for the odds ratios (i.e., are the odds of IgG lower in females?)

- Supplemental table 4. Please indicate what is the referent group for age the odds ratios i.e., is it age 2-5 years compared to all other age groups? You may consider making 0-2 the referent group for subsequent age groups)

- Line 290. This sentence about viral infections being more severe in children could be softened. Chickenpox, for example, is more severe in adults.

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

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Reviewer #2: No

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

Data Requirements:

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

Reproducibility:

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

23 Oct 2020

Submitted filename: Response to Reviewer comments.docx

Click here for additional data file.

13 Dec 2020

Dear Dr. Nkenfou,

Thank you very much for submitting your manuscript "ENHANCED PASSIVE SURVEILLANCE OF DENGUE INFECTION AMONG FEBRILE CHILDREN: PREVALENCE, CO-INFECTIONS AND ASSOCIATED FACTORS IN CAMEROON" 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. Reviewers continue to believe that the topic of your paper is relevant and of interest to our audience, but this revision did not satisfactorily address comments noted on the first submission.

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

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

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

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

Important additional instructions are given below your reviewer comments.

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

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

Sincerely,

Hannah E Clapham

Associate Editor

PLOS Neglected Tropical Diseases

Emily Gurley

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: See below

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

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: See below

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

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: See below

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

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: See below

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

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 made some significant improvements in the precision with which results are presented compared to the previous drafts. However, significant ambiguity and inconsistency remain with the inter-mixing of how dengue ‘cases’ are defined (for example, how IgM and IgG results are interpreted). Further, numerous typological / grammatical errors remain.

MAJOR COMMENTS.

- There remains some mixing of definitions of a dengue ‘case’ or dengue-infected individual. For example, in Table 2, a case seems to be defined as IgG or IgM (+). This is in contradiction to the primary aim of the study, which is to define ‘the prevalence’ (incidence?) of dengue in a cohort of children presenting with fever. IgG would reflect past infection (seroprevalence, not necessarily active infection) while IgM would reflect current or recent infection. Table 6, IgG antibodies are not necessarily suggestive dengue infection (as in the table heading) and it is confusing to consider them as such in the table. For the purposes of the study, would define a dengue ‘case’ as IgM only and use these definitions more consistently.

- Relatedly, please be consistent with your definition of ‘seroprevalence’ – Discussion, lines 196-197 – authors use seroprevalence to refer to both IgM and IgG. This is confusing and, again, reflects a mixing of definitions for acute and historical infection.

- The authors may switch to using terms such as ‘the proportion of children presenting with fever who were found to have acute dengue infection was XX’ rather than ‘the prevalence of dengue’ – this is an acute infection (in contrast to malaria) and the over-reliance on the term throughout the manuscript to describe acute infections in the study is confusing when inter-mixed with references to seroprevalence.

INTRODUCTION:

- Lines 53-54: recommend shortening to “dengue…is the most prevalent mosquito-borne viral disease worldwide and is described as a ‘neglected’….”

- Line 59-60. There is a licensed dengue vaccine available in many countries, but safety concerns and screening requirements limit its widespread use and impact

- Line 65-66: A study of prevalence and distribution – looking at what population? Adults / children / all presenting with fever? This influences interpretation.

- Line 66: A study conducted in 2009 – what kind of study?

- Line 68: What type of climate? (Tropical? Subtropical?)

- Line 72. Two serotypes identified (DENV-1 and DENV-2)

- Lines 75-76 – would make very clear: the objective of the study was to determine…and associated risk factors in a pediatric population presenting with fever in Yaounde, Cameroon.

METHODS:

- Years of the study? Duration of the study? April to October of one year?

- Any limits to the duration of fever for inclusion criteria? (i.e., within 7 days of onset of fever?)

- Lines 102-103: Please refine sentence such as “Infants were enrolled only when their parent or guardian gave written proxy-consent” – but was this true only for infants? Assent obtained from older minors?

- Lines 134-138. Would move these definitions to the methods. Please rephrase “Were considered exposed” and “Were considered dengue infected” to be something like “Therefore, current or recent dengue infection was defined on the basis of a positive result for NS1 Ag and / or IgM.”

RESULTS:

- Clinical characterization: please provide information on the duration of fever, if known

- Line 123: “Highest temperature was 41”, “Average temperature was…”

- Line 123: Did you collect data only on active symptoms, or history of symptoms? For example, “12.6% reported a history of vomiting” versus “12.6% reported current symptoms of vomiting at the time of enrollment”

- Line 141.” The average CD4 count was 969 cells/mm3” “Children have a lower CD4 count were more likely to be positive”(Statistical test used, p value?)

- Line 144. “The rate of Malaria – dengue coinfection was 19.5%”

- Line 145. “4.3% of the study population had evidence of current or recent dengue infection, as well as both malaria and HIV” (is this correct?

- Line 152. What does “small raining season mean” – it was a low year for rainfall?

- Table 4. If the lowest age group is referent, why is the OR not 1?

- Table 5. What is referent? Presumably, the absence of the other infection?

- Table 6. Presence of dengue IgG antibody is not consistent with your definition of ‘dengue infection’ rather ‘prior dengue exposure’. Please consider removing this comparison from the table – why would we think that historical exposure to dengue would be associated with diarrhea, for example?

- Lines 178-179. As above.

DISCUSSION:

- Lines 196-197. Confusing to use seroprevalence to refer to both IgM and IgG.

- Line 205. Please comment specifically on how your methodology may underestimate coinfection compared to the prior study.

- Line 214. Recommend you remove the sentence beginning “In such setting”

- Line 216. “Viral coinfections”

- Line 219. Capitalize Aedes and Anopheles

- Line 224-225. Aedes and Anopheles don’t exactly breed in the same conditions, but both may be increased during rainy seasons and thus may co-occur temporally

- Line 226-227. Please refine final sentence, it is a little confusing

- Please comment on the finding that sleeping under a bednet or living in an area without stagnant water was protective against dengue. These aren’t classically thought of as protective against Aedes – is this reflective of some other, related risk factor? (Socioeconomic status, etc?)

- You may consider commenting that the seroprevalence increased steadily with age (in fact, this may be a good figure to include, perhaps combining tables 3 and 4 to make space for a new figure) – this suggests that transmission is somewhat high and endemic (rather than epidemic, where we may be more even distribution across ages)

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

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Reviewer #2: No

Figure Files:

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

Data Requirements:

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

Reproducibility:

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

11 Jan 2021

Submitted filename: Responses to Reviewers comments on R2 final.docx

Click here for additional data file.

23 Feb 2021

Dear Dr. Nkenfou,

Thank you very much for submitting your manuscript "ENHANCED PASSIVE SURVEILLANCE OF DENGUE INFECTION AMONG FEBRILE CHILDREN: PREVALENCE, CO-INFECTIONS AND ASSOCIATED FACTORS IN CAMEROON" 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.

We thank the authors for their revisions.

There remain some sections of the manuscript that are still in need of refinement. Please see the comments at the end of this email message.

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,

Hannah E Clapham

Associate Editor

PLOS Neglected Tropical Diseases

Emily Gurley

Deputy Editor

PLOS Neglected Tropical Diseases

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

We thank the authors for their revisions.

There remain some sections of the manuscript that are still in need of refinement. Please see the comments below.

"Past" not "passed"

The following is still not a full sentence, please edit to a full sentence, for example “The highest temperature observed was…etc”: Highest temperature was 41°C, lowest temperature was 37.8°C and the average temperature was 38.9°C.

“Children having the lower CD4 count (240-400 CD4 cells/mm3) were more likely to be positve for malaria (20% compared to 10.8%; X2=0.71, p=0.4), HIV (40% compared to 0%; X2=31.08; p<0.001) or dengue (20% compared to 5.3%; X2 = 2.96, p=0.085) .” This only appears to be positive to HIV, so I would suggest rephrasing these results as it currently reads like you are suggesting that there are relationships with all HIV, dengue and malaria.

“the reason being that they are less at risk of been bitten by flies”. This should be in the discussion, and the word flies needs to be replaced with either mosquito or vector. Similar for later reference to “flies”.

“Stagnant water provide breeding conditions for Anopheles and Aedes.” This also should be in the discussion.

“IgG represents cases of previous exposure to dengue” I would suggest removing “cases of” here for clarity.

Discussion:

Please add to the comment about bednets being protective against dengue too, as bednets are generally thought to have less impact on dengue, due to the day biting of dengue transmitting mosquito. As the review suggested, could this also be a soci-economic factor or something else here?

“Among the children enrolled, 12.6% were HIV infected. Cameroon is said to be in a generalized HIV epidemic, although with decreasing prevalence, from 5.5 to 3.4% 21-23. Pediatric HIV is also a major public health problem although transmission is reduced due to the implementation of option B+ (Treat all HIV infected pregnant or breastfeeding mother despite CD4 count or viral load) for the prevention of 235 mother to child transmission 24. The percentage of HIV obtained in this study is in agreement with the national prevalence at the time of the study. Our study identified 21 out of 349 cases of dengue-HIV co infections, thus a prevalence of 6%.” I don’t understand the two % for HIV prevalence here- please rephrase.

“The result obtained in the present study is higher than the 9.8% previously reported by Demanou et al in Yaoundé among adults 12.” Please give more details on this study and which of the previous reported results from your study this is being compared to.

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:

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Reproducibility:

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References

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4 Mar 2021

Submitted filename: Responses to Reviewers comments.docx

Click here for additional data file.

16 Mar 2021

Dear Dr. Nkenfou,

We are pleased to inform you that your manuscript 'ENHANCED PASSIVE SURVEILLANCE OF DENGUE INFECTION AMONG FEBRILE CHILDREN: PREVALENCE, CO-INFECTIONS AND ASSOCIATED FACTORS IN CAMEROON' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

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

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

Best regards,

Hannah E Clapham

Associate Editor

PLOS Neglected Tropical Diseases

Emily Gurley

Deputy Editor

PLOS Neglected Tropical Diseases

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

3 Apr 2021

Dear Dr. Nkenfou,

We are delighted to inform you that your manuscript, "ENHANCED PASSIVE SURVEILLANCE OF DENGUE INFECTION AMONG FEBRILE CHILDREN: PREVALENCE, CO-INFECTIONS AND ASSOCIATED FACTORS IN CAMEROON," 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.

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

Best regards,

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases